Current Topics in Membranes and Transport
Volume 18
MEMBRANE RECEPTORS
Advisory Board
M . I? Blaustein A . Essig
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Current Topics in Membranes and Transport
Volume 18
MEMBRANE RECEPTORS
Advisory Board
M . I? Blaustein A . Essig
R . K . H . Kinne I? A. Knauf Sir H . L. Kornberg
P. Lauger C. A . Pasternak W. D. Stein W. Stoeckenius K. J . Ullrich
Contributors
Michael C. Lin Suzanne K. Beckner Robert Dale Brown B. Richurd Martin Steen E. Pedersen Brian A. Cooke Dermot M . E Cooper John I? Perkins W. D. Rees Manjitsri Das Elliott M . Ross Vincent A. Florio J . Gliemann Paul Schlesinger Larry M . Gordon Virginia Shepherd Jeffrey M . Stadel Miles D. Hoidslay Serge Jard Philip Stahl Duvid A . Johnson Palmer Taylor Robert J . Leflowitz A . M . Tolkovsky Simon van Heyningen
Current Topics in Membranes and Transport Edited by
Arnost Kleinzeller Department of Physiology University of Pennsylvania Philadelphiu. Pennsylvania
Volume 18
MEMBRANE RECEPTORS Guest Editors
Arnost Kleinzeller
B. Richard Martin
Department of Physiology University of Pennsylvania Philadelphia, Pennsylvania
Department of Biochemistry Uniwrsitv of Cumbridge Cumbridge. England
1983
@
ACADEMIC PRESS
A Siihlidion of Hon owi Bruce J o ~ u n oiii h Pihlidirr,
New York London Paris San Diego San Francisco Slo Paulo Sydney Tokyo Toronto
COPYRIGHT @ 1983, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS,INC.
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United Kingdom Editiott pubtisfzed by ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road. London NWl7DX
LIBRARY OF CONGRESS CATALOG CARD NUMBER: 70-1 17091 ISBN 0-12-153318-2 PRINTED IN THE UNITED STATES OF AMERICA 83 84 85 86
9 8 1 6 5 4 3 2 1
Contents Contributors, xi Preface, xiii Yale Membrane Transport Processes Volumes, xv Contents of Previous Volumes, xvii
PART I .
ADENYLATE CYCLASE-RELATED RECEPTORS
Hormone Receptors and the Adenylate Cyclase System: Historical Overview
B. RICHARD MARTIN Text. 3 References. 8
The Elucidation of Some Aspects of Receptor Function by the Use of a Kinetic Approach A. M. TOLKOVSKY
I. Introduction, II 11. Signal-Response Coupling and Receptor Theory, 13 111. Applying Kinetic Theory to Data Generated by Turkey Erythrocyte Adenylate Cyclase. 22 IV. Conclusions, 40 References. 43
The p-Adrenergic Receptor: Ligand Binding Studies Illuminate the Mechanism of Receptor-Adenylate Cyclase Coupling
JEFFREY M . STADEL and ROBERT J. LEFKOWITZ
I. 11. Ill. IV.
Introduction. 45 Development of Radioligands Specific for Adrenergic Receptors, 47 Study of Adrenergic Receptors in Membranes. 49 Characterization of Detergent-Solubilized Adrenergic Receptors, 57 References. 63 V
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CONTENTS
Receptor-Mediated Stimulation and Inhibition of Adenylate Cyclase
DERMOT M. F. COOPER I. 11. 111. IV.
V. VI. VII. VIIl. IX. X.
Introduction. 67 Stimulation of Adenylate Cyclase, 68 GTP-Dependent Inhibition of Adenylate Cyclase. 70 Bimodally Regulated Adenylate Cyclase Systems, 71 Receptor Binding of Inhibitory Ligands, 73 The Role of GTP Hydrolysis in Inhibition of Adenylate Cyclase, 75 The Relationship between N, and N,, 76 Structural Studies on Dually Regulated Adenylate Cyclase Systems, 77 Future Directions. 78 Conclusion, 81 References, 8 I
Desensitization of the Response of Adenylate Cyclase to Catecholamines
JOHN I? PERKINS 1. Introduction. 85 11. Scope of the Review, 87 111. Catecholamine-Induced Desensitization of Intact Cells, 88 IV. Catecholamine-Induced Changes in Adenylate Cyclase and in PAR Binding Properties, 93 V. Separation of Native and Desensitized PAR, 95 VI. Receptor Endocytosis as a Mechanism for Agonist-Induced Desensitization, 97 V11. A Kinetic Model for Agonist-Induced Desensitization, 98 VIII. Differential Expression of PAR during Growth of 1321NI Cells, 100 IX. Down-Regulation of PAR and the Recovery of Lost Receptors. 100 X. Isoproterenol-Induced Changes in Agonist Binding Properties of Intact 1321N1 Cells, 103 XI. Conclusions, 104 References. 106
Hormone-Sensitive Adenylate Cyclase: Identity, Function, and Regulation of the Protein Components
ELLIOTT M. ROSS, STEEN E. PEDERSEN, and VINCENT A. FLORIO
I. 11. 111. IV.
Overview, 109 Protein Components of Hormone-Sensitive Adenylate Cyclase, I 10 Protein-Protein Interactions and the Regulation of Adenylate Cyclase, 127 Assessment of Progress, 137 References, 137
CONTENTS
The Regulation of Adenylate Cyclase by Glycoprotein Hormones
BRIAN A. COOKE
I. Introduction. 143 11. Nature of the Hormones. 144
111. Nature of the Receptors, 145 IV. Involvement of Cyclic AMP in Hormone Action. 149 V. Important Features of the Hormone Receptor-Adenylate Cyclase System, 151 VI. Desensitization and Down-Regulation by Homologous Hormone, 152 References. 172
The Activity of Adenylate Cyclase Is Regulated by the Nature of Its Lipid Environment
MILES D. HOUSLAY and LARRY M . GORDON 1. Structure of Biological Membranes. 180 11. Structural Aspects of Hormone Receptor-Adenylate Cyclase Interaction. 183 111. Membrane Fluidity as a Regulator of Adenylate Cyclase Activity, 185 1V. Selective Modulation of Adenylate Cyclase by Asymmetric Perturbations of the Membrane Bilayer. 20X V. Phospholipid Headgroup Composition and Adenylate Cyclase Activity. 223 VI. Disease States. 225 References. 226
The Analysis of Interactions between Hormone Receptors and Adenylate Cyclase by Target Size Determinations Using Irradiation Inactivation
B. RICHARD MARTIN 1. Irradiation Inactivation: General Considerations, 233 11. Practical Considerations in irradiation Inactivation Sttidies
on Membranes, 236 111. Analysis of Data, 237
IV. The Application of Target Size Analysis to Rat Liver Plasma Membrane Adenylate Cyclase, 239 V. Model of Hormone Action. 241 VI. Effects of Fluoride, 246 V11. Evaluation of the Model in Relation to the Results of Other Approaches. 248 References, 253
vi i
...
CONTENTS
Vlll
PART 11.
RECEPTORS NOT INVOLVING ADENYLATE CYCLASE
Vasopressin lsoreceptors in Mammals: Relation to Cyclic AMP-Dependent and Cyclic AMPIndependent Transduction Mechanisms SERGE JARD I. Introduction, 25.5 11. Methodological Basis for the Characterization of Vasopressin Isoreceptors, 256 111. Kinetics of Hormone Binding to Vasopressin Receptors, 259 1V. Transduction Mechanisms Triggered by Vasopressin Receptors, 265 V. VI. VI1. VIII.
Effects of Nucleotides and Other Putative Effectors on Vasopressin Receptors, 272 Physicochemical Characteristics of Solubilized Vasopressin Receptors, 274 Recognition Patterns of Vasopressin Isoreceptors, 275 Summary and Conclusions. 279 References. 280
Induction of Hormone Receptors and Responsiveness during Cellular Differentiation MICHAEL C. LIN AND SUZANNE K. BECKNER 1. Introduction, 287 11. Model Systems, 290 111. Conclusion, 307 References, 310
Receptors for Lysosomal Enzymes and Glycoproteins VIRGINIA SHEPHERD, PAUL SCHLESINCER, and PHILIP STAHL
I. Introduction, 317 11. The Phosphomannosyl Recognition Pathway, 3 19 111. Role of Oligosaccharide Moiety in Recognition of Extracellular Lysosomal
Enzymes and Glycoproteins, 323 IV. Lysosomal Enzymes and the Mannosyl Recognition System, 324 V. Receptor-Mediated Endocytosis of Glycoconjugates, 327 VI. Conclusion, 335 References, 335
The Insulin-Sensitive Hexose Transport System in Adipocytes
J. GLIEMANN and W. D. REES
I. Summary of the Present Status, 339 11. Historical Background, 340
CONTENTS
111. IV. V. V1.
VII. VIII. IX. X. XI. XII. XIII. XIV.
Critical Steps in the Methodology, 342 Kinetic Approaches to the Study of Hexose Transport. 348 Transport of Nonmetabolizable Sugars and Sugar Analogs in the Adipocyte, 355 The Requirements for D-Glucose Binding to t h e Adipocyte Hexose Transport System, 359 Nontransported Competitive Inhibitors of Transport. 360 Sugars Which Are Both Transported and Phosphorylated-Rate-Limiting Steps. 362 Modulation of the Transport System by Glucose Metabolites, 366 Mechanism of Insulin’s Ability to Increase V,,,, 367 Human Adipocytes. 371 The Transport System in Obesity and Diabetes, 371 Reconstitution of the Hexose Transporter, 372 Concluding Remarks, 373 References, 373
Epidermal Growth Factor Receptor and Mechanisms for Animal Cell Division MANJUSRI DAS I. 11. 111. IV. V.
Introduction, 381 Properties of EGF, 382 The EGF Receptor, 383 The Pathway to Nuclear DNA Replication. 393 A Family of EGF-like Polypeptides and Their Role in Animal Development and Growth, 398 References. 400
The Linkage between Ligand Occupation and Response of the Nicotinic Acetylcholine Receptor PALMER TAYLOR. ROBERT DALE BROWN, and DAVID A. JOHNSON
I. Introduction, 407 11. Structure of the Isolated Receptor, 408 111. Biophysical Properties of the Receptor Channel, 412 IV. The Behavior of Partial Agonists, Antagonists, and Anesthetics in Relation to Channel Activation. 414 V. Desensitization of the Receptor, 415 VI. Ligand Occupation and Transitions in Receptor State, 416 V11. Other Ligands Affecting Receptor Function, 423 VIII. Analysis of Receptor Activation. 425 1X. Toward the Understanding of Coupling between Occupation of the Receptor and the Permeability Response. 426 X. Occupation and Activation by Agonists. 427 XI. Association of Antagonists with the Receptor and Functional Antagonism, 430 XII. Quantitation of Antagonist Occupation and Functional Antagonism, 434
ix
CONTENTS
X
X111. Structural Implications and Arrangement of Subunits, 435 XIV. Analysis of the Bound Ligand States. 437 References, 438
The Interaction of Cholera Toxin with Gangliosides and the Cell Membrane
SIMON VAN HEYNINGEN I. Structure and Action of Cholera Toxin, 446 11. The Role of Ganglioside GMl as a Cell-Surface Receptor, 450 111. The Nature of the Reaction between Ganglioside and Toxin, 455
IV. Transport of Cholera Toxin across the Cell Membrane and the Role of Binding to Ganglioside. 460 V. Other Compounds That Bind to Gangliosides, 465 References, 466
Index, 473
Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin. Suzanne K. Beckner, Laboratory of Cellular and Developmental Biology, National Institute of Arthritis. Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda. Maryland 20205 (287) Robert Dale Brown, Division of Pharmacology. Department of Medicine, University of California, San Diego, La Jolla. California 92093 (407) Brian A. Cooke, Department of Biochemistry, Royal Free Hospital School of Medicine, University of London. London WClN IBE England (143) Dermot M. F. Cooper,' Section on Membrane Regulation. Laboratory of Nutrition and Endocrinology, National Institute of Arthritis. Diabetes. Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205 (67) Manjusri Das, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine. Philadelphia. Pennsylvania 19104 (381) Vincent A. Florio, Department of Pharmacology, University of Texas Health Science Center at Dallas. Dallas, Texas 75235, and Department of Pharmacology, University of Virginia, Charlottesville, Virginia (109) J. Gliemann, Institute of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark (339) Larry M. Gordon, California Metabolic Research Foundation. La Jolla. California 92038, and Rees Stealy Research Foundation. San Diego, California 92101 (179) Miles D. Houslay, Department of Biochemistry. University of Manchester Institute of Science and Technology, Manchester, England. and California Metabolic Research Foundation, La Jolla, California 92038 (179) Serge Jard, Centre CNRS-INSERM de Pharmacologie-Endocrinologie, 34033 Montpellier Cedex. France (255) David A. Johnson, Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla. California 92093 (407) Robert j. Lefkowitz, Department of Medicine (Cardiology), Howard Hughes Medical Institute, Duke University Medical Center, Durham. North Carolina 27710 (45) Michael C. Lin, Laboratory of Cellular and Developmental Biology, National Institute of Arthritis. Diabetes. Digestive and Kidney Diseases. National Institutes of Health, Bethesda, Maryland 20205 (287) B. Richard Martin, Department of Biochemistry. University of Cambridge. Cambridge CB2 IQW, England (3. 233) Steen E. Pedersen, Department of Pharmacotogy, University of Texas Health Science Center at Dallas, Dallas. Texas 75235, and Department of Biochemistry, University of Virginia. Charlottesville, Virginia (109)
Present address: Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262. xi
xii
CONTENTS
John P. Perkins, Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27514 (85) W. D. Rees, Institute of Physiology. University of Aarhus. DK-8000 Aarhus C, Denmark (339) Elliott M. Ross, Department of Pharmacology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 (109) Paul Schlesinger, Department of Physiology and Biophysics, Washington University School of Medicine. St. Louis. Missouri 631 10 (317) Virginia Shepherd, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 (317) Jeffrey M. Stadel, Department of Medicine (Cardiology), Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 (45) Philip Stahl, Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 631 10 (317) Palmer Taylor, Division of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, California 92093 (407) A. M. Tolkovsky, Department of Pharmacology. Hadassah Medical School, The Hebrew University, Jerusalem, Israel ( I I) Simon van Heyningen, Department of Biochemistry, University of Edinburgh, Edinburgh EH8 9XD, Scotland (445)
Preface The articles in this volume are intended to provide a survey of current ideas about the mechanisms by which plasma membrane receptors function. Part I deals with hormone receptors that modulate the activity of adenylate cyclase and, hence, the concentration of the second messenger cyclic 3',5'-AMP inside the cell. The discovery of hormone-sensitive adenylate cyclase in the early 1960s by Sutherland and his colleagues was perhaps the single most important advance in our understanding of the mechanism of hormone action. It led to an explanation of the action of a large class of hormones. It also led to the formulation of the second messenger hypothesis, which was an important concept in the study of hormone action in general. We now know a great deal about the mechanisms by which hormones activate adenylate cyclase. Rodbell and his colleagues showed that the activation requires the presence of GTE and it has since been shown, notably by the work of Gilman and his colleagues, that the effects of GTP and other guanine nucleotides are mediated through a separate protein subunit distinct from the hormone receptor and the catalytic subunit. Thus the system contains at least three components, and the major focus of interest at the present time is to determine precisely how these three components interact to modulate adenylate cyclase activity. More recently it has been shown that certain hormones and other agents, such as prostaglandins, directly inhibit adenylate cyclase in some tissues. It has also been found that prolonged exposure to activator hormones can lead to a loss of response, a process commonly referred to as desensitization. These effects are less well characterized than the activation process. The aim of Part I is to provide an overview of a single system in some depth. The aim of Part I1 is to give an impression of the diversity of mechanisms by which membrane receptors are thought to act. We have attempted to cover as many aspects as possible, ranging from the acetylcholine receptor, which functions by altering the membrane ion permeability in a very specific rapid and direct way, to toxins and growth factors, which ultimately enter the cell after binding and require comparatively long periods to act. We hope that the selection of contributors has resulted in a reasonably xiii
xiv
PREFACE
comprehensive overview of both aspects. However, the authors were asked to concentrate primarily on the work of their own laboratories rather than to attempt comprehensive reviews of the literature. As a result there are inevitably many topics equally worthy of consideration that have been omitted. Such deficiencies are entirely the responsibility of the Editors, ARNOSTKLEINZELLER B. RICHARD MARTIN
Yale Membrane Transport Processes Volumes
Joseph F. Hoffman (ed.). (1978). “Membrane Transport Processes,” Vol. 1. Raven, New York. Daniel C. Tosteson, Yu. A. Ovchinnikov, and Ramon Latorre (eds.). (1978). “Membrane Transport Processes,” Vol. 2. Raven, New York. Charles F. Stevens and Richard W. Tsien (eds.). (1979). “Membrane Transport Processes,” Vol. 3: Ion Permeation through Membrane Channels. Raven, New York. Emile L. Boulpaep (ed.). (1980).“Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Trunsport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume I5 of Ciirrcnt Topics in Mernhrcincs rind Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Cirrrent Topics in Mernhrrinrs rind Trrinsport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York.
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Contents of Previous Volumes Volume 1
Volume 3
Some Considerations about the Structure of Cellular Membranes MAYNARD M. DEWEY AND LLOYDBARR The Transport of Sugars across Isolated Bacterial Membranes H. R. KABACK Galactoside Permease of Escherichia cnli ADAMKEPES Sulfhydryl Groups in Membrane Structure and Function ASER ROTHSTEIN Molecular Architecture of the Mitochondrion H. MACLENNAN DAVID Author Index-Subject Index
The Na+, K+-ATPase Membrane Transport System: Importance in Cellular Function ARNOLDSCHWARTZ, GEORGE E. LINDENMAYER, AND JuLius C. ALLEN Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function ANTHONYMARTONOSI The Role of Periaxonal and Perineuronal Spaces in Modifying Ionic Flow across Neural Membranes W . J. ADELMAN, JR. AND Y. PALTI Properties of the Isolated Nerve Endings GEORGINA RODRiGUEZ DE LORES ARNAIZ AND EDUARDO DE ROBERTIS Transport and Discharge of Exportable Proteins in Pancreatic Exocrine Cells: I n Vitro Studies J . D. JAMIESON The Movement of Water across Vasopressin-Sensitive Epithelia M. HAYS RICHARD Active Transport of Potassium and Other Alkali Metals by the Isolated Midgut of the Silkworm AND WILLIAM R. HARVEY KARLZERAHN Author Index-Subject Index
Volume 2 The Molecular Basis of Simple Diffusion within Biological Membranes w. R. LIEBA N D w . D. STEIN The Transport of Water in Erythrocytes ROBERTE. FORSTER Ion-Translocation in Energy-Conserving Membrane Systems AND M. MONTAL B. CHANCE Structure and Biosynthesis of the Membrane Adenosine Triphosphatase of Mitochondria ALEXANDERTZAGOLOFF Mitochondria1Compartments: A Cornpanson of Two Models HENRY TEDESCHI Aurhor Index-Subject Index
Volume 4 The Genetic Control of Membrane Transport W.SLAYMAN CAROLYN xvii
xviii Enzymic Hydrolysis of Various Components in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Regulation of Sugar Transport in Eukaryotic Cells HOWARD E. MORGANA N D CAROL F. WHITFIELD Secretory Events in Gastric Mucosa RICHARD P. DURBIN Author Index-Subjeci Index
Volume 5 Cation Transport in Bacteria: K+, Na+, and H+ FRANKLIN M. HAROLD AND KARLHEINZ ALTENDORF Pro and Contra Camer Proteins: Sugar Transport via the Periplasmic GalactoseBinding Protein WINFRIED Boos Coupling and Energy Transfer in Active Amino Acid Transport ERICHHEINZ The Means of Distinguishing between Hydrogen Secretion and Bicarbonate Reabsorption: Theory and Applications to the Reptilian Bladder and Mammalian Kidney AND WILLIAM A. BRODSKY THEODORE P. SCHILB Sodium and Chloride Transport across Isolated Rabbit Ileum G. SCHULTZ AND STANLEY PETER F. CURRAN A Macromolecular Approach to Nerve Excitation ICHIll TASAKI AND EMILIO CARBONE Subject Index
Volume 6 Role of Cholesterol in Biomembranes and Related Systems MAHENDRA KUMAR JAIN Ionic Activities in Cells A. A. LEVAND W. MCD. ARMSTRONG Active Calcium Transport and Ca*+-Activated ATPase in Human Red Cells H. J. SCHATZMANN The Effect of Insulin on Glucose Transport in Muscle Cells TORBEN CLAUSEN
CONTENTS OF PREVIOUS VOLUMES
Recognition Sites for Material Transport and Information Transfer HALVOR N. CHRISTENSEN Subject Index
Volume 7 Ion Transport in Plant Cells E. A. C. MACROBBIE H+ Ion Transport and Energy Transduction in Chloroplasts AND RICHARD A. DILLEY ROBERT T. G~AQUINTA The Present State of the Camer Hypothesis PAULG . LEFEVRE Ion Transport and Short-circuit Technique S. REHM WARREN Subjeci Index
Volume 8 Chemical and Physical Properties of Myelin Proteins M. A. MOSCARELLO The Distinction between Sequential and Simultaneous Models for Sodium and Potassium Transport P. J. GARRAHAN AND R. P. GAMY Soluble and Membrane ATPase of Mitochondria, Chloroplasts, and Bacteria: Molecular Structure, Enzymatic Properties, and Functions RIVKAPANET AND D. h0 SANADI Competition, Saturation, and InhibitionIonic Interactions Shown by Membrane Ionic Currents in Nerve, Muscle, and Bilayer Systems ROBERT J. FRENCH AND WILLIAM J. ADELMAN, JR. Properties of the Glucose Transport System in the Renal Brush Border Membrane R. KINNE Subject Index
Volume 9 The State of Water and Alkali Cations within the Intracellular Fluids: The Contribution of NMR Spectroscopy AND MORDECHAI SHPORER MORTIMER M. CIVAN
xix
CONTENTS OF PREVIOUS VOLUMES
Electrostatic Potentials at Membrane-Solution Interfaces STUART MCLAUGHLIN A Thermodynamic Treatment of Active Sodium Transport S. ROYCAPLAN AND ALVINESSIG Anaerobic Electron Transfer and Active Transport in Bacteria WIL N. KONINGS AND JOHANNES BOONSTRA Protein Kinases and Membrane Rosphorylation M. MARLENEHOSEYAND MARIANO TAO Mechanism and Physiological Significance of Calcium Transport across Mammalian Mitochondria1 Membranes LEENAMELA Thyroidal Regulation of Active Sodium Transport F. ISMAIL-BEIGI Subject Index
Volume 10 Mechanochernical Properties of Membranes E. A. EVANS AND R. M.HOCHMUTH Receptor-Mediated Protein Transport into Cells. Entry Mechanisms for Toxins, Hormones, Antibodies, Viruses, Lysosomal Hydrolases, Asialoglycoproteins, and Carrier Proteins JR. A N D DAVID M. NEVILLE, TA-MINCHANG The Regulation of Intracellular Calcium CARAFOLI AND ERNESTO MARTINCROMITON Calcium Transport and the Properties of a Calcium-Sensitive Potassium Channel in Red Cell Membranes L. LEWA N D VIRGILIO HUGOG. FERREIRA Proton-Dependent Solute Transport in Microorganisms A. A. EDDY Subject Index
Volume 11 Cell Surface Glycoprotelns: Structure, 6losynth~ls,and Blologlcal Functions
The Cell Membrane-A Short Historical Perspective ASER ROTHSTEIN The Structure and Biosynthesis of Membrane Glycoproteins JENNIFER STURGESS, MARIOMOSCARELLO, AND HARRY SCHACHTER Techniques for the Analysis of Membrane Glycoproteins R. L. JULIANO Glycoprotein Membrane Enzymes JOHNR. RIORDAN AND GORDON G. FORSTNER Membrane Glycoproteins of Enveloped Viruses W. COMPANS AND RICHARD MAURICEC. KEMP Erythrocyte Glycoproteins MICHAELJ. A. TANNER Biochemical Determinants of Cell Adhesion LLOYDA. CULP Proteolytic Modification of Cell Surface Macromolecules: Mode of Action in Stirnulating Cell Growth KENNETHD. NOONAN Glycoprotein Antigens of Murine Lymphocytes MICHELLELETARTE Subject h d e x
Volume 12 Carriers and Membrane Transport Protelns
Isolation of Integral Membrane Proteins and Criteria for Identifying Carrier Proteins MICHAELJ . A. TANNER The Carrier Mechanism S. B. HLADKY The Light-Driven Proton Pump of Halobacterium halobium: Mechanism and Function MICHAELEISENBACH AND S. ROY CAPLAN Erythrocyte Anion Exchange and the Band 3 Protein: Transport Kinetics and Molecular Structure PHiLip A. KNAUF
xx The Use of Fusion Methods for the Microinjection of Animal Cells R. G. KULKA AND A, L ~ Y T E R Subject Index
Volume 13 Cellular Mechanlsms of Renal Tubular
Ion Transport PART I: ION ACTIVITY AND ELEMENTAL COMPOSITION OF INTRAEPITHELIAL COMPARTMENTS lntracellular pH Regulation WALTERF. BORON Reversal of the pH,-Regulating System in a Snail Neuron R. C. THOMAS How to Make and Use Double-Barreled Ion-Selective Microelectrodes THOMAS ZUETHEN The Direct Measurement of K, CI, Na, and H Ions in Bullfrog Tubule Cells MAMORUFUJIMOTO, KUNIHIKO KOTERA, AND YUTAKAMATSUMURA Intracellular Potassium Activity Measurements in Single Proximal Tubules of N e c turus Kidney TAKAHIRO KUBOTA, BRUCE BIAGI,AND GERHARD GIEBISCH Intracellular Ion Activity Measurements in Kidney Tubules RAJAN. KHURI Intracellular Chemical Activity of Potassium in Toad Urinary Bladder JOEL DELONG AND MORTIMERM. CIVAN Quantitative Determination of Electrolyte Concentrations in Epithelial Tissues by Electron Microprobe Analysis ROGERRICK,ADOLFDORGE, RICHARD BAUER,FRANZBECK, JUNEMASON,CHRISTIANE ROLOFF, AND KLAUS THURAU PART 11: PROPERTIES OF INTRAEPITHELIAL MEMBRANE BARRIERS IN THE KIDNEY
CONTENTS OF PREVIOUS VOLUMES
Hormonal Modulation of Epithelial Structure JAMESB. WADE Changes in Cell Membrane Surfaces Associated with Alterations of Transepithelial Ion Movement MICHAELKASHGARIAN The Dimensions of Membrane Bamers in Transepithelial Flow Pathways LARRY w . WELLING AND DANJ. WELLING Electrical Analysis of Intraepithelial Barriers AND EMI LE L. BOULPAEP HENRY SACKIN Membrane Selectivity and Ion Activities of Mammalian Tight Epithelia SIMON A. LEWIS,NANCYK. WILLS, A N D DOUGLAS C. EATON Ion Conductances and Electrochemical Potential Differences across Membranes of Gallbladder Epithelium Luis REUSS A Kinetic Model for Ion Fluxes in the Isolated Perfused Tubule BRUCE BIAGI, ERNESTO GONZALEZ, A N D GERHARD GIEBISCH The Effects of Voltage Clamping on Ion Transport Pathways in Tight Epithelia ARTHURL. FINN AND PAULA ROGENES Tubular Permeability to Buffer Components as a Determinant of Net H Ion Fluxes G . MALNIC, v. L. COSTA SILVA, s. s. CAMPIGLIA, M. DE MELLO AIRES, AND G. GIEBISCH Ionic Conductance of the Cell Membranes and Shunts of Necturus Proximal Tubule AND GENJIRO KIMURA KENNETH R. SPRING Luminal Sodium Phosphate Cotransport as the Site of Regulation for Tubular Phosphate Reabsorption: Studies with Isolated Membrane Vesicles HEINIMURER,REINHARD STOLL, CARLA EVERS,ROLFKINNE, JEAN-h1LIPPE BONJOUR, AND HERBERT FLEISCH The Mechanism of Coupling between Glucose Transport and Electrical Potential in the Proximal Tubule: A Study of Potential-
xxi
CONTENTS OF PREVIOUS VOLUMES
Dependent Phlorizin Binding to Isolated Renal Microvillus Membranes PETERS. ARONSON Electrogenic and Electroneutral Na Gradient-Dependent Transport Systems in the Renal Brush Border Membrane Vesicle BERTRAM SACKTOR PART 111: INTRAMEMBRANE CARRIERS AND ENZYMES IN TRANSEPITHELIAL TRANSPORT Sodium Cotransport Systems in the Proximal Tubule: Current Developments A N D H. MURER R. KINNE,M. BARAC, ATPases and Salt Transport in the Kidney Tubule DE LA MARCARITA PEREZ-GONZALEZ MANNA, FULGENCIO PROVERBIO, AND GUILLERMO WHITEMBURY Further Studies on the Potential Role of an Anion-Stimulated Mg-ATPase in Rat b o x imal Tubule Proton Transport AND R. KINNE E. KINNE-SAFFRAN Renal Na+- K+-ATPase: Localization and Quantitation by Means of Its K+-Dependent Phosphatase Activity 111 AND REINIER BEEUWKES SEYMOUR ROSEN Relationship between Localization of N+K+-ATPase, Cellular Fine Structure, and Reabsorptive and Secretory Electrolyte Transport STEPHEN A. ERNST, CLARA v. RIDDLE, AND JR. KARLJ. KARNAKY, Relevance of the Distribution of Na+ Pump Sites to Models of Fluid Transport across Epithelia JOHNW. M u s AND DONALD R. DIBONA Cyclic AMP in Regulation of Renal Transport: Some Basic Unsolved Questions THOMAS P. DOUSA Distribution of Adenylate Cyclase Activity in the Nephron F. MOREL,D. CHABARDBS, AND M. IMBERT-TEBOUL Subject Index
Volume 14 Carrlers and Membrane Transport Protelns
Interface between Two Immiscible Liquids as a Tool for Studying Membrane Enzyme Systems L. I. BOGUSLAVSKY Criteria for the Reconstitution of Ion Transport Systems ADILE. SHAMOO AND WILLIAM F. TIVOL The Role of Lipids in the Functioning of a Membrane Protein: The Sarcoplasmic Reticulum Calcium Pump J . P.BENNETT, K. A. MCGILL,AND G.B. WARREN The Asymmetry of the Hexose Transfer System in the Human Red Cell Membrane w. F. WIDDAS Permeation of Nucleosides, Nucleic Acid Bases, and Nucleotides in Animal Cells PETER G . w. K A G E M A " AND ROBERT M. WOHLHUETER Transmembrane Transport of Small Peptides D. M. MAITHEWS ANDJ.W. PAYNE Characteristics of Epithelial Transport in Insect Malpighian Tubules S. H. P. MADDRELL Subject Index
Volume 15 Molecular Mechanlsms of Photoreceptor Transductlon
PART I: THE ROD PHYSIOLOGICAL RESPONSE The Photocurrent and Dark Current of Retinal Rods G. M A ~ H E W AND S D. A. BAYLOR Spread of Excitation and Background Adaptation in the Rod Outer Segment K.-W. YAU. T. D. LAMB,A N D P. A. MCNAKJGHTON Ionic Studies of Vertebrate Rods W. GEOFFREY OWENAND VINCENT TORRE
xxii Photoreceptor Coupling: Its Mechanism and Consequences GEOFFREY H. GOLD PART 11: THE CYCLIC NUCLEOTIDE ENZYMATIC CASCADE AND CALCIUM ION First Stage of Amplification in the CyclicNucleotide Cascade of Vision LUBERT STRYER, JAMES B. HURLEY, A N D BERNARD K.-K. FUNG Rod Guanylate Cyclase Located in Axonemes DARRELLFLEISCHMAN Light Control of Cyclic-Nucleotide Concentration in the Retina G. EBREY,PAULKILBRIDE, THOMAS JAMES B. HURLEY, ROGERCALHOON, AND MOTOYUKITSUDA Cyclic-GMP Phosphodiesterase and Calmodulin in Early-Onset Inherited Retinal Degenerations G. J. CHADER, Y. P. LIU, R. T. FLETCHER, G . ACUIRRE, R. SANTOS-ANDERSON, A N D M. T’so Control of Rod Disk Membrane Phosphodiesterase and a Model for Visual Transduction P. A. LIEBMAN AND E. N. WGH, JR. Interactions of Rod Cell Proteins with the Disk Membrane: Influence of Light, Ionic Strength, and Nucleotides HERMANN KUHN Biochemical Pathways Regulating Transduction in Frog Photoreceptor Membranes M. DERICBOWNDS The Use of Incubated Retinas in Investigating the Effects of Calcium and Other Ions on Cyclic-Nucleotide Levels in Photoreceptors ADOLPHI. COHEN Cyclic AMP Enrichment in Retinal Cones DEBORA B. FARBER Cyclic-Nucleotide Metabolism in Vertebrate Photoreceptors: A Remarkable Analogy and an Unraveling Enigma M. W. BITENSKY, G. L. WHEELER, A. YAMAZAKI, M. M. RASENICK, AND P. 3. STEIN
CONTENTS OF PREVIOUS VOLUMES
Guanosine Nucleotide Metabolism in the Bovine Rod Outer Segment: Distribution of Enzymes and a Role of GTP HITOSHI SHICHI Calcium Tracer Exchange in the Rods of Excised Retinas ETE z. SZUTS The Regulation of Calcium in the Intact Retinal Rod: A Study of Light-Induced Calcium Release by the Outer Segment GEOFFREY H. GOLDAND JUANI. KORENBROT Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP ROBERT T. SORBI PART 111: CALCIUM, CYCLIC NUCLEOTIDES, AND THE MEMBRANE POTENTIAL Calcium and the Mechanism of Light Adaptation in Rods BRUCEL. BASTIAN AND GORDON L. FAIN Effects of Cyclic Nucleotides and Calcium Ions on Bufo Rods AND JOELE. BROWN GERALDINE WALOGA The Relation between Caz+and Cyclic GMP in Rod Photoreceptors STUART A. LIFTONAND JOHNE. DOWLING Limits on the Role of Rhodopsin and cCMP in the Functioning of the Vertebrate Photoreceptor SANFORD E. OSTROY, EDWARD P. MEYERTHOLEN, PETERJ. STEIN, ROBERTAA. SVOBODA, AND MEEGAN J . WILSON [Ca2+],Modulation of Membrane Sodium Conductance in Rod Outer Segments BURKSOAKLEY I1 A N D LAWRENCE H. PINTO Cyclic-GMP-Induced Depolarization and Increased Response Latency of Rods: Antagonism by Light WILLIAM H. MILLERAND GRANT D. N~COL
xxiii
CONTENTS OF PREVIOUS VOLUMES
PART IV: AN EDITORIAL OVERVIEW Ca2+and cGMP WILLIAM H. MILLER Index
Volume 16 Electrogenic Ion Pumps PART I. DEMONSTRATION OF PUMP ELECTROGENICITY IN EUKARYOTIC CELLS Electrophysiology of the Sodium Pump in a Snail Neuron R. C. THOMAS Hyperpolarization of Frog Skeletal Muscle Fibers and of Canine Purkinje Fibers during Enhanced Na+-K+ Exchange: Extracellular K+ Depletion or Increased Pump Current? DAVIDC. GADSBY The Electrogenic Pump in the Plasma Membrane of Nitella ROGERM. SPANSWICK Control of Electrogenesis by ATP, Mg2+. H+, and Light in Perfused Cells of Cham MASASHI TAZAWA AND TERUO SHIMMEN PART 11. T H E EVIDENCE IN EPITHELIAL MEMBRANES An Electrogenic Sodium Pump in a Mammalian Tight Epithelium S. A. LEWIS A N D N. K. WILLS A Coupled Electrogenic Na+- K+ Pump for Mediating Transepithelial Sodium Transport in Frog Skin ROBERTNltLSEN Transepithelial Potassium Transport in Insect Midgut by an Electrogenic Alkali Metal Ion Pump MICHAEL G . WOLFERSBERGER, AND WILLIAM R. HARVEY. MOIRAClOFFl The ATP-Dependent Component of Gastric Acid Secretion G. SACHS, B. WALLMARK, E. RABON, G . SACCOMANI, H. B. STEWART. D. R. DIBONA,AND T. BERGLINDH
PART 111. REVERSIBILITY: ATP SYNTHESIS DRIVEN BY ELECTRIC FIELDS Effect of Electrochemical Gradients on Active H+ Transport in an Epithelium QAISAL-AWQATI AND TROYE. DIXON Coupling between H+Entry and ATP Synthesis in Bacteria PETERC. MALONEY Net ATP Synthesis by H+-ATPase Reconstituted into Liposomes YAW0 KAGAWA Phosphorylation in Chloroplasts: ATP Synthesis Driven by A$ and by ApH of Artificial or Light-Generated Origin PETERGRABER PART IV. SOME THEORETICAL QUESTIONS Response of the Proton Motive Force to the Pulse of an Electrogenic Proton Pump ERICHHEINZ Reaction Kinetic Analysis of CurrentVoltage Relationships for Electrogenic Pumps in Neurospora and Acetabularia DIETRICH GRADMANN, AND ULF-PETERHANSEN, CLIFFORD L. SLAYMAN Some Physics of Ion Transport J. MOROWITZ HAROLD PART V. MOLECULAR MECHANISMS OF CHARGE SEPARATION An H+-ATP Synthetase: A Substrate Translocation Concept I. A. KOZLOVA N D V. P. SKULACHEV Proton Translocation by Cytochrome Oxidase MARTENWIKSTROM Electrogenic Reactions of the Photochemical Reaction Center and the UbiquinoneCytochrome b / c z Oxidoreductase P. LESLIEDUTTON,PAULMUELLER, P. O'KEEFE, DANIEL NIGELK. PACKHAM, ROGERc. PRINCE, AND DAVIDM. TIEDE
CONTENTS OF PREVIOUS VOLUMES
xxiv Proton-Membrane Interactions in Chloroplast Bioenergetics R. A. DILLEY. L. J. PROCHASKA, G. M. BAKER,N. E. TANDY, AND P. A. MILLNER Photochemical Charge Separation and Active Transport in the Purple Membrane BARRY HONK Mitochondrial Transhydrogenase: General Principles of Functioning I. A. KOZLOV Membrane Vesicles, Electrochemical Ion Gradients, and Active Transport H. R. KABACK PART V1. BlOLOGlCAL SIGNIFICANCE OF ELECTROGENIC ION PUMPS The Role of Electrogenic Proton Translocation in Mitochondrial Oxidative Phosphorylation JANNA P. WEHRLE Electrogenic Reactions and Proton Pumping in Green Plant Photosynthesis WOLFGANG JUNCE The Role of the Electrogenic Sodium Pump in Controlling Excitability in Nerve and Cardiac Fibers MARIO VASSALLE
Pumps and Currents: A Biological Ferspective FRANKLIN M. HAROLD Index
Volume 17 Membrane Lipids of Prokaryotes Lipids of Prokaryotes-Structure and Distribution HOWARD GOLDFINE Lipids of Bacteria Living in Extreme Environments A. LANGWORTHY THOMAS Lipopolysaccharides of Gram-Negative Bacteria OTTO LUDERITZ, M A R I N A A. FREUDENBERG, CHRIS GALANOS, VOLKER LEHMANN. AND ERNSr TH. RIETSCHEL, DEREK H. SHAW Prokaryotic Polyterpenes: Phylogenetic Precursors of Sterols GUY OURISSON AND MICHELROHMER Sterols in Mycoplasma Membranes SHMUEL RAZIN Regulation of Bacterial Membrane Lipid Synthesis CHARLES 0. ROCKAND JOHNE. CRONAN. JR. Transbilayer Distribution of Lipids in Microbial Membranes SHLOMO ROTTEM Lipid Phase Transitions and Regulation of Membrane Fluidity in Prokaryotes D O N A L D L. MELCHIOR Effects of Membrane Lipids on Transport and Enzymic Activities RONALDN. MCELHANEY Index
Current Topics in Membranes and Transport
Volume 18
MEMBRANE RECEPTORS
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Part I
Adenylate Cyclase-Related Receptors
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME IS
Hormone Receptors and the Adeny late Cyclase System: Histo rical Overview B . RICHARD MARTIN Deparrment of Biochemistry University of Cambridge Cambridge, England
Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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When Sutherland and his co-workers first demonstrated the existence of hormone-stimulated adenylate cyclase a very simple model was sufficient to explain the available information on hormone regulation of cell function. It could be suggested that the enzyme consisted of a single protein which spanned the plasma membrane. The catalytic site responsible for the conversion of ATP to cyclic 3',5'-AMP would be located on the inner surface of the plasma membrane and a recognition or receptor site for the hormone on the outer surface of the plasma membrane. The binding of the hormone to the recognition site on the receptor would then lead to a conformational change resulting in an increase in the catalytic activity of the enzyme (Robinson et al., 1967). Thus, it was suggested that adenylate cyclase functioned in a fashion similar to any other regulatory enzyme with the additional feature that the regulatory and catalytic sites were located asymmetrically on opposite sites of the plasma membrane. This would allow the enzyme to be used as a means of transfer of information from the outer surface of the cell to the cytoplasm. This relatively simple formulation was rapidly shown to be inadequate, and we now know that the hormone-sensitive adenylate cyclase system consists of at least three distinct protein components. The nature of the physical interactions between these components is the subject of many of the articles in this volume. 3
Copynght Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153318-2
4
E. RICHARD MARTIN
Among the most significant early observations were those of Rodbell and his colleagues (for review see Rodbell et al., 1975). They showed that in rat fat cell plasma membranes several different hormones were capable of activating adenylate cyclase. In their initial study they examined the effects of P-adrenergic agonists, glucagon, ACTH, and secretin, but the enzyme is also activated by a number of other hormones. They found that the effects of the hormones were not additive. If adenylate cyclase was maximally activated by the most effective agonist, isoproterenol, the addition of any combination of the other hormones had no effect on either the activation or inhibition of the enzyme. In view of the very marked dissimilarities between the structures of the different agonists it seemed unlikely that they were competing for the same receptor, and, indeed, a large body of evidence from many groups has shown that each hormone has its own distinct receptor protein, Thus, it seemed likely that the hormone recognition site was on a protein component distinct from that containing the catalytic site of adenylate cyclase and that several different hormone receptors were able to compete for the activation of a common pool of adenylate cyclase catalytic units. To explain the interaction of a number of different receptors with a common pool of catalytic units, the mobile receptor hypothesis was proposed (Birnbaumer, 1973; Bennett et al., 1975). This suggested that in the absence of hormone, the catalytic unit of adenylate cyclase and the hormone receptor were separate from each other but able to move in relation to each other, laterally within the fluid matrix of the membrane lipid. The binding of the hormone then results in a conformational change in the hormone receptor, which increases its affinity for the adenylate cyclase catalytic unit so that when they make contact a long-lived association is set up. It was proposed that this association results in the activation of the catalytic unit. Direct evidence for the existence of separate components responsible for hormone binding and adenylate cyclase catalytic activity came from a number of studies in which plasma membranes were solubilized using nonionic detergents. When this was done it was found that the capacity of the enzyme to be activated by hormone was invariably lost, and in some cases it was possible to separate physically the hormone binding activity from the adenylate cyclase catalytic activity (Welton et al., 1977, 1978). Direct evidence for the mobile receptor hypothesis came from the studies of Schramm and his colleagues (Orly and Schramm, 1976). They selected two distinct cell lines, a Friend erythroleukemia cell which contained adenylate cyclase catalytic activity but no P-adrenergic receptor and a turkey erythrocyte which contained both adenylate cyclase catalytic activity and a P-adrenergic hormone receptor. The catalytic activity of adenylate cyclase in the turkey erythrocytes was then selectively destroyed by treatment with N-ethylmaleimide, leaving the specific P-adrenergic binding activity intact. Thus, they had one cell line which contained a hormone receptor but no adenylate cyclase catalytic activity and a second cell line which contained cata-
HISTORICAL OVERVIEW
5
lytic activity but no hormone receptor. The two cell lines were then fused using Sendai virus and an isolated plasma membrane fraction. The hybrid plasma membranes were found to contain adenylate cyclase, which was activated by catecholamines. This provided a convincing demonstration that the two separate entities, the receptor and the enzyme, were able to migrate within the plane of the plasma membrane and to produce a productive functional interaction. The same group has been able to extend this approach using a number of different sources to supply the catalytic unit and receptors for a number of different hormones again derived from several different tissues (Schramm et al., 1977; Schulster ef al., 1978). This has conclusively demonstrated that the recognition site for the hormone and the catalytic site of adenylate cyclase are located on distinct protein components. It also incidentally shows that the structure of the sites on the two different components which interact must be very highly conserved since it is possible to generate hybrid membranes in which the adenylate cyclase will demonstrate a hormone response and in which the catalytic unit and the hormone receptor are derived from completely different species. Rodbell and his colleagues were also responsible for a second major advance in our understanding of the mechanism of activation of adenylate cyclase by hormones. They showed that in rat fat cell plasma membranes and rat liver plasma membranes, adenylate cyclase was activated by GTP at very low concentrations of the order of lo-’ M .They also showed that GTP and hormone used in combination gave a markedly synergistic activation (for review see Rodbell et al., 1975). Subsequent studies have shown that if care is taken to exclude GTP from the system and, in particular, to ensure that the ATP used as substrate for adenylate cyclase is not contaminated with GTP then there is very little activation by hormone alone and the hormonal activation becomes largely dependent upon added GTP (Kimura er al., 1976). The next stage was to consider the possible role of transfer of the terminal phosphate of GTP in the activation process. This was approached by making use of an analog of GTP, guanylyl imidodiphosphate [p(NH)ppG], in which the terminal phosphate linkage is resistant to hydrolysis or transfer. Far from being less effective as an activator of adenylate cyclase than GTP, p(NH)ppG gave a much larger and essentially irreversible activation. In the case of adenylate cyclase, in most plasma membranes from mammalian sources p(NH)ppG is capable of activating the enzyme to its maximum extent and the effect of addition of hormone is only to increase the rate at which activation is achieved. In the widely studied turkey erythrocyte plasma membrane and in other avian erythrocyte plasma membranes the activation of the enzyme by either GTP or p(NH)ppG is largely dependent upon the presence of hormone and is much slower than in mammalian membranes (Rodbell et al., 1975; Tolkovsky and Levitzki, 1978). This has been taken advantage of in the kinetic studies of Tolkovsky and Levitzki which are discussed in Part I of this volume. The involvement of GTP as an additional component required for
6
B. RICHARD MARTIN
hormonal activation led Rodbell to postulate the existence of a third component of the hormone-sensitive adenylate cyclase. He suggested that the primary activator of the enzyme was GTP and that the role of the hormone receptor complex was to render the activation of adenylate cyclase by GTP more effective. The existence of a component which he designated the transducer was proposed to act as a link between the hormone receptor and the catalytic unit and to mediate the effects of GTP. The greater effectiveness of p(NH)ppG as an activator and the irreversible nature of the activation were taken to suggest that the binding of GTP was responsible for the activation of adenylate cyclase and that the reversal of activation required the hydrolysis of GTP to GDP and Pi. More recent studies have shown that at least in outline both of these suggestions are correct. Using turkey erythrocyte plasma membranes Cassel and Selinger (1976) demonstrated the existence of a specific GTPase which was activated by catecholamines. They suggested that the increase in the rate of hydrolysis of GTP resulted from an increase in the rate of formation of an enzyme GTP complex which was promoted by the hormone receptor complex. Cholera toxin was known to activate adenylate cyclase irreversibly, the effect being both time dependent and dependent upon the presence of NAD (Gill, 1975). They were able to show that pretreatment of turkey erythrocyte plasma membranes with cholera toxin in the presence of NAD caused an irreversible loss of hormonesensitive GTPase activity and that the activation of adenylate cyclase by cholera toxin was dependant upon the presence of GTP (Cassel and Selinger, 1977). Later it was demonstrated that in both avian erythrocyte plasma membranes and liver plasma membranes cholera toxin catalyzed the ADP-ribosylation of a protein with a molecular weight of 42,000. In the case of the avian erythrocyte plasma membranes the same protein could be shown to be covalently labeled by a photoreactive analog of GTP, whereas in liver plasma membranes the ADPribosylation was shown to be dependent upon the presence of GTP (Cassel and Pfeufer, 1978; Doberska et al., 1980). It seems likely, therefore, that the protein is a component of the hormone-activated GTPase. Thus, the effect of cholera toxin is to modify this GTPase covalently by the incorporation of ADP-ribose using NAD as donor and resulting in the inhibition of the GTPase activity. This has the effect of rendering GTP more effective as an activator since the rate of reversal of activation by hydrolysis of GTP to GDP and Pi is reduced. Subsequent work using a variety of guanine nucleotide analogs and examining the rate of release of GDP from the membranes led to the conclusion that the role of the hormone receptor complex is to open the guanine nucleotide binding site and to allow the exchange of one guanine nucleotide for another (Cassel and Selinger, 1978). The effect of this under physiological conditions will be to allow the exchange of bound GDP remaining at the end of a cycle of activation for GTP to initiate a fresh cycle of activation. We should now address the question of the location of the guanine nucleotide
HISTORICAL OVERVIEW
7
binding site. There are two possibilities: it could be located on the same protein component as the catalytic site of adenylate cyclase or it could be located on a distinct "transducer" protein as suggested by Rodbell. In most cases detergentsolubilized preparations of adenylate cyclase retain the ability to respond to p(NH)ppG, and in early studies of this type of preparation it was not found to be possible to resolve the catalytic activity of adenylate cyclase from the guanine nucleotide binding activity of the preparation (Pfeufer and Helmreich, 1975). On the basis of this type of circumstantial evidence it seemed possible that the guanine nucleotide binding site was located on the same protein component as the catalytic site of adenylate cyclase. Subsequent work by Gilman and his coworkers has demonstrated conclusively that the guanine nucleotide binding site and the adenylate cyclase catalytic site are on separate protein components. They made use of a strain of S49 lymphoma cells known as cyc- . In contrast to the wild type, these cells appear to lack a hormone-sensitive adenylate cyclase. It can be shown, however, that the binding of catecholamines to the cell surface is normal. Furthermore, it can be shown that the plasma membranes contain a normal adenylate cyclase catalytic unit. This can be demonstrated by making use of the activation of the enzyme by Mn2+ ions. In the presence of Mn2+ the activity of adenylate cyclase in cyc- cell plasma membranes is the same as the activity in wild-type membranes under the same conditions. Thus, these membranes contain a functional hormone receptor and a functional catalytic site, but the two components lack the ability to interact. The component which is either missing or impaired in function is the guanine nucleotide binding site. The membranes lack the 42,000-dalton protein which is ADP-ribosylated by cholera toxin. The guanine nucleotide response can be restored by fusion with membranes which contain a functional guanine nucleotide binding protein (Ross er al., 1978). In fact, the transfer of guanine nucleotide binding protein between membranes may be much simpler than the transfer of hormone receptors, which requires membrane fusion. Lad et al. (1980) have reported that the guanine nucleotide binding protein can be transferred to turkey erythrocyte plasma membranes from human erythrocyte plasma membranes which, while lacking the other components of the system, appear to contain the guanine nucleotide binding component. A recent report by Bhat er al. (1980) showed that the guanine nucleotide response can be restored by fractions derived from the cytoplasm of cells as well as by fractions derived from the plasma membrane. These observations imply that the guanine nucleotide binding protein may be a membrane peripheral protein rather than a membrane integral protein. In any case the use of cyc - membranes as a means of detecting the presence of the guanine nucleotide binding protein is well established and has allowed considerable progress toward the purification of this component of the system. At this point, in order to explain the activation of adenylate cyclase by hormones we must take account of the role of at least three separate protein compo-
8
B. RICHARD MARTIN
nents: (1) the hormone receptor usually designated (R), (2) the catalytic unit usually designated (C), and (3) a guanine nucleotide binding component usually designated (N) or, by some workers, ( G ) .The situation may well be even more complex with other components responsible for the action of inhibitory hormones and for the down-regulation of adenylate cyclase. However, at the present time there seems to be no reason to invoke any protein components in the activation process other than those discussed above. Obviously, it is of great interest to determine the sequence of interactions between these three components; that is, which interacts with which and at which stage of the cycle of activation and reversal of activation of adenylate cyclase. A number of approaches to this question have been used. Tolkovsky and Levitzki have made use of a detailed analysis of the kinetics of activation by hormones. Houslay and his colleagues have examined the effects of alterations in the membrane lipid mobility on adenylate cyclase activity in the presence of different activators. Both of these studies are described in Part I of this volume. We have used the method of target size analysis by irradiation inactivation in an attempt to obtain a direct physical measure of size changes of the components of the hormone-sensitive adenylate cyclase and, accordingly, of the association and dissociation of the different components. The approaches discussed above represent significant steps toward the elucidation of the molecular mechanisms involved in the interaction of hormones and membrane receptors and the adenylate cyclase system, REFERENCES Bennett, V., O’Keefe, E., and Cuatrecasas, P. (1975). Mechanism of action ofcholera toxin and the mobile receptor theory of hormone receptor adenylate cyclase interactions. Proc. Natl. Acud. Sci. U.S.A. 72, 33-37. Bhat, M. K., Iyengar, R., Abramowitz, J., Bordelon-Riser, M. E., and Birnbaumer, L. (1980). Naturally soluble components that confer guanine nucleotide and fluoride sensitivity to adenylate cyclase. Proc. Nafl. Acad. Sci. U.S.A. 77, 3836-3840. Birnbaumer, L. (1973). Hormone sensitive adenylate cyclases. Useful models for studying hormone receptor functions in cell free systems. Biochim. Biophys. Acra 300, 129-158. Cassel, D., and Pfeufer, T . (1978).Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide binding protein of the adenylate cyclase system. Proc. Nurl. Acad. Sci. U.S.A. 75, 2669-2673. Cassel, D., and Selinger, Z. (1976).Catecholamine stimulated GTPase activity in turkey erythrocyte membranes. Biochim. Biophys. Acra 452, 538-551. Cassel, D., and Selinger, Z. (1977). Mechanism of activation of adenylate cyclase by cholera toxin. Inhibition of GTP hydrolysis at the regulatory site. Proc. Nafl. Acud. Sci. U.S.A. 74, 3307-331 I . Cassel, D., and Selinger, Z. (1978). Mechanism of activation of adenylate cyclase through the padrenergic , receptor: Catecholamine-induced idisplacement of bound GDP by GTP. Proc. Natl. Acad. Sci. U.S.A. 75, 4155-4159. Doberska, C. A., Macpherson, A. J. S., and Martin, B. R. (1980). Requjrement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase. Biochem. J . 186, 749-754.
HISTORICAL OVERVIEW
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Gill, D. M. (1975). Involvement of nicotinamide adenine dinucleotide in the action of cholera toxin in vitro. Pror. Natl. Acad. Sci. U.S.A. 72, 2064-2068. Kimura, N., Nakane, K., and Nagata, N. (1976). Activation by GTPof liveradenylate cyclase in the presence of high concentrations of ATP. Bi{Jchm.Biophvs. Res. Commun. 70, 1250- 1256. Lad, P. M., Nielsen, T. B., and Rodbell. M. (1980). A probe for the organisation of the P-adrenergic receptor-regulated adenylate cyclase system in turkey erythrocyte plasma membranes by the use of a complementation assay. FEBS Lett. 122, 179-183. Orly, J., and Schramm, M. (1976). Coupling of catecholamine receptor from one cell with adenylate cyclase from another cell by cell fusion. Proc. Nail. Acad. Sci. U.S.A. 73, 4410-4414. Pfeufer. T., and Helmreich, E. J . M. (1975). Activation of pigeon erythrocyte plasma membrane adenylate cyclase by guanyl nucleotide analogues and separation of a nucleotide binding protein. J . B i d . Chem. 250, 867-876. Robinson, G. A., Butcher, R. W., and Sutherland, E. W. (1967). Adenyl cyclase as an adrenergic receptor. Ann. N.Y. Acad. Sci. 139, 703-723. Rodbell, M., Lin, M. C., Salomon, Y.,Londos, C., Harwood, J . P., Martin, B . R., Rendell, M., and Berman, M. (1975). Role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones. Evidence for multisite transition states. Adv. Cyclic Nucleotide Res. 5, 3-29. Ross, E. M., Howlett, A. C., Ferguson, K. M . , and Gilman. A. G. (1978). Reconstitution of a hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J . B i d . Chem. 253, 6401-6412. Schulster, D., Orly, J., Seidel, G., and Schramm, M. (1978). lntracellular cyclic AMP production enhanced by a hormone receptor transferred from a different cell. J . B i d . Chem. 253, I201 - 1206. Schramm, M., Orly, J., Eimerl, S . , and Komer, M. (1977). Coupling of hormone receptors to adenylate cyclase of different cells by cell fusion. Nature (London) 268, 310-313. Tolkovsky, A , , and Levitzki, A. (1978). Collision coupling of the P-adrenergic receptor with adenylate cyclase. In “Hormones and Cell Regulation” (J. Dumont and J. Nunez, eds.), Vol. 2, pp. 89-105. North-Holland, Amsterdam. Welton, A. F., Lad, P. M., Newby, A. C., Yamaniura, H., Nicosia, S . , and Rodbell, M. (1977). Solubilisation and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide sensitive states. J . Biol. Chem. 252, 5947-5950. Welton, A. F., Lad. P. M., Newby, A. C., Yamamura, H.. Nicosia, S . , and Rodbell. M. (1978). The characteristics of lubrol solubilised adenylate cyclase from rat liver plasma membranes. Biorhim. Biophys. Acra 522, 625-639.
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CLlRRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 111
The Elucidation of Some Aspects of Receptor Function by the Use of a Kinetic Approach A.
M . TOLKOVSKY
Department of Pharmacology Hadassah Medical School The Hebrew University Jerusalem, Israel
............................. r Theory. . . . . . . . . ....... A. The Basic Kinetic Formulation: One-Step Model .................. B. The Separation of Sensation and Function . . . . . .................. e Cyclase . . . . . . . . . . C. Guanylnucleotides and the Rate of Activation of 111. Applying Kinetic Theory to Data Generated by Turkey Erythrocycte Adenylate Cyclase ............................................. A. Methodological Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Slow Activation versus Rapid Equilibrium in a Precoupled System.. . . . . . . . . . C. Slow Activation by a Sequential Mechanism: The Collision Coupling Model. . . D. The Role of GppNHp as Modulator and the Position of Subunit in the Activation Pathway.. . . . . . . . . . . . . . . . .
.......................................... List of Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
I1 13 13 15 20 22 22 23 29 35 40 42 43
INTRODUCTION
The purpose of this article is to describe what we have learned in the past few years about the mechanism whereby hormone receptors and guanylnucleotides cause the activation of adenylate cyclase, by applying a kinetic approach. What use, if any, are kinetic analyses of complex biological phenomena? Kinetic analyses enable one to come to terms with quantitative aspects of phe11 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-153318-2
12
A. M . TOLKOVSKY
nomena which evolve with time. They also provide a framework for creating molecular models which yield predictions concerning quantitative aspects of temporal events. It is fundamental to the kinetic approach that such predictions be subjected to experimental tests. A prediction which does not provide the means to test its own validity is of no value or consequence. As such, kinetics define and predict the behavior of a molecular mechanism of action. The power of refutation, a fundamental component of kinetics, lies in the hand of the experimentalist. The particular problem of the activation of adenylate cyclase by hormone receptors belongs in the realm of signal-response relationships. In the past few years, the chemical models which pertain to the adenylate cyclase response system have become much more complex than the first model of receptorresponse coupling as formulated by Clark (1937). This development did not occur in one step. Structural and functional studies which were rapidly forthcoming from many laboratories (Rodbell et af., 197 1; Bourne et al., 1975; Orly and Schramm, 1976; Neer, 1976; Ross and Gilman, 1977; Ross et al., 1978) provided strong evidence for the multicomponent nature of the hormone receptor adenylate cyclase system. These studies served as conceptual catalysts, which forced the kinetic studies to seek out and formulate more complex models. New structural entities had to be integrated into old models. As a result, new modes of coupling were devised, and tested experimentally. The experiments in turn suggested the participation of new structural or isomeric entities in the activation process. This developmental, iterative process is the essence of the methodology used in the kinetic approach. In this artkle I have chosen to trace the interplay of facts and concepts which has led to the stepwise reapplication of more and more complex kinetic formulations to the problem of adenylate cyclase activation by receptors. Perhaps this description will promote the understanding of kinetics on a more intuitive basis and shed some light on the unique solutions which kinetics can provide for the study of signal-response coupling. I also hope that this will create a feel for the valid use of theory in presenting new and valuable experimental approaches which can be used to derive facts concerning signal transmission from the outside environment into the cell. Finally, a word about paradigms. Some years ago, Kuhn (1970) advanced the idea that the development of a particular field of science occurs by the periodic reformulation of a leading paradigm or a shared example. I wish to pay homage to what may be identified as a paradigm in the receptor field-the hypothesis of the mobile receptor (Cuatrecasas, 1974a,b). This paradigm, I believe, has probably been the most influential element in creating a new conceptual framework for relating the kinetic approach to the problem of adenylate cyclase activation by hormones.
13
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
II.
SIGNAL-RESPONSE COUPLING AND RECEPTOR THEORY
A. The Basic Kinetic Formulation: One-Step Model The kinetic approach to the study of receptor function and to receptor-response coupling originates in the formulation by Clark (1937) and by Ariens (1954) of the idea that the response of a biological system to a ligand is proportional to the occupancy of a receptor by that ligand. This statement is formally equivalent to the following scheme: L
I
u
+ R S LR + response 7
(1)
in which L is the ligand, R the receptor, LR the ligand-receptor complex, 1 the ligand receptor association rate constant, 2 the ligand receptor dissociation rate constant, and a a proportionality constant between LR and the response. What constitutes a quantitative solution to this formal statement depends on our ability to express the magnitude of the response in terms of the components which cause its creation. In this case the components are L,, the total ligand concentration, R,, the total receptor concentration, and the rate constants, 1 , 2, and a . For example, when equilibrium between L and R is achieved and L, = Lfree the following expression is obtained ( t is time):
response
= dRTtl(211
+ L)
(2)
Three parameters are sufficient to characterize the behavior of this system: the equilibrium constant 2/1, the total receptor concentration R,, and the proportionality constant a. The ability to formulate an idea and to translate it into a chemical statement which defines and predicts the temporal nature of events which ensue from the moment of signal initiation to the final point at which a response is generated establishes the fundamental principles and boundaries of the kinetic discipline as relating to receptor function. 1 . TESTINGTHE FORMULATION AND
FOR
HIDDEN ASSUMPTIONS
LIMITATIONS
One then asks, does this model provide a sufficient foundation upon which to build a fundamental molecular mechanism of action which both defines and predicts how a ligand may cause a response? Is this formulation exclusive and complete? On purely kinetic grounds the answer is yes. On conceptual grounds the answer is no. A choice exists between these two answers. Choosing involves
14
A. M. TOLKOVSKY
(1) understanding and accepting all the assumptions of the model, those upon which the formulation is based, and those which are consequential to the basic hypothesis (often elusive and hidden from cognitive assessment) and (2) accepting the limitations which are set by the assumptions. Thus, the foundation provided by Eq. (1) is sufficient and complete in sofur as one has chosen to formulate a prediction about the nature of response systems which concerns only L and R, on the one hand, and the response, on the other hand. What are the assumptions that were made? The formulation of this model was founded on the principle that the response is proportional to the concentration of LR. But the model also contains an implied assumption, which stems from the nature of the chemical statement: that the receptor molecule is not only the acceptor of L but also the exclusive physical vehicle for the response which it creates. Figuratively speaking, it would mean that the acetylcholine receptor would also be the carrier of ion fluxes, that the insulin receptor would also be the glucose transport system, and that the P-adrenergic receptor would also be an adenylate cyclase. What limitations are therefore imposed? The simple, one-step model limits the extent to which the temporal mechanism of informational transfer from the receptor to an effector or response-carrying molecule can be examined, because it simply does not consider, nor describe in physicochemical terms, the separate existence of any of the molecules which participate in the response. Also, because of the underlying assumption regarding the role of the receptor in generating the response as well as the signal, the nature of a is obscure. Is it really the rate constant which defines the rate at which the response is generated’? If so, is this the rate-limiting step which one is interested in pursuing? 2, THE VALUEOF THE BLACK Box APPROACH TO RECEPTORFUNCTION What then is the strength and the weakness of Clark’s model? The strength of the formulation lies in its power to distill the idea of receptor-response coupling to a point where a pure and focused conceptual framework is obtained. This model thus becomes the most abstract expression of the idea of receptor-response coupling. All the events which occur between the input and output are buried in a black box. As such, this model has had great impact on our current understanding of ligand-receptor interactions and receptor function. From the abstract, probabalistic nature of a,the concept of efficacy was derived to explain partial agonist action (Stephenson, 1956). The major conceptual weakness of this model lies in its failure to account more precisely for an entity which carries the functional properties of the response system. Thus, in order to arrive at chemical statements of consequence concerning the separate, sequential nature of signals and responses, a black box approach
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
15
is insufficient. Models must tie formulated which will allow functional entities to be separate.
B. The Separation of Sensation and Function The separation of sensation and function to two distinct molecular entities was first suggested by Cuatrecasas (1974a,b) in the form of the mobile receptor hypothesis. This hypothesis, in which it was proposed that a nonstoichiometric relationship exists between receptors which activate adenylate cyclase and the catalytic moiety, arose in response to newer concepts in membrane biology which were advanced by Singer and Nicolson (1 972) that the membrane is a fluid solvent of finite dimensions in which proteins are free to move laterally in the plane of the membrane. The lateral nature of the membrane matrix, on the one hand; the perpendicular nature of the information whose signal originated on the outside of the cell but the response to which was generated inside the cell, on the other hand; and the concept of separate molecules which contain sensory and response roles were the basis of a paradigm concerning receptor function which has had a great impact in the field of adenylate cyclase research. The reason that the hypothesis of the mobile receptor was first quantitatively applied to research on receptor-adenylate cyclase coupling (and not to membrane receptors whose physical and structural characterization was much more advanced, such as the nicotinic acetylcholine receptor) was probably due to three factors. (1) The identity of the first biochemical event following receptor occupancy was thought to be most clearly defined, namely, CAMPelevation. Therefore, the molecular formulation found fertile grounds in the reciprocal, biochemical nature of the adenylate cyclase response system. Allosteric properties and enzyme kinetics could be adapted to this system with ease. (2) The output of this system was slow enough to be easily measured under the same conditions as one could measure ligand binding. Faster response systems, such as ion gating responses to acetylcholine, and the quanta1 nature of this system left the single ion channel firmly attached to the acetylcholine receptor (Werman, 1975). (3) A qualitative experiment was designed and executed by Orly and Schramm (1976) which established that the catalytic units belonging to Friend erythroleukemiacells could be coupled to P-adrenergic receptors belonging exclusively to turkey erythrocyte cells. This experiment of cell fusion provided tangible evidence for the concept of the mobile receptor by crossing experimental techniques which belonged to the discipline of membrane biology with biochemical techniques of enzyme catalysis. 1. FORMULATION OF THE MOBILERECEPTOR HYPOTHESIS
The actual formulation of the mobile receptor hypothesis in molecular terms was presented by Jacobs and Cuatrecasas (1976) in the following form:
16
A. M. TOLKOVSKY
LRE
response
in which E generates the response. As observed by Jacobs and Cuatrecasas (1976), the outstanding implication of this model is the prediction of a nonlinear relationship between ligand binding as a function of ligand concentration and the response generation as a function of ligand concentration. This model also predicts that the binding function of L has a complex pattern from which a highaffinity-like site and a low-affinity-like site can be derived. The two classes of sites which emerge result from L binding to two species of the receptor, R and RE, and from the random, boxlike sequence of interactions which occurs. The response is found to be proportional to the high-affinity site alone, thereby dissociating the binding behavior from the response. This model constitutes the most general formulation of the mobile receptor hypothesis, barring additional subsidiary hypotheses concerning state transitions of the receptor from R to R‘ and of the catalytic unit from E to E’ as proposed by De Haen (1976).
2. TWO-STEPSEQUENTIAL MODELSOF THE MOBILERECEPTOR
A critical analysis of this model entails simplifying it to limiting cases. Boeynaems and Dumont (1975, 1977a,b) actually preceded the most general formulation presented above by suggesting in its stead three separate linear sequences of L to R to E interactions, all of which could be applied to account for adenylate cyclase activation by hormones. Upon inspection, these models turn out to be special cases of the general mobile receptor formulation. The three models are L
+ RE
LRE
response
(4)
KI
L t R
s LR
+ E e LRE Ir, response
KI
L
(5)
K2
+ RE e LRE s LR + E 5 response Kl
K?
In Eq. (4) R and E are always in a complex, and only LRE can generate a response. Kinetically, it is a degenerate, overformulated form of Clark’s model. In Eq. (5) R and E are separate and the union of LR with E to form a complex LRE gives rise to a response. In Eq. (6) RE is preassociated but E is inactive.
BINDING A N D RESFWNSE FUNCTIONS Ok ONE-STEP Function
Binding
TABLE I A N D TWO-STEP S E Q U E N T I A L RAPID EQLJILIERIUM MODELSO Response
L + R E S LRE Kl
LRE 3 response
LR
+E
LRT LRE = _ _ L + Kl
C LRE KI e,
2
,
C LRE K,
+
4 5
+ RE
g
L
2 response
E
LRE
W
E 1;response The solutions to the two-step models are approximations of square root functions, derived by expansion into a power series. Only the first terms were taken. Concentrations are designated by italics.
18
A. M. TOLKOVSKY
The binding of L causes a dissociation, producing an active form of E which generates a response. In contrast to the general case, each of the limiting cases allows only one binding step involving L, similar to the one-step model. Can such simple models, in which in addition to the ligand binding step a second step occurs which involves either the association or the dissociation of R and E, generate patterns of behavior which deviate from the one-step model? If so, there may be room to consider such two-step models in the testing of the mobile receptor hypothesis. Therefore, one would also like to know whether these two-step models are at all distinguishable from each other. In order to answer these questions, it is necessary to solve the equations in terms of binding and response patterns as a function of the parameters L, K , K,, R,, and E,. Approximate solutions to all three models are presented in Table I. The binding and response patterns as a function of L are also illustrated in Fig. 1, for the particular case where (R,) = (E,) = K, = K , = 1. Complete simulations are provided by Boeynaems and Dumont (1977a,b). From inspection of the formulations presented in Table I one can conclude that, indeed, each sequential two-step model predicts a different pattern of bebavior. The dependence of the response on the concentration of the ligand is different. Also, the binding patterns are different from the response patterns for each model. In each case, the terms which include the species E, and R , are instrumental in determining the differential patterns of each model. To what factor can this difference between models be ascribed? The answer does not entail structural arguments. All three models were set up to contain the
binding
response
v/F
B/F
111
I
V
B
FIG. 1 . Theoretical binding and response curves of one-step and two-step sequential rapid equilibrium models. Models which are presented in Table I are shown as Eadie plots ( v . rate of response output; F , free L concentration) or Scatchard plots ( E , bound ligand; F . free L concentration) in the particular case where K , = Kz = ET = RT = 1 . L concentration varies from 0.2 to 50 K units. I, Precoupled; 11, sequential association; 111, sequential dissociation.
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
19
same number of components. The functional roles of the components are identical. Also, the models share similar combinations of structural elements. Therefore one must conclude that it is not structure alone which determines a specific pattern of signal-to-response coupling. Rather, the different patterns of behavior stem from the fact that different sequences of events, and a different number of steps, ensue from the moment of signal initiation (concomitant in all models with L occupancy by definition) to the point at which the response is generated. Each sequence creates new, transient structural entities, providing the system with unique properties. Since each system is composed of sequential events, a kinetic analysis will distinguish between these models. Kinetics will therefore allow a glimpse into functional relationships between predefined structures. A structural analysis will, at best, establish their existence.
3. ASSUMPTIONS AND LIMITATIONS OF THE SEQUENTIAL MODELS: THE RAPIDEQUILIBRIUM The sequential models depart from the one-step models in two important respects. 1. The ligand has changed its role. In the one-step models the role of the ligand is to initiate a signal and a response. The nature of the signal vis-h-vis the response in molecular terms remains obscure. In the sequential models, the role of the ligand is to alter the affinity between R and E. This in itself gives the concept of efficacy a sound physical basis: each ligand would have its own potential to alter the affinity between R and E. Thus, while cx controls the efficacy parameter in the Stephenson formulation (Stephenson, 1956) in the twostep models the efficacy will be expressed by a term which involves the affinity constant between R and E. 2. The mere separation of sensation and function defines and allows one to focus on an event which follows ligand binding but which occurs prior to response evolution. We shall term this event coupling. Coupling in its most elementary form may occur by more than one pathway. In fact, by assuming one coupling step, three minimal models replace the singular formulation of the precoupled systems. The multiplicity is a necessary consequence of the added degree of functional freedom we have allowed the system. Each of these models displays unique properties. Each could account for events which occur subsequent to the binding of the ligand to the receptor.
Thus, the foundation provided by the sequential models is almost sufficient to begin to examine experimentally the evolution of coupling events. In one important respect, though, the sequential models still impose a barrier to the elucidation of coupling phenomena. The barrier stems from the fact that all these models bury the temporal nature of the coupling event beneath a rapid equilibrium assumption.
20
A. M. TOLKOVSKY
The rapid equilibrium assumption implies that the mechanism of coupling between R and E is not time consuming compared to the slowest step in signal generation. Under rapid equilibrium conditions between L and R and between LR and E, the rate of the slow step is still controlled by a. In essence, the sequential and the precoupled models propose a concerted mechanism of action: the binding event of L is synonymous in time with the formation of LRE and RE. We shall illustrate this with an example: In the mechanism L t R
e LR KI
+ E s LRE K:
u
response
(7)
when K , is small relative to L and R , and K , is large relative to LR and E , most of the receptor is in the LR form. The binding of L is almost exclusively to the LR form, and is therefore controlled by K , . The response, on the other hand, is still generated at a rate defined by a and by the extent of LRE which is formed. Thus, although LRE takes up a negligible portion of R, its formation is crucial for the response to occur. The formation of LRE occurs concomitant with the binding of L, but it cannot be probed by L since it does not accumulate. The coupling step is buried and lost. In summary, molecular models which allowed the separation of sensation and function paved the way for focusing on coupling events. Understanding the coupling process would lead to an appreciation of crucial aspects of the mechanism whereby a receptor causes a response, namely, functional interactions. At the same time equilibrium assumptions somewhat destroyed the potential of sequential models to enable the elucidation of the coupling process because they converted an inherently dynamic process which occurs sequentially in time into a static, concerted process. In some respects, then, sequential models were still not providing a good enough foundation for the study of signal-response coupling. In practice as well as in theory, rapid equilibrium had to be dissected further into rate processes.
C. Guanylnucleotidesand the Rate of Activation of Adenylate Cyclase For many years there was no experimental evidence in the field of adenylate cyclase research which could be applied to contradict the assumption of a concerted mechanism, namely, that a hormone-receptor adenylate cyclase ternary complex accumulated prior to the generation of CAMP. The first experimental evidence to the contrary was provided from observing the effects of guanylnucleotides on adenylate cyclase activation (Rodbell ef al., 1971). GTP was shown to elevate the catalytic activity of adenylate cyclase synergistically with the hormone (Salomon el af., 1975). Rodbell (1975) then proposed that
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
21
GTP works by binding to a subunit which he termed a transducer molecule. The concept of a guanylnucleotide-sensitivetransducer molecule which was interposed between R and E upset the foundation provided by the sequential models as pertaining to a molecular mechanism of receptor coupling to an effector as applied to adenylate cyclase research in three basic respects. 1. It upset the contention that the RE complex needs to accumulate (or dissociate) in order to make E active. It suggested, on the contrary, that LR and E need not ever accumulate. The transducer molecule could conceivably shuttle between the two entities. Thus, E would become a second effector in the signal-response system of adenylate cyclase. Its activation would be a product of tbe primary interaction between R and N (N will be used throughout to represent the guanylnucleotide subunit), the transducer molecule. As we shall see, the level of complexity in formulating such models increases considerably, just as when the separation of R and E replaced one model with three alternative models. 2 . It upset the basic hypothesis that the rate of formation of the active form of the catalytic unit is rapid. In the presence of guanylyl imino-py-diphosphate (GppNHp), a nonhydrolyzable analog of GTP, cAMP accumulation was no longer a linear function of time. Rather, cAMP now appeared with a considerable lag time. Thus, cx could not be the determinant of the rate-limiting step. The lag time in AMP accumulation in the presence of GppNHp appeared in three very different experimental systems: in the P-adrenergic receptor-activated system of turkey erythrocytes (Sevilla et al., 1976) and the P-adrenergic receptor-activated system of the frog erythrocyte (Schramm and Rodbell, 1975); in the glucagon receptor-activated system of liver membranes (Salomon ef af., 1975); and in the P-adrenergic receptor-activated system of S49 lymphoma cell mernbranes (Ross et a/., 1977). The fact that the slow activation phenomenon was shared by different systems implied that it may have some fundamental mechanistic significance. 3. The concept of a guanylnucleotide-activated transducer molecule also upset the contention that the role of L is to allow a tighter or looser association between R and E and thereby to amplify the extent (or concentration) of the active form of the catalytic unit. In contrast, early studies by Rodbell (1975) suggested that the role of the activating ligand L is to promote a faster rate of appearance of the active form of E. The extent of amplification of catalytic activity seemed to be controlled by the type of guanylnucleotide used.
The use of GppNHp as an experimental handle in probing receptor-adenylate cyclase relationships changed the nature of the simple, sequential two-step formulations even before they were applied rigorously to test the mechanism of adenylate cyclase activation by hormones. Instead, new fonnulations which departed from rapid equilibrium assumptions were introduced (Sevilla et a/.,
22
A. M. TOLKOVSKY
1976; Ross et al., 1977; Tolkovsky and Levitzki, 1978a,b). These formulations now contained an irreversible step whicb defined the slow transition from an inactive to an active form of E following receptor-effector complex formation. It is this step which now assumed the identity of a coupling event. For example, even the precoupled mechanism L
+ RE + LRE 5 response
(8)
could be formulated into an irreversible coupling-containing process such as L
slow + RE + LRE + LRE’ + response K ( I
(9)
The irreversible nature of this slow step was based on experimental evidence which suggested that once the enzyme was activated in the presence of GppNHp, it remained in an active form even after washing or in the presence of an antagonistic ligand which is bound to the receptor (Schramm and Rodbell, 1975). By introducing an irreversible step, an added level of rigor in quantitation of temporal phenomena in relation to adenylate cyclase was established. In practice, defining the rate law by which such activation is achieved and defining the parameters which control the rate of activation enabled the examination and testing of the mechanism of adenylate cyclase coupling to a P-adrenergic receptor and to an adenosine receptor in the simple system of the turkey erythrocyte membrane.
111.
APPLYING KINETIC THEORY TO DATA GENERATED BY TURKEYERYTHROCYTEADENYLATECYCLASE
A. Methodological Considerations The historical survey in the preceding sections is a biased description of some of the developments which occurred in the field of adenylate cyclase research presented in conjunction with receptor theory. It was intended as a developmental exposition of concepts which have led to the application of kinetic theory and terminology as tools for the elucidation of the mechanism of activation of adenylate cyclase by receptors. We have seen that in order to analyze the kinetics of coupling events in relation to the activation of adenylate cyclase by hormones one wants to (1) define a molecular mechanism of action, (2) convert it to a statement which includes all of the species which participate in the response pathway, (3) allow coupling events to be separate from signaling events on the receptor, and (4) eliminate rapid equilibrium assumptions, replacing them with rate constants unless a rapid equilibrium can be verified experimentally.
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
23
In order to test such modcls experimentally one needs to (5) evaluate critically all the implications of each model, (6) look for vulnerable points which make the model conflict with a simpler version of itself, (7) use conflicts in order to test the models against the experimental system, and (8) reject inconsistent models and reapply processes (1) to (7) to an extended form of the model which is most consistent with the data, by altering one variable. This process is theoretically complete at a point where the most extended model recapitulates precisely all the predictions of the previous model. Such a protocol will be followed in this section. We will first formulate and compare the simplest rapid equilibrium model with its congener version in which a slow activation step occurs. The salient features of each model are listed in Table 11. A simulation is presented side by side in Fig. 2. We shall then set up discriminatory criteria to distinguish between both models. These criteria are also listed in Table 11. The resultant functions are portrayed in Fig. 3. Data derived from an analysis of the activation of turkey erythrocyte adenylate cyclase by an adrenergic receptor in the presence of GppNHp are presented and examined in relation to each model in Fig. 4. It will be shown that all the sequential models in which slow activation occurs after the formation of the complex LRE are not consistent with the data (Table 111). The precoupled slow activation model is the only model to which the data conform. This model is then reformulated by altering one variable-the position of the slow step in the activation process. The new model so derived' (termed collision coupling) is compared with the parent model in Table IV. It will be shown that the new model is indistinguishable from the parent model by the first discriminatory criteria which were established (otherwise it would have to be rejected outright). New rejection criteria to distinguish between the two models are also described in Table 1V as a second level discrimination and are portrayed in Fig. 5. Experiments which were designed to test the new model are shown in Fig. 6 . Four probes are used to test each model: (1) the dependence of the response evolution in time on the concentrations of the interacting species L, R, and E (2) the relation of the binding function to the response function; (3) the dependence of the apparent rate constant which determines the rate-limiting step on the concentrations of the interacting species and on the equilibrium constants; and (4) the rate law which governs the activation process. Detailed comment regarding each figure and table follows. 6. Slow Activation versus Rapid Equilibrium in a
Precoupled System Table 11 compares the mechanisms depicted in Eq. (8) and (9) in terms of the response evolution and the intermediate accumulation. The mathematical solutions to the molecular formulations and the general features are also listed. Four
24
A. M. TOLKOVSKY
features, all inherently kinetic, make these models distinguishable from each other: ( I ) the pattern of LRE or of LRE‘ accumulation, or of the response output as a function of time, (2) the levels of LRE and of LRE’ at infinite time, (3) the rate law of LRE or of LRE’ accumulation, and (4) the patterns of each feature (1)-(3) as determined by the ligand concentration and by K , the dissociation constant of LR. These are illustrated graphically in Fig. 2. In Fig. 2A and B the accumulation of the response as a function of time at different ligand concentrations is described. The rapid equilibrium model (A) predicts a linear accumulation of response at all ligand concentrations. The slow activation model (B) predicts that the response will accumulate with a lag time. The lag time will increase when the ligand concentration is reduced. At long times the response becomes independent of ligand concentration, and all the slopes are equivalent. The response reflects the extent of the active enzyme which has been formed (Fig. 2C and D). In the rapid equilibrium model the active form of the enzyme was established instantaneously. Therefore the extent of active enzyme does not change as a function of time at any ligand concentration (C). It is this behavior which generates (upon integration) the linear response output in Fig. 2A. In the slow activation model, the time-consuming reaction is the formation of active enzyme (D). Since each active enzyme molecule will produce a response output as function of time, the response is generated in a cooperative rather than in a linear manner (B). At long times, say 10 half-lives, all the active enzyme has been formed. Therefore, independent of ligand concentration, the response output is linear and maximal. The behavior of L is determined largely by the magnitude of its dissociation constant. How is the dissociation constant for L derived from such data? This is shown in Fig. 3. If the slopes of the response output (which are equivalent to response units divided by time, or normalized response units) are plotted as a function of L, a saturation function for L (Fig. 3A) is obtained. In the case of the rapid equilibrium model, because response output is linear with time, one such function will be generated regardless of the time at which the data were collected (A, dashed line). This function can then be linearized ( C , dashed line). The slope of the linear plot is equal to the negative value of the affinity constant and so K, the dissociation constant, is obtained. In the case of the slow activation model, each time point at which the value of the normalized response units as a function of ligand concentration is calculated will yield a different curve (A). The slopes of the linear expressions of these curves ( C ) are seen to change as a function of time. But, if the slope is always K , it should remain invariable, since K is a constant and not subject to change in time. Since that is not the case, the slopes are not K, and therefore such plots cannot be used to derive K for a mechanism involving slow activation. On the other hand, precisely for this reason such plots can be used as a very sensitive probe for nonlinear response output.
TABLE 11 A COMPARISON OF THE ONE-STEP RAPIDEQUILIBRIUM MODELWITH Mechanism
ONE-STEP SLOW ACTIVATION MODEL"
Rapid equilibrium
+ RE
Formulation
L
Concentration of intermediate
LRE=
Response evolution
THE
LRE
5 response
RETL K+L (Fig. 2C)
dETL K+L (Fig. 2A)
Slow activation
L
+ RE e LRE J, LRE' 5
LRE' = RET{~- exp[-sLr/(K
response
+ L)])
(Fig. 2D) d E ~ {exp[-sLr/(K + L)I - 1) sLI(K + L ) (Fig. 2B)
dET1 + Discriminating criteria
Accumulation of LRE or LRE'
So rapid that it is fully formed at zero time
Slow, rate determined by s, L, and K (Fig. 2D)
(Fig. 2C) Levels of LRE or LRE' at infinite time
Equivalent to zero time levels (Fig. 2C)
One common maximal level equivalent to RET (Fig. 2D)
Rate law of LRE or LRE' accumulation
Zero order
First-order, slope of semilogarithmic plot gives sL/(K + L) (Fig. 3D)
Measurable factors which are determined by L and by K
LRE (Fig. 2C) Slope of response output (Figs. 2A and 3A)
Extent of LRE' before r is infinite (Fig. 2D) Lag time in response output (Figs. 2 9 and 3B-D)
A simulation of each mode! is presented in Figs. 2 and 3. These are indicated in parentheses
A.
26
60
K=l
-c "7
L
60-
K-1
M.TOCKOVSKY
L
-
a 0 y1 0
-
n
T I M E , min
Fic. 2. Theoretical response output and active enzyme accumulation curves of a one-step rapid equilibrium model compared to a one-step slow activation model. The behavior of each system is shown as a function of ligand concentration. Ligand concentration is given in K units. K is the LRE dissociation constant. (A, C) Rapid equilibrium; ( B , D) slow activation. Details are given in Section
111,B.
In order to derive the dissociation constant in this case, the fractional activation of the enzyme, which is a calculated entity E',/EfT, can be plotted as a function of time in a sernilogaritbrnic plot (Fig. 3D). Such plots are linear because the saturation function for L appears in an exponential expression (Fig. 2D). When the slopes so derived are replotted as a function of ligand concentration (Fig. 3B) a curve which is identical to the dashed curve in Fig. 3A is obtained. The ordinate of this plot (Fig. 3B) no longer describes the normalized response units as in Fig. 3A. It describes kobs, or the values of the slopes derived from the semilogarithmic plots. Thus, at infinite ligand concentrations a measure of total active enzyme concentration, R&, is not obtained. Instead one obtains the value of the rate constant for the conversion of LRE to LRE',, a (Table 11). The half
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
27
FIG. 3. A critical analysis of the one-step slow activation model. The slow activation model (Fig. 2D) yields nonequivalent patterns of enzyme activation (E‘,/E’T)as a function of L at different times (A). In contrast, thc pattern of enzyme activation as a function of L which is predicted by a rapid equilibrium model is indifferent to time (dashed line). This function yields a linear Scatchard plot (C). A slow activation model predicts linear semilogarithmic plots (D). (D) is actually a replot of the - E‘,)/ErTat different L concentrations lines in Fig. 2D. The slopes of these lines which depict are the values of k,,,,, characteristic of each ligand concentration and are used to construct a kobs versus L plot (B). Although this curve is identical to the dashed line in (A), the ordinate units of each plot are different: at infinite time, E’, + ElT in (A), but kobs + a in (B). Thus one can derive the values of EIT and of a , the rate constant for activation. In addition to the values of E’T or a. the parameter which is derived from (A) and (B) is K . the ligand dissociation constant. K can be derived from the slope of the Scatchard-like plot shown in (C). Note how time causes a change in the apparent slope of the lines which are derived from the curves depicted in (A) and how this is rectified in (B). Further details are given in Section 1II.B. EIT. total active enzyme concentration at infinite time; E’,, active enzyme concentration at time I ; E’,/EIT,fraction of active enzyme; (E’T-E’,)IE’T. fraction of inactive enzyme. (ElT
saturation constant derived from this function is now equivalent to the dissociation constant. Figure 4 shows data generated by following the activation of adenylate cyclase of turkey erythrocytes at various L-epinephrine concentrations, in the presence of 0.1 mM GppNHp. The data describing CAMP accumulation as a function of epinephrine concentration and time were collected at 25°C (Fig. 4A). The lines
28
A. M. TOLKOVSKY B
A
4
I
c
K.37uM
I
min
I
1
60 80
0
0
I
1
120
rnin
.3.
- .2 c E
;.l-
_*
0I
i
5
0 L-epinephrine. pM
FIG.4. Activation of turkey erythrocyte adenylate cyclase by L-epinephrine in the presence of GppNHp. Activation is at 25OC (A) or at 37°C (B-D). In all the experiments 0.1 mM GppNHp is present. In (A), the accumulation of cAMP is shown as a function of time and hormone concentra3 @I; 0 , 15 p M . The lines all fit to one equation (Fig. 2B). The tion. A, 0.5 pM; A,1 @I; 0, parameters derived from fitting the data to the model are shown in the figure. Note curvature. In (B), data concerning the accumulation of the E’ form is shown at various hormone concentrations: 0 , O .I phf; 0.0.4 @I; A, 1 pM; 5 pM; 17,100 pM. Linear replots are shown in (C) from which the values of k&s are derived and used to construct the lines which are drawn into the data in (B): 0, 0.035 min-I; 0 , 0.066 min-1; A, 0.154 min-I; A,0.245 min-I. Each slope is dependent on hormone concentration (D). From the kobs versus L curves one can derive the values of K ,the ligand dissociation constant, and a, the rate constant for the activation of E to E’.
A,
through all the points were derived by fitting the data to the function which was used to construct Fig. 2B. The following parameters are obtained: REi-,., the maximal catalytic activity (Vmax); a, the slow-step rate constant; and K , the dissociation constant. The rest of the data (B-D)were collected by initially incubating the membranes with epinephrine and GppNHp. At each point in time a sample was removed into propranolol (a potent 6-adrenergic receptor antagonist). This stops the activation process instantaneously. The extent of active enzyme is then measured by following cAMP accumulation for a fixed amount of time (Tolkovsky and Levitzki, 1978b). The cAMP will evolve linearly.
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
29
In Fig. 4B an entire time course of enzyme activation was collected at different hormone concentrations. Each curve was linearized by the semilogarithmic plot (Fig. 4C). The slopes derived from the semilogarithmic plot are apparent rate constants, kobs. The calculated values of kohs (each slope in Fig. 4C) are given in the legend to Fig. 4. Each curve was constructed using this value and the equation given above Fig. 2D. The values of kobs are also replotted as a function of hormone concentration in Fig. 4D. The curve through the points was theoretically derived from fitting the data to the following expression, which relates kohs to L and to K (Fig. 3B): kohs
=
0.295 X L K + L
where 0.295 min- I is the value of a. At this point one would conclude that the activation of turkey erythrocyte adenylate cyclase by epinephrine and by GppNHp is consistent with the precoupled slow activation model. There is no instance in which the precoupled rapid equilibrium mechanism could be shown to conform to the data. On the other hand, there are also no data which are consistent with similar two-step slow activation models (Table 111; Tolkovsky and Levitzki, 1978b, 1980). Were the mechanism of adenylate cyclase activation related to the two-step sequential models as formulated in Table 111, linear semilogarithmic plots would not have been observed when the fractional activation of the enzyme as a function of time was examined. Also, the binding of the hormone was observed to be simple and not apparently negatively cooperative, as these models would predict. In addition, the binding was also completely independent of the state of the enzyme.
C. Slow Activation by a Sequential Mechanism: The Collision Coupling Model The conclusion that the P-receptor and the enzyme are tightly coupled did not fit with results of the fusion experiments which showed that the turkey erythrocyte P-receptor will form an active complex with a foreign catalytic unit. But none of the sequential models could account for the activation data either. This disturbing conflict made us seek a model which would accommodate the idea that the receptor need not be precoupled tightly to the enzyme, and yet remain consistent with the data. In designing this model two criteria of consistency had to be fulfilled (this is the first level of discrimination): (1) Simple binding of the ligand. The binding of the ligand must be governed by the same constant as the half saturation constant which is derived from the activation data. (2) First-order activation. In order to allow the model an added degree of freedom, we added the assumption that the receptor is no longer required to bind to the active catalytic
TABLE 111 GENERALFEATURESOF THE SEQUENTIAL, SLOWACTIVATION MODELS* Formulation
L+R=
LR+E=LRE
Kl
LRE
L + R E = LRE= LR + E Kl
K2
i LRE’ 5 response
E
5
Kl
E‘ 5 response
Binding
Simple (note equivalence in affinity of L to R and RE forms)
Two-component (apparent negative cooperativity )
Two-component (apparent negative cooperativity)
Rate law
Non-first-order (deviates more at low hormone concentrations)
Second-order
Neither first- nor second-order
Effect of reducing ET
Reduces extent of activation
Reduces rate and extent of activation
Reduces rate and extent of activation
Effect of reducing
Reduces rate of activation, not extent
Reduces extent of activation
Reduces extent of activation
RT
aThe solutions are correct for LT = Lf,,; s is the rate constant governing the slow step
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
31
unit in order to maintain its activity in the presence of GppNHp. In order to retain first-order behavior we introduced the idea that both LRE and LRE’ maintain a steady-state concentration, similar to the Briggs-Haldane assumption so often used in enzyme kinetics. In effect, we converted the receptor into a catalyst, and obtained a mechanism which contained both an association and a dissociation between R and E: L
+ R+ LR + E .-(LRE-LRE‘) K “ slow
rani
+ LR h
a + E’ + response
(1 1)
Three conditions make this formulation distinct from all the previous two-step slow activation models: (1) LRE and LRE‘ must never accumulate to any significant extent compared to LR. These conditions are met by assuming a to be much smaller than b. (2) LR dissociates from E’. E’ can maintain its activity indefinitely as long as ample GppNHp is present. (3) The slow step is no longer sequential to the formation of the LRE complex; it precedes LRE formation. The catalytic role assigned to the receptor is the most extreme case of nonstoichiometric coupling. R is the catalyst, E‘ is the product of the catalysis, and both LRE and LRE’ are negligibly small compared to LR. Because LR remains attached to E for a very short time, we called this mechanism of activation “collision coupling.” The accumulation of the active enzyme is predicted to occur via a first-order process:
The discriminatory features of this model as compared to the precoupled, slow activation model are presented in Table IV. The rejection criteria focus on the altered role of the receptor, which is reflected in the position of the receptor in Eq. (12) compared to Eq. (9). The way this changes the kinetics of the activation process is illustrated in Fig. 5. The activation of the enzyme as a function of time and as a function of receptor concentration is shown in Fig. 5A and B. By reducing the receptor concentration, the precoupled model predicts a reduced extent of activation (A). The collision ‘coupling model predicts that the rate of activation becomes slower but that the final extent of activation remains unaltered and maximal. Response generation is given in Fig. 5C and D. For both models there is a lag time in response output, because both include an exponential form. For the precoupled slow activation model the final response is proportional to the concentration of the receptor in a linear manner. For the collision coupled model, the kinetics are very similar to those shown for reduced ligand concentration in Fig. 2B: the receptor, like the ligand in that system, increases the lag time of the response, but
TABLE IV A COMPARISON OF THE COLLISION COUPLING MODEL WITH Mechanism Formulation
THE
PRECOUPLED SLOWACTIVATION MODEL"
Recoupled slow activation L
+ RE
LRE
2
Collision coupled
L
LRE'
+ R sK LR + E 2
(LRE ~ L R E ' )
1LR + E' ( b >> a ) First level of discrimination Rate law
First-order
First-order
E' or LRE' at t + =
RET (independent of L)
ET (independent of L)
Reduction in L concentration
Slows rate of activation. depends on LI(K
+ L)
Slows rate of activation. depends on L/(K + L )
Second level of discrimination Apparent rate constant of activation
aL (Fig. 5A) K+L
K + L
(Fig. 5B)
CAMP accumulation (Fig. 5C) Reduction in
RT
Reduction in ET
(Fig. 5D)
Limits extent (Fig. 5A and '.-'
Limits rate of activation, does not limit extent (Fig. 5B and D)
Limits extent (Fig. 5A and C )
Limits extent (Fig. 5B and D)
Simulation of each model is given in Fig. 5, indicated in parentheses.
33
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
I
I
r
1
1
1
0
C
-
RT
6o
1
r 0
1
I
1
I
60
0
I
1
60
TIME ,min FIG. 5 . Theoretical response output and fractional activation curves of a one-step slow activation model versus a collision coupling model. The behavior of each system is identical with respect to dependence on ligand concentration. The test to distinguish between them i s given by observing the dependence of the activation on the receptor concentration. ( A . 0 Slow activation; (B.D) collision coupling. Note how reducing RT reduces the extent of activation in (A), but reduces the rate of activation in (B). A detailed analysis is given in Section 1II.B.
at longer times all responses will be generated at an equal rate. Reduction of the extent of E will reduce, in both cases, the extent of activation and response. Data to test the collision coupling model were obtained with the aid of an irreversible P-adrenergic antagonist affinity label which was synthesized by Dr. D. Atlas (Atlas et a/., 1976). The treatment of turkey erythrocyte membranes with this ligand caused a reduction in the number of receptors which could bind epinephrine. After treatment with this ligand, adenylate cyclase was activated in the presence of 0.1 mM GppNHp and a saturating concentration of L-epinephrine (0.1 mM). The data were derived as previously described and are given in Fig. 6A. The data were replotted on a semilogarithmic scale and the apparent rate constant was derived (as in Fig. 4C). The value of kobs derived for each curve is given beside Fig. 6A. The lines are theoretical constructions according to the collision coupling equation. The relationship between the maximal number of receptors, remaining after affinity label treatment as determined by a binding
34
A. M. TOLKOVSKY A kobs, r n d
.033 0
,052
A
,094 -217 374
A v
I
1
0
50
I
100
B
t
I
0 'obs
1
I
.4
0
1
i
30
min
FIG.6. Activation of turkey erythrocyte adenylate cyclase by limiting receptor concentration. Data are derived at 37°C. in the presence of 0.1 mM CppNHp and 0. I mM (saturating) L-epinephrine. Each curve in (A) is based on the values of kobs given in the figure. These values are derived from a semilogarithmic plot of the data generated in (A) (like Fig. 4C). Note how similar (A) and Fig. 4B are. This is due to the fact that both RT and the expression LI(L + K ) modify the exponential term in a linear manner (Fig. SB,or Table IV). In (B), the kobsvalues are compared with the RT values. Note the linear dependence. In (C), CAMPaccumulation is shown as a function of ET. Since ET determines the extent of activation, not the rate, one should compare these data with the theoretical behavior depicted in Fig. 5C. The value of a, the rate constant remains unaltered.
experiment, and the measured kobs is given in Fig. 6B. The linear relationship between these two measured entities attests to one further prediction of the collision coupling model: that the rate of activation is dependent on the receptor concentration in a linear rather than in a saturable manner. We also examined the effect of reducing the enzyme concentration by treating membranes with the mercurial PHMB (Fig. 6C). It can be observed that reducing the enzyme concentration altered the extent of activation but had no influence on the rate constant, which remained at a value of 1 min- even after the activity of the catalytic unit was reduced to 32% of its value in native membranes (these results conform to the pattern presented in Fig. 5C). Thus a collision coupling mechanism can account for all the experimental situations encountered so far.
KINETIC APPROACH TO THE STUDY
OF RECEPTOR FUNCTION
35
D. The Role of GppNHp as Modulator and the Position of the Guanylnucleotide Subunit in the Activation Pathway The process of progressive reformulation of simple models in order to account for complex data has gone through one cycle. No doubt, consistency of models with data is the pitfall of the modeling procedure. Analogies are drawn by conjecture. For example, we have seen that data could be generated which were consistent with a simple precoupled slow activation model, in which R is precoupled to E. But, to conclude that R and E are precoupled would have been wrong. The internal consistency between data and the precoupled slow activation model failed upon further scrutiny. Actually, this model would have been rejected independently of structural data had it occurred to the experimentalist to test whether R is precoupled to E by altering the concentration of R independently of the concentration of E. Since slow activation sequential models did not fit the data, it was difficult to arrive at the idea of such an experiment unless one could conceive of a mechanism in which R assumes other than a modifier role. The conclusion is methodological: in testing such models one must formulate, modulate, and probe experimentally all the species involved in the signal response pathway. This then allows one to determine the position and independence of each component. Clearly, then, one serious omission in the collision coupling model remains. This model considered a two-component system in which informational transfer from R to E occurred. At the same time, we know that this information is mediated in an obligatory fashion by the guanylnucleotide subunit. Without GppNHp no activation of E is obtained. Therefore, it is necessary to account for a stable activation of the adenylate cyclase subunit by GppNHp and the hormone receptor, where the receptor, which is the promoter of this process, acts in a catalytic manner. In order to do so it must be assumed that N, the guanylnucleotide subunit to which GppNHp is bound, is the message of the coupling process. When N now is in its active mode, and is coupled to E, the E form of the catalytic subunit is transformed to E’. This is an obligatory process; the act of E to E’ transition is a passive reflection of events which occur in N. What is the molecular nature of the arrangement between R, N , and E which allows this process to occur? How does N assume the role of messenger and message? In order to answer the first of these questions one would like to know (1) whether N is separate from R and from E; (2) whether the hormone receptor causes the activation of N in a ternary complex which involves RNE, which would then dissociate upon activation; (3) whether alternatively R can cause the activation of N in a binary complex, RN; and (4) whether the site for GppNHp exists independently of the state of the receptor, or whether the site is created only upon the interaction between R and N, and therefore GppNHp binding follows the activation of the guanylnucleotide subunit by the receptor. We begin by formulating models in which N assumes an independent role. We
TABLE V ( T H E GUANVLNUCLEOTIDE SLIBUNIT) ASSUMES A N INDEPENDENT ROLP
A COMPARISON OF COLLISION COUPLING MODELSI N WHICHN
Mechanism
NE are always coupled
z
36
-.
N is activated first, it then couples to E (N’ is the message from R to E)
Rate-limiting step
Formulation (A) LR
A
+ NE LR
f
(LRNE
= LRNE’)
Order of reaction second-level test
Rate law
Reducing N and E third-level test
~.LR.NE
In F = -uRTI
First-order
Rate remains constant for anyNorE levels
u*RT.N
In F
First-order
Slows rate because N’ to E becomes rate limiting (Fails secondlevel test) (Fails secondlevel test) (Fails secondlevel test)
N’E’
+ N 5 LRN LRN A LR + N’
(B) LR
N’ + E 1,N‘E’ ( I ) R to N rate iimiting
=
-uR+
Zero-order
(2) N to N’ conversion rate limiting
Complex Zero-order to first-order
(iii) NT > R7
k
I
(iv) NT << RT In F2 = -br (3) N' to E rate limiting
In
(FlIF2) = (NT
[1
(4)R to N rate limiting N' in equilibrium with E
$
N interacts with R as in (B). but activation of N occurs as in ( A )
(C) LR
+N
F?
N'E' =
u.LRN.E
LRN
-
Complex
(Fails secondlevel test)
Second-order with respect to N and E
Slows rate
Largely firstorder except when CT << NT
Slightly alters rate. but E' activity # Mn2+ activity
Non-first-order
(Fails secondlevel test) Reduction in NT alters rate of activation
b +[ I - exp(-R,t)]
RT
- ET) {ct -
eXp( -d,f)]/Ut?~} ETNT[I - eXp( - d ~ f ) ] KNE + ET + NT[l - exp(-uRTr)]
(i) See footnote b
KKN
LRN
+ E 5 (LRNE -
LRN'E'
LR
+ N'E'
First-order
UThe following symbol has been added: N', which signifies the activated species of N to which GppNHp is bound. By dissociating N from E, three-step sequential models are formulated. These create complex kinetic patterns and complex mathematical formulation. In an attempt to simplify treatment of these models, the rate laws are given before integration. The fractional activation of the enzyme assumes three forms depending on the model:
Also, except where explicitly stated as a rapid equilibrium step (for instance model B, case 2 and 4)all steps are given as unidirectional fluxes. This means that steps a, b, and c will be complex functions of rate constants. Only third-level discriminatory criteria are given in this table (last column). Models which do not predict firstorder kinetics must be rejected. Those which do will be tested only by a reduction of N and E concentrations. hFor a complete solution see Tolkovsky and Levitzki (1981).
38
A. M. TOLKOVSKY
are now forced to elevate the hierarchy of the kinetic formulation by one level, in order to consider three-component systems. A summary of such models is presented in Table V. A detailed mathematical analysis of each model is presented elsewhere (Tolkovsky and Levitzki, 1981). Three types of interactions are described in Table V. (A) N and E are precoupled. This model is a redundant formulation of the collision coupling model, a necessary link between the twocomponent and the three-component formulation. (9) N is an independent subunit which is activated by LR in a binary complex and then proceeds, in the form of NL, to couple to E. (C) N is an independent subunit but activation occurs in a ternary complex involving LR, N, and E which then dissociates as in the precoupled system. This model, then, is somewhat of a hybrid complex halfway between models A and 9. Because the three-step model B contains three steps which occur after ligand binding we are obligated to consider three positions for the rate-limiting step of the activation process: in BI, the formation of the RN complex is postulated to be rate limiting, in BII the transition from N to the active N’ form is postulated to be rate limiting, and in BIll the coupling between N‘ and E to form the active complex N’E’ is postulated to be rate limiting. Model BII is probably the most interesting of the models, because the activation process of N alone is suggested to be the rate-limiting step. Thus, RN would accumulate. Therefore, model BII is further analyzed by testing the rate law change at different N to R ratios, a situation which can be manipulated experimentally. One can observe from the analysis presented in Table V that a rate law change would be predicted to occur due to the alteration of the N to R ratios. This situation would be encountered experimentally if the N and E concentrations are more or less equal in the native membranes and if, at the same time, the guanylnucleotide subunit is in a 10-fold excess over the total receptor concentration. On the one hand, by limiting N , through adding limiting GppNHp concentrations, the ratio of NT to R , would change. On the other hand, if N’E’ is the active form of E, E would continually deplete free N’ from the system. Thus there would be two pressures on the participating molecules: one imposed internally by the activation process (the N’E‘ formation), and the other imposed externally by reducing NT a priori. Clearly, the number of possibilities to account for coupling between three interacting components is augmented considerably as compared to the two-component system. Each acceptable model must now meet two criteria even before it is subjected to experimental test. The first criterion was established when we selected the slow activation model over the rapid equilibrium model (Table 11): all acceptable models must predict that the activation occurs by a first-order process where the ligand concentration limits the rate of activation in a saturable manner. This is the first-level test. The second criterion was established when we selected the collision coupling model over the precoupled model (Table IV): all acceptable models must predict that the rate of activation is limited by the receptor concentration in a linear manner. Also, the active receptor must not accumulate with
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
39
the E' form of the enzyme, and the mechanism of activation must be formulated in the collision coupling manner. This is the second-level test. A third-level test is all that remains to differentiate between models which conform to the first and second criteria: the effect of reducing the total guanylnucleotide subunit concentration, N T , and the effect of reducing the concentration of the catalytic unit, ET, on the rate and the pattern of activation. TESTINGTHREE-STEP SEQUENTIAL MODELS
Of the three models and four subclasses of the sequential binary complex model, only A, B1 and BIV, and Cii withstand the stringent requirement for a first-order process of activation. These models can be distinguished from each other on the basis of their behavior when NT concentration and ET concentration are made very small as compared to their concentrations in the native state of the receptor. Theoretically, when such conditions are achieved, models B1 and C predict a change in the rate of activation. BIV predicts that if K,, is very small relative to the concentrations of N and E, and the equilibrium between them is established before the response is generated, the rate of activation is not altered. But then the levels of N'E', do not correspond in a linear manner to the levels of E', which could be measured by a direct probing of the catalytic activity using manganese. This is probably the most subtle level of discrimination. The reason for the discrepancy between the two measurements of E l , stems from there being an equilibrium between E and N. Only the precoupled model A predicts no change in the rate of activation even when N and E concentrations are very small. Experimentally this is what occurs. In Figs. 7 and 8 data which were generated I \,irying the hormone concentration at fixed but varied GppNHp concentration are presented. Figure 7 shows two features: (1) GppNHp will limit the extent of activation when it is present at subsaturating concentrations (0.05 pM causes 50% activation compared to 0.5 fl). ( 2 ) The rate of activation is controlled by the hormone concentration alone, and is independent of the GppNHp concentration. This point is further elaborated upon in Fig. 8A. At saturating hormone concentrations a 100-fold change in GppNHp concentrations does not alter the first-order nature of the activation process. Therefore, one can derive a saturation curve for the hormone (B) independently of the final extent to which GppNHp achieved activation of the catalytic unit. It must be concluded, therefore, that the hormone and GppNHp have nonequivalent roles in the activation, and that their respective acceptor proteins, R and N, fulfill their roles by a different mechanism of action. There is no doubt that the receptor promotes a change in N. But the nature of this change is not to induce a GppNHp site. Rather, GppNHp must remain bound in order for N ' to remain in the activating mode for any length of time. It would seem, therefore that N and E are precoupled. Nevertheless, on purely kinetic grounds, there is an alternative explanation. NT may be so abundant relative to R that a remaining 10%is sufficient to maintain the rate of interaction I,
40
A. M. TOLKOVSKY 0
L
10
20
I
1
30
40
W
40 1
$
1-
--
.8 .t-
20
0
1-
6-
.y \
7
.I-
w
Y
-
.4-
. .2
-
.2
1
0
-
I
,
20
0
40
20
40
TIME, rnin FIG. 7. Activation of turkey erythrocyte adenylate cyclase by varying hormone concentration as a function of limiting GppNHp concentrations. The role of GppNHp as an obligatory modifier in the activation process is tested by repeating experiments like those presented in Fig. 4 but at different, fixed GppNHp concentrations. Limiting GppNHp is seen to lower the extent of activation (lower panels) but does not change the rate of activation which is still controlled exclusively by the hormone and receptor concentrations. Also. semilogarithmic plots remain linear (upper panels), hence the process is first order. The lines in the lower panels are obtained from the kobs values derived in the upper panels using model A, Table V.
between N’ and E to form N’E’ as non-rate-limiting. In this case model BII, in which a collision coupling between R and N occurs, is a viable alternative. But from the data, this would mean that since 0.1 X N, > R, and 0.1 X NT < E,, ET >>R,. We have no clue as to whether this is at all likely to be the case. This question remains open to further investigation and so opens the possibility of examining a whole new class of models.
IV. CONCLUSIONS In this article I have presented evidence obtained by kinetic studies which was used to evaluate the mechanism of activation of turkey erythrocyte adenylate cyclase by hormones receptors and guanylnucleotides. I have attempted to give a stepwise account of how such evidence is assimilated and understood. I believe that in turkey erythrocytes, in the presence of GppNHp, it is likely that the
41
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
a
B .3
-
::.2
-
.1
-
I -
.+
,
W
- .I .* W
=
- .01 I
.k
W
.01
.5
.c5 .1
2 . 5 p M , K for L-epinephrine 0.3/min, a
1
0
10 min
1
2
3
4
5
PM
C
I
8
I
0
4
8 mi n
12
Activation of turkey erythrocyte adenylate cyclase by limiting guanylnucleotide concentrations. N , the guanylnucleotide subunit, must be firmly attached to the catalytic unit in turkey erythrocyte membranes, in situ. The reasons are as follows: (1) The rate of activation of the enzyme is independent of GppNHp concentrations (A). All semilogarithmic plots are linear, therefore the process of activation is first order. Thus, although the extent of W Tis reduced 10-fold, no switch in the rate-limiting step is observed. (2) GppNHp does not alter the behavior of the receptor (B). This would indicate that the site for GppNHp must preexist independently of the hormone. (3) The pattern of activation remains unmodified even after a 140-fold reduction in both N and E concentrations. Thus it is unlikely that the interaction of N with E is rate limiting. (See also Tolkovsky e t a / . (1982).)
following process occurs: L
+ R z LR
box ra*1 + NGE*low e (LRNGE e LRNkE’) LR + NbE‘ 4
Kl
The turkey erythrocyte and other enucleated erythrocytes seem to have been designed by nature for the exclusive use of biochemists. It is designed so that no leaks in information occur. E is transformed measurably slowly from an entirely inactive state to a highly active and stable state. This transition occurs only by some kind of interaction with the activated receptor and is mediated exclusively by guanylnucleotides: GppNHp or GTP (Cassel et al., 1977) or GMP. The state of E is a passive image of the state of N. It is most clearly a “two-state system”
42
A. M. TOLKOVSKY
insofar as the catalytic unit is concerned (Iyengar et al., 1980). Even phosphodiesterase activity is virtually absent. Nevertheless, not all the problems relating to the mechanism of activation of adenylate cyclase by hormones in turkey membranes have been solved. We lack an understanding of the nature of LRNE and the LRN’E’ complexes. This is the heart of the matter-the activation step per se-and should be approached by transition state probes. A transition state probe will cause LRNE and LRN‘E’ states to accumulate and become vulnerable to analysis. One such probe is probably GppNHp, which keeps part of the system, the N‘E‘ complex, “stuck” in a highly active form. No doubt the guanylnucleotide subunit, being the molecule which conveys the coupling message, is the point of focus. We would like to have an understanding of N in its active modes. We would also like to know more about the actual concentrations of N and E in the membrane and study the potential of N to be a modulator of other membrane functions. Many of these problems cannot be solved and cannot benefit from kinetic analysis. The final proof of any molecular mechanism lies in our ability to isolate the components and reconstitute them in an active mode. It is not clear whether, in addition to biochemists, turkeys take advantage of this system and put it to biological use. It may be a relic of reticulocyte development but unfunctional in mature erythrocytes. Other systems derived from turkey (turkey brain for instance; Tamir and Tolkovsky, in preparation) and other mammalian systems in which regulation of CAMP levels in the cell is important for the function, regulation, and viability of the cell are not so simply designed. There are numerous leaks in the process of information transfer. GppNHp and GTP activate the catalytic subunit independently of the receptor. Even in the absence of GppNHp or GTP highly active basal activities are seen. Often, no lag time in the activation by GppNHp occurs. Other modulators, such as calmodulin, interact with the catalytic subunit as well. I believe that the tools developed through the investigation of the turkey erythrocyte can now be applied to these more complex systems. We would like to delineate the mechanism of activation and of inhibition of adenylate cyclase in systems where these may have consequences to the well-being of the cell and the organism at large. With care, the kinetic approach may be used to answer questions pertaining to complex phenomena which occur in a noisy, multiregulated system. LIST OF SYMBOLS
L an activatory ligand R receptor E catalytic moiety of adenylate cyclase (or a response-generating moiety)
L free ligand concentration R or RT free or total receptor concentration E or ET free or total concentrations of the catalytic moiety
KINETIC APPROACH TO THE STUDY OF RECEPTOR FUNCTION
an activated form of E
43
E’ o r E;
guany lnucleotide-sensitive component an activated form of N to which GppNHp is bound equilibrium dissociation constants dissociation cunstant of the RN complex dissociation constant of the NE complex apparent rate constants which define the flux through a chemical reaction an apparent first-order rate constant a rate constant which determines response output time
free or total cuncentrations of the activated catalytic moiety N or NT free or total concentrations of the inactive forms of N N ’ or N+ free or total concentrations of the activated forms of N RE, total concentration of a precoupled receptor-effector complex NE or N‘E’ free concentration of the inactive ur activated forms of the guanylnucleutide-scnsitive component coupled to the catalytic moiety LRN free Concentration of the ternary complex ligand-receptorguanylnucleotide-sensitive component
REFERENCES Ariens, E. J. (1954). Affinity and intrinsic activity in the theory of competitive inhibition. I . Problems and theory. Arch. Inr. Pharmucodyn. 99, 32-49. Atlas. D., Steer, M. L., and Levitzki, A . (1976). Affinity label for beta adrenergic receptor in turkey erythrocytes. Proc. Natl. Acud. Sci. U.S.A. 73, 1921-1925. Boyenaems, J . M.. and Dumont, J . E. (1975). Quantitative analysis of the binding of ligands to their receptors. J . Cvclic. Nucleotive Res. 1, 123- 142. Boeynaems. J. M., and Dumont, J. E. (1977a). The twu step model of ligand-receptor interaction. Mol. Cell. Endocrinol. 7, 33-47. Boeynaems, J . M . , and Dumont, 1. E. (1977b). Models of dissociable receptors applicable to cyclic AMP dependent protein kinases and membrane receptors. Mol. Cell. Endocrinol. 7,275-295. Bourne, H. R.. Coffino, P., and Tomkins, G . M. (1975). Selection of a variant lymphoma cell deficient in adenylate cyclase. Science 187, 950-952. Cassel, D., Levkovitz, H., and Selinger, Z . (1977). The regulatory GTPase cycle of turkey erythrocyte adenylate cyclase. J . Cvclic Nucleotide Rex. 3, 393-406. Clark. A. J . (1937). General pharmacology. I n “Handbuch der Experimentalen Pharmakologie” (A. Haffter and H. Heubner, eds.), Vol. 4. Springer-Verlag, Berlin and New York. Cuatrecasas, P. ( I974a). Insulin receptors. cell membranes and hormone action. Biochem. Pharmacol. 23, 233-2361, Cuatrecasas, P. (1974b). Membrane receptors. Annu. Rev. Bittchem. 43, 169-214. De Haen, C. ( 1976). The nonstoichiometric, floating receptor model for hormone-sensitive adenylyl cyclase. J . Theor. B i d . 58, 383-400. lyengar, R., Abramowitz, J . . Bordelon-Riser, M., Blume, A., and Birnhaumer, L. (1980). Regulation of hormone-receptor coupling to adenylyl cyclase: Effects of GTP and GDP. J . B i d . Chem. 255, 10312-10321. Jacobs. S . , and Cuatrecasas, P. (1976). The mobile receptor hypothesis and cooperativity o f hormone binding. Application to insulin. Biochim. Biophvs. Aria 433, 482-495. Kuhn, T. S . (1970). “The Structure of Scientific Revolutions,” 2nd ed. Univ. of Chicago Press, Chicago, Illinois.
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Neer, E. J. ( I 976). Two soluble forms of guanosine 5’4imino)triphosphate and fluoride activated adenylate cyclase. J . Biol. Chem. 251, 5831-5834. Orly, J . , and Schramm, M. (1976). Coupling of catecholamine receptors from one cell with adenylate cyclase of another cell by cell fusion. Proc. Natl. Acad. Sci. U . S . A . 73, 4410-4414. Rodbell, M. (1975). On the mechanism of activation of fat cell adenylate cyclase by guanylnucleotides. J . B i d . Chem. 250, 5826-5834. Rodbell, M., Birnbaumer, L., Pohl, S. C., and Krans, H. M. J. (1971). The glucagon sensitive adenylate cyclase system in plasma membranes of rat liver. IV. An obligatory role of guanylnucleotides in glucagon activation. J . Bid. Chem. 246, 1877- 1882. Ross. E. M., and Gilman, A. G. (1977). Resolution of some components of adenylate cyclase necessary for catalytic activity. J , B i d . Chem. 252, 6966-6969. Ross, E. M., Maguire, M. E., Sturgill, T. W., Biltonen, R. L., and Gilman, A. G . (1977). Relationship between the beta-adrenergic receptor and adenylate cyclase: Studies of ligand binding and enzyme activity in purified membranes of S49 lymphoma cells. J . B i d . Chem. 252, 5765-5775. Ross, E. M., Howlett, A. C., Ferguson, K. M.. and Gilman. A. G . (1978). Reconstitution of hormone sensitive adenylate cyclase activity with resolved components of the enzyme. J . Biol. Chem. 253, 6501-6512. Salomon. Y., Lin, M. C., Londos, C., Rendell, M., and Rodbell, M. (1975). The hepatic adenylate cyclase system: Evidence for transition states and structural requirements for guanine nucleotide activation. J . B i d . Chem. 250, 4239-4245. Schramm, M., and Rodbell, M. (1975). A persistent active state of the adenylate cyclase system produced by the combined actions of isoproterenol and guanylimidophosphate in frog erythrocyte membranes. J . B i d . Chem. 250, 2232-2237. Sevilla, N.. Steer, M. L., and Levitzki, A. (1976). Synergistic activation of adenylate cyclase by guanylylimido phosphate and epinephrine. Biochemistry 15, 3493-3499. Singer, S. J., and Nicolson, G . L. (1972). The fluid mosaic model of the structure of cell membranes. Science 175, 720-731. Stephenson. R. P. (1956). A modification of receptor theory. Br. J . Phurmacol. Chemother. 11, 379-393. Tamir. A., and Tolkovsky, A . M. (1981). Early burst kinetics and transition states of the guanylnucleotide subunit in relation to activation of adenylate cyclase in brain. In preparation. Tolkovsky, A. M., and Levitzki, A. (1978a). Collision coupling of the beta adrenergic receptor with adenylate cyclase. In “Hormones and Cell ,Regulation” (J. Dumont and J. Nunez, eds.), Vol. 2, pp. 89-105. Elsevier, Amsterdam. Tolkovsky, A. M., and Levitzki, A. (1978b). Mode of coupling between the beta adrenergic receptor and adenylate cyclase in turkey erythrocytes. Biochemistry 17, 3795-3810. Tolkovsky, A. M., and Levitzki, A. (1980). Molecular models for receptor to adenylate cyclase coupling. I n “Mathematical Models in Molecular and Cellular Biology” (L. A. Segel. ed.) pp. 89-1 11. Cambridge Univ. Press, London and New York. Tolkovsky, A. M.. and Levitzki, A. (1981). Theories and predictions of models describing sequential interactions between the receptor, the GTP regulatory subunit and the catalytic unit of hormone dependent adenylate cyclases. J . Cyclic Nitcleotide Res. 7, 139- 150. Tolkovsky, A. M., Braun, S . , and Levitzki, A. (1982). The kinetics of interaction between beta receptors, the GTP protein and the catalytic unit of turkey erythrocyte adenylate cyclase. Proc. Natl. Acad. Sri. U.S.A. 79, 213-217. Werman, R. (1975). The transduction of chemical signals into electrical information at synapses, I n “Stability and Origin of Biological Information” (1. R. Miller, ed.), pp. 226-244. Wiley, New York.
CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME i a
The p-Adrenergic Receptor: Ligand Binding Studies Illuminate the Mechanism of Recepto r-Adenylate Cyclase Coupling JEFFREY M. STADEL AND ROBERT J . L E F K O W I Z Department of Medicine (Cardio1og.v). Howard Hughes Medical Institute Duke University Medical Center Durhum, North Carolina
Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of Radioligands Specific for Adrenergic Receptors . . . . . . . . . . . . . . . . . 111. Study of Adrenergic Receptors in Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Characterization of Detergent-Solubilized Adrenergic Receptors. . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.
11.
1.
45 47 49 57 63
INTRODUCTION
The concept of specific receptors or discriminators that recognize a particular hormone or drug and thereby initiate the biological action of these agents has been evolving for nearly a century (Dale, 1906). A wealth of information has been generated through the investigation of the metabolic effects of pharmacologically active agents, both in vivo and in vitro, and these studies have provided indirect evidence for the existence of discrete receptor moieties. Considerable investigative effort has been directed toward understanding the biological specificity as well as the mechanism of action of catecholamines, since these compounds regulate cellular metabolism in a wide variety of tissues. Evaluation of pharmacological data on the actions of catecholamines in several organs pointed to a logical division of their effects. Ahlquist (1948) proposed the existence of at least two types of adrenergic receptors. His studies, which utilized several catecholamine agonisfs, showed that organ responses could be grouped 45 Copyright 0 1983 by Academic RCW. Inc All right$ of reproduction in any form reserved. ISBN a-12-153xn-2
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JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
according to the order of potency of these agents in evoking a characteristic response. The first type of response, which he termed “alpha,” followed the agonist potency series epinephrine > norepinephrine > isoproterenol. The second or “beta” group of responses followed the order isoproterenol > epinephrine > norepinephrine. A typical a-adrenergic response is smooth muscle contraction. Smooth muscle relaxation, inotropic and chronotropic regulation in the heart, and metabolic effects such as lipolysis are mediated through P-adrenergic receptors. Further support for the concept of two discrete types of adrenergic responses was provided by the development of highly potent antagonist compounds. aAdrenergic responses to catecholamines are competitively blocked by drugs such as phentolamine, phenoxybenzamine, and the ergot-alkaloids. P-Adrenergic responses are inhibited by a different set of drugs which has little affinity for the areceptors, e.g., propranolol, alprenolol, and pindolol. The characteristics of high potency and biological specificity make antagonist compounds particularly useful in the classification of adrenergic responses and in the direct study of adrenergic receptors (see below). Recent, extensive studies using a large arsenal of synthetic compounds specific for adrenergic receptors have pointed to a further division of both a-(Berthelson and Pettinger, 1977) and P- (Lands et al., 1964) adrenergic responses into pharmacologically defined subtypes. The subclassification of P-adrenergic receptors is based on the relative potency of epinephrine and norepinephrine (Lands et al., 1964). p I-Receptors demonstrate approximately equal affinity for epinephrine and norepinephrine, whereas P,-receptors recognize epinephrine with higher affinity than norepinephrine. More recently it has been realized that a-adrenergic receptors can also be divided into receptor subtypes (Berthelson and Pettinger, 1977; Hoffman and Lefkowitz, 1980). a,and a,-Adrenergic receptors are differentiated by their affinities for a variety of subtype selective agents. For example, a,-receptors have very high affinity for the antagonist prazosin, whereas a,-receptors have high affinity for the antagonist yohimbine. It is also possible to distinguish between a-and P-adrenergic responses based on the biochemical events necessary to produce the physiological changes that accompany adrenergic stimulation. (3-Adrenergic responses are directly linked to the activation of adenylate cyclase in the plasma membranes of target cells, suggesting that cyclic AMP (CAMP) mediates the regulatory effects of P-adrenergic agonists (Robison el a/., 197 1). This is true of the responses mediated by both PI- and p,-receptor subtypes. a-Adrenergic responses, on the other hand, are not always linked to adenylate cyclase. Agonist binding to a,-adrenergic receptors does not affect adenylate cyclase activity directly. However, alterations in Ca2 fluxes have been implicated in aI-adrenergic responses (Exton, 1979). a,-Adrenergic receptors do appear to be linked to adenylate cyclase activity, +
THE 6-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
47
which they inhibit (Jakobs et al., 1976). This is in contrast to P-adrenergic receptors, which always stimulate the production of CAMP. The purpose of this article is to relate the study of adrenergic receptors to the regulation of adenylate cyclase activity. The ubiquitous nature of the adrenergic responses has made these systems prototypes or models for the study of receptor-effector coupling in general. Progress is being made in the elucidation of the molecular mechanisms of transmembrane signaling via adrenergic receptors. We will focus primarily on studies of the P-adrenergic receptor-adenylate cyclase complex. Work on P-adrenergic stimulation of adenylate cyclase is now proceeding toward the purification and reconstitution of the molecular components of the receptor-cyclase complex. This will be necessary to define precisely the chain of events which links agonist occupancy of the receptors to enzyme activation. The components currently identified include the agonist binding sites or receptors, the enzyme catalytic unit, and the guanine nucleotide regulatory protein (for reviews see Ross and Gilman, 1980; Stadel et al., 1982). Although in this article we rely heavily on the work derived from investigations in our own laboratory, we also relate our observations to those of other investigators. In addition, we will point out, where appropriate, how understanding of receptor-cyclase coupling distilled from the investigations of the stimulatory p-adrenergic receptor-adenylate cyclase system may also be applicable in understanding inhibition of adenylate cyclase activity, as mediated, for example, by a,-adrenergic receptors.
II. DEVELOPMENT OF RADIOLIGANDS SPECIFIC FOR ADRENERGIC RECEPTORS Pharmacological studies using intact tissues or broken cell preparations have indicated the existence of adrenergic receptor subtypes. Although such pharmacological studies suggested the existence of discrete receptor moieties, more direct experimental approaches were required to document this fact. As so often occurs in science, technical innovations led the way to an explosion of information about the nature of hormone and drug receptors. In the late 1960s and early 1970s procedures were developed to incorporate radionuclides covalently into hormone structures with little or no alteration in the biological activity and specificity of these hormones (Roth, 1973). These procedures were first applied to peptide hormones such as ACTH (Lefkowitz el al., 1970) and angiotensin (Lyn and Goodfriend, 1970). In 1974 three laboratories reported the successful development of radioligands specific for the P-adrenergic receptor (Atlas et al., 1974.;Aurbach et al., 1974; Lefkowitz et al., 1974). All three groups utilized P-
48
JEFFREY
M. STADEL AND ROBERT J. LEFKOWITZ
adrenergic antagonists, albeit three different compounds, to take advantage of the high affinity and specificity of these agents. Of the three original radioligands, ( +)[3H]propranolol (Atlas er al., 1974), ( -)[3H]dihydroalprenolol (DHA) (Lefkowitz et al., 1974), and ( +)'2sI-labeled hydroxybenzylpindolol (HYP) (Aurbach et af., 1974), the latter two, [3H]DHA' and '2sI-labeled HYP, are still widely used to characterize P-adrenergic receptors in a variety of tissues. Recently two more antagonist radioligands have been developed, ( +-)'251-labeled cyanopindolol (Engle, 1980) and (*)I2Tlabeled pindolol (Barovsky and Brooker, 1980). The utility of all five of these radioligands rests in the fact that they are of very high specific radioactivity (50-2000 Ci/mmole) and high affinity ( K , < 10 nM). Although specific for P-adrenergic receptors, these radioligands do not discriminate between P I - and P,-receptor subtypes. Shortly after the development of P-adrenergic specific radioligands, Williams and Lefkowitz (1976) reported that a tritiated derivative of an ergot alkaloid, ( -+)[3H]dihydroergocryptine, could be used to identify a-adrenergic receptors. [3H]Dihydroergocryptineis a potent a-adrenergic antagonist as are many of the subsequently developed radioligands for characterization of a-adrenergic receptors such as (?)[3H]WB4101 (Greenberg et al., 1976), (*)['H]prazosin (Greengrass and Brenner, 19791, and (+)[3H]yohimbine (Tharp er al., 1981). Unlike [3H]dihydroergocryptine, which does not discriminate between a-adrenergic receptor subtypes, [3H]prazosin and ['Hlyohimbine are highly selective for a,and a,-receptors, respectively. Although radiolabeled antagonist ligands have been valuable tools for determining the properties of adrenergic receptors, an underlying goal of direct receptor studies is to understand how agonist binding to receptors initiates a biological response. The study of receptor binding characteristics by employing radiolabeled antagonists in competition with unlabeled adrenergic agonists has provided indirect information as to how agonist drugs might work to activate intracellular signaling mechanisms (see below). However, binding studies with radiolabeled agonists would clearly provide an extra dimension to the investigation of how agonists interact with their receptors. Lefkowitz and Williams (1977) described the successful labeling of P-adrenergic receptors in frog erythrocyte membranes with the agonist ligand (+-)[3H]hydroxybenzylisoproterenol(['HIHBI). This agonist radioligand has been an important tool for probing the properties of Padrenergic receptors and particularly P,-receptors since the ligand is more potent in binding to this receptor subtype. [3H]HBI appears to be virtually unique as an 'Abbreviations used: (3H]DHA, (-)~3H]dihydroalprenolol: [12sI]HYP, (2)1251-labeled hydroxybenzylpindolol; ['HIHBI, ( ~)[3H]hydroxybenzylisoproterenol;Gpp(NH)p, guanyl-5'-yl imidodiphosphate; GTPyS, guanosine S'-O-(3-thiotriphosphate): ACTH, adrenocorticotrophic hormone; NEM, N-ethylmaleimide, SDS, sodium dodecyl sulfate; H, hormone or agonist; R , receptor; N , guanine nucleotide regulatory protein; C, adenylate cyclase catalytic unit; X, an unspecified additional membrane component.
THE P-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
49
agonist radioligand specific for P-adrenergic receptors, although [ 3H]epinephrine has been reported to identify f3-adrenergic receptors in brain (U’Prichard et a / ., 1978). [3HH]Epinephrinehas, in fact, been more extensively used to investigate aadrenergic receptors (U’Prichard and Snyder, 1977; U’Prichard et d.,1977). In addition to [3H]epinephrine, [3HH]clonidine(U’Prichard et at., 1977), a partial 01adrenergic agonist, can be used in some systems. These agonist ligands have been used to confirm and extend early indirect observations of receptor binding properties employing radiolabeled antagonists in competition with unlabeled adrenergic agonist agents. The development of radioligands that identify specific receptors for drugs and hormones has had a far reaching impact on the investigation of the mechanisms of action of these agents. Radiolabeled ligands have now been developed for nearly every type of receptor known to the pharmacologist. The concept of “receptor” now implies a specific molecular entity whose properties can be probed and evaluated. Many different receptors have been extensively characterized with direct binding studies and the development of these sensitive assay methods has made possible the goal of receptor purification and biochemical analysis.
111.
STUDY OF ADRENERGIC RECEPTORS IN MEMBRANES
Radioligands have been most extensively used to characterize receptors on the surface of target cells or in plasma membrane preparations derived from these cells. It is necessary that a radioligand fulfill several essential criteria for identification of physiologically relevant receptor sites (Williams and Leflcowitz, 1978). (1) The binding of the radioligand to receptor preparations should be saturable, reflecting binding to a finite number of receptor sites. ( 2 ) The concentration range over which the ligand binds to the receptor should be comparable to the concentration range over which the ligand initiates or inhibits a biological response. (3) The kinetics of binding should likewise reflect the kinetics of the biological response. (4)The receptor sites labeled should demonstrate the appropriate specificity of the biological response. In the case of the P-adrenergic receptor this last criterion means that the ability of different agonists to compete for the binding of the radioligand must follow the same potency series which is characteristic of the stimulation of adenylate cyclase by catecholamines, namely, isoproterenol > epinephrine > norepinephrine. In addition, P-adrenergic antagonists should potently compete for the binding of the radioligand, reflecting their ability to inhibit cyclase stimulation. Furthermore, the biologically active (-) stereoisomers of adrenergic agonists and antagonists should be more potent in competing for the binding sites than the less active (+) stereoisomers. The radioligands currently employed to identify adrenergic receptors (see above)
50
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
have been shown to fulfill these criteria and therefore label physiologically relevant receptors in a variety of tissues. One of the earliest applications of the radioligand binding technique to the study of P-adrenergic receptors was the investigation of the interaction of a wide variety of unlabeled P-adrenergic agonist and antagonist compounds with the receptors. This is accomplished through competition binding experiments in which the dose-dependent inhibition of binding of a fixed concentration of radioligand is examined. By comparing the affinities of these unlabeled agents for the ligand binding sites with their abilities to modulate adenylate cyclase activity, it could be convincingly shown that agonists and antagonists compete for the same set of P-adrenergic receptor binding sites. The affinity of both agonists and antagonists for the P-receptors is primarily determined by their stereo configuration and the substitutions on the amino nitrogen. These studies also reinforced the notion that agonist “activity” is not simply related to binding affinity, but rather involves additional interactions not triggered by antagonists. Following the validation of the radioligand binding approach for studying adrenergic receptors, investigative emphasis shifted toward utilizing these new tools to explore the mechanism of agonist activation of adenylate cyclase. Agonist agents interact with P-adrenergic receptors in a fundamentally different way than do antagonists in order to initiate the chain of events leading to characteristic biological responses. Radioligand binding assays provided an opportunity to examine these differences at the receptor level. One of the first unique properties of agonist binding to (3-adrenergic receptors which was documented was that guanine nucleotides modulate receptor affinity for agonists but not antagonists (Maguire et al., 1976; Lefkowitz et al., 1976). The rationale for exploring the effects of guanine nucleotides on adrenergic receptor binding properties was based on the studies of Rodbell er al. (1971), who demonstrated that these nucleotides were essential regulators of the glucagon receptor-adenylate cyclase complex in rat liver membranes. For (3-adrenergic receptors, the initial demonstrations of guanine nucleotide regulation of agonist binding were accomplished using partially purified plasma membranes prepared from C6 glioma cells (Maguire et al., 1976) and frog erythrocytes (Lefkowitz et al., 1976). It was found that agonist competition binding curves vs radiolabeled antagonists were “shallow” in the absence of guanine nucleotides but became steeper and shifted toward the right, indicating a lower apparent affinity of the receptor for agonist, in the presence of exogenously added guanine nucleotides such as GTP or Gpp(NH)p (Fig. 1). The addition of guanine nucleotides to the binding assay did not affect the binding of the radiolabeled antagonist nor did it affect the shape or position of the competition binding curves generated by unlabeled antagonists (Fig. 2). These curves are “steep” and uniphasic under all experimental conditions. The guanine nucleotide-dependent ‘‘shift to the right” of the agonist binding curves, i.e., to lower receptor affinity, was observed for a series of p-
51
THE (3-ADRENERGICRECEPTOR: LIGAND BINDING STUDIES
9
8
7
-loglo
6
5
I-)Isoproterenol]
4
3
2
(M)
FIG. I . Computer modeling of competition binding data of isoproterenol for r3H]DHA in frog erythrocyte membranes. Competition of the agonist isoproterenol for ['HIDHA in the absence (0) and presence (0) of CTP. The curve in the absence of nucleotide was significantly ( p < 0.00 I ) better fit by a model for two binding states of the receptor. See text for details. (From Kent et a / ., 1979.)
25 5
5 100
-
-
F
0
u
0
s
80-
(-1 Alprenolot
0
UL= KH * 12nM
m
-
5
60-
C
ea
0
40-
-s5
20-
e
D %
I
.-.. I
0
1
I
1
1
I
,. r
FIG. 2. Computer modeling of competition binding data of alprenolol for ['HIDHA in frog erythrocyte membranes. The competition curve of the antagonist alprenolol for ["IDHA is adequately modeled to a homogeneous class of binding sites. (From Kent er a / . . 1979.)
52
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
adrenergic agonists (Lefiowitz et a / ., 1976). Interestingly the magnitude of this “nucleotide-dependent shift” correlates with the intrinsic activity of the agonist for activation of the adenylate cyclase. Intrinsic activity is a term used to quantitate the maximum ability of an agonist to stimulate a biological response, such as adenylate cyclase activity. The correlation of intrinsic activity and nucleotide effects on agonist binding suggests a relationship between guanine nucleotide regulation of receptor affinity for an agonist and the drug’s ability to stimulate the cyclase enzyme. Thus agonists, but not antagonists, have the ability to form a high-affinity complex with the P-adrenergic receptor, and this agonist-receptor complex is modulated by guanine nucleotides. Computer-aided analysis of radioligand binding data has added a new quantitative dimension to our understanding of the differences between agonist and antagonist binding to P-adrenergic receptors. As described above, the competition binding curves of adrenergic agonists for a radiolabeled antagonist such as [3H]DHA are shallow (slope factors < 1) indicating complex binding interactions between agonists and the receptors. In contrast, the competition binding curve of unlabeled antagonists for [3H]DHA is always steep (slope factor = I ) indicating a uniform affinity of the receptors for antagonists. Computer modeling of the shallow agonist competition binding curves indicated a statistically significant improvement in the fit of the binding data by a model based on two binding states of the receptor (Fig. I ) (Kent er al., 1979). This two-state model was found to be appropriate for all agonists tested. The two affinity states of the receptor were characterized by specific dissociation constants (K,, KL) and the proportion of the total receptor population in each state was determined (RH, RL). Using a series of full and partial agonists, a significant correlation was shown to exist between the ability of an agonist to stimulate the adenylate cyclase (intrinsic activity) and the ratio of the dissociation constants of the agonist for the high- and low-affinity states of the receptor (KLIKH).A significant correlation was also found to exist between agonist intrinsic activity and the proportion of the receptors binding the agonist with high affinity (% R H ) (Kent et ai., 1979). Quantitative analysis of radioligand binding data thus provides additional evidence for the important role of agonist high-affinity binding in the process of transmembrane signaling by receptor-cyclase complexes. The ability of an agonist to form a high-affinity, nucleotide-sensitive complex with the P-adrenergic receptor is dependent upon the ionic environment of the membranes during the binding assay. Although neither receptor binding properties nor adenylate cyclase activity shows a significant dependence on ionic strength, divalent cations can be shown to be required for both of these activities. Concentrations of Mg2+ or Mn2+ in the millimolar range are necessary for adenylate cyclase activity and these same cations are also required for agonist high-affinity binding to receptors (Williams et al., 1978). Monovalent cations cannot substitute for these divalent metal ions. The regulation of receptor affinity
'THE 5-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
53
by divalent cations is observed only with agonist binding; antagonist interactions with the P-adrenergic receptor are unperturbed by the presence or absence of metal ions in the binding medium. Subsequent radioligand binding studies have demonstrated several additional unique binding properties of agonists to P-adrenergic receptors. Pike and Lefkowitz (1978) reported that decreasing the temperature of the binding assay incubation increased the apparent affinity of turkey erythrocyte P-adrenergic receptors for agonists without a significant effect on receptor affinity for antagonists. This observation has recently been extended to show a similar specific temperature effect on agonist binding to (3-adrenergic receptors in a variety of mammalian tissues containing both p,- and @,-receptor subtypes (Weiland e l a / . , 1980). Briggs and Lefkowitz (1980) were able to show that when assayed below physiological temperatures, agonists are not able to induce the high-affinity, nucleotide-sensitive state of the P-adrenergic receptor in turkey erythrocyte membranes and that this observation correlates with an inhibition of the ability of agonists to stimulate the adenylate cyclase in these membranes. Treatment of the turkey erythrocyte membrane with the unsaturated fatty acid cis-vacennic acid, which increases the flujdity of these membranes (Rimon et al., 1978), led to reappearance of the ability of agonists to form a high-affinity complex with the receptor and concomitantly facilitated agonist activation of the adenylate cyclase at low temperature. These effects of temperature on agonist binding characteristics and activation of adenylate cyclase may reflect a specific agonist-induced conformational change in the receptor, specific receptor-lipid interactions, and/ or necessary lateral mobility of the receptor-cyclase components within the lipid matrix. Additional evidence supporting an agonist-induced conformational change in the p-adrenergic receptor comes from studies using N-ethylmaleimide (NEM) in turkey erythrocyte membranes. N-Ethylmaleimide reacts specifically and irreversibly with free sulfhydryl groups of proteins (Means and Feeney, 1971). Pretreatment of turkey erythrocyte membranes with NEM alone does not affect the ability of (3-adrenergic receptors to bind the radiolabeled antagonist [3H]DHA, but pretreatment of these membranes with NEM in the presence of a p-adrenergic agonist resulted in a loss of up to 50% of the (3-receptors as determined by a decrease in binding capacity for ["IDHA (Bottari et al., 1979). Simultaneous treatment of membranes with NEM and antagonist did not result in receptor loss, indicating that agonist binding induces a specific conformational change in the receptor which exposes a cysteine residue to NEM. This agonist-specific effect appears to be related to the mechanism by which agonist binding to receptor results in activation of adenylate cyclase. The rate at which NEM inactivates turkey erythrocyte (3-receptors correlates with the intrinsic activity of the agonist which occupies the receptor (Vauquelin et al., 1979). Guanine nucleotides which
54
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
regulate receptor affinity for agonists and are required for adenylate cyclase activation also inhibit the inactivation of receptors by NEM in the presence of agonists (Vauquelin et al., 1980). The effect of NEM in the presence of agonist is not restricted to the P ,-adrenergic receptor in turkey erythrocyte membranes. Similar results have been obtained by treating membranes from S49 mouse lymphoma cells (P,-receptor subtype) with NEM and agonist (Vauquelin and Maguire, 1980). It is noteworthy that membranes from S49 mutant cell clones that are functionally devoid of components necessary for guanine nucleotide regulation of receptor affinity failed to show an effect with NEM in the presence of agonists, suggesting that productive coupling between the P-adrenergic receptor and the guanine nucleotide regulatory protein of the adenylate cyclase complex may be necessary for the exposure of the critical sulfhydryl group (Vauquelin and Maguire, 1980). A perplexing question raised by these studies concerns the observation that the maximal effect of NEM and agonist reduces the receptor population only by 50%. Complete inactivation of the receptors was never achieved by this approach. “Heterogeneous” populations of receptors have been offered as a possible explanation, but further investigation will be required to clarify this observation. An important advance in the characterization of agonist binding to P-adrenergic receptors was achieved through the development of an effective radiolabeled agonist ligand. Lefiowitz and Williams (1977) reported the properties of the binding of (+)[ 3H]hydroxybenzylisoproterenolto the P-adrenergic receptor of frog erythrocyte membranes. It became evident that this radioligand would be useful in the direct investigation of P,-adrenergic receptors in purified plasma membrane preparations. Availability of this ligand permitted, for the first time, a direct examination of agonist binding to the P-adrenergic receptor. Previously such studies had been performed by examining agonist competition with radioligand antagonist binding. High-affinity [3H]HBI binding to the P-adrenergic receptor is characterized by a very slow rate of dissociation that is not affected by the addition of competing adrenergic ligands (Williams and Lefiowitz, 1977). In contrast, guanine nucleotides promote the rapid and complete dissociation of [3H]HBI from the membrane receptors. Thus [3H]HBI labels exclusively the high-affinity state of the P-adrenergic receptor in frog erythrocyte membranes. The effect of guanine nucleotides to lower receptor affinity for agonist appears to relate to their ability to destabilize the agonist high-affinity binding state resulting in rapid release of the agonist from the receptor. Chemical treatments of frog erythrocyte membranes which interfere with the ability of agonists to stimulate the adenylate cyclase generally prevent the ability of agonists to form a highaffinity, slowly dissociable complex with the receptor (Williams and Lefkowitz, 1977). Stimulation of adenylate cyclase by guanine nucleotides is therefore associated with a decrease in affinity of the receptor for agonists and rapid dissociation of the high-affinity agonist-receptor complex to free agonist and receptor. Formation of the tight complex between agonist and receptor thus
THE P-ADRENERGIC RECEPTOR:LIGAND BINDING STUDIES
55
appears in some way to facilitate activation of the cyclase enzyme by regulatory nucleotides. It is possible to demonstrate experimentally that the high-affinity, slowly dissociable agonist-receptor complex which is sensitive to guanine nucieotides is an intermediate state on the pathway of coupling between the receptor and adenylate cyclase activation (Stadel et al., 1980). The high-affinity state of the receptor can be isolated by preincubation of purified frog erythrocyte membranes with agonist. These membranes were then washed extensively to remove the free agonist and then assayed for adenylate cyclase activity in the presence of the antagonist propranolol. Basal and NaF-stimulated cyclase activity were unaffected by the preincubation procedures, but the ability of the nonhydrolyzable guanine nucleotide analog Gpp(NH)p to stimulate the enzyme directly was significantly enhanced in membranes preexposed to the agonist compared to membranes preincubated in buffer alone. Experiments of this type suggest that the increased stimulation of the adenylate cyclase by Gpp(NH)p in membranes pretreated with agonist is the result of high-affinity agonist binding to the receptor that persists through the washing procedures. Moreover, they demonstrate that this agonist-receptor complex is an intermediate for agonist activation of cyclase activity. These procedures may be repeated using turkey erythrocyte membranes with qualitatively similar results. Thus the mechanism by which agonists activate adenylate cyclase is similar for both a P I - and a P,-adrenergic receptor (Stadel er af., 1980). Additional insights into the molecular mechanisms of receptor-cyclase coupling can also be gained through the application of computer modeling techniques to radioligand binding data. Quantitative analysis of agonist competition binding curves for [”IDHA in the presence and absence of guanine nucleotides is compatible with the notion that nucleotides mediate a transition between highand low-affinity states of the receptors (Kent er al., 1979). Thus, the agonist competition binding curve is steep in the presence of guanine nucleotides (slope factor = 1) and the uniform dissociation constant for agonist binding to the receptor is identical to the low-affinity dissociation constant ( K J determined for the same agonist in the absence of the nucleotide (Fig. 1). The extent of the transition from high- to low-affinity state is dependent on the concentration of guanine nucleotide in the binding assay. The observations that guanine nucleotide mediates a transition of the agonist high-affinity state of the receptor to the low-affinity state without a similar effect in antagonist binding, and that partial agonists induce differing proportions of the receptor into the high- and low-affinity state at equilibrium (% R H ) ,represent strong evidence that the highand low-affinity states of the agonist-occupied receptors are interconvertible (Kent et al., 1979). A systematic comparison of the ability of several mechanistic models to fit and reproduce agonist competition binding data in the presence and absence of guanine nucleotides led De Lean er af. (1980) to propose a “ternary complex”
56
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
(precoupled)
FIG.3. Schematic diagram of the ternary complex model. The model involves the interaction of the hormone (H), the receptor (R), and an additional membrane component (X). Nucleotide-dependent coupling between the ternary complex (HRX) and activation of adenylate cyclase (E) is also shown. (From De Lean er al., 1980.)
model as an explanation for the agonist-specific binding properties of P-adrenergic receptors. This model involves the interaction of the receptor (R) with an additional membrane component (X) in the presence of agonist (H) to form a high-affinity ternary complex HRX (Fig. 3). Agonist initially binds to the receptor to form a low-affinity binary complex HR which precedes the ternary complex formation. The modeling indicates that the stoichiometry between the receptor and the component X is close to I:]. The intrinsic activity of an agonist correlates with the affinity constant (L) for the combinations of the agonistreceptor complex (HR) with the additional membrane component X: HR
+X
L
HRX
(Fig. 3). This correlation is entirely consistent with the relationships alluded to above between agonist intrinsic activity and other quantitative parameters of the agonist-promoted high-affinity state (KL/K,l, % RH). The computer-aided modeling of the binding data is independent of the nature of the additional membrane component X, but several lines of evidence suggest that X is a guanine nucleotide regulatory protein (N). The computer analyses of binding data indicate that the presence of guanine nucleotides in the binding assay specifically decreases the ability of an agonist to stabilize the ternary complex between HR and X (De Lean ef al., 1980). Biochemical experiments using high concentrations of Mn2+ ( > l o mM) to uncouple agonist binding to receptors from activation of adenylate cyclase (Limbird et al., 1979) or using the specific sulfhydryl reagent N-ethylmaleimide to inactivate adenylate cyclase catalytic activity (Howlett et al., 1978; Stadel and Lefkowitz, 1979) have demonstrated that a functional cyclase enzyme is not
THE B-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
57
required for agonists to promote formation of a high-affinity, nucleotide-sensitive complex with the receptor. However, Stadel and Lefkowitz (1979) have shown that an additional membrane component necessary for agonist high-affinity binding was also sensitive to NEM, but at concentrations 100-fold greater than that necessary to inactivate cyclase catalytic activity. The effect of NEM on agonist high-affinity binding is distal to the ligand binding site of the receptor, since agonist low-affinity binding (in the presence of guanine nucleotide) and the binding of the competitive antagonist [ 3H]DHA were unaffected. Preformation of the agonist high-affinity state of the receptor protected the complex against the effects of NEM, and the complex was still fully sensitive to modulation by guanine nucleotides (Stadel and Lefkowitz, 1979). It is therefore unlikely that the ternary complex (HRX) contains the cyclase enzyme. However, the observation that agonist binding to the P-adrenergic receptor is uniquely modulated by guanine nucleotides is consistent with the notion that component X is a guanine nucleotide regulatory protein. It is of interest to note that a-adrenergic agonist binding to a,-receptors in platelets (Tsai and Lefkowitz, 1979) or neural cell lines (Haga and Haga, 1981) is also characterized by shallow competition binding curves with radiolabeled antagonists that steepen and shift to the right in the presence of guanine nucleotides (Hoffman et al., 1980). The guanine nucleotide sensitivity of the a,receptor is also an agonist-specific property since the affinity of antagonists appears to be unperturbed by the addition of nucleotides. The nucleotide sensitivity of agonist binding to a,-adrenergic receptors again provides clues to understanding the mode of action of a-adrenergic agonists in inhibiting adenylate cyclase, since guanine nucleotides are also stringently required for coupling of these receptors to the catalytic moiety of the enzyme (Jakobs ef al., 1978). Although computer modeling using the ternary complex model (De Lean el al., 1980) has not been applied to binding data for a-adrenergic agonists, it appears likely that such a complex is in fact an intermediate for inhibition as well as stimulation of adenylate cyclase.
IV. CHARACTERIZATION OF DETERGENT-SOLUBILIZED ADRENERGIC RECEPTORS A first step toward the biochemical characterization of membrane-bound hormone receptors is the solubilization of the binding activity from the lipid bilayer through the use of detergents. The P-adrenergic receptor was first solubilized from frog erythrocyte membranes using the plant glycoside digitonin (Caron and Lefkowitz, 1976). Although many different detergents were tested, digitonin was found to be uniquely capable of extracting the receptor in an active form. The binding properties of the soluble receptor sites were in most respects essen-
58
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
tially the same as those of the membrane-bound receptors (Caron and Lefkowitz, 1976). The soluble receptors demonstrate the appropriate potency order for pzadrenergic receptors (isoproterenol > epinephrine > norepinephrine) and strict stereoselectivity for binding both agonist and antagonist ligands. The single difference in the properties of the soluble receptors is that agonist binding to soluble receptors is of uniformly low affinity. Thus agonists do not promote formation of the high-affinity state of the receptor in soluble preparations. This observation correlates with the inability of agonists to stimulate adenylate cyclase activity in these soluble preparations (Caron and Lefkowitz, 1976). Fractionation of detergent extracts by gel filtration (Limbird and Lefkowitz, 1977) or on sucrose gradients (Haga et al., 1977) has led to a clear resolution of receptor binding activity from adenylate cyclase activity, thus demonstrating that these two activities reside on different polypeptide chains. Studies of soluble receptor preparations have shed light on the unique interactions of P-adrenergic receptors with agonist agents. Soluble extracts from frog erythrocyte or rat reticulocyte membranes which were prelabeled with either the radiolabeled agonist [3H]HBI or the radiolabeled antagonist [3H]DHAwere fractionated by gel filtration over AcA34 resin (Limbird and Lefkowitz, 1978; Limbird et al., 1980b). The ['HIHBI prelabeled receptor was resolved from the antagonist-occupied receptor and appeared to elute with an apparent larger molecular size (Fig. 4A). Several explanations are consistent with this observation including an agonist-promoted asymmetric conformational change in the receptor, agonist-induced receptor aggregation, or the stable association of the agonist-occupied receptor with an additional membrane component. This latter explanation is, of course, consistent with the computer modeling described above. The proposed additional component of the agonist-receptor high-affinity state did not appear to be the cyclase enzyme itself, since the enzyme activity eluted several fractions removed from the receptor binding activity. Additional experiments using rat reticulocyte membranes implicated the nucleotide regulatory protein as a constituent of the agonist-receptor high-affinity complex (Fig. 4B). Prelabeling of rat reticulocyte membranes with [3H]HBI in the presence of guanine nucleotide allows agonist binding to the low-affinity form of the receptor. This low-affinity agonist-receptor complex survives the gel filtration procedures and now coelutes with the smaller antagonist prelabeled receptor. Thus guanine nucleotides which destabilize the high-affinity state of the receptor in the membrane also convert the larger molecular form of the agonist-receptor complex to a smaller species that coelutes with antagonistoccupied receptor. More direct evidence as to the molecular compositions of the agonist-promoted ternary complex was obtained by radioactively labeling of the nucleotide regulatory protein of the adenylate cyclase complex (Limbird et al., 1980a). Cholera toxin catalyzes the covalent transfer of ADP-ribose from NAD to the +
THE p-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
59
FIG.4. Gel exclusion chromatography of P-adrenergic receptors solubilized from rat reticulocyte membranes with digitonin after prelabeling with agonist ['HIHBI or antagonist [ 3H]DHA. The column material was AcA34. (A) Prelabeling in the absence of nucleotides. (B) Prelabeling conducted in the presence of 0. I mM Gpp(NH)p. (From Limbird et a / . , 1980b.)
42,000 M , subunit of the guanine nucleotide regulatory protein (for reviews see Ross and Gilman, 1980; Stadel et al., 1982). If 32P-labeledNAD+ is used as the cofactor for the toxin, a radioactive tag is covalently incorporated into the nucleotide regulatory protein. Limbird et al. (1980a) were able to show that agonist pretreatment of rat reticulocyte membranes prior to solubilization resulted in the coelution of the 32P-labeled 42,000 M , protein in the [3H]HBI-receptor region from the gel filtration column. Similar pretreatment of these membranes with antagonist did not cause the labeled subunit of the nucleotide regulatory protein to associate with the receptor. These experiments provide biochemical evidence that agonist occupancy of the P-adrenergic receptor promotes the association of the receptor with the guanine nucleotide regulatory protein of the adenylate cyclase complex.
60
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
Several recent reports provide additional evidence that the high-affinity, nucleotide-sensitive agonist-receptor complex represents a ternary complex of HRN. Cholate-solubilized extracts of membranes prepared from wild-type S49 lymphoma cells successfully reconstituted hormonally sensitive adenylate cyclase activity in membranes from S49 mutant cell clones that are functionally uncoupled (Sternweis and Gilman, 1979). The critical factor in the detergent extract that allows recoupling of the receptors to the cyclase is the guanine nucleotide regulatory protein (Ross et al., 1978). The P-adrenergic receptors of the mutant cell membranes reconstituted with the cholate extracts of S49 wildtype membranes also demonstrate nucleotide sensitivity of receptor affinity for agonists. It was not possible to separate the component necessary for recoupling of hormonal activation of the cyclase from the component required for restoration of nucleotide-sensitive agonist high-affinity binding to the P-adrenergic receptors. These experiments are consistent with the notion of a single membrane component regulating receptor affinity for agonists and for coupling agonist occupancy of the receptor to activation of the cyclase enzyme. Using reconstitution of lubrol-solubilized components, Stadel et al. (198 1) were able to isolate the nucleotide regulatory protein associated with the 6receptor as a result of agonist binding and subsequently show that this N protein conveyed nucleotide-dependent adenylate cyclase activity to a suitable catalytic unit acceptor. The high-affinity ternary complex HRN was solubilized from frog erythrocyte membranes in the nonionic detergent lubrol and then bound to wheat germ agglutinin immobilized on Sepharose. The ternary complex is bound to the lectin through the carbohydrate moieties of the receptor, which is a glycoprotein (Shorr et al., 1980). After extensive washing of the lectin gel the resin was eluted in the presence of GTPyS. The guanine nucleotide destabilizes the ternary complex HRN resulting in the release of an N-GTPyS complex. The GTPyS eluate from the lectin-Sepharose conveyed nucleotide-sensitive adenylate cyclase to a soluble catalytic unit acceptor. The ability of the GTPyS eluate of the lectin-resin to reconstitute adenylate cyclase activity was strictly dependent on the preformation of the agonist high-affinity state in frog erythrocyte membranes prior to solubilization. The notion that the nucleotide regulatory protein associated with the P-adrenergic receptor by the binding of agonist is the same N that modulates adenylate cyclase activity was supported by additional experimentation (Stadel et at., 1981). Radioactive labeling of the N protein by 32P-labeledNAD+ in the presence of cholera toxin allows the observation of the N protein throughout the solubilization, lectin chromatography, and elution procedures. The amount of 32P-labeled42,000 M, subunit associated with the lectin-resin in soluble extracts from membranes pretreated with agonist or antagonist correlated with the ability of these extracts to stimulate adenylate cyclase activity in the soluble reconstitution assay. These experiments bring together both structural and functional evi-
61
THE B-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
p- Adrenergic Receptor Cycle
Nucleotide Regulatory
Adenylate Cyclase
Protein Cycle
Catalytic Moiety Cycle
FIG. 5 . Schematic model of hormonal activation of adenylate cyclase involving agonist (H), receptor (R), nucleotide regulatory protein (N), and enzyme catalytic unit ( C ) . See text for details.
dence that a single nucleotide regulatory protein acts as a “coupler” conveying information from the agonist-occupied receptors to the adenylate cyclase. The observations characterizing the unique binding properties of agonists to the P-adrenergic receptor in both membrane and soluble studies are consistent with a model for receptor-cyclase coupling shown in Fig. 5 . This model is based on the information contained in the experiments reviewed above as well as additional investigations of the properties of the enzyme adenylate cyclase reported by other investigators. In this model agonist occupancy of the receptor (Step 1) promotes or stabilizes the formation of the high-affinity HRN complex (Step 2). As a consequence, GDP is released from N, creating a vacant guanine nucleotide binding site (Step 3). The binding of a guanine nucleotide triphosphate to N (Step 4)results in dissociation of the HRN complex, and N-GTP now associates with C (Step 5 ) to stimulate catalytic activity (Pfeuffer, 1977, 1979). As shown by Cassel and Selinger (1976), hydrolysis of GTP by a GTPase associated with the NC complex (Step 6) is the “turn off” mechanism for adenylate cyclase activity and returns the system to the basal state (Step 7). Binding of agonist to the receptor reinitiates the cycle. The major features of the model are ( 1 ) agonist binding results in the stabilization of the high-affinity ternary complex HRN which facilitates the exchange of nucleotides bound to N; ( 2 ) the guanine nucleotide regulatory protein acts as a coupler between the receptor and the enzyme catalytic unit; (3) the GTPase activity associated with the NC complex deactivates enzyme catalytic activity and dissociates this complex. This model may also provide a starting point for investigating the mechanism of inhibition of adenylate cyclase mediated by qadrenergic receptors. As described above from radioligand binding studies it is apparent that a,-adrenergic receptors are capable of forming a high-affinity, nucleotide-sensitive complex with agonists but not antagonists. Recent studies (Michel er al., 1981; Smith and
62
JEFFREY M. STADEL AND ROBERT J. LEFKOWITZ
Limbird, 1981) also show that agonist occupancy of platelet a,-receptors similarly induces an increase in the apparent molecular size of the receptor compared to antagonist-occupied receptors as assessed by centrifugation through sucrose gradients. The soluble agonist-receptor complex is also sensitive to guanine nucleotides, consistent with the notion that the increased molecular size of the agonist-receptor complex is due to the agonist-promoted association of the receptor with a guanine nucleotide regulatory protein (Smith and Limbird, 1981). These data suggest that a-adrenergic inhibition of adenylate cyclase shows many features in common with, and may be analogous to, the mechanism of P-adrenergic stimulation of the cyclase. Further investigation will be necessary to determine how the formulations in the model shown in Fig. 5 for the stimulation of adenylate cyclase might apply to the mechanism of inhibition of the enzyme. A long-range goal of studies of the mechanism of receptor-cyclase coupling is the purification and reconstitution of the individual components of the systems in a functional way. In the past 2 years considerable progress has been made in this regard. The development of sensitive assays for detergent-solubilized components of the complex has allowed the application of biochemical techniques for purification. Purification of these components is a major undertaking since the constituents of the receptor-adenylate cyclase complex exist in very small quantities in the plasma membranes of target cells. Recently, the guanine nucleotide regulatory protein has been purified to apparent homogeneity by classic biochemical techniques (Northup et al., 1980). The purified protein is composed of three heterologous subunits with approximate molecular weights of 52,000, 45,000, and 35,000. The purified guanine nucleotide regulatory protein reconstitutes guanine nucleotide-, hormonal-, and sodium fluoride-dependent stimulation of adenylate cyclase activity in membranes prepared from mutant S49 lymphoma cells that lack a functional regulatory unit. All three subunits appear to be required for successful reconstitution. A key step in the purification of the P-adrenergic receptor was the development of an efficient affinity chromatography gel (Caron et a!., 1979). Alprenolol was immobilized on Sepharose 4B through a hydrophilic spacer arm. The biospecific nature of the interaction of the digitonin-solubilized P-adrenergic receptor with the affinity gel could be demonstrated. Both the adsorption of the soluble frog erythrocyte P-receptor to the resin and its subsequent elution demonstrated typical P-adrenergic specificity. For both processes (blocking adsorption and promoting elution), the agonist potency order was isoproterenol > epinephrine > norepinephrine and stereoselectivity was preserved for both agonist and antagonist agents. The resin adsorbed up to 95% of the receptor in the soluble preparations, and 60% was ultimately specifically eluted. By recycling the soluble receptor preparation through the affinity resin the purification was over 15,000fold from the original membranes (Caron et al., 1979). By coupling the affinity chromatography procedures to ion exchange chro-
THE P-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
63
matography the ligand binding site of the P-adrenergic receptor has been purified 55,000-fold (Shorr et al., 1981). The purified receptor demonstrates a 58,000 M, band on SDS-polyacrylamide gel electrophoresis. The purified 58,000Mr protein comigrates with soluble radioligand antagonist prelabeled receptor on sucrose gradients and in isoelectric focusing procedures. Ligand binding experiments using I3H]DHA and the purified protein demonstrate the affinity, specificity, and stereoselectivity expected for the P-adrenergic receptor (Shorr et al., 1981). The purification of the P-adrenergic receptor and the nucleotide regulatory protein leaves the catalytic unit of adenylate cyclase as the only known component remaining to be purified. The rapid progress that has been made in the isolation and characterization of the molecular components of the P-adrenergic receptor-adenylate cyclase complex raises expectations that functional reconstitution of this system will be achieved in the not too distant future. REFERENCES Ahlquist, R. P. (1948). A study of the adrenotropic receptors. Am. J . Phvsiol. 153, 586-600. Atlas, D.. Steer, M. L., and Levitzki, A. (1974). Stereospecific binding of propranolol and catecholamines to the beta-adrenergic receptor. Proc. Narl. Acad. Sci. U.S.A. 71, 4246-4248. Aurbach, G. D., Fedak, S. A , , Woodard, C. J., Palmer, J . S.. Hauser, D., and Troxler, F. (1974). The beta-adrenergic receptor: Stereospecific interaction of an iodinated beta-blocking agent with a high affinity site. Science 186, 1223-1224. Barovsky, K..and Brooker, G . (1980). (-)('2sI]IodopindoloI, a new highly selective radioiodinated P-adrenergic receptor antagonist: Measurement of (3-receptors on intact rat astrocytoma cells. J. Cyclic Nucleoride Res. 6, 297-307. Berthelson, S., and Pettinger, W. A. (1977). A functional basis for the classification of alphaadrenergic receptors. Ljfe Sci. 21, 595-606. Bottari. S . , Vauquelin, 0.. Durien, O., Klutchko, C., and Strosberg, A. D. (1979). The P-adrenergic receptor of turkey erythrocyte membranes: Conformation modification by P-adrenergic agonists. Biochem. Biophys. Res. Commun. 86, 131 1-1318. Briggs, M. M., and Lefkowitz, R. J. (1980). Parallel modulation of catecholamine activation of adenylate cyclase and formation of the high-affinity agonist-receptor complex in turkey erythrocyte membranes by temperature and cis-vaccenic acid. Biochemisrv 19, 4461-4466. Caron, M. G . , and Lefkowitz, R . J. (1976). Solubilization and characterization of the P-adrenergic receptor binding sites of frog erythrocytes. J. B i d . Chem. 251, 2374-2384. Caron, M. G., Srinivasan, Y.,Pitha, J., Kociolek, K., and Letkowitz, R. J. (1979). Affinity chromatography of the P-adrenergic receptor. J. B i d . Chem. 254, 2923-2927. Cassel, D.,and Selinger, 2. (1976). Catecholamine-stimulated GTPase activity in turkey erythrocyte membrane. Biochim. Biophys. Acta 452, 538-55 I . Dale, H. H. (1906). On some physiological actions of ergot. J. Physiol. (London) 34, 165-206. De Lean. A . , Stadel, J. M., and Letkowitz, R. J . (1980). A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled P-adrenergic receptor. J. B i d . Chem. 255, 7108-71 17. Engle, G . ( 1 980). Identification of different subgroups of beta-receptors by means of binding studies in guinea-pig and human lung. Triangle 19, 69-76. Exton, J. H. (1979). Mechanisms involved in effects of catecholamines on liver carbohydrate metabolism. Biochem. Pharmacol. 28, 2237-2246.
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Greenberg, D. A , , U'Prichard, D. C., and Snyder, S. H. (1976). Alpha-noradrenergic receptor binding in mammalian brain: Differential labelling of agonist and antagonist states. Life Sci. 191, 69-76. Greengrass, P.. and Brenner, R. (1979). Binding characteristics of [3H]prazosin to rat brain alphaadrenergic receptors. Eur. J. Pharmacol. 55, 323-325. Haga, T., and Haga, K . (1981 ). Characterization by [3H]dihydroergocryptine binding of alphaadrenergic receptors in neuroblastomd X glioma hybrid cells. J. Neurochem. 36, 1152- 1159. Haga. T., Haga, K., and Gilman, A. G. (1977). Hydrodynamic properties of the f3-adrenergic receptor and adenylate cyclase from wild-type and variant S49 lymphoma cells. J . Biol.Chem. 252, 5776-5782. Hoffman, B. B., and Lefkowitz, R. J. (1980). Radioligand binding studies of adrenergic receptors. Annu. Rev. Pharmaml. Toxicol. 26, 581 -608. Hoffmann, B . B . , Mullikin-Kilpatrick, D., and Lefkowitz, R. J. (1980). Heterogeneity of radioligand binding to a-adrenergic receptors. J. B i d . Chem. 255, 4645-4652. Howlett, A. C., Van Arsdale, P. M., and Gilman, A. G . (1978). Efficiency of coupling between the beta-adrenergic receptor and adenylate cyclase. Mol. Pharmarol. 14, 53 1-539. Jakobs, K. H., Saur, W . , and Schultz, G . (1976). Reduction of adenylate cyclase activity in lysates of human platelets by alpha-adrenergic component of epinephrine. J. Cyclic. Nucleotide Res. 2, 381-392. Jakobs, K. H., Saur, W . , and Schultz, G. (1978). Inhibition of platelet adenylate cyclase by epinephrine requires GTP. FEBS Lett. 85, 167-170. Kent, R. S . , De Lean, A., and Lefkowitz, R. J. (1979). A quantitative analysis of beta-adrenergic receptor interactions: Resolution of high and low affinity states of the receptor by computer modeling of ligand binding data. Mol. Pharmacol. 17, 14-23. Lands, A. M., Arnold. A.. McAuliff, J. P., Luduena, F. P., and Braun, T. G . (1964). Differentiation of receptor systems activated by sympathomimetic amines. Nature (London) 214, 597-598. Lefkowitz, R. J., and Williams, L. T. (1977). Catecholamine binding to the beta-adrenergic receptor. Proc. Natl. Acad. Sci. U.S.A. 74, 515-519. Lefkowitz, R. J., Roth, I . , Pricer, W., and Pastan. I. (1970). ACTH receptors: Specific binding of ACTH-['2sI] and its relation to adenyl cyclase. Proc. Natl. Acad. Sci. U.S.A. 65, 745-752. Letkowitz. R . J . , Mukherjee, C., Coverstone, M . , and Caron, M. G. (1974). Stereospecific [3Hl(-)alprenolol binding sites, beta-adrenergic receptors and adenyl cyclase. Biochem. Biophys. Res. Commun. 60, 703-709. Lefkowitz, R. J., Mullikin, D.. and Caron, M. G . (1976). Regulation of beta-adrenergic receptors iw guanyl-5'-yl imidophosphate and other purine nucleotides. J. B i d . Chem. 251, 4680 4692. Limbird, L. E . , and Lefkowitz, R. J. (1977). Resolution of f3-adrenergic receptor lmiding and adenylate cyclase activity by gel exclusion chromatography. J. Biol. Chem. 252, 799-802. Limbird, L. E., and Lefkowitz, R. J. (1978). Agonist-induced increase in apparent P-adrenergic receptor size. Proc. Natl. Acad. Sci. U.S.A. 75, 228-232. Limbird, L. E., Hickey, A. R., and Lefkowitz, R. J. (1979). Unique uncoupling of the frog erythrocyte adenylate cyclase system by manganese. J. Biol. Chem. 254, 2677-2683. Limbird. L. E., Gill. D. M., and Lefkowitz, R. J . (1980a). Agonist-promoted coupling of the padrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system. Proc. Nail. Acad. Sri. U.S.A. 77, 775-779. Limbird, L. E., Gill, D. M., Stadel, J. M., Hickey, A. R., and Lefkowitz, R. J . (1980b). Loss of padrenergic receptor-guanine nucleotide regulatory protein interactions accompanies decline in catecholamine responsiveness of adenylate cyclase in maturing rat erythrocytes. J . Biol. Chem. 255, 1854-1861. Lyn. S. Y., and Goodfriend, T. L. (1970). Angiotensin receptors. Am. J. Phvsiol. 218, 1319-1328.
THE p-ADRENERGIC RECEPTOR: LIGAND BINDING STUDIES
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Maguire. M. E., Van Arsdale, P. M., and Gilman, A. G . (1976). An agonist-specific effect of guanine nucleotides on binding to the beta-adrenergic receptor. Mol. Pharmacol. 12, 335-339. Means, ti. E..and Feeney, R. E. (1971). “Chemical Modification of Proteins.” Holden-Day, San Francisco, California. Michel,T., Hoffman. B. B., Lefkowitz, R. J., andcaron, M. G . (1981). Differential sedimentation properties of agonist- and antagonist-labelled platelet alphaz-adrenergic receptors. Biochem. Biophys. Res. Cornmun. 100, 1 I3 I - 1 135. Northup, J. K.,Sternweis, P. C.. Smigel, M. D . , Schleifer, L. S., Ross, E. M., andGilman, A. G. (1980). Purification of the regulatory component of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 77, 6516-6520. Pfeuffer, T. (1977). GTP-binding proteins in membranes and the control of adenylate cyclase activity. J . Biol. Chem. 252, 7224-7234. Pfeuffer, T. ( 1979). Guanine nucleotide-controlled interactions between components of adenylate cyclase. FEBS Lett. 101, 85-89. Pike, L. J . , and Lefkowitz, R. I. (1978). Agonist specific alterations in receptor binding affinity associated with solubilization of turkey erythrocyte membrane beta-adrenergic receptors. Mol. Pharmacol. 14, 370-375. Rimon. G.. Hanski, E., Braun. S., and Levitzki, A. (1978). Mode of coupling between hormone receptors and adenylate cyclase elucidated by modulation of membrane fluidity. Nature (London) 276, 394-396. Robison, G. A,, Butcher, R . W . , and Sutherland, E. W . (1971). “Cyclic AMP.” Academic Press, New York. Rodbell, M., Birnbaumer, L., Pohl. S. L., and Krans. H. M. (1971). The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver: An obligatory role of guanyl nucleotides in glucagon action. J . Bio/. Chem. 246, 1877-1882. Ross, E. M., and Gilman, A. G. (1980). Biochemical properties of hormone-sensitive adenylate cyclase. Annu. Rev. Biochem. 49, 533-564. Ross. E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. 0 . (1978). Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J . Biol. Chem. 253, 6406-6412. Roth. J. (1973). Peptide hormone binding to receptors: A review of direct studies in vitro. Merab. Clin. Exp. 22, 1059-1073. Shorr, R. G. L., Caron, M . G., and Lefkowitz, R. J . (1980). Isolation and characterization of betaadrenergic receptors from frog erythrocyte membranes. Fed. Proc., Fed. Am. Soc. Exp. Biol. 39, 1616. Shorr, R . G. L., Lefkowitz, R. 1 . . and Caron, M. G . (1981). Purification of the p-adrenergic receptor: Identification of the hormonal binding subunit. J . B i d . Chem. 256, 5820-5826. Smith, S. K.,and Limbird, L. E. (1981). Solubilization of human platelet a-adrenergic receptors: Evidence that agonist occupancy of the receptor stabilizes receptor-effector interactions. Proc. Nail. Acad. Sci. U.S.A. 78, 4026-4030. Stadel, I. M.. and Lefkowitz, R. J. (1979). Multiple reactive sulfhydryl groups modulate the functions of adenylate cyclase-coupled P-adrenergic receptors. Mol. Pharmacol. 16,709-71 8. Stadel, I. M.,De Lean, A . , and Lefkowitz, R. J . (1980). A high affinity agonist P-adrenergic receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase in turkey and frog erythrocyte membranes. J . B i d . Chem. 255, 1436-1441. Stadel, J. M . , Shorr. R . G . L., Limbird. L. E., and Lefkowitz, R. I. (1981). Evidence that padrenergic receptor associated guanine nucleotide regulatory protein conveys guanosine 5’-O-(3-thiotriphosphate)dependent adenylate cyclase activity. J . Biol. Chem. 256, 8718-8723.
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Stadel, J. M..De Lean, A., and Lefkowitz, R. J. (1982). Molecular mechanisms of coupling in hormone receptor-adenylate cyclase systems. Adv. Enzyrtol. 53, 1-43. Sternweis, P. C., and Gilman, A. G. (1979). Reconstitution of catecholamine-sensitive adenylate cyclase. f . Biol. Chem. 254, 3333-3340. Tharp, D. M., Hoffman, B. B.. and Lefkowitz, R. J . (1981). a-Adrenergic receptors in human adipocyte membranes: Direct determination by ["]yohimbine binding. J. Clin. Endorrinol. Merah. 52, 709-714. Tsai. B. S., and Lefkowitz, R. J. (1979). Agonist-specific effects of guanine nucleotides on alphaadrenergic receptors in human platelets. Mol. Pharmacol. 16, 61-68. U'Prichard, D. C., and Snyder, S. H. (1977). 13H]Epinephrine and [3H]norepinephrine binding to alpha-noradrenergic receptors. L$e Sci. 20, 527-533. U'Prichard, D. C., Greenberg. D. A,, and Snyder, S. H. (1977). Binding characteristics of a radiolabelled agonist and antagonist at central nervous system alpha-noradrenergic receptors. Mol. Pharmacol. 13, 454-473. U'Prichard, D. C., Bylund, D. B., and Snyder. S . H. (1978). (+)-[3H]Epinephrine and ( -)-[7H]dihydroalprenolol binding to P I and Pz-noradrenergic receptors in brain, heart, and lung membranes. J. B i d . Chem. 253, 5090-5102. Vauquelin, G., and Maguire. M. E. (1980). Inactivation of 0-adrenergic receptors by N-ethylmaleimide in S49 lymphoma cells: Agonist induction of functional receptor heterogeneity. Mol. Pharmacol. 18, 362-369. Vauquelin, G., Bottari, S . , and Strosberg. A. D. (1979). Inactivation of P-adrenergic receptors by Nethylmaleimide: Permissive role of P-adrenergic agents in relation to adenylate cyclase activation. Mol. Pharmacol. 17, 163-171. Vauquelin, G., Bottari, S.. Andre, C.. Jacobson. B., and Strosberg, A. D. (1980). Interaction between P-adrenergic receptors and guanine nucleotide sites in turkey erythrocyte membranes. Proc. Nail. Acad. Sci. U.S.A. 77, 3801-3805. Weiland. G. A., Minneman, K. P., and Molinoff, P. B. (1980). Thermodynamics of agonist and antagonist interactions with mammalian P-adrenergic receptors. Mol. Pharmarol. IS, 34 1-347. Williams, L. T., and Lefkowitz, R. J . (1976). Alpha adrenergic receptor identification by 13H]dihydroergocryptine binding. Science 192, 791-793. Williams, L. T., and Letkowitz. R. J. (1977). Slowly reversible binding of catecholamine to a nucleotide-sensitive state of the beta-adrenergic receptor. J. Biol. Chem. 252, 7207-72 13. Williams, L. T., and Lefkowitz, R. J. (1978). "Receptor Binding Studies in Adrenergic Pharmacology." Raven, New York. Williams L. T., Mullikin. D., and Lefkowitz, R . J . (1978). Magnesium dependence of agonist binding to adenylate cyclase-coupled hormone receptors. J. Biol. Ckem. 253, 2984-2989.
CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I X
Receptor-Mediated Stimulation and Inhibition of Adenylate Cyclase DERMOT M. F . COOPER' Section on Membrane Regulation Laboratory of Nutrition and Endocrinology National Institute of Arthritis. Diabetes. Digestive and Kidney Diseases National lnstitutes of Health Bethesda. Maryland
I. 11.
Ill.
1v. V. VI. VII. VIII.
IX . X.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stimulation of Adenylate Cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GTP-Dependent inhibition of Adenylate Cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bimodally Regulated Adenylate Cyclase Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptor Binding of Inhibitory Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Role of GTP Hydrolysis in Inhibition of Adenylate Cyclase . . . . . . . . . . . . . . . . . The Relationship between N, and N , . . . . . .......... Structural Studies on Dually Regulated Ad A. Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cholera Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Calmodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Ultrastructural Studies. . . . . . . . . . . . . . . . . . . . .. Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
67 68 70 71 73 75 76 77 77 77 78 78 78 81 81
INTRODUCTION
The major site of action of many hormones is the adenylate cyclase regulatory complex, which generally responds to these stimuli by increasing cyclic AMP production. In recent years it has become apparent that a variety of hormonal 'Present address: Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262.
67 Copyright ,Q 1983 by Academic Prcsr. Inc. All rights of mpmduction in any f0rm rcierved ISBN 0-12-153318-2
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DERMOT M. F. COOPER
agents are also capable of inhibiting the enzyme, which increases the regulatory flexibility of these systems. Thereby the effects of stimulatory hormones can be attenuated and, in addition, basal cyclic AMP levels can be reduced. GTP plays a central role in determining which regulatory options are available to the enzyme. Evidence accumulated to date would support the notion that distinct GTP regulatory proteins mediate the opposing effects. This article will summarize current studies on dual regulation of adenylate cyclase and contrast these with what is known of the more intensively investigated stimulatory systems, and thus attempt to project future progress in this area.
II. STIMULATION OF AOENYLATE CYCLASE The role of GTP in the stimulation of adenylate cyclase has received intense attention in the last 10 years. A number of excellent reviews of this area have recently appeared (Rodbell, 1980; Ross and Gilman, 1980; Limbird, 1981). As a prelude to a discussion of inhibition of adenylate cyclase it is appropriate to present a brief perspective on stimulation of adenylate cyclase. Many hormones interact with cell surface receptors to transmit a stimulatory signal to the catalytic unit of adenylate cyclase through the intervention of a GTP regulatory protein (termed N,).2 By a mechanism that remains unclear (due largely to a limited knowledge of the components of the system), GTP both decreases the affinity of hormones for their receptors and synergistically amplifies hormonal stimulation of activity (Figs. 1 and 2). In general, the nonhydrolyzable GTP analog Gpp(NH)p promotes the latter action more effectively than the native compound. These and allied findings have led to the development of a general hypothesis, which proposes that hormone binding to receptors leads to the release of previously bound GDP (an ineffective stimulator), which allows occupancy by GTP and, consequently, attainment of a more active R,N,C complex. On hydrolysis of GTP to GDP, the complex reverts to an inactive form, which coincides with the release of hormone (Rodbell, 1980; Ross and Gilman, 1980; Limbird, 1981). Considerable gaps exist in our knowledge of stimulatory adenylate cyclase systems. Quantitative information is lacking on the relationship between hormone occupancy, GTP hydrolysis, and active complex formation. Similarly the relative stoichiometry of R,:N,:C is a subject for speculation. Whether the vari’The following abbreviations are functional assignments which may be represented by one or more distinct proteins: R, and R, are receptors for hornionesineurotransmittersevoking either stimulation o r inhibition, respectively. o f the catalytic activity, C: N, and Ni are the GTP regulatory elements mediating either the stimulation or inhibition of activity. Other abbreviations: CAMP, cyclic 3 ’ 5 adenosine monophosphate; Gpp(NH)p, guanylyl irnidodiphosphate; GP(CH*)P, guanylyl-a,P-methylene phosphonate.
69
STIMULATION AND INHIBITION OF ADENYLATE CYCLASE
-
+ Hormone
Basal
I 10-8
I
I
I
10-7
I 10-6
I 10-5
(GTP)
F w . 1 . Dependence of stirnulatory hormone on GTP for the stimulation of adenylate cyclase. This schematic presentation of the actions of a stimulatory hormone on a "typical" stirnulatory adenylate cyclase shows that, in the absence of GTP, hormone alone (top curve) cannot elicit significant stimulation of activity, but with increasing GTP concentrations, marked amplification is observed. In the absence of hormone (bottom curve) increasing concentrations of GTP elicit little if any increase in activity.
ous elements exist in a preformed complex which is stabilized on interaction with regulatory ligands, or whether there is some degree of independent movement or collision, is unclear. Regulatory components in addition to those already identified may exist. For example, cytoskeletal elements are candidates for supporting roles in these systems. The number of proteins comprising the N, unit appears to
[Hormone]
FIG.2. Effect of GTP on receptor binding of a stirnulatory hormone. This schematic presentation shows displacement of a fixed concentration of a labeled hormone by increasing concentrations of unlabeled hormone in the absence and presence of GTP. A typical effect of GTP on the binding of a stirnulatory hormone to its receptor is shown, i.e., GTP decreases the apparent K d for the hormone. (Compare this effect of GTP with that of the nucleotide on binding to inhibitory receptors: Fig. 5.)
70
DERMOT M . F. COOPER
be either two or three, depending on the source of the purified component. In addition an ADP-ribosylation factor, which permits the N, unit to be ADPribosylated by cholera toxin, may also be an integral component of the N, unit (Sternweis et al., 1981). Notwithstanding the unanswered questions, the central role of the N, unit in mediating the stimulatory effects of hormones is clearly established, and progress in understanding the functioning of this component provides a yardstick against which our understanding of inhibitory regulation can be evaluated.
111.
GTP-DEPENDENT INHIBITION OF ADENYLATE CYCLASE
Early observations on fat cell membranes indicated that GTP concentrations exceeding 1 pM could reduce adenylate cyclase activity (Cryer et n l . , 1969; Harwood et al., 1973; Ebert and Schwabe, 1974). Evidence had also been accumulating that cyclic AMP production could be decreased by a number of agents, such as adenosine and PGE, in adipocytes (Fain er al., 1972), or norepinephrine in platelets (Moskowitz et al., 1971). Several reports demonstrated that adenylate cyclase activity could be inhibited in various broken cell preparations by, for example, muscarinic cholinergic drugs (Murad et al., 1962), norepinephrine (Moskowitz et al., 1971), and opiates (Collier and Roy, 1974). A common factor linking these observations became apparent when it was shown that inclusion of GTP in concentrations exceeding those required for the stimulation of adenylate cyclase by hormones permitted inhibition of the enzyme by
, Stirnulatory Phase
I
Inhibitory Phase
GTP)
FIG. 3. Response of the fat cell adenylate cyclase to GTP in the absence and presence of a stimulatory hormone. This schematic presentation of the effects of GTP on fat cell adenylate cyclase demonstrates characteristic biphasic behavior of a dually regulated adenylate cyclase with GTP. As in Fig. 1, a stirnulatory range of GTP concentrations is required for full amplification of the stirnulatory response. However, after reaching a peak, activity declines in what is referred to as the inhibitory response to GTP.
STIMULATION AND INHIBITIONOF ADENYLATE CYCLASE
71
various putative neurotransmitters, for example, epinephrine in platelets (Jakobs et d.. 1978) and in neuroblastoma x glioma hybrids (Sabol and Nirenberg,
1979) and adenosine in fat cells (Londos et d . , 1978). The GTP requirement for these effects clearly established adenylate cyclase inhibition as a receptor-mediated event, which was somewhat analogous to the stimulation of adenylate cyclase by hormones. Indications that distinct GTP regulatory proteins might mediate stimulation and inhibition of adenylate cyclase came from a series of studies with the fat cell. This enzyme displayed a pronounced biphasic response to GTP; GTP concentrations up to 40 nM increased activity, whereas higher concentrations evoked a steady decline in activity (Fig. 3). The nonhydrolyzable analog Gpp(NH)p did not share the inhibitory response (Cooper et af., 1979). Treatment of fat cell membranes (or cells prior to membrane preparation) with either trypsin (Yamamura et a / . , 1977) or cholera toxin and NAD, or assaying in the presence of Mn2+ resulted in the abolition of the inhibitory response to GTP. In contrast, treatment of membranes with p-hydroxymercuriphenylsulfonic acid eliminated stimulation but retained inhibition by GTP (Cooper e f a / . , 1979). The functional association between the inhibitory response to GTP and GTP-mediated inhibition by adenosine analogs was established by the observation that conditions which eliminated inhibition by GTP also led to the loss of the ability of adenosine analogs to inhibit the enzyme in a GTP-dependent manner (Cooper et al., 1979).
IV. BIMODALLY REGULATED ADENYLATE CYCLASE SYSTEMS A rapid growth in reports of GTP-mediated inhibition of adenylate cyclase by many putative neurotransmitter receptors in a variety of tissues has occurred in recent years. These include opiate (Blume ef a/., 1979), muscarinic cholinergic (Lichahtein et a / . , 1979), and a,-adrenergic (Sabol and Nirenberg, 1979) in neuroblastoma X glioma hybrids; muscarinic cholinergic in myocardium (Jakobs et u l . , 1979; Watanabe et a / ., 1978); dopamine (via a D, receptor) in intermediate pituitary (Cote et a!. , 198 1); adenosine, PGE, , and nicotinic acid in adipocytes (Londos et a/., I98 1 ; Schimrnel et a/., I98 I ; Aktories et a/., 1980); a*adrenergic in platelets (Jakobs et a/., 1978; Cooper and Rodbell, 1979); opiate and adenosine in hippocampus (Girardot et a / . , 1981); opiates in striatum (Law et a / ., 198 I ; Cooper et a/., 1982); angiotensin and a,-adrenergic in liver (Jard et a / ., I98 1); and adenosine in brain cortex (Cooper et a / . , 1980). Common features shared by most of these systems are as follows:' 1 . Biphasic GTP kinetics are almost always encountered (Cooper and Lond-
os, 1982). 'The summary o f the properties of dually regulated adenylate cyciasc systems is drawn from studies in many laboratories. Detailed references are available in the review articles cited in this section.
72
DERMOT M. F. COOPER
FIG. 4. Effect of sodium ion and phenylisopropyladenosine(PIA) on !he response of the fat cell adenylate cyclase to GTP. ( A ) In the absence of NaCI. but in the presence of a stirnulatory hormone. the typical biphasic response of the fat cell enzyme is observed; PIA inhibits activity in the inhibitory GTP phase only (cf. Fig. 3 ) . ( B ) Inclusion of NaCl almost totally reverses the inhibition evoked by GTP alone. so that the response to GTP becomes like that of a stirnulatory system (cf. Fig. 1). However, activity measured in the presence of PIA is virtually unchanged by the presence ofthe salt. The net result is an increase in the absolute inhibition evoked by PIA.
2. GTP concentrations beyond those in the stirnulatory range (ca. l o p 7 M) promote inhibition by putative neurotransmitters and related compounds (Jakobs, 1979; Cooper and Londos, 1982). 3. Where GTP, in the absence of other ligands, causes a decrease in activity, sodium ion (up to 100 mM) reverses this effect (Londos et al., 1981; see Fig. 4). 4. Sodium ion amplifies inhibition by ligands in direct proportion to its reversal of the inhibition promoted by GTP alone (Blume el al., 1979; Londos et a / ., I98 I ) . 5. Gpp(NH)p does not promote inhibition by inhibitory hormones or neurotransmitters, even when it evokes a transient inhibition at early incubation times (Jakobs, 1979; Cooper and Londos, 1982). 6. Inhibition by neurotransmitters is generally less than 60%. except when directed against basal activities, when it may reach 80%. 7. Where multiple inhibitory effectors operate, their effects are nonadditive (Sabol and Nirenberg, 1979; Londos et u l . , 1981). 8. Divalent cations (most effectively MnZ ) selectively abolish GTP-mediated inhibition of activity (Cooper et al., 1979). 9. As with stimulatory ligands, the binding of inhibitory ligands is modulated by GTP (see Section V). 10. Sodium ion and Gpp(NH)p modulate binding of inhibitory ligands, even though these agents may not affect the ability of the inhibitory ligands to attenuate activity (see Section V). +
STIMULATION AND INHIBITIONOF ADENYLATE CYCLASE
73
Deviation from these properties is not generally encountered in a wide range of dually regulated systems, unless technical difficulties arise due to, for example, endogenous GTP or inhibitory agent (see Cooper and Londos, 1982).
V.
RECEPTOR BINDING OF INHIBITORY LIGANDS
Extensive studies have been performed on the binding of inhibitory ligands to their receptors. The early studies of Pert and Snyder (1974) on the binding of opiates to brain receptors provided valuable insights for later studies on the inhibition of adenylate cyclase by opiates. In particular, monovalent cations (most effectively sodium) were shown to increase the receptor binding of antagonists and decrease that of agonists. Sub,sequently Blume et al. (1979) found that sodium ion amplified the inhibition of adenylate cyclase in neuroblastoma X glioma cells by opiates. Sodium ions have now been shown to modify the binding of inhibitory ligands in diverse systems, even in situations in which the cation does not modify inhibition of adenylate cyclase by these ligands. For example, in platelets, a decrease in the affinity of the a-adrenergic receptor was encountered (Tsai and Lefkowitz, 1979; Michel et al., 1980), whereas the cation did not modify the inhibition by epinephrine of adenylate cyclase activity (Jakobs, 1979). This latter observation correlates with the lack of inhibition by GTP in the absence of inhibitory ligand in platelet membranes. By contrast, in the fdt cell, a striking effect of sodium on inhibition is seen due to the marked inhibition of enzyme activity by GTP in the absence of inhibitory ligand (Londos et al., 1981). The retention of a sodium ion effect on binding in situations in which it shows no effect on activity may indicate separate loci for these two regulatory events; alternatively, an excess of receptors over catalytic elements would permit discrepant regulation of receptor binding and the function mediated by the receptors. The other alkali metals share these effects of sodium with the following potency: Na+ > K + > Cs+ (Pert and Snyder, 1974; Blume ef a / . , 1979). The sodium effect on binding is not constant with respect to all inhibitory ligands. For example, in brain, the binding of both opiate agonists and antagonists is regulated by sodium (Blume, 1978), whereas only a-adrenergic agonist binding is modified (Greenberg et ul.. 1978). In contrast to the monovalent cations, magnesium ion generally increases agonist affinity (Tsai and Lefkowitz, 1979; U’Prichard and Snyder, 1980). Guanine nucleotides are more consistent in the regulation of the binding of inhibitory ligands. GTP and Gpp(NH)p generally decrease the affinity of inhibitory ligands for their receptors (Tsai and Lefkowitz, 1979; U’Prichard and Snyder, 1980). This is directly analogous to the effects of guanine nucleotides on the binding of stimulatory ligands to their receptors. However, this observation is in conflict with the inability of the nonhydrolyzable GTP analog to promote
74
DERMOT M. F. COOPER
20 mM Mg2+
0 Mg"
PIA(nMI
PIAhM)
PIAbMI
MgC12h M I
FIG. 5 . The effect of GTP on binding to the adenosine receptor of fat cell membranes in the presence of a range of magnesium ion concentrations. Fat cell membranes (30 kg) were incubated with 3HH-labeled Nh-cyclohexyladenosine (CHA) ( 2 nM) in the presence of the indicated concentrations of MgClz in the absence (0)or presence (0) of 20 pM GTP. Note that, in the absence of magnesium ion, GTP decreases binding to the adenosine receptor. However, in the presence of the cation. GTP increases binding to the receptor. A Scatchard analysis of this data reveals that the total number of binding sites is increased by GTP rather than a change in the receptor affinity (Cooper and Gill, in preparation).
inhibition of adenylate cyclase. It might have been anticipated that, since only GTP, and not Gpp(NH)p, can promote adenylate cyclase inhibition by these ligands, the latter compound might not have shared the ability of GTP to modulate binding. The fact that this prediction is not fulfilled again raises the possibility of either separate loci for the regulation of binding compared with function or an excess of inhibitory receptors not in association with catalytic activity, which permits discrepant regulation of total binding compared with a small pool of receptors associated with the enzyme. In tissues in which it has been studied, it appears that magnesium ion can modify the effect of guanine nucleotides on inhibitory ligand binding. In both brain (a,-adrenergic receptor binding) and fat cell membranes (adenosine receptor binding), at magnesium concentrations above 1 mM GTP increases the number of binding sites for the inhibitory ligands, whereas at lower cation concentrations GTP decreases affinity for the ligands (U'Prichard and Snyder, 1980; Cooper and Gill, in preparation, Fig. 5 ) . The apparently wide diversity which exists in the regulation of the binding of inhibitory ligands to their receptors compared with that of stimulatory agents
STIMULATION AND INHIBITION OF ADENYLATE CYCLASE
75
emphasizes our rather primitive understanding of both the interaction of inhibitory receptors with the putative inhibitory GTP regulatory components and the precise transduction mechanisms which translate the binding event into an inhibitory response.
VI.
THE ROLE OF GTP HYDROLYSIS IN INHIBITION OF ADENYLATE CYCLASE
The most persuasive evidence that GTP hydrolysis plays a role in hormonally mediated inhibition of adenylate cyclase is the observation that Gpp(NH)p, the nonhydrolyzable analog, will not promote inhibition under any assay conditions. A characteristic of dually regulated systems is a transient inhibitory response to low concentrations of Gpp(NH)p (Ebert and Schwabe, 1974; Girardot et a / . , 1981; Cooper and Londos, 1982; Sulakhe et al., 1977). Under such conditions, in the absence or presence of sodium ion, inhibitory ligands will not affect activity. An obvious interpretation of this widely encountered finding is that GTP hydrolysis is an absolute requirement for inhibition. The hydrolysis product, GDP, is not required, since GDP (or GP(CH,)P], under conditions in which care is taken to prevent phosphorylation to the triphosphate, will not promote inhibition (Cooper and Schlegel, unpublished; Cooper and Londos, 1982). These observations contrast with the ready interchange of GTP with Gpp(NH)p in stimulation of adenylate cyclase, where GTP hydrolysis does not seem to be a stringent requirement for enzyme stimulation. Supportive evidence for an obligatory role for GTP hydrolysis is available from studies with cholera toxin. As mentioned in Section 111, cholera toxin treatment abolishes both GTP inhibition and inhibition promoted by adenosine in a GTP-dependent manner in fat cell membranes (Cooper ef a / . , 1979). Cholera toxin invariably enhances hormonal stimulation in stimulatory systems by a mechanism believed to involve the inhibition of a specific GTPase activity associated with the stimulatory (N,) unit. Consequently, the observations with the fat cell could be interpreted to imply that toxin treatment also inhibited a GTPase activity associated with the inhibitory (N,) unit. However, this effect of cholera toxin is not universally encountered in dually regulated systems. Inhibitory effects were retained following toxin treatment of Chinese hamster ovarian (CHO), neuroblastoma x glioma hybrid cells, and platelets (Evain and Anderson, 1979; Propst and Hamprecht, 1981; Jakobs and Schultz, 1979). Very recently the first direct evidence pertaining to this issue has been presented. In neuroblastoma X glioma and platelet membranes, a GTPase activity has been detected which can be stimulated by opiates and a-adrenergic agents, respectively (Koski and Klee, 198 1; Aktories and Jakobs, 198 I ) . Substrate spec-
76
DERMOT M. F. COOPER
ificities were not examined in these studies, thus a specific GTPase activity was not unequivocally demonstrated. Nevertheless, a twofold stimulation was achieved, and it is tempting to speculate that the GTPase activity measured is relevant to the inhibition of adenylate cyclase mediated by the receptors in these tissues. The rather complex assay mixtures utilized may allow transphosphorylation of the terminal phosphate of GTP to ATP, which is included in the assay. Neither study has attempted to determine the source of the Pi released into the medium; therefore, the possibility must be considered that inhibitory ligands may be stimulating an ATPase activity in plasma membranes in a manner analogous to that reported for insulin and catecholamines (Resh et al., 1980; Titheradge et al., 1979). However, notwithstanding the reservations raised concerning these recent studies, the accumulated evidence from more indirect studies would perhaps have anticipated a more central role for GTPase in inhibitory regulation than in the case of stimulatory regulation.
VII. THE RELATIONSHIP BETWEEN N, AND N, Definitive evidence is lacking on whether N, and Ni are distinct proteins or merely functional notations. The broadly descriptive options which might be considered include ( I ) that the N unit (or complex, see Section 11) is constant in all adenylate cyclase systems and that the functions associated with N, and Ni merely reflect the association of the N unit with R, or Ri, respectively, (2) N, and N, are distinct regulatory protein complexes which share a common catalytic TABLE 1 S U M M A R Y Ol- THE P R O P E R T l t S ASSOC‘lATtD WITH T H t
TWO GTP RLCUIATOKY FUNCTIONS“
Property
Ni
111 IV 111 111 111, IV
I pM”
GTP requirement (EDs,)) Sodium ions Cholera toxin Me2 Gpp(NH)p Cholera toxin labeled bands Mild trypsin treatment Effect of GTP, Gpp(NH)p on binding GTP on basal cyclase Sodium effect on binding
-. T -. 5 1
+
.
Section
- 3
5
Additional bands -3
.1 Kci.
1 1 Kd
.
7
R
Vlll 111 V 111 V
ampiification of the function; 1 dampening of the function. “Section” refers -, No effect; to sections in the text in which these features are discussed. When stirnulatory systems are considered.
STIMULATION AND INHIBITIONOF ADENYLATE CYCLASE
77
unit, or (3) Ni may be a modified form of N, (an oligomeric form; an association with an exogenous factor or protein, such as calmodulin; etc.) which may or may not be stabilized or promoted upon interaction with Ri. Differences in properties between the functional entities referred to as N, and Ni are summarized in Table 1. These differences justify the functional assignments and, with other evidence discussed here, support the view that fundamental differences exist between the two regulatory systems. Nevertheless a full appreciation of their properties will become available only by further structural studies .
VIII.
STRUCTURAL STUDIES ON DUALLY REGULATED ADENYLATE CYCLASE SYSTEMS
A. Receptors
Apart from the preliminary studies on the fat cell indicating selective abolition and retention of one of the two effects mediated by GTP (see Section IV), most attention has focused on the receptors in dually regulated systems. For instance, opiate receptors have been solubilized with full retention of their agonist specificity (Simonds et al., 1980). Another solubilized opiate receptor preparation retained the ability of sodium ion to modify binding (Ruegg et al., 1981), although no effects of GTP were detected. This important observation suggests that the sodium site may be associated with the receptor rather than the GTP regulatory unit. The a-adrenergic receptor from liver has been solubilized and partially purified (Guellaen et al., 1979). The irreversibly binding antagonist, phenoxybenzamine, was utilized to monitor the presence of the receptor through various purification proccclures. Although phenoxybenzaminc cannot readily be removed from the receptor preparation, this material is quite suitable for generating antibodies which may be utilized to identify and purify unoccupied receptors. Prior incubation of platelet plasma membranes with a,-adrenergic agonists stabilizes a higher molecular weight form of the receptor than that observed following incubation with antagonists. This data may suggest that agonists stabilize interaction between receptor and Ni unit (Smith and Limbird, 1981). 8. Cholera Toxin
Since cholera toxin, with NAD, modifies the functions ascribed to both N, and N,, it is conceivable that with [32P]NAD, protein bands additional to those encountered in stimulatory systems might be detected on sodium dodecyl sulfate (SDS) electrophoresis following exposure to the toxin of dually regulated sys-
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DERMOT M. F. COOPER
tems. When a range of plasma membrane preparations was compared, those subject to dual regulation showed additional labeled bands; in fat cell and CHO cells, a 52,000 and a 54,000 M, band were detected; in platelet, a 58,000 M, band was detected, in addition to the widely observed 42,000 M , band (Cooper et af.,1981). The relationship of these additional proteins to the N, unit, or Ni function. remains to be established.
C. Calmodulin The hippocampal adenylate cyclase is a dually regulated system (Section IV) which can be inhibited by opiates and adenosine analogs in a GTP-dependent manner. Calmodulin appears to play a role in this system, since its removal by EGTA treatment results in the loss of inhibition by the opiates and adenosine. Addition of calmodulin restores the effect (Girardot et af., 1981). This association does not seem to be generally applicable to inhibitory systems, since the platelet and fat cell systems, for example, are not affected by EGTA treatment (Cooper and Londos, 1982). However, in the case of the hippocampal system (and possibly other neural systems), calmodulin may provide a means for identifying inhibitory components.
D. Ultrastructural Studies Recent electron microscopic studies indicate that both opiate and cholinergic receptors occur in clusters on the cell surface (Hazum er a / ., 1979; Peng et a / ., I98 1). Irradiation inactivation studies also indicated that very large structures mediated stimulation and inhibition of fat cell adenylate cyclase (Schlegel et a/., 1980). Such findings are readily accommodated in view of the multiplicity of stimulatory and inhibitory neurotransmitters converging on a common pool of catalytic activity (see Section IV). These structures, which if composed of heterogeneous stimulatory and inhibitory receptors with their associated N units, would resemble multienzyme complexes and would provide a ready means of achieving the nonadditive stimulation and inhibition of adenylate cyclase by different neurotransmitters discussed in Section IV.
IX. FUTURE DIRECTIONS A number of putative neurotransmitters and peptides which have been identified in brain as yet do not have a measurable function in isolated membrane preparations. It seems likely that some of these compounds, including histamine, serotonin, GABA, glutamine, ACTH, substance P, VIP, a-MSH, and others, will turn out to utilize GTP inhibitory pathways.
STIMULATION AND INHIBITIONOF ADENYLATE CYCLASE
79
Whether the catalytic unit of adenylate cyclase is identical in dually regulated and in stimulatory systems must be determined. It is conceivable that the complexity of catalytic units varies as a function of the degree of regulation to which they are subject. Further progress in understanding the structural nature of these systems will require a combination of approaches, including ultrastructural studies, solubilization and reconstitution of components, identification of mutants lacking one or another of the regulatory elements, coupled with the fusion/complementation approach pioneered by Orly and Schramm ( 1976). A perplexing question, which must reflect a fundamental property of inhibitory systems, arises from the partial inhibition evoked by inhibitory neurotransmitters. Since inhibition ranges from 20 to 80% maximally (Cooper and Londos, 1982; Jakobs, 1979) and it is generally assumed that a gross overproduction of cAMP is produced in response to stirnulatory hormones, the physiological significance of this regulation may be doubted. However, partial inhibition of adenylate cyclase combined with the presence of phosphodiesterase in intact cells can result in substantial reduction of intracellular cAMP levels. The striking inhibition of cAMP production in the fat cell by adenosine and its analogs (leading to a marked inhibition of lipolysis) and in platelets by epinephrine (resulting in platelet aggregation) must reflect such a situation. A question related to that raised above pertains to the different consequences of inhibiting basal versus hormone-stimulated activity. Clearly, inhibition of hormone-stimulated adenylate cyclase will result in attenuation of the process(es) stimulated by the elevated cAMP levels. However, it is conceivable that in the absence of stimulatory hormone the basal cAMP levels may maintain distinct processes in an activated state due to different sensitivity to phosphorylation. Thus inhibition of basal cAMP production would result in modulation of processes separate from those governed by stimulatory hormones. Potential examples of the types of process maintained by basal cAMP levels might be fundamental cellular mechanisms concerned with maintenance of normal functions, such as ion transport. Thus it is of interest to examine the results of the exposure of intact cells to inhibitory agents, regardless of whether the processes concerned have previously been implicated with cAMP as a second messenger. The recent finding of angiotensin I1 and a-adrenergic inhibition of adenylate cyclase in liver (Jard et al., 1981), which had previously been considered a simple stimulatory system, raises some intriguing possibilities. Inclusion of high concentrations of EDTA was required during all stages of the preparation of the plasma membranes for the observation of this effect. It has also been known for some time that both GTP and sodium ion affect angiotensin binding to adrenal cortex membranes (Glossmann ef al., 1974) (without EDTA treatment), although angiotensin does not inhibit adrenal cortical adenylate cyclase. Thus the possibility is raised that an R N complex existed for angiotensin, which had not been linked to adenylate cyclase prior to the chelator treatments, but to some
80
DERMOT M. F. COOPER
other process. Certainly a number of receptors mediating inhibition of adenylate cyclase have also been implicated in either calcium transport or phosphatidylinositol metabolism, e.g., muscarinic cholinergic, a,-adrenergic, and angiotensin (Jones and Michell, 1978). The findings discussed above raise the possibility that switching of the function served by an RN complex can occur physiologically in addition to the experimental means presented above. An alternative interpretation of these results would be that in normal liver membranes, the expression of inhibitory activity is suppressed by interaction of the Ni unit with divalent cation, as discussed in Section IV, and revealed upon chelator treatment. Sharma el al. (1975) described increased adenylate cyclase activity following prolonged exposure of neuroblastoma X glioma hybrid cells to morphine without alteration in receptor number. The situation was presented as a model system for tolerance and addiction to opiates. Current appreciation of the existence of stimulatory and inhibitory GTP regulatory protein interactions may provide a means of understanding the basis of this observation. Recently, receptor-mediated inhibition of Gpp(NH)p-activated adenylate cyclase activity by progesterone in Xenopus oocytes and by a-mating factor in yeast (Finidori-Lepicard er al., 198 1; Sadler and Maller, 198 1; Liao and Thorner, 1980) has been reported. In both cases fundamental developmental changes correlate with these inhibitions. It is possible that modified forms of Ni with less severe restrictions on the terminal diphosphate bond of the guanine nucleotide mediate these effects.
FIG.6. Schematic representation of dually regulated adenylate cyclase with suggested sites of action of various regulators. In this scheme broken arrows indicate inhibition or suppression of a function: solid arrows indicate promotion or enhancement of a function; the heavy directional lines between R and N units identify the guanine nucleotides which promote communication from R to N or from N to R, respectively.
STIMULATION AND INHIBITIONOF ADENYLATE CYCLASE
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X. CONCLUSIONS The functional components involved in dual regulation of adenylate cyclase and the site of action of some modulators of activity are presented schematically in Fig. 6. The central role of guanine nucleotides is evident. Appreciation of the potential importance of GTP has proved to be the key to our current understanding of these systems. Evidence is accumulating from measurements of both activity and regulation of binding of the two classes of receptor that separate GTP regulatory proteins are associated with inhibition and stimulation. In the next few years there will be an increasing awareness of the physiological importance of these systems as well as insights into their structural composition. ACKNOWLEDGMENTS
I would like to acknowledge the useful comments of my colleagues Drs. D.L. Gill, R. Honnor, and R. T . Simpson on this manuscript. I would also like to thank Dr. M. Rodbell for his continuing support and encouragement and Ms. Bonnie Richards for her expert secretarial assistance. REFERENCES Aktories, K., and Jakobs, K. H. (1981). Epinephrine inhibits adenylate cyclase and stimulates a CTP-ase in human platelet membranes via a-adrenoceptors. FEBS Left. 130, 235-238. Aktories. K.. Jakobs, K . H.. and Schultz, G . (1980). Nicotinic acid inhibits adipocyte adenylate cyclase in a hormone-like manner. FEBS Lert. 115, 11-14. Blume, A. J . (1978). Interaction of ligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides. Proc. Natl. Acad. Sci. U.S.A. 75, 1713-1717. Blume. A. J . . Lichtshtein, D.. and Boone, G . (1979). Coupling of opiate receptors to adenylate cyclase: Requirement for Na+ and GTP. Proc. Nad. Acad. Sci. U.S.A. 76, 5626-5630. Collier. H. 0. J., and Roy, A. C . (1974). Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature (London) 248, 24-27. Cooper, D. M. F.. and Londos. C. (1982). GTP-dependent stimulation and inhibition of adenylate cyclase. I n “Horizons in Biochemistry and Biophysics” (L. D. Kohn, ed.), Vol. 6, pp. 309- 333. Cooper, D. M. F., and Rodbell, M. (1979). ADP is a potent inhibitor of human platelet plasma membrane adenylate cyclase. Nature (London) 282, 5 17-5 18. Cooper, D. M. F.. Schlegel, W . . Lin, M. C . , and Rodbell. M. (1979). The fat cell adenylate cyclase system. Characterization and manipulation of its bimodal regulation by GTP. J . Biol. Chem. 254, 8927-8930. Cooper. D.M. F.. Londos, C . , and Rodbell, M. (1980). Adenosine-receptor-mediated inhibition of rat cerebral cortical adenylate cyclase by a GTP-dependent process. Mol. Pharmacol. 18, 598-601. Cooper, D. M. F.. Jagus, R., Somers, R . L., and Rodbell, M. (1981). Cholera toxin labels diverse CTP-modulated regulatory proteins. Biochem. Biophvs. Res. Commun. 101, I 179-1 185. Cooper. D. M. F.. Londos, C., Gill, D. L., and Rodbell, M. (1982). Opiate receptor-mediated inhibition of adenylate cyclase in rat striatal plasma membranes. J . Neurocliem. 38, 1164-1167. Cote. T. E . , Grewe, C . W., and Kebabian, J. W. (1981). Stimulation of a D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland decreases the responsiveness of the P-adrenoceptor: Biochemical mechanism. Endocrinology 108, 420-426.
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Cryer, P. E., Jarrett. L., and Kipnis, D. M. (1969). Nucleotide inhibition of adenylate cyclase activity in fat cell membranes. Biochim. Biophy. Acta 177, 586-590. Ebert. R., and Schwabe, U. (1974). Biphasic effect of 5’-guanylylimidodiphosphateon fat cell adenylate cyclase. Naunyn-Srhmiedebergs Arch. Pharmarol. 296, 297-313. Evain, D., and Anderson, W. B. (1979). Inhibitory effect of guanyl nucleotides toward adenylate cyclase activity of Chinese hamster ovary cell membranes activated in vitro by cholera toxin. J . B i d . Chem. 254, 87268729. Fain, J. N., Pointer, R. H., and Ward, W. F. (1972). Effects of adenosine nucleosides on adenylate cyclase, phosphodiesterase, cyclic adenosine monophosphate accumulation, and lipolysis in fat cells. J . Biol. Chem. 247, 6866-6872. Finidori-Lepicard, I., Schorderet-Slatkine, S., Hanoune. J . , and Baulieu, E. E. (1981). Progesterone inhibits membrane-bound adenylate cyclase in Xenopus laevis oocytes. Narure (London) 292, 255-257. Girardot, J. M., Cooper, D. M. F., and Kempf, J. (1981). Regulation of rat hippocampal adenylate cyclase by guanyl nucleotides. Adv. Cyclic Nurleotide Res. 14, 657. Glossmann, H.. Baukal, A., and Catt. K. I. (1974). Properties of angiotensin I1 receptors in the bovine and rat adrenal cortex. J. Biol. Chem. 249, 664-666. Greenberg, D. A., U’Prichard, D. C., Sheehan. P., and Snyder, S. H. (1978). a-Noradrenergic receptors in the brain: Differential effects of sodium on binding of [3H] agonists and pH] antagonists. Brain Res. 140, 378-384. Guellaen, G., Aggerbeck, M., and Hanoune, J. (1979). Characterization and solubilization of the adrenoreceptor of rat liver plasma membranes labeled with (3H]phenoxyhenzamine. J . Biol. Chem. 254, 10761-10768. Hanvood, J. P., Low, H., and Rodbell, M. (1973). Stimulatory and inhibitory effects of guanyl nucleotides on fat cell adenylate cyclase. J. Biol. Chem. 248, 6239-6245. Hazum, E., Chang. K. J., and Cuatrecasas, P. (1979). Opiate (enkephalin) receptors of neuroblastoma cells: Occurrence in clusters on the cell surface. Srienre 206, 1077-1079. Jakobs, K. H. (1979). Inhibition of adenylate cyclase by hormones and neurotransmitters. Mol. Cell. Endocrinol. 16, 147- 156. Jakobs, K. H., and Schultz, G. (1979). Different inhibitory effect of adrenaline on platelet adenylate cyclase in the presence of GTP plus cholera toxin and of stable GTP analogues. NaunynSchmiedeberg’s Arch. Pharmacol. 310, 121-127. Jakobs, K. H., Saur, W., and Schultz, G. (1978). Inhibition of platelet adenylate cyclase by epinephrine requires GTP. FEES Len. 85, 167-170. Jakobs, K. H., Aktories, K., and Schultz, G. (1979). GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists. Naunyn-Srhmiedeberg’s Arch. Pharmarol. 310, 113-1 19. Jard, S., Cantau, B . , and Jakobs, K. H. (1981). Angiotensin I1 and a-adrenergic agonists inhibit rat liver adenylate cyclase. J . B i d . Chem. 256, 2603-2606. Jones, L. M., and Michell, R. H. (1978). Stimulus-response coupling at a-adrenergic receptors. Biochem. Sor. Trans. 6, 673-693. Koski, G., and Klee, W. A. (1981). Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis. Proc. Natl. Acad. Sci. U.S.A. 78, 4185-4189. Law, P. Y.,Wu, J., Koehler, I . E., and Loh, H. H. (1981). Demonstration and characterisation of opiate inhibition of the striatal adenylate cyclase. J . Neurochem. 36, 1834-1846. Liao, H., and Thomer, J. (1980). Yeast mating pheromone a factor inhibits adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 77, 1898-1902. Lichtshtein, D., Boone, G., and Blume, A. J. (1979). Muscarinic receptor regulation of NG108-IS adenylate cyclase: Requirement for Na+ and GTP. J . Cyclic Nucleoride Res. 5, 367-375. Limbird, L. E. (1981). Activation and attenuation of adenylate cyclase. Biochem. J . 195, 1-13. Londos, C., Cooper, D. M. F., Schlegel, W., and Rodbell, M. (1978). Adenosine analogs inhibit
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adipocyte adenylate cyclase by a GTP-dependent process: Basis for actions of adenosine and methylxanhines on cyclic AMP production and lipolysis. Proc. Nut/. Aiwd. Sci. U . S . A . 75, 5362-5366. Londos. C.. Cooper. D. M. F.. and Rodbell, M. (1981). Receptor-mediated stimulation and inhibition of adenylate cyclases: The fat cell as a model system. Adv. Cvclic Nucleotide Res. 14, 163- I7 I. Michel, T . . Hoffman. B . B.. and Lefkowitz, R . J. (1980). Differential regulation of a,-adrenegic receptor by Na+ and guanine nucleotides. Nature (London) 288, 709-71 I . Moskowitz, J.. Harwood. J. P.. Reid, W . D.. and Krishna. 0. (1971). The interaction of norepinephrine and prostaglandin E, on the adenyl cyclase system of human and rabbit blood platelets. Biochim. Biophys. Actu 230, 279-285. Murad, F., Chi. Y.-M.. Rall, T. W., and Sutherland. E. W. (1962). Adenyl cyclase 111. The effect of catecholamines and choline esters on the formation of adenosine 3',5'-monophosphate by preparations from cardiac muscle and liver. J . B i d . Chem. 237, 1233-1238. Orly, J . . and Schramm. M . (1976). Coupling of catecholamine receptor from one cell with an adenylate cyclase from another cell by cell fusion. Proc. Nut/. Acud. Sci. U.S.A. 73, 44104414. Peng, H. B.. Cheng. P.-C., and Luther, P. W. (1981). Formation of ACh receptor clusters induced by positively charged latex beads. Nature (London) 292, 831-834. Pert. C. B . , and Snyder. S. H. (1974). Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol. Phurmucol. 10, R68-879. Propst. F., and Hamprecht. B . (1981). Opioids, noradrenaline and GTP analogs inhibit cholera toxin activated adenylate cyclase in neuroblastoma X glioma hybrid cells. J . Neurochem. 36, 580-588. Resh. M. D., Nemenoff. R . A., and Guidotti, G . (1980). Insulin stimulation of (Na+,K+)-adenosine triphoaphatase-dependent H'Rb+ uptake in rat adipocytes. J . Biol. Chem. 255, 10938-10945. Rodbell, M. (19x0). The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nuture (London) 284, 17-22, Ross, E. M.. and Gilman. A. G. (1980). Biocheniical properties of hormone-sensitive adenylate cyclase. Annu. Rev. Biochem. 49, 533-564. Ruegg, U. T . , Cuenod, S., Hiller, J. M.. Gioannini, T., Howells, R . D.. and Simon, E. J. (1981). Characterisation and partial purification of solubilized opiate receptors from toad brain. Proc. Nut/. Acud. Sci. U . S . A . 78, 4635-4638. Sabol. S . L.. and Nirenberg, M. (1979). Regulation of adenylate cyclase of neuroblastoma X glioma hybrid cells by a-adrenergic receptors. 1. Inhibition of adenylate cyclase mediated by areceptors. J . B i ~ l Chem. . 254, 1913-1920. Sadler. S . E., and Maller. J. L. (1981). Progesterone inhibits adenylate cyclase in Xenopits oocytes. Action on the guanine nucleotide regulatory protein. J . Biol. Chem. 256, 6368-6373. Schimmel. R . J., McMahon. K. K.. and Serio, R . (1981 j . Interactions between alpha-adrenergic agents. prostaglandin E l , nicotinic acid and adenosine in regulation of lipolysis in hamster epididynial adipocytes. Mol. Phurmucol. 19, 248-255. Schlegel. W.. Cooper, D. M. F.. and Rodbell, M. (1980). Inhibition and activation of fat cell adenylate cyclase by GTP is mediated by structures of different size. Arch. Biochem. Biophys. 201, 678-6x2. Sharma, S. K.. Klee, W. A.. and Nirenberg, M. (1975). Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc. Nut/. Acud. Sci. U.S.A. 72, 3092-3096. Simonds. W . F . , Koski. G.. Streaty, R . A.. Hjelmeland, L. M.. and Klee, W. A. ( 1980). Solubilisation of activc opiate receptors. Proc. Nail. Acud. Sci. U . S . A . 77, 4623-4627. Smith. S . K.. and Linibird. L. E. (1981). Solubilization of human platelet a-adrenergic receptors:
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Evidence that agonist occupancy of the receptor stabilizes receptor-effector interactions. Proc. Nut/. Acad. Sci. U.S.A. 78, 4026-4030. Sternweis, P. C.. Northup, I . K., Hanski. E., Schleifer, L. S . . Smigel. M. D., and Gilman, A. G . (1981). Purification and properties of the regulatory component (GIF) of adenylate cyclase. Adv. Cyclic Nucleotide Res. 14, 23-36. Sulakhe, P. V.. Leung, N. L. K., Arbus, A. T., Sulakhe, S . J., Jan. S. H.. and Narayanan. N. ( 1977). Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides. Biochem. J. 164, 67-74. Titheradge, M . A., Stringer. J. L., and Haynes, R. C.. Jr. (1979). The stimulation of the mitochondrial uncoupler-dependent ATP-ase in isolated hepatocytes by catecholamines and glucagon and its relationship to gluconeogenesis. Eur. J . Biochenz. 102, 117-124. Tsai. B. S . , and Lefkowitz. R. J . (1979). Agonist-specific effects of guanine nucleotides on alphaadrenergic receptors in human platelets. Mol. Phurmacol. 16, 61-68. U’Prichard. D. C., and Snyder, S. H . (1980). Interactions of divalent cations and guanine nucleotides at az-noradrenergic receptor binding sites in bovine brain mechanisms. J . Neurochem. 34, 385-394. Watanabe. A. M., McConnaughey, M. M.. Strawbridge. R. A.. Fleming. J . W . , Jones, L. R.. and Besch, H. R.. Jr. ( 1978). Muscarinic cholinergic receptor modulation of P-adrenergic receptor affinity for catecholamines. J . B i d . Chern. 253, 4833-4836. Yamamura, H., Lad. P. M., and Rodbell. M. (1977). GTP stimulates and inhibits adenylate cyclase in fat cell membranes through distinct regulatory processes. J . B i d . Chem. 252, 7964-7966.
CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME I 8
Desensitization of the Response of Ade ny Iate Cyclase to CatechoIamines JOHN P . PERKINS Department of’Pharmacology University of North Curolinu Chapel Hill. N m h Carolina
I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Scope ofthe Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catecholatnine-Induced Desensitization of Intact Cells . . . . . . . . A. Origin and Characteristics of the Human Astrocytorna Cell B. Analysis of Rates of Synthesis and Degradation of Cyclic AMP in Whole Cells IV. Catecholamine-Induced Changes in Adenylate Cyclase and in PAR Binding Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Separation of Native and Desensitized PAR. . . . . . . . . . VI . Receptor Endocytosis as a Mechanism for Agonist-lndu VII. A Kinetic Model for Agonist-Induced Desensitization. . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Differential Expression of PAR during Growth of 1321Nl Cells . . . . . . . . . . . . . . . . . 1x. Down-Regulation of PAR and the Recovery of Lost Receptors . . . . . . . . . . . . . . . . . . X . Isoproterenol-Induced Changes in Agonist Binding Properties of Intact 132lN I Cells XI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . ..................... 11. 111.
1.
85 87 88 88 91 93 95 97 98 100 100 103 104
I06
INTRODUCTION
The initial effect of catecholamines on the j3-adrenergic receptor (PAR) linked adenylate cyclase system is to increase the rate of formation of cyclic AMP. This stimulatory effect occurs essentially instantaneously in most cells. However, secondary effects of exposure to catecholamines can be detected in certain cells within 1 minute. The functional consequence of these secondary reactions is a reduction in the rate and/or extent of cyclic AMP accumulation. Recent studies of such inhibitory events have revealed a complicated series of reactions, in85 Copyrlgh! 0 1983 by Academic Prerr. Inc. All rights 0 1 reproduction in any form rcscrved. ISBN 0-12-IS331R-2
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duced by receptor agonist, designed to down-regulate responsiveness of the cell to continued or subsequent challenge with a catecholamine. The purpose of such a cellular capability is not yet known, but the process of ligand-induced downregulation of receptor function has been shown to be general in nature; for example, the receptor systems for insulin, glucagon, epidermal growth factor (EGF), thyroid-stimulating hormone (TSH), leutinizing hormone (LH), folliclestimulating hormone (FSH), prostaglandins, histamine, acetylcholine, and adenosine all exhibit agonist-induced modification in function. However, while such down-regulating or desensitizing reactions appear to occur in general, the specific molecular mechanisms involved may be quite different for different receptor systems. About the only property shared in common by the receptor systems listed above is that in each case the receptor is a cell surface protein involved in transduction of the effects of an extracellular information molecule. Our studies of this general phenomenon have focused on those receptors that mediate the stimulatory effects of catecholamines on adenylate cyclase. The
Y
GDP
FIG. 1. This model indicates the hypothetical interactions of a catecholamine (H), the P-adrenergic receptor (R), a guanine nucleotide binding protein (N), the catalytic protein (C), and guanine nucleotides (GDP and GTP). The model indicates that RH is able to bind to N, resulting in the release of free GDP and the formation of HRN. The RN complex has higher affinity for H than does R. The formation of HRN is rate limiting in the activation process, and, in the presence of GTP, HRN is rapidly coverted to N-GTP and HR; thus, the role of the hormone receptor system is to effect the conversion of N-GDP to N-GTP. Once formed, N-GTP interacts with C to form the enzymically active complex C-N-GTP. The lifetime of the active complex is determined by the activity of a GTPase (probably an integral part of N) which hydrolyzes the bound GTP to release Pi, with the subsequent regeneration of C and N-GDP. In the absence of GTP, addition of H leads to formation of HRN in amounts sufficient to change the apparent K, of the system for H. Thus, in the absence of GTP, agonists (H) exhibit binding characteristic of interaction at two sites (R and RN). In the intact cells. or upon addition of GTP to membranes, the amount of HRN would be small because its rate of formation is postulated to be the limiting step in the intact system. Under these conditions, agonists (H) would exhibit binding characteristic of the reaction R + H RH, namely low-affinity binding to a single type of site. (Modified from Su et al., 1980.)For detailed reviews of the hormone-sensitive adenylate cyclase system see Ross and Gilman (1980) and Lirnbird (1981).
*
LIGAND-INDUCED CHANGES IN P-RECEPTOR FUNCTION
87
most compelling reasons for this choice of system include
I . The identities of the major molecular components of the hormone-sensitive adenylate cyclase have been established (see Fig. I ) . 2. A plausible working model for the activation sequence initiated by hormones has been described (see Fig. 1). 3. The amount and functional state of the three major components of the system can be determined by independent assay in subcellular membrane fractions and by reconstitution of the previously resolved components. 4. Assays exist for measurement in intact cells of PAR binding properties, the rate of formation of cyclic AMP, and the rate of cyclic AMP degradation. Thus, as compared to polypeptide hormone receptor systems for insulin and EGF, which have been well studied in terms of down-regulation, the receptorlinked adenylate cyclase systems allow one to investigate the relation between changes in binding events and changes in functional events that occur essentially instantaneously with ligand binding. The PAR-linked adenylate cyclase is the most thoroughly studied receptor-linked adenylate cyclase system, due in large part to the variety of agonist, partial agonist, and antagonist ligands for the PAR that are available for use as experimental probes (e.g., see Minneman et al.. 1981). A variety of high-specific-activity, selectively binding radioligands are available commercially or readily synthesized from available starting materials (Aurbach et al., 1974; Williams and Lefkowitz, 1978; lnnis et a / . , 1979; Barovsky and Brooker, 1980; Engel et d., 1981; Staehelin and Simon, 1982.)
II. SCOPE OF THE REVIEW The observation that chronic exposure to catecholamines of the PAR-linked adenylate cyclase system leads to a reduction in responsiveness has been verified in studies of intact animals and humans (reviewed in Perkins er al., 1979, 1982; also see Aarons et al., 1980; Fraser et a/.. 1981). In addition, it can be shown that chronic underexposure to catecholamines in intact animals leads to an increase in responsiveness. Thus, the concept has evolved that this receptor-enzyme system exists in vivo in various states of responsiveness that depend, inversely, on the recent level of exposure to catecholamines. We will not attempt here to provide evidence of the physiological significance of ligand-induced adaptive changes in the responsiveness of cells to hormones; however, readers are referred to other recent reviews which provide evidence of this nature (Perkins er al., 1979, 1982; Lefkowitz et a/.. 1980). In this article, the biochemical mechanisms involved in such adaptive responses will be reviewed, primarily as they have come to be understood from experiments utilizing either cultured mammalian cells or erythrocytes from mammals, birds, and frogs. The review
88
JOHN P. PERKINS
will further focus on experiments carried out over the past 10 years in my laboratory, the past 6 years in collaboration with T. K . Harden. Work by other investigators will be referred to primarily to demonstrate general points or to highlight areas of controversy.
111.
CATECHOLAMINE-INDUCED DENSENSITIZATION OF INTACT CELLS
A. Origin and Characteristics of the Human Astrocytoma Cell Line 1321N1 Since 1971 we have utilized cultured human astrocytoma cells as a convenient experimental system in which to study the regulation of cyclic AMP metabolism by hormones. The original source of the cell line was a primary culture of a Grade 111 astrocytoma-glioblastoma (multiform) isolated by Ponten and Maclntyre (1968) and designated 118 MG-C. A subclone ( 1 181Nl) of this culture that maintained a more consistent spindle morphology was isolated (MacIntyre er a / ., 1972) and characterized in terms of its adenylate cyclase, cyclic AMP-phosphodiesterase, and cyclic AMP-dependent protein kinase activities (Perkins et al., 1971). For the past 7 years we have primarily studied a subclone of the 1 1 8 1N I line designated 1321 N 1 . These cells respond to catecholamines (Clark and Perkins, 197 I), adenosine (Clark et al., 1974), and prostaglandins (Ortmann and Perkins, 1977), with a rise in cyclic AMP. The cells also express muscarinic cholinergic receptors that mediate inhibitory effects of acetylcholine on cyclic AMP accumulation (Gross and Clark, 1977). The response of 1321NI cells to addition of a catecholamine to the growth medium is shown in Fig. 2. The response is clearly biphasic; the initial rapid rise in cyclic AMP is followed by a decline to near precatecholamine levels. This response is typical of most, if not all, cells that respond to catecholamines. If the cells are washed free of catecholamine, the cellular content of cyclic AMP allowed to fall, and then catecholamine added again, the response observed declines in a manner that is related to the concentration of catecholamine and the time of the initial exposure (Fig. 2). Since 1321N1 cells respond to both catecholamines and prostaglandins, we were able to determine if agonist-induced desensitization was selective' for the inducing hormone or if cross-desensitization occurred (Su er a / . , 1976a). One experimental approach to this question is illustrated in Fig. 3. Cells were first exposed to either isoproterenol or prostaglandin E, for increasing periods of time; the response to the same agonist or the alternate agonist was then tested in a second incubation. Numerous such experiments in our laboratory and others have clearly indicated that the protocol involving a homologous pre- and post-
LIGAND-INDUCEDCHANGES IN f3-RECEPTOR FUNCTION
0
m
20
Y)
40
89
50
60
'ID
Minuter FIG. 2 . The effect of time and agonist concentration on desensitization. Cells were incubated with 100 )uM norepinephrine ( 0 )or 10 phf norepinephrine (m) and 3H-labeled cyclic AMP (CAMP) accumulation was measured. At the times indicated, norepineprhine was removed by washing. The decline of 'H-labeled cyclic AMP content is shown by the dashed line. The cells were subsequently challenged with 100 pA4 norepinephrine for 5 minutes. (From Su ef a/..1976a.)
incubation leads to a more rapid and greater extent of loss of response than does the heterologous protocol. The specificity of the desensitization process of 1321Nl cells is high at short times of incubation, but eventually a 40-60% heterologous desensitization is observed. In this protocol the agonist concentrations used were high enough to fully saturate the receptors and fully activate cyclic AMP production. If instead cells were incubated with low concentrations (1 -5 nM) of catecholamine (Perkins et al., 1979) [or prostaglandin (Leightling et a/.. 1976)j for extended periods of time (12-24 hours), a highly agonist-specific desensitization was observed. The results described above suggested to us that desensitization could occur by more than one mechanism and that the processes might exhibit different time courses and different concentration-effect relationships. Since the concentration-effect relations for catecholamine-induced cyclic AMP formation and for catecholamine-induced desensitization (at 60 minutes) were similar, it seemed reasonable that cyclic AMP might mediate desensitization. Incubation of 1321N1 cells with dibutyryl or 8-methyl thio analogs of cyclic AMP caused a loss of response to both isoproterenol and prostaglandin E, (Su er al., 1976a).
90
JOHN P. PERKINS
2.5
-
5
A 4
2.0
a:
-
4
3
x
QS
0
-
2
-
1
..........................................
ti 0
~~
I
I
I
2
3
0
1
2
3
Hours FIG.3. The time course of loss of homologous and heterologous responsiveness. Cells were first exposed to 10 ph4 isoproterenol (ISO) or 10 ph4 PGE, for varying periods of time as shown on the abscissa. The agonists were then removed by washing and the cells incubated in the absence of agonist for 10 minutes to allow 3H-labeled cyclic AMP content to decline. ( A ) The cells were then challenged with 10 j&f isoproterenol for 5 minutes and the 'H-labeled cyclic AMP content was measured. The homologous response (IS0 then ISO) is represented by the solid circles ( 0 )and the (B) The cells were challenged with heterologous response (PGE, then ISO) by the open circles (0). 10 PGE, for 5 minutes and the 3H-labeled cyclic AMP content was measured. The homologous and the heterologous responsiveness (PGE, then PGE,) is represented by the solid squares Dashed lines represent the basal level of responsiveness (IS0 then PGE,) by the open squares (0). 3H-labeled cyclic AMP in the cells. (From Su et a / . . 1976a.)
(m),
The time courses were similar as were the extents of loss (40-50%), and in this regard the effects of cyclic AMP resembled heterologous desensitization as illustrated in Fig. 3. If cyclic AMP acts as a feedback inhibitor it is reasonable to expect that its action would be nonspecific with regard to the inducing agonist. Our early results and the more extensive studies of Brooker and co-workers (Terasaki et al., 1978; Nickols and Brooker, 1979) are consistent with the conclusion that cyclic AMP mediates a hormone-induced heterologous desensitization by effecting an inhibition of the adenylate cyclase reaction at a site distal to the hormone receptor interaction. A more complete discussion of the evidence in support of this contention can be found in Terasaki et al. (1978) and Perkins et al. (1982).
91
LIGAND-INDUCED CHANGES IN P-RECEPTOR FUNCTION
B. Analysis of Rates of Synthesis and Degradation of Cyclic AMP in Whole Cells If a rise in cellular cyclic AMP caused a rise in cyclic AMP-phosphodiesterase activity, a loss in the capacity of hormones to raise cyclic AMP levels also would result. Such an induction of phosphodiesterase activity has been shown to occur in a variety of cells (D’Armiento et al., 1972; Manganiello and Vaughan, 1972). In order to determine the relative roles of changes in synthesis and degradation of cyclic AMP as the basis of desensitization, we developed techniques for such assessments in intact cells. A pulse-labeling technique was used to determine the relative rates of synthesis of cyclic AMP during a series of 3-minute periods throughout a 2-hour exposure of human astrocytoma cells to catecholamines (Su et al., 1976b). The results provided evidence that, within 3 minutes, catecholamines induce a reduction in the rate of cyclic AMP synthesis that further declined over the 2-hour period. In related experiments the initial velocity of accumulation of cyclic AMP (5- to 10-second intervals for 40 seconds) was measured as an indication of changes in the rate of synthesis of cyclic AMP during desensitization. The results of both types of experiments indicated that a rapid loss in the capacity of the cells to synthesize cyclic AMP occurs within several minutes of exposure to catecholamines. Clark and Butcher (1979) have used a related approach to detect, continuously, changes in the rate of cyclic AMP synthesis during exposure of W1-38 fibroblasts to catecholamines. From the results of such studies it has been concluded that a change in the rate of synthesis of cyclic AMP is the primary basis for the loss of response of intact human astrocytoma and WI-38 cells to catecholamines. In order to measure the rate of degradation of cyclic AMP, it was first necessary to raise the intracellular content by exposure of the cells to a catecholamine. The synthesis of cyclic AMP was then reduced rapidly to basal levels by adding the antagonist propranolol. The subsequent decay of cyclic AMP levels appeared to obey first-order kinetics and the rate constant for degradation (K,) was determined as shown in Fig. 4. No significant change in K , was observed during a 60minute incubation with norepinephrine. During this time span the cellular cyclic AMP level underwent a typical biphasic change. No detectable change in the content of cyclic AMP in the medium occurred after addition of propranolol; thus, we could assume that the rate of loss of cyclic AMP was a reflection of the rate of phosphodiesterase activity in the intact cells. We carried out approximately 30 determinations of Kd at various times during the first 60 minutes of exposure of the cells to norepinephrine or isoproterenol. The mean + SD for 30 determinations was 0.32 0.05. Since there was no significant difference in the values of Kd determined after exposure of the cells to
*
92
JOHN P. PERKINS
i . . ’ ” i . ’ r i . *o
Mnute.
rope=-.n
-.2
-
aopc=-.J2
rbp=-.3a
rope= -.u
-.4
2
*P
-.6
-.E -1.0
0
1.0 1.5 Minutes
0.5
2.0
FIG.4. Determination of the rate constant for degradation of‘cyclic AMP. Cells were challenged with norepinephrine (NE) to elevate intracellular 3H-labeled cyclic AMP concentration (upper panel). At the times indicatcd propranolol was added to block completely the siniulatory effect of NE on 3H-labeled cyclic AMP formation. The content of 3H-labeled cyclic AMP in the cells was measured over the next 2 minutes to determine the rate of decline. The results are expressed as the natural logarithm of the fraction of ”-labeled cyclic AMP (A/Ao)remaining versus the time after addition of propranolol. The first-order rate constant of degradation is proportional to the negative slope. See Su rt a / . (1976b) for details of experimental procedure.
norepinephrine or isoproterenol, we concluded that rapidly induced, agonistspecific effects on phosphodiesterase activity did not occur. The idea that desensitization primarily involves a decrease in adenylate cyclase activity rather than an increase in phosphodiesterase activity is supported by these observations. Agonist-induced increases in phosphodiesterase activity have not been observed with less than 90 minutes of exposure to either PGE, or the catecholamines. Thus, the increase in degradative activity (1.5- to 2.0-fold after 2 hours) could play only a minor role late in the overall desensitization process. Based on studies with intact cells we were able to surmise that at least three different processes are induced by receptor agonists that lead to a loss of cellular responsiveness to hormones. The processes exhibit different time courses that allow their distinction. The most rapid process involves agonist-specific modifications and at high hormone concentrations is clearly evident by 3 minutes. At lower agonist concentrations this process is slower but does appear to proceed to
93
LIGAND-INDUCEDCHANGES IN f3-RECEPTOR FUNCTION
near-complete desensitization. A second process results in a nonspecific loss of hormonal responsiveness that can be detected after 30-60 minutes of exposure of 1321N1 cells to either catecholamines or prostaglandins; cyclic AMP analogs appear to induce a similar process over the same time course. Even under optimal conditions nonspecific or heterologous desensitization does not exceed 40-60% inhibition. The third agonist-induced process contributing to loss of response is the induction of phosphodiesterase activity. In 1321N1 cells this occurs after 90- I20 minutes of exposure to an agonist and results in a 50% reduction in the steady-state level of cyclic AMP. It is probable that the three processes are additive in their effects since they appear to occur by distinct mechanisms. Our subsequent studies have focused on an investigation of the mechanism responsible for agonist-specific desensitization.
IV. CATECHOLAMINE-INDUCED CHANGES IN ADENYLATE CYCLASE AND IN PAR BINDING PROPERTIES As an initial hypothesis it seemed reasonable to propose that agonist-specific desensitization would involve changes in the number or the functional state of the PAR. Thus, our first experiments (Su et al., 1980) compared the loss of whole cell response to isoproterenol, the loss of response of adenylate cyclase to isoproterenol, and changes in the number of PAR, during a 24-hour exposure of 1321Nl cells to isoproterenol. PAR were measured with a high-affinity, highly
16
0
24
HOURS
FIG. 5 , Time courses of decrease in PAR density, isoproterenol-stimulated adenylate cyclase activity. and isoproterenol-stimulated cyclic AMP accumulation during exposure to isoproterenol. Data are presented as the percentage of activities expressed in untreated cells assayed at the same time in culture life. (From Su er a/..1980.)
94
JOHN P.PERKINS
specific receptor antagonist, [ 1251]iodohydr~xybenzylpindolol (IHYP). The results of the comparison are shown in Fig. 5. The most striking observation was that loss of response to isoproterenol measured either in whole cells or membranes, was a much more rapid process than was loss of PAR. In fact, detectable loss of PAR did not occur before 1 hour. It also is apparent that the loss of responsiveness of adenylate cyclase to isoproterenol cannot fully account for the loss of response of intact cells. This latter discrepancy is to be expected since by 30-40 minutes of exposure to isoproterenol, heterologous desensitization should begin to contribute to the overall process of desensitization. Also, by 90-120 minutes the effects of a twofold rise in phosphodiesterase activity would contribute to the reduction in whole cell response. Other investigators have observed discrepancies between the magnitude of agonist-induced PAR loss and the extent of desensitization of hormone-stimulated adenylate cyclase activity (Shear et al., 1976; Johnson et al.. 1978; Wessels et al,, 1978, 1979). The distinct lag in PAR loss, while quite evident in most of our experiments with 1321Nl cells, is less evident or not observed in other cell types. Nonetheless, careful kinetic analyses uniformly have exposed a significant discrepancy between the extent of loss of hormone-stimulated enzyme activity and reduction in PAR number. A more detailed kinetic analysis (Su ef al., 1980) led to the conclusion that incubation of 1321NI cells with isoproterenol results in a rapid (t1,2 = 3 minutes) decrease in isoproterenol-stimulated adenylate cyclase activity. This reaction appeared to reach a steady state in which about 50% of control responsiveness remained. Upon removal of isoproterenol full responsiveness was regained with a I,,, for reversal of about 7 minutes. During the first 30-45 minutes of exposure of cells to isoproterenol the only detectable change in the functional status of the adenylate cyclase system was the partial loss of response to catecholamines. Basal enzyme activity and NaF-, Gpp(NH)p (guany 1-5’-yl imidodiphosphate)-, and PGE, -stimulated activities remained unchanged. We have used the term “uncoupled” to describe the state of the desensitized system during this stage of the process, i.e., PAR have not been lost nor have the components of adenylate cyclase been altered; nonetheless, receptor agonists do not stimulate enzyme activity. Our current understanding of the adenylate cyclase system suggests that a change in one of only two of the components of the enyzme system could account for the selective loss of hormone responsiveness; namely, alterations in the receptor per se or in the guanine nucleotide binding component (N) (see Fig. 1). The work of Gilman and co-workers (Ross et al., 1978; Sternweis and Gilman, 1978) suggests that a single class of N-proteins mediates activation of adenylate cyclase by both catecholamines and prostaglandins. Since the loss of responsiveness induced by isoproterenol is agonist specific, it seems less tenable
95
LIGAND-INDUCED CHANGES IN p-RECEPTOR FUNCTION
that changes in the N-protein could account for the observed change. However, the possibility remains that the N-protein could contain independent domains of interaction for each type of receptor expressed in a particular cell. A number of observations led us to propose (Su et al., 1980) that reaction schemes for agonist-induced activation of adenylate cyclase and agonist-induced desensitization share a common intermediate. For example, the uncoupling reaction and activation of cyclic AMP production in whole cells both exhibit a for isoproterenol of 0.03 pM. Also, partial agonists have about equivalent partial effects on the degree of uncoupling and the degree of activation of cyclic AMP synthesis. In addition, agonist-induced loss of PAR does not occur in the cycmutant of the S49 lymphoma cell; such cells exhibit normal PAR and adenylate cyclase but lack functional N-protein; thus, cyc- cells are “uncoupled. However, recent experiments by Green and Clark (1 98 l ) cast some doubt on our proposal. They exposed cyc - S49 cells to isoproterenol then supplemented membranes from such cells with N-protein extracted with detergent from wildtype S49 cells. Such extracts were sufficient to “recouple” membranes from naive cyc- cells but did not cause complete recoupling of the response to isoproterenol in cyc membranes from cells previously exposed to isoproterenol. They concluded that isoproterenol-induced desensitization does not require activation coupling of PAR and the N-protein. Experiments by Iyengar et al. (1981) had previously shown that the N-protein extracted from desensitized S49 wild-type cells was capable of “recoupling” cyc- membranes to the same extent as did N-protein from naive wild-type cells. Thus, while there is general agreement that it is the PAR that is modified during desensitization, it is not clear that this reaction requires normal coupling of PAR and N-protein. ”
-
V.
SEPARATION OF NATIVE AND DESENSITIZED PAR
In 1980 we reported that PAR from homogenates of 1321N1 cells exposed to isoproterenol for 15-30 minutes could be separated into two classes based on their respective migration patterns during centrifugation through sucrose density gradients (Harden et al., 1980). The basic observation is illustrated in Fig. 6. Isoproterenol-stimulated adenylate cyclase activity is recovered as a single peak migrating as a dense particle (45-50% sucrose) from both control and desensitized cells. PAR from control cells also are found predominantly in these fractions. However, the PAR from desensitized cells distribute in about equal proportions in the light peak and the heavy peak. The shift in PAR to the light peak appears to be a highly selective process. We have measured the various parameters of adenylate cyclase activity, muscarinic receptors, EGF receptors, Na-KATPase, and total protein in the light fraction before and after desensitization
96
JOHN P. PERKINS
C
.-0 u
e . .L
C
E a \
z
200-
Q, W -
0
a E
FRACTION NUMBER
Fir;. 6 . Sucrose density gradient distribution of P-adrenergic receptors and adenylate cyclase activity after short-term incubation of cells with isoproterenol. Cells were incubated with 1 mM sodium ascorbate ( 0 )or I mM sodium ascorbate plus I isoproterenol (0) for 15 minutes. The cells were treated with concanavalin A, then lysed and centrifuged in a sucrose density gradient. (A) '2s1HYP was used to determine P-receptor density in gradient fractions. ( B ) Isoproterenol-~timulated adenylate cyclase activity was determined. (From Harden ef a / . . 1980.)
with isoproterenol. In no case did these activities shift in their migration pattern. This analysis is not exhaustive, but it is sufficient to indicate a high degree of specificity for the PAR shift reaction. The time course of appearance of receptors in the light vesicle fraction (PAR,,) is similar to the time course of the uncoupling reaction with a t,,* of about 3 minutes; in addition, the rates of reversal of both processes are similar. Also, both processes appear to proceed in a fashion similar to a steady state in which 50% of the receptors remain in the native state (PAR,) and 50% are altered (PAR, or PAR,,). Such correlations suggest a functional role for PAR,, in the reactions leading to loss of responsiveness to isoproterenol.
LIGAND-INDUCED CHANGES IN PRECEPTOR FUNCTION
97
The ligand binding properties of PAR,, have been studied in some detail (Harden et al., 1980). In terms of antagonist binding characteristics no change in Kd values has been detected. Conversely, agonist binding affinity is reduced about 10-fold compared to agonist affinity for PAR, or for receptors in the heavy peak. A similar reduction in the affinity of agonist binding was observed in homogenates of desensitized cells (Harden et al., 1979b). It appears that such homogenates contain receptors with native affinity for agonists as well as modified receptors with lower affinity for agonist. Centrifugation of the homogenate over sucrose gradients separates these two affinity states on the basis of physical differences in the membranes with which they are associated. Electron micrographs of the light fractions of the gradient reveal primarily small vesicles, whereas the heavy peak contains primarily large open sheets of plasma membrane. The plasma membrane fragments behave as open sheets due to a treatment of the cells with concanavalin A prior to lysis. Such treatment appears to stabilize the plasma membrane and reduce fragmentation and vesiculation (see Lutton et al., 1979). Enzymatic markers for the plasma membrane and for the Golgi apparatus migrate exclusively in the heavy and light peaks, respectively (Lutton et al., in preparation). Recent experiments in our laboratory (Hertel and Wakshull, unpublished results) indicate that 1321N1 cell surface EGF receptors migrate exclusively in the heavy peak whereas EGF-induced, internalized receptors migrate primarily in the light peak, corresponding to the migration of PAR,,.
VI.
RECEPTOR ENDOCYTOSIS AS A MECHANISM FOR AGONIST-INDUCED DESENSITIZATION
The results cited above and the analogy to polypeptide hormone-induced receptor endocytosis (Pastan and Willingham, 1981) as a first step in receptor down-regulation support the idea that catecholamine-induced endocytosis of PAR, to yield PAR,, occurs during the first few minutes of agonist exposure to 1321N1 cells. Recent experiments in our laboratory (Toews et af.,in preparation) have examined the binding characteristics of [ '2sl]iodopindolol ( 12sI-Pin)at 4°C to intact cells before and during the desensitization process, and to fractions from sucrose density gradients. Briefly stated, the results indicate that at 4°C '2sl-Pin does nor bind to PAR,, but will bind to the PAR, remaining in the heavy peak. Consistent with this finding is the observation that the number of i2sI-Pin binding sites at 4°C on intact cells is reduced to about 50% of control cells by prior incubation of 1321N1 cells at 37°C with isoproterenol. The loss of binding sites occurs with kinetics of onset and reversal that are consistent with the kinetics of formation and reversal of PAR, and PAR,,. We have tentatively concluded that at 4°C 12sI-Pinbinds only to cell surface receptors, i.e., it does
98
JOHN P. PERKINS
not cross membrane barriers effectively. Such a diffusion limitation would offer a plausible explanation for the results if PAR are lost via agonist-induced endocytosis. Other workers in this field have presented evidence in favor of agonist-induced internalization of PAR. For example Chuang and Costa (1979) reported that incubation of bullfrog erythrocytes with isoproterenol results in the appearance of a soluble fraction of PAR in cell lysates. Staehelin and Simons (1982) have carried out a series of studies of catecholamine-induced desensitization in C6 glioma cells. They utilized a nonhydrophobic radioligand for the PAR, 'Hlabeled CGP-12177, to study cell surface binding much as described above for Iz5I-Pin. Their results from studies carried out in Basel with C6 cells and similar studies carried out in collaboration with us by Dr. C. Hertel working in Chapel Hill using 1321Nl cells all are consistent with an endocytosis model for PAR desensitization. Unfortunately, none of the results reported to date are definitive for such a mechanism.
VII.
A KINETIC MODEL FOR AGONIST-INDUCED DESENSITIZATION
The kinetics of onset and reversal of the early stage of agonist-induced desensitization have been studied in some detail. In summary four different aspects of the process can be measured: (1) the loss of adenylate cyclase activity, (2) the change in agonist binding affinity, (3) the formation of PAR,,, and (4) the loss of whole cell binding to 1251-Pin(or 3H-labeled CGP- 12177) when measured at 4°C. These results all are consistent with the following simple relationship (ISO, isoproterenol): t IS0
BARN C PAR (modified) -IS0
where the forward reaction has a t,,2 at receptor saturation of 2-3 minutes and the recovery reaction has a t,,2 of 6-8 minutes. Recently we have shown that prior exposure of 132I N 1 cells to concanavalin A (Con A) completely prevents the formation of PAR,, but has no effect on the loss of responsiveness of adenylate cyclase to isoproterenol (Waldo, Perkins, and Harden, in preparation). Recovery of PAR, to PAR, occurs upon removal of isoproterenol in the continued presence of Con A. This result suggests the following modification of the kinetics model: 11) t IS0
I21 t
IS0
PARN G PARu S P A R L ~ -IS0
-IS0
99
LIGAND-INDUCED CHANGES IN B-RECEPTOR FUNCTION
0
I
I
I
I
I
I
1
2
3
4
5
6
Time ( h r )
FIG. 7. Recovery of isoproterenol-stimulated adenylate cyclase activity following desensitization. Cells were incubated with 1 p M isoproterenol. At the times indicated the culture dishes were washed with fresh medium. Membrane fractions were then prepared or were prepared after a 20minute further incubation in the absence of isoproterenol. The open circles indicate the loss (00) then recovery (0- -0) of adenylate cyclase activity. The solid triangles indicate the loss then recovery of IHYP binding sites. (From Su el d..1980.)
-A)
(A-
(A--A)
and allows the speculation that a prior modification of PAR leads not only to an inability to couple with the nucleotide binding protein, but initiates conversion of the receptor to PAR,,, possibly by way of endocytosis. The nature of the initial receptor modification is currently under investigation. Reactions ( 1 ) and ( 2 ) have not been distinguished by kinetic analysis and thus occur with similar rates. At early times of desensitization these reactions appear to be completely reversible; however, when loss of PAR binding sites for antagonist radioligands occurs, complete recovery of responsivenss of adenylate cyclase to isoproterenol does not occur (Su et al., 1980). Nonetheless, rapid recovery of enzyme responsiveness does occur (t,,z = 7-10 minutes) to the extent that receptor binding sites remain. This relationship is illustrated in Fig. 7. Such observations are consistent with the following overall kinetic model: (I)
I?)
t IS0
PAR,
t
BARci -IS0
(31
IS0
1
IS0
PAR,, Q PARL
~-Iso
- IS0
where reaction (3) is essentially irreversible within the time span of experimentation. The study of the reversal of reaction (3), to be described below, demonstrates that the reaction does actually reverse with a t,,z of about 12-14 hours. The model does not explicitly explain the lag in the formation of PAR, since the amount of PAR,,, the apparent precursor of PAR,, rises rapidly to a steady-
100
JOHN P. PERKINS
state value long before the rate of PAR, formation is at its maximum. Thus, the formation of PAR, is not a simple first-order reaction with respect to the concentration of PAR,, and must involve at least one intermediate reaction. Alternatively, we must leave open the possibility that PAR, is formed by a set of reactions that does not require prior formation of PAR, or PAR,,, i.e., PAR, may be formed by an independent pathway.
VIII.
DIFFERENTIAL EXPRESSION OF PAR DURING GROWTH OF 1321N1 CELLS
When 1321N1 cells are passaged in culture in the traditional manner, the specific activity of isoproterenol-stimulated adenylate cyclase and the number of PAR per cell vary significantly and in parallel as the dilute cultures ( 5 X 10’ cells/cm2) grow to confluence (3 X lo5 cells/cm2). Basal enzyme activity and NaF- and PGE, -stimulated activities remain relatively constant irrespective of culture density. A series of experiments from our laboratory established that the number of PAR per cell is regulated in a reproducible manner with respect to cell density (Harden et al., 1979a). Cells taken from postconfluent cultures and seeded at low density in fresh culture dishes usually exhibit 2000-3000 PAR/ cell. After a 20- to 24-hour lag the cells begin to accumulate receptors at the rate of 12- 13 X lo3 PAR/cell/cell division until a steady-state receptor density of 10 X 10’ PAR/cell is reached, whereupon the rate declines to 10 X lo3 PAR/celI/ cell division. Upon reaching confluence, PAR synthesis in the culture appears to cease, and, because the cells continue to grow, the number of PAR declines to 2-3 X 103/cell. It is at this point that the culture is usually passaged. Thus, the cells appear to “sense” that the number of PAR is less than lo4 PAR/cell and, if growing at less than confluent density, will begin to make PAR at an accelerated rate. If the receptor depleted cells are plated at confluent or greater density the cells will not activate receptor synthesis. The cells also appear to ‘‘sense” when they attain lo4 PAWcell since the synthesis rate declines to maintain the preconfluence steady-state rate of lo4 PAR/cell/division. A third signal appears to be initiated by cell contact (not by conditioned medium) and terminates receptors synthesis. The physiological significance of the growth-related regulation of PAR is not apparent, but cognizance of the phenomenon allowed us to examine the process of PAR loss and recovery more effectively.
IX.
DOWN-REGULATION OF PAR AND THE RECOVERY OF LOST RECEPTORS
Exposure of 1321N 1 cells to catecholamines for 12-24 hours leads to greater than 90% loss of PAR. If the catecholamine is removed and the cells washed
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FIG.8. Recovery of down-regulated PAR in pre- and postconfluent cultures. Cells were exposed to isoproterenol (ISO) for 12 hours. The cultures were then washed and the incubation continued in fresh growth medium in the absence or presence of cycloheximide (CHX) or tunicamycin (TUN). At the time indicated the density of PAR was determined by I25IHYP binding. (A) Preconfluent cultures. (B) Postconfluent cultures.
0
102
JOHN P. PERKINS
with fresh medium, PAR reappear as does catecholamine responsiveness of adenylate cyclase. Depending on the cell density of the cultures two patterns of recovery have been observed (Doss et al., 1981). These patterns are illustrated in Fig. 8 . When cells were down-regulated and then allowed to recover under preconfluent growth conditions, recovery could not be differentiated from new receptor synthesis. As shown in Fig. 8A, the cultures regained PAR at a rate and to an extent identical to control cells. However, if recovery was allowed to occur in the presence of cycloheximide, PAR returned only after a lag and at a reduced rate; nonetheless, PAR eventually recovered to the level present in the cultures at the time of exposure to isoproterenol. It should be noted that cycloheximide added to control cultures stopped cell growth immediately and completely inhibited the synthesis of PAR (Doss ef al., 198I). The effects of cycloheximide on both cell proliferation and PAR synthesis were completely reversible even after 48 hours of exposure to the protein synthesis inhibitor. Our initial conclusion from such studies (Fig. 8A) was that although PAR, were not detectable by standard binding assays, the primary amino acid structure of the receptor was not destroyed during down-regulation and apparently the full complement of PAR, could be recovered in the absence of protein synthesis. We also have utilized tunicamycin, which inhibits dolichol phosphate-mediated protein glycosylation, in a similar set of experimental protocols (Doss et al., 1982). Like cycloheximide, tunicamycin added to control cultures completely inhibits the appearance of PAR; however, it does not completely block cell proliferation. In fact, cells continue to grow at about half the normal rate. Recovery of down-regulated PAR in the presence of tunicamycin occurs more rapidly than in the presence of cycloheximide but to about the same extent, i.e., to the level present in the cultures at the time of first exposure to isoproterenol. The most obvious interpretation of the recovery experiments carried out with preconfluent cultures is that essentially all of the lost receptors are recovered even in the presence of agents that completely block PAR synthesis. Thus, PAR, does not represent a receptor modified in its primary amino acid sequence and probably it is not modified in terms of its core glycosylation. An unexpected result was obtained when down-regulated receptors were allowed to recover in postconfluent cultures; namely, recovery was completely blocked by cycloheximide (Fig. 8B). This result suggested that new receptors were synthesized during recovery in postconfluent cells. However, in contrast to cycloheximide, tunicamycin had little or no effect on either the rate or extent of receptor recovery. We attempted to resolve the dichotomy of the contrasting effects of tunicamycin and cycloheximide by a method capable of a direct identification of newly synthesized proteins. Postconfluent cultures were treated with isoproterenol for 12 hours, then allowed to recover in the presence of heavy isotope 2H,15N,13C-containingamino acids. Any newly synthesized PAR would necessarily be of increased mass and could be identified by their behavior during
LIGAND-INDUCED CHANGES IN O-RECEPTOR FUNCTION
103
centrifugation in sucrose-D,O gradients (see Fambrough, 1979, and Reed and Lane, 1980, for similar application of this technique to other receptor systems). The receptors that recovered under such conditions, however, migrated on the gradients like native, 1H-,14N-,12C-containingPAR. Taken together the results suggest that whereas the recovery of PAR in postconfluent cells does not require synthesis of the receptor per se, other proteins involved in the recovery process must be synthesized during the recovery period for recovery of PAR to occur. If the turnover of such proteins was greater in post- than in preconfluent cultures it would provide an explanation for the differential effects of cycloheximide (compare Fig. 8A and B). It is of some interest that 1321N1 cells seem not to turn over PAR at any appreciable rate. Inhibition of new PAR synthesis with tunicamycin or cycloheximide does not result in the degradation of previously synthesized receptors. Even when the number of PAR per cell is declining as in postconfluent cultures, the total number of PAR per culture remains constant. The cells even preserve PAR upon down-regulation; as noted above, PAR recover to the level present in the cell at the time of initiation of desensitization. It is not clear what purpose is served by the stringent maintenance of cellular PAR once they are synthesized. In contrast, insulin receptors and nicotinic cholinergic receptors turnover with a /, 7 -9 (Reed and Lane, 1980) and 17-21 hours (Fambrough, 1979), 1 cspectively . The results presented above also suggest that PAR are not continuously synthesized during long-term exposure to isoproterenol; at least the number of PAR, that can be recovered is related directly to the number present upon exposure to isoproterenol (see Fig. 8). This relationship was examined (Doss, unpublished observations) quantitatively by exposing cells in preconfluent culture to isoproterenol for increasing periods of times (12, 24, 36, and 48 hours). In each case the number of PAR that reappeared upon removal of isoproterenol was the same. If PAR synthesis had continued at the same rate as in control cells, and if these receptors had been shuttled into the form PAR,, approximately three times as many receptors should have been recovered after 24 hours of exposure than after 12. Thus the desensitization process appears to inhibit new PAR synthesis as well as initiate functional loss of PAR.
X.
ISOPROTERENOL-INDUCEDCHANGES IN AGONIST BINDING PROPERTIES OF INTACT 1321N1 CELLS
The formation during the early stage of desensitization of a PAR variant (PAR,,) with lowered affinity for agonists is a change that theoretically should be demonstrable in whole cells. The problem with such an experiment is that the typical binding reaction requires 30-60 minutes to come to equilibrium at 37°C
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JOHN P. PERKINS
and the desensitization reaction has a t,,, for induction of about 2-3 minutes. Thus we were required to design binding protocols that would allow determination of changes in the affinity of agonists for the PAR of intact cells during the first few minutes of exposure to an agonist. An accurate equilibrium dissociation constant for a competing agonist can be obtained in nonequilibrium binding assays if initial velocity conditions are met for the radioligand binding reaction, and if the competing agonist is at equilibrium with the receptor throughout the time of radioligand binding. Using '251-Pin we have been able to carry out experiments to determine the initial binding parameters for the interaction of isoproterenol and epinephrine with PAR on intact cells (Toews et a l . , 1982). The binding competition is conducted over short times (10-60 seconds) at low 1251Pin concentrations. Using this protocol it can be demonstrated that isoproterenol binds to naive I32 IN 1 cells with a Kd of 0.1 pM; however, after exposure of the cells to isoproterenol the Kd of binding of isoproterenol shifts from 0.1 to 20-40 IJ.n with a tIlz for the agonist-induced change of 1-2 minutes. Upon removal of isoproterenol the Kd returns to about 0.1 pM with a c , , ~of 6-8 minutes. The shift is not induced by antagonists and the Kd values for antagonists are not changed by exposure of the cells to isoproterenol. Thus the rates of change in PAR function during the onset and reversal of desensitization are similar when assessed using intact cells or membrane fractions. The proportion of PAR in lowand high-affinity states cannot be accurately assessed from the data of such binding experiments; however, it would appear that a 20-minute exposure of cells to receptor-saturating concentrations (>1 pM) of isoproterenol results in the conversion of about 70% of PAR to the low-affinity state.
XI.
CONCLUSIONS
Exposure of intact 1321N 1 cells to isoproterenol not only causes the activation of adenylate cyclase and the accumulation of cyclic AMP but sets in motion a complex series of events that results in the down-regulation of responsiveness if exposure to the catecholamine is extended in time. Three general categories of down-regulating responses have been identified: ( I ) a rapid uncoupling of the PAR-adenylate cyclase system with subsequent loss of PAR; (2) a slower, nonspecific desensitization of adenylate cyclase to the effects of all classes of receptor agonists by a process that is mediated by cyclic AMP; and (3) a slow induction of phosphodiesterase activity mediated by cyclic AMP. The overall process of agonist-induced desensitization to the further effects of agonists is probably the summation over time of these three processes. The receptor-specific process of desensitization is currently best described
105
LIGAND-INDUCEDCHANGES IN p-RECEPTOR FUNCTION
kinetically by the following set of reactions: PARN
(1)
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(3)
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+IS0
+ IS0
-IS0
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Reaction ( 1 ) results in a reversible modification of native receptors that prevents their interaction with the nucleotide binding protein of the adenylate cyclase system; this results in an “uncoupling” of the effects of receptor binding from activation of adenylate cyclase. Reaction (2) involves conversion of the receptor to a form that behaves as if the PAR or its environment had been physically altered. We have speculated that PAR,, are formed by a process of endocytosis (see Fig. 9). The reactions to this point in the sequence are rapidly and completely reversible. Reaction (3) represents a slowly reversible further change in the properties of the receptor. PAR, no longer bind to antagonist ligands; however, it probably is not a degraded protein since full recovery of PAR, to PAR, occurs even in the presence of cycloheximide or tunicamycin, each of which blocks new synthesis of PAR,. It is possible that the receptor is recycled through the Golgi apparatus and, after refurbishing, returned to the plasma membrane along the normal pathway of receptor insertion. This idea and the details of the sequence of events depicted in Fig. 9 are speculative, but clearly our results to date are compatible with such a model, which is similar to the models proposed for the downregulation of polypeptide hormone receptors (Pastan and Willingham, 198 1) and the internalization and recycling of LDL-receptor proteins (Goldstein et al., 1979).
FIG.9. Agonist-induced receptor internalizationas a model for catecholamine-induced desensitization. The model depicts the binding, uncoupling, endocytosis, and receptor loss steps of catecholamine-induced down-regulation in graphic terms commonly used to illustrate down-regulation of polypeptide hormone cell surface receptors.
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REFERENCES Aarons, R. D.. Nies. A. S.. Gal, J.. Hegstrand, L. R.. and Molinoff, P. B. (1980). Elcvation of P-adrenergic receptor density in human lymphocytes after propranolol administration. J. Clin. Invest. 65, 949-957. Aurbach, G . D., Fedak, S. A., Woodard, C. J . , Palmer, I . S., Hauser, D., and Troxler, T. (1974). The beta-adrenergic receptor: Stereospecific interaction of iodinated beta-blocking agent with a high affinity site. Science 186, 1223-1224. Barovsky. K., and Brooker, G. (1980). 12slodopindolol. a ncw highly selective radioiodinated P-adrenergic receptor antagonist: Measurement of P-receptors on intact rat astrocytoma cells. J . Cyclic Nuclentide Res. 6 , 297-307. Chuang. D. M.. and Costa, E. (1979). Evidence for internalization of the recognition site of P-adrenergic receptors during receptor sub-sensitivity induced by (-)-isoproterenol. Proc. Nail. Acad. Sci. U.S.A. 76. 3024-3028. Clark, R. B., and Butcher, R. W . (1979). Desensitization of adenylate cyclase in cultured fibroblasts with prostaglandin El and epinephrine. J. B i d . Chem. 254, 9373-9378. Clark, R. B., and Perkins, J . P. (1971). Regulation of adenosine 3’:s’-cyclic monophosphate concentrations in cultured human astrocytoma cells by catecholamines and histamine. Proc. Nail. Acad. Sci. U.S.A. 68, 2757-2760. Clark, R . B., Gross, R., Su, Y. F., and Perkins, J. P. (1974). Regulation of adenosine 3‘5’monophosphate content in human astrocytoma cells by adenosine and the adenine nucleotides. J. Bid. Chem. 249, 5296-5303. D‘Armiento, M., Johnson, G. S., and Pastan. I. (1972). Regulation of adenosine 3’:5’-cyclic monophosphate phosphodiesterase activity in fibroblasts by intracellular concentrations of cyclic adenosine monophosphate. P roc. Nail. Arad. Sci. U.S.A. 69, 459-62. Doss. R. C., Perkins. J. P.. and Harden, T. K. ( 1981). Recovery of P-adrenergic receptors following long temi exposure of astrocytoma cells to catecholamine. J. B i d . Chem. 256, 12281- 12286. Doss. R. C., Harden, T. K.. and Perkins, J. P. (1982). Role of protein glycosylation in the synthetic processing of P-adrenergic receptors (PAR). Fed. Proc. Fed. Am. Soc. Exp. Eiol. 41, 7392 abs. a new Engel, G., Hoyer, D., Berthold, R., and Wagner, H. (1981). ( 2 ) I~~lodocyanopindolol, ligand for P-adrenoceptors: Identification and quantitation of subclasses of P-adrenoceptors in guinea pig. Nauyn-Schmied. Arch. Pharmacoi. 317, 277-285. Fambrough, D. M. (1979). Control of acetylcholine receptors in skeletal muscle. Phvsiol. Rev. 59, 165-227. Fraser, J . , Nadeau, J., Robertson, D., and Wood, A. J. J. (1981). Regulation of human leukocyte beta receptors by endogenous catecholamines. J. Clin. Invest. 67, 1777-1 784. Goldstein, J. L., Anderson, G. W., and Brown, M. S. (1979). Coated pits, coated vesicles. and receptor-mediated endocytosis. Nature (London) 279, 679-685. Green, D. A,, and Clark, R. B. (1981). Adenylate cyclase coupling proteins are not essential for agonist-specific desensitization of lymphoma cells. J. B i d , Chem. 256, 2105-2108. Gross, R. A., and Clark, R. B. (1977). Regulation of adenosine 3’5’-monophosphate content in human astrocytoma cells by isoproterenol and carbachol. Mof. Pharmacol. 13, 242-250. Harden, T. K., Foster, S. J., and Perkins, J. P. (1979a). Differential expression of components of the adenylate cyclase system during growth of astrocytoma cells in culture. J. Eiol. Chem. 254, 4416-4422. Harden, T. K., Su Y-F, and Perkins, J. P. (l979b). Catecholamine-induced desensitization involves an uncoupling of baa-adrenergic receptors and adenylate cyclase. J. Cvclic Nucleotide Res. 5 , 99- 106. Harden, T. K., Cotton, C. U.,Waldo, G. L., Lutton, J. K.. and Perkins, J. P. (1980). Cate-
LIGAND-INDUCED CHANGES IN P-RECEPTOR FUNCTION
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cholamine-induced alteration in the sedimentation behavior of membrane-bound P-adrenergic receptors. Science 210, 44 1-443. Innis, R. B.. Cornea, F. M. A.. and Snyder, S. (1979). Carazolol, an extremely potent p-adrenergic blocker: Binding to P-receptors in brain membranes. Life Sci. 24, 2255-2264. Iyengar, R., Bhat, M. K . , Riser, M. E., and Bimbaumer. L. (1981). Receptor-specific desensitization of the S49 lymphoma cell adenylyl cyclase. J . B i d . Chem. 256, 4810-4815. Johnson, G. L. Wolfe, B. B., Harden, T. K.. Molinoff, P. B., and Perkins, J. P. ( 1978). Role of padrenergic receptors in catecholamine-induced desensitization of adenylate cyclase in human astrocytoma cells. J. B i d . Chem. 253, 1472- 1480. Lefkowitz. R . J., Wessels, M. R., and Stadel. J. M. (1980). Hormones, receptors, and cyclic AMP: Their role in target cell refractoriness. Current TO\>. Cell. Regul. 17, 205-230. Leichtling. B. J . , Drotar, A. M., Ortmann, R.. and Perkins, J. P. (1976). Growth of astrocytoma cells in the presence of prostaglandin E l : Effect on the regulation of cyclic AMP metabolism. J . Cvclic, Nideotide Res. 2, 89-98. Linibird. L. E. (1981). Activation and attenuation of adenylate cyclase: The role of GTP-binding proteins as macromolecular messengers in receptor-cyclase coupling. Biochcm. J . 195, I- 13. Lutton, J. K.. Frederich, R. C., and Perkins, J. P. (1979). Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A. J. B i d . Chem. 254, I 1181-11184. Maclntyre, E. H.. Ponten, J.. and Vatter, A. E. (1972). The ultrastructure of human and murine astrocytes and of human fibroblasts in culture. Actu Pathol. Microbiol. Scand. 80, 267-283. Mangdniek. V . . and Vaughan. M. ( 1972). Prostaglandin El effects on adenosine 3':5'-cyclic monophosphate concentration and phosphodiesterase activity in fibroblasts. Proc. Nail. Acad. Sci. U . S . A . 69, 269-273. Minneman, K . P . . Pittman. R. N., and Molinoff, R. B. (1981). p-Adrenergic receptor subtypes: Properties. distribution and regulation. Annic. Rei.. Neurosci. 4, 419-462. Nickols, 0. A , . and Brooker, G. (1979). Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin. J . Cyclic Nurleotide Rrs. 5 , 435-447. Ortniann. R.. and Perkins. J. P. ( 1977). Stimulation of adenosine 3'3-monophosphate formation by prostaglandins in human astrocytoma cells. J . B i d . Chcm. 252, 6018-6025. Pastan, I . H., and Willingham, M. C. (1981). Receptor-mediated endocytosis of hormones in cultured cells. Annu. Rev. Phvsiol. 43, 239-250. Perkins, J. P.. Maclntyre, E. H., Riley. W. D., and Clark, R. B. (1971). Adenylate cyclase, phosphodiesterase. and cyclic AMP dependent protein kinase of malignant glial cells in culture. Life Sci. 90 (Part l ) , 1069-1080. Perkins, J . P.. Su Y.-F., and Harden, T . K. (1979). Adaptive changes in the responsiveness of hol 4, 279-294. adenylate cyclase to catecholamines. Drug A l ~ ~ ~Depend. Perkins. J . P., Harden, T. K . , and Harper, J . F. (19x2). Acute and chronic modulation of the responsiveness of receptor-associated adenylate cyclases. Hundh. Exp. Pharmacol. 58, 185-224. Ponten. J . . and Maclntyre, E. H. (1968). Long term culture of normal and neoplastic human glia. A m Pathol. Microbiol. 74, 465-486. Reed, B. C.. and Lane, M. D. (1980). Insulin receptor synthesis and turnover in differentiating 3T3LI preadipocytes. Proc. Nail. Acud. Sci. U.S.A. 77, 285-289. Ross. E. M.. and Gilman, A. G. (1980). Biochemical properties of hormone-sensitive adenylate cyclase. Annri. Rev. Biochem. 49, 533-564. Ross. E. M.. Howlett, A. C . , Ferguson. K. M., and Gilman, A. G . (1978). Reconstitution of hormone-benbitive adenylate cyclase activity with resolved components of the enzyme. J . B i d . Chcm. 253, 6401-6412.
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Shear, M.. Insel, P. A., Melmon, K. L., and Coffino, P. (1976). Agonist-specific refractoriness induced by isoproterenol. J . B i d . Chem. 251, 7572-7576. Staehelin, M.. and Simons, P. (1982). Rapid and reversible disappearance of P-adrenergic cell surface receptors. EMBO J . I, 187-190. Sternweis. P. C.. and Gilman, A. G. ( 1979). Reconstitution of catecholamine-sensitive adenylate cyclase. J . B i d . Chem. 254, 3333-3340. Su. Y .-F., Cubeddu-Ximenez, L., Perkins, J . P. (1976a). Regulation of adenosine 3’:5’-monophosphate content of human asrrocytoma cells: Desensitization to catecholamines and prostaglandins. J . Cvclic. Nucleoride Res. 2, 257-270. Su. Y.-F., Johnson, G. L., Cubeddu-Ximenez. L.. Leichtling, B. H., Ortmann, R.. and Perkins, I. P. ( 1976b). Regulation of adenosine 3’5’-monophosphatecontent of human astrocytoma cells: Mechanism of agonist-specific desensitization. J . C.vclic Nucleotide Res. 2, 271-285. Su, Y.-F., Harden, T. K . , and Perkins, J . P. (1980). Catecholamine-specific desensitization of adenylate cyclase: Evidence for a multistep process. J . Biol. Chem. 255, 7410-4719. Terasaki. W. L.. Brooker, G . , de Vellis, J . , Inglish, D., Hsu. C.-Y.. and Moylan. R. D. (1978). Involvement of cyclic AMP and protein synthesis in catecholamine refractoriness. Adv. Cyclic Nuclcotide Res. 9 , 33-52. Toews, M. L., Harden, T. K . , and Perkins. J . P. (1982). Detection of high-affinity agonist binding to P-adrenergic receptors on intact cells. Fed. Proc. Fed. Am. Soc. Exp. Eiol. 41, 7393 (Abstr.). Wessels. M. R., Mullikin, D., and Lefkowitz, R . J. (1978). Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization. J . Biol. Chrm. 253, 3371-3373. Wessels, M. R., Mullikin, D., and Lefkowitz, R. J . (1979). Selective alteration in high affinity agonist binding: A mechanism of beta-adrenergic receptor desensitization. Mol. Pharmucol. 16, 10-20. Williams, L. T., and Lefkowtiz, R. J . (1978). “Receptor Binding Studies in Adrenegic Pharmacology.” Raven, New York.
CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 18
Hormone-Sensitive Adenylate Cyclase: Identity, Function, and Regulation of the Protein Components ELLIOTT M . ROSS," STEEN E . PEDERSEN,".' AND VINCENT A . FLORIO*,i *Department of Pharmacology University c$ Texas Health Science Center at Dallas Dallas. Texas and Depurtments of' tBiochemistr?, and $Pharmacology University of Virginia Charlottesville. Virginia
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Protein Components of Hormone-Sensitive Adenylate Cyclase A. Identities of the Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Resolution of the Catalytic and Regulatory Proteins of Adenyla C. Catalytic Component of Adenylate Cyclase: C . . . . . . . . . . . . . . D. The Stimulatory GTP-Binding Regulatory Protein: G/F. . . . . . . . . . . . . . . . . . . . . E. Cell Surface Receptors That Stimulate Adenylate Cyclase . . . . . . . . . . . . . . . . . . 111. Protein-Protein Interactions and the Regulat lase.. . . . . . . . . . . . A. General Considerations. . . . . . . . . . . . . ..... ..... B. Activation of C by GI F . . . . . . . . . . . . . . . . . . . . . . . . . . ................ C. Regulation of the Activation of GIF by Receptor and Hormone. . . . . . . . . . . . . . IV. Asscssment of Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
109
11.
1.
121 I25 127 127 128 131 I37 137
OVERVIEW
It is now clear that the hormone-sensitive adenylate cyclase system in the plasma membrane of target cells consists of at least three distinct protein species. There may be as many as seven. This complexity is magnified when multiple receptors for different hormones, both inhibitory and stimulatory, exist. Our 109 Copyright 0 1983 by Academic Press. Inc All righis of reproduclion in any [om reserved ISBN 0-12-153318-2
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ELLIOTT M. ROSS ET AL.
laboratory has decided to approach the study of this complex network by trying first to identify and separate these individual protein components, to study them in isolation using increasingly pure preparations, and then to study their interactions and regulation after suitable reconstitution. This is in marked contrast to the approach of studying adenylate cyclase in intact plasma membranes and inferring mechanistic information from the modulation of that activity by regulatory ligands. In this article we will discuss results both from our own laboratory and from others, but it will stress our basic analytical approach to a multienzyme regulatory complex. We will implicitly assume that all vertebrate, membranebound, hormone-sensitive adenylate cyclases are qualitatively similar in their regulation and composition. It follows that observed differences among different cells will merely reflect differences in the concentration of each protein or quantitative differences in their kinetic or equilibrium constants rather than qualitative differences in mechanism. As adenylate cyclase components prepared from different cells are shown to be capable of interacting with each other, this assumption is increasingly supported.
II. THE PROTEIN COMPONENTS OF HORMONE-SENSITIVE ADENYLATE CYCLASE A. Identities of the Proteins Much of the recent research activity in the field of adenylate cyclase has involved the enumeration and identification of the proteins that compose the system. From the early 1970s, most investigators have at least tacitly assumed that the protein that catalyzes the adenylate cyclase reaction on the inner face of the plasma membrane is distinct from the receptor that binds hormone on the cell surface. This assumption rested primarily on kinetic and developmental studies (reviewed by Perkins, 1973) and gained increasing support from chemical and genetic approaches to the resolution of receptor and enzyme (reviewed by Maguire et al., 1977; Ross and Gilman, 1980). In 1977, both Limbird and Lefkowitz (1977) and Haga et al. (1977a) chromatographically separated (3-adrenergic receptors from adenylate cyclase after detergent solubilization of plasma membranes. The receptors were measured by assaying the binding of appropriately specific radioactive ligands , and adenylate cyclase was assayed according to its enzymatic activity. This experiment has now been duplicated in several laboratories using receptors for various hormones. It is thus clear that the population of cell surface receptors that activate adenylate cyclase constitutes a large family of proteins that, on a simple level, interact with adenylate cyclase in a common fashion.
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111
Although hormonal stimulation of activity is lost upon the disruption of the plasma membranes, the adenylate cyclase activity that is solubilized by detergents usually retains its characteristic responsiveness to guanine nucleotides and to fluoride. Ross and Gilman (1977b; also Ross et af., 1978) and Pfeuffer (1977) showed that the activity that is stimulated by fluoride or by analogs of GTP reflects the interaction of two separate proteins (see below). The isolated catalytic protein, referred to as C, is insensitive to these compounds. Its activity is stimulated by these ligands only in the presence of a regulatory GTP-binding protein, referred to as GIF (and also as G, N , etc.). These three proteins-receptor, catalyst (C), and regulatory protein (G/F)constitute the essential hormone-sensitive adenylate cyclase. A fourth protein, calmodulin (calcium-dependent regulatory protein), has been shown to mediate the Ca2 -dependent stimulation of adenylate cyclase in membranes of brain and of C6 glioma cells (Cheung et af., 1978; Wolff and Brostrom, 1979). The mechanism of calmodulin’s effects on adenylate cyclase, and even its site of action, is still in dispute and will not be discussed here (see Toscano et al., 1979; Salter et af., 1981; Sano and Drummond, 1981). In addition, Schleifer et al. (1982) have suggested that yet another protein may interact with G/F. A physiological function for this protein, which so far is assayable only by its effects on the interaction of G/F with cholera toxin, is unknown. Hormonal inhibition of adenylate cyclase further suggests the presence of one or two other proteins that are involved with regulation of adenylate cyclase. It has been known for 20 years that a number of hormones inhibit the enzymatic activity of adenylate cyclase (Murad et al., 1962), and each presumably has a unique receptor in the plasma membrane of target cells. Since the receptormediated inhibitory effects also depend upon the presence of GTP, Rodbell and his colleagues suggested that there might be a distinct, inhibitory GTP-binding protein associated with this process (see Cooper, this volume). The groups of Limbird and Ui have tentatively supported this idea (Limbird et af., 1981; Limbird, 1981; Hazeki and Ui, 1981; Katada and Ui, 1981, 1982). Of the seven proteins mentioned above, only one, G/F, has been purified in useful quantity such that functional studies may be carried out (Sternweis et af., 1981). Other components may remain to be identified. Thus, one should consider hormone-sensitive adenylate cyclase to represent almost a rnultienzyme, information-transducing organelle, in the same category as the enzymes of oxidative phosphorylation. It cannot be likened to the simpler nicotinic cholinergic receptor that has been studied in such detail. This article will concentrate on the structures, activities, and interactions of C, GIF, and the stimulatory receptors that compose one segment of this organelle. The other proteins referred to above are discussed more fully in the works cited. +
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ELLIOTT M. ROSS ET AL.
B. Resolution of the Catalytic and Regulatory Proteins of Adenylate Cyclase The complex kinetic behavior of hormone-sensitive adenylate cyclase led Rodbell (1972) and others to propose the existence of a separate protein that acts to couple hormone receptors to adenylate cyclase. Since the site at which fluoride and guanine nucleotides stimulate the activity of adenylate cyclase seemed to be more closely linked to the enzyme than were hormone receptors, it was also suggested that these ligands might act upon such a regulatory protein or coupling factor. The existence of a coupling factor was also supported by the isolation of a variant S49 lymphoma cell in which adenylate cyclase activity and the ligandbinding activity of the receptor were both essentially at normal levels, although hormone binding no longer stimulated adenylate cyclase activity (Haga et ul., 1977b). These variant cells, referred to as UNC (for uncoupled), seemed to be defective in such a coupling factor. Direct evidence for the existence of a distinct regulatory protein in the adenylate cyclase system came from the work of Pfeuffer (1977) and Ross and Gilman (1977b). Pfeuffer found that chromatography of a detergent extract of pigeon erythrocyte plasma membranes on GTP-agarose decreased its responsiveness either to fluoride or to Gpp(NH)p.' If the GTP-agarose was washed with GTP or Gpp(NH)p, a factor was eluted that partially restored these responses. While neither the resolution of the two fractions nor the reconstitution of stimulation was quantitative, these experiments were the first to argue strongly for physically dissociable regulatory and catalytic components of adenylate cyclase. Ross and Gilman (1977b) demonstrated the presence of these two proteins by taking advantage both of complementary cell lines that were deficient in one or the other protein and of the proteins' differential stabilities to denaturation. These authors originally attempted to reconstitute hormonal stimulation of adenylate cyclase activity by reincorporating detergent-solubilized enzyme into membranes that already contained hormone receptors. They showed that a detergent extract of plasma membranes that contained adenylate cyclase activity could, under appropriate conditions, recombine with membranes of a phenotypically adenylate cyclase-deficient S49 lymphoma cell (denoted cyc - ) to yield hormonesensitive activity (Fig. 1) (Ross and Gilman, 1977a). The cyc- variant cells retain P-adrenergic receptors (Insel et al., 1976) but lack adenylate cyclase activity that is assayable in the presence of Mg2+ and ATP alone (Fig. 1). These experiments suggested that the mechanism of the reconstitution might be the interaction of solubilized enzyme with hormone receptors in or on the cyc-
'Abbreviations used: Gpp(NH)p, guanyl-5'-yl irnidodiphosphate; GTPyS, guanosine-5'-(3-thiotriphosphate); GDPpS, guanosine-5'-(2-thiodiphosphate).
113
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
400-
300-
[AC- Membranes]
(mglrnl)
FIG. I . Reconstitution of hormone-sensitive adenylate cyclase in membranes of cyc- S49 lymphoma cells. Adenylate cyclase activity was solubilized from plasma membranes of 882 fibroblasts using the detergent Lubrol 12A9. These cells have no P-adrenergic receptors. A fixed amount of the detergent extract was diluted into increasingly concentrated suspensions of cyc - plasma membranes. The membranes contain P-adrenergic receptors but essentially no adenylate cyclase activity when assayed in the presence of Mg* and the absence of Mn2 . Adenylate cyclase activity in the reconstituted mixture was assayed in the presence of Mg2+ plus the activators shown. Also shown is the stimulation of activity by isoproterenol relative to the GTP basal (dashed line). In retrospect, the increased activity stimulated by NaF or Gpp(NH)p probably reflects the contribution of extra C by the cyc membranes. The decline in activity with higher concentrations of cyc- membranes is probably caused by the addition of insufficient detergent to promote reconstitution. AC- is the original name of the cyc- variant. (From Ross and Gilman, 1977a.) +
+
~
membranes. However, thermal denaturation of the soluble enzymatic activity at 30°C led to only slightly decreased levels of activity in the reconstituted mixture. Thus, a heat-inactivated detergent extract from originally active plasma membrane could combine with the inactive cyc- S49 membranes to yield relatively high levels of adenylate cyclase activity that could be stimulated by fluoride, Gpp(NH)p, or hormone (Fig. 2) (Ross and Gilman, 1977b; Ross et a l ., 1978). Similarly the heated extract could reconstitute soluble fluoride- or Gpp(NH)pstimulatable activity upon combination with a detergent extract of cyc - membranes, These authors argued that the cyc - membanes (or extracts therefrom) were supplying a heat-labile factor intrinsic to adenylate cyclase that was destroyed during the heating of the complementary extract. The heated extract was hypothesized to provide a second, more stable component in which the cyc-
114
ELLIOTT M. ROSS ET AL. 100
Donor a1
5
IS
10
20
25
30'C
30
Time (min)
FIG. 2. Selective thermal denaturation of the catalytic component of adenylate cyclase. Adenylate cyclase activity was solubilized from plasma membranes of wild-type S49 lymphoma cells using Lubrol 12A9, and the extract was heated at 3OoC for various times. Activity in the extract was assayed after dilution in detergent-free buffer, and the decay of activity with time is shown by the dashed lines [upper line, Gpp(NH)p-stimulated; lower line, NaF-stimulated]. Aliquots of extract were also mixed with cyc- plasma membranes and the mixtures were assayed for activity (solid lines). Assays were performed in the presence of the stimulating ligands shown in the figure. Note that after all assayable activity in the extract is lost (20-30 minutes of heating), its ability to reconstitute activity in the cyc- membranes is decreased only slight if at all. INE, (-) Isoproterenol (isopropylnorepinephrine). (From Ross er a/., 1978.)
cells were deficient. Sensitivity to proteases, heat, and sulfhydryl reagents suggested that both factors were proteins (Ross and Gilman, 1977b). In addition to establishing the existence of the two proteins, this work also provided novel, reconstitutive assays for their activities, thereby allowing their continued study and fractionation. Studies with the crude preparations described above led these authors to propose that the more thermostable protein, which was missing in cyc- cells, served a regulatory function and mediated the effects of guanine nucleotides, since its activity was stabilized by either GTP or Gpp(NH)p and it bound to Pfeuffer's GTP affinity matrix. The identification of the more labile protein in cyc- membranes as the catalyst was facilitated by the finding that these membranes contain a Mn2 -stimulated adenylate cyclase activity which had not previously been observed. This Mn2 -stimulated enzyme displayed hydrodynamic properties similar to the protein that was active in reconstitution with the thermostable factor. The reconstitutive and catalytic activities +
+
115
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
also displayed similar thermostabilities that could be varied in parallel by ATP, Mn’ , or MgZ+ . These studies, taken together, demonstrated that the catalytic protein, C, and the regulatory protein are indeed separate. They also showed that guanine nucleotides and fluoride (hence the name “G/F”), as well as hormone receptors, exerted their stimulatory effects on the regulatory protein rather than directly upon C. G/F was further implicated as a true coupling factor between catalyst and receptor by the observation that it is also responsible for mediating the negatively cooperative binding interaction between hormones and guanine nucleotides. The affinity of agonist, but not antagonist, ligands of adenylate cyclase-linked receptors is usually decreased in the presence of those purine nucleotides that permit hormonal activation of adenylate cyclase (see, for example, Ross et al., 1977; or Ross and Gilman, 1980). Ross et al. (1977) noted that receptors from cell lines that had unaltered G/F showed this interaction, but that loss or chemical modification of G/F destroyed the effect of nucleotides on the affinity of hormone binding. Sternweis and Gilman (1979) confirmed the role of G/F in this effect by showing that reconstitution of cyc- or UNC plasma membranes with G/F restored the negative heterotropic interaction of hormone and nucleotide. +
C. Catalytic Protein of Adenylate Cyclase: C 1. PREPARATIONS A N D PROPERTIES Physical and enzymological studies of C have been limited by the difficulty of preparing C that is free from G/F and that still retains reasonable stability. Most of the initial observations related to C came from studies of plasma membranes from cyc- S49 lymphoma cells or of crude detergent extracts therefrom. Hydrodynamic studies suggested that the molecular weight of C from cyc- cells is about 1.9 X los and that it binds a significant amount of detergent (Ross et a!., 1978). The latter property is suggestive of a large hydrophobic surface area that is characteristic of intrinsic membrane proteins (Clarke, 1975). These early results are roughly consistent with more recent data on cholate-solubilized preparations (Strittmatter and Neer, 1980; Ross, 1981), but the tendency of C to aggregate, even in the presence of detergent, requires more detailed study using the purified protein. Preliminary characterization of Lubrol-solubilized C from cyc- cells also showed that C is an extremely heat-labile protein, and that it is stabilized somewhat against denaturation by Mn2 plus ATP (Ross and Gilman, 1977b). It is also stabilized by high ionic strength (Ross, 1981). phosphatidylcholine (Strittmatter and Neer, 1980; Ross, 1981, 1982), and forskolin (E. M. Ross, unpublished). C contains at least two sulfhydryl residues that are sensitive to N-ethylmaleimide, the more reactive of which is required for interaction with G/F but not for catalytic activity (Ross et a / . , 1978). +
116
ELLIOTT M. ROSS ET AL.
fE I
10
15
20
Elution Volume (ml)
FIG. 3. Separation of C and G/F by gel filtration of a cholate extract of hepatic plasma membranes. C was assayed either in the presence of Mn2+ ( 0 )or of added G/F, M g z + , and GTPyS (A), GIF was assayed according to its ability to mediate the activation of added C by GTPyS (0). The separation was carried out in buffer containing 0.5 M (NH&S04 and 12.5 mM cholate. In more recent preparations of C, the activity recovered has been increased up to fourfold. (From Ross, 1981.)
More recently, other preparations in which active C has been resolved from G/F have become available. Strittmatter and Neer (1980) and Ross (1981) demonstrated that C could be resolved by gel filtration in cholate solution if manipulations were carried out at high ionic strength (Fig. 3). Hepatic C, prepared in this way, displayed properties essentially identical to those of the enzyme from cyc- S49 cells. It was judged to be free of contamination by G/F according to (1) a high ratio of Mn2+-stimulated activity to basal activity in the presence of Mg2+, (2) lack of stimulation by either GTPyS or fluoride, both of which responses could be restored by the addition of pure G/F, (3) undetectable levels of G/F activity as assayed by the stimulation of a large excess of added C (see below), and (4) the absence of measurable amounts of the 45,000-dalton substrate for cholera toxin (Table I). Little progress has been made toward purification of C, primarily because it appears to be quite unstable in detergent solution. The t,,* for denaturation of soluble, resolved C is 24 hours at best. Storm's group has reported a specific activity of 15 nmoles min- I mg- I for a partially purified fraction from cerebral cortex (Westcott et al., 1979), but this value is at best about 0.01 of that which might be expected if the enzyme were homogeneous. The purification of C is clearly necessary for the study of its regulation, and the development of strategies to deal with its apparent lability deserves a high priority.
117
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
TABLE 1 ADENYLATE CYCLASEACTIVITIES OF C PREPARATION FROM RABBITLIVEROR CYC- S49 LYMPHOMA CELLSO Specific activity (nmole min- I mg-
1)
C
Additions None
Plus GIF
Plus cyc- membranes
Assay conditions MnCI, MgC12 MgC12. NaF MgC12, GTPyS MgCl2 MgC12, NaF MgC12. GTPyS MgC12, NaF MgC12, CTPyS
Rabbit liver
cyc- S49 cells
61
174 12 9 7 12 I39 256
8 7 7 7 57 151 4 8 ~
Liver (C
58
I1 89 276 10 86 25 1 757 1335
3 N.D.“ ~~
+ CIF)
~~
Adenylate cyclase activities in preparations of C from rabbit liver or from cyc- S49 cells, both added as cholate solutions. are compared with those of cholate-solubilized. unfractionated hepatic plasma membranes. Activities were assayed either in the absence of additions, after addition of a saturating amount of hepatic GIF, or after addition o f a large excess of cyc- plasma membranes. The latter assay gives an estimate of total GIF activity. Data are from Ross (1981). N.D., Not determined. ‘I
2. REGULATION
C was originally described as being totally inactive in the presence of Mg2+ and active only in the presence of Mn2+ or of G/F. It is now clear that the catalytic activity of C is exquisitely regulated by at least four ligands and that the response to these ligands is modulated by the hydrophobic environment in which C is located. While G/F is presumably the physiologically most important ligand, C is also stimulated by Mn2+ and by forskolin (7P-acetoxy-8,13-epoxyla,6P,9a-trihydroxylabd-14-en-I I-one) (Seamon and Daly, 1981). Neer and Salter (1981) and our laboratory (Florio and Ross, 1982a,b) have also shown that adenosine acts directly upon C at the so-called P site to inhibit activity. The interaction of C with G/F will be dealt with in Section III,B and regulation by small molecules will be considered here. Divalent manganese was first shown to stirnulate C in plasma membranes of cyc- S49 cells. Relative to an extremely low “basal” activity measured in the presence of Mg2+, Mn2+ could stimulate activity up to 20-fold. However, the extent of stimulation by Mn2 was variable, particularly in native membranes. +
118
ELLIOTT M. ROSS ET AL.
The source of this variability has not been defined, but data obtained using soluble preparations are much more uniform. In a Lubrol 12A9 extract of cycplasma membranes, free Mn2+ in the 0.5-2 mM range reproducibly stimulates activity 15- to 20-fold (Ross et al., 1978). A 7- to 10-fold stimulation by Mn2 is more typical of resolved hepatic C (Ross, 1981), in agreement with data obtained for cerebral cortical C (Strittmatter and Neer, 1980). Careful study of the stimulation of isolated C by Mn2+ has not been undertaken. However, it appears that free Mn2 is the activating ligand as opposed to MnATP acting as a preferred substrate. Stimulation is maximal at calculated concentrations of free Mn2+ of 0.5-2 mM over a wide range of ATP concentrations (20-2000 and the addition of 10 mM MgCI, or 1 mM EDTA does not alter activity significantly (V. A. Florio and E. M. Ross, unpublished data). Neer (1979) suggested further that Mn2+ is also necessary for the stimulation of C by G/F, citing the ability of Mn2+ to reverse the blockade of G/F-mediated stimulation that is caused by EGTA. It will be difficult to substantiate this claim using currently available preparations. Possibly, a second low-molecular-weight activator of C has recently been identified by Seamon and Daly (1981). Forskolin, a substituted diterpene extracted from Coleus forskohlii, had been identified originally according to its cardiovascular regulatory activities (Lindner et d . , 1978). Seamon et d. (1981) demonstrated that forskolin in the 1-100 pkif range was active in elevating adenylate cyclase activities in numerous tissues, and Seamon and Daly (198 1) found that forskolin also activated adenylate cyclase in plasma membranes of cyc- S49 cells. Since this preparation lacks G/F, these authors suggested that forskolin directly activates C. We have recently shown that forskolin also stimulates resolved rabbit hepatic C (Ross, 1982). Thus, while none of these test systems is pure, it is likely that forskolin is a ligand of C or of a closely associated protein. In our hands, forskolin reversibly stimulates the activity of C up to &fold in the presence of either Mn2 or Mg2 . Quantitation of stimulation is difficult, however. Forskolin is not soluble in water at concentrations greater than about 0.2 mM, and it may be difficult to demonstrate maximal activation due to this limited solubility. The potency of forskolin is also hard to quantitate, since forskolin presumably can partition into the lipid phase of membranes or into detergent micelles. Regardless, forskolin frequently activates adenylate cyclase in membranes more than any other agent. Given these experimental qualifications, forskolin also seems to activate C with greater potency in the presence of G/F, suggesting that forskolin and activated G/F bind with positive cooperativity to independent sites. Forskolin is slightly less potent in activating adenylate cyclase in cyc- S49 plasma membranes as compared to wild type, and the addition of GTPyS-activated G/F to resolved hepatic C increases forskolinstimulated activity and decreases the concentration of forskolin that gives halfmaximal activation (E. M. Ross, unpublished). +
+
w),
+
+
119
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
Negative regulation by adenosine and by its ribose-modified analogs has also been observed in all preparations of C studied so far. Such inhibition was noted previously in plasma membranes from numerous tissues. Since this inhibition is specific for analogs with an unmodified purine moiety, this site has been called the P site to distinguish this function from the inhibition and stimulation of adenylate cyclase caused by ribose-specific analogs that act on cell surface receptors (R sites) (Londos and Wolff, 1977; Londos et al., 1979). Wolff et al. (1978) proposed that the P site might exist on C because P site-mediated inhibition was retained after solubilization of membranes by detergent and because it did not require guanine nucleotides. This idea was supported by Premont et al. ( 1979), who observed P site-mediated inhibition after functionally uncoupling C and GIF using cholate. We have confirmed that the P site resides on C or on a closely associated protein by demonstrating inhibition of activity by 2' ,5'-dideoxyadenosine (DDA), a P site-specific compound using either plasma membranes of cyc- S49 cells or our preparation of resolved hepatic C (Florio and Ross, 1982a,b). Adenosine also has no direct effect on the rate of activation or deactivation of GIF, although it may promote the binding of activated G/F to C. The inhibition of C by P site agents is complex. Fractional inhibition of activity of resolved C is greatly increased in the presence of activators. Thus, DDA can decrease the activity of C nearly to basal values whether activity is initially stimulated 7- to 10-fold by M n * + , 30- to 50-fold by forskolin plus M g 2 + , or over 100-fold by either activated G/F or by forskolin plus Mn2+
"
10-8
10-6
10-2
[2',5'-Dideoxyadenosine] (M)
FIG. 4. Inhibition of C in cyc- plasma membranes by 2'5'-dideoxyadenosine (DDA). Adenylate cyclase activity in plasma membranes of cyc - S49 lymphoma cells was assayed in the presence of increasing amounts of DDA. Reaction volumes contained 25 mM MgC12 (a),2.5 mM MnCI2 (0). or MnClz plus 0.3 mM forskolin (A).
I
4 1.0
i1:: 0.8
Q6
0
Y
'3%
0
1u3
[Forskolin] (M) FIG.5 . Forskolin makes DDA a more potent inhibitor of C. The concentration dependence upon DDA as an inhibitor of Mn2+-stimulated adenylate cyclasc activity in plasma membranes of cyccells was determined at different concentrations of forskolin. Two measures of potency are shown: the concentration of DDA at which total activity is decreased by 20% (0)and the concentration of For DDA at which inhibition is 50% of the maximum inhibition observed with 3 mM DDA (0). comparison, the stimulation of adenylate cyclase activity by forskolin under similar conditions is also Note that forskolin by itself causes no increase in activity at concentrations below lo-" shown M .A similar disparity exists for GTPyS-activated GIF between the concentration needed to stimulate the activity of C and the concentration needed to potentiate inhibition by DDA.
(A).
C'A
--
KIO
CoA
FIG. 6. A minimal three state model for the action of P site inhibitors. Activating ligand (L) is assumed to bind preferentially to an active state of the enzyme (C*). Analogs of adenosine (A), in the presence of L. bind preferentially to the third, inhibited state (Co). The activities of the basal state (C) and inhibited state (C") are assumed to be zero. Note that the preferential binding of P site agents to basal C will yield apparent competitive inhibition with respect to L. Cooperative binding of L and A to the Co state is required to simulate experimental data. This model and the derivable constants for DDA and for Mn2+, forskolin, or GTPyS-activated G/F are described in more detail elsewhere (Florio and Ross, 1982b).
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
121
(Fig. 4). Inhibition of basal activity (i.e., Mg2+ alone) is rarely greater than 30%. This pattern would be consistent with a simple, two-state allosteric model in which activators bind preferentially to an active state of C and DDA binds to an inactive basal state, except that this simple scheme would predict an apparent competition between activator and inhibitor. As shown in Fig. 4, DDA inhibits at lower concentrations in the presence of activators, and the increase in apparent affinity for DDA at the P site is directly related to the maximum efficacy of the activator. By this we mean that DDA is most potent in the presence of activated G/F or forskolin (K& = 0.5-2 pM) and least potent at inhibiting basal activity (I& > 5 mM). The effect of activating ligands of C on its apparent affinity for DDA is reciprocal, as would be predicted thermodynamically. Thus, while forskolin stimulates the activity of C half-maximally at about 50 pk! (Seamon and Daly, 1981; Florio and Ross, 1982a,b), it potentiates the inhibitory activity of DDA half-maximally at less than 1000-fold lower concentrations (Fig. 5). We have noted a similar, quantitatively reciprocal interaction of DDA with activated G/F, and Mn2+ appears to display a similar effect. Unless a previously undetected protein that mediates the response of one ligand or the other exists, these data minimally demand a three-state model for C (Fig. 6), in which a nonactivated (basal) state, an active state, and an inhibited state coexist. P site agents bind with low affinity to the basal or active state and most tightly to the inhibited state. Activators alone bind preferentially to the active state. To fit our data quantitatively, it must be assumed further that activator and P site agent bind positively cooperatively to the inhibited state. If the shifts in apparent affinity shown in Figs. 5 and 6 represent true changes in K,s for the ligands, then the free energy of coupling for this interaction is a strikingly large number (AGA.L = -6 kcal) (Weber, 1975). It should be stressed that this formalism, although adequate, does not demonstrate that a simple three-state, cooperative model is correct. The large, negative AGA,L almost indicates that the mechanism must be more complex. Fortunately, the observed interactions should allow us to develop ligandbinding assays to explore the mechanism in greater detail. The major conclusion is that, regardless of detail, the entity (or entities) that we now refer to as C is a highly regulated and complex enzyme rather than a mere mirror of the activation of G/F.
D. The Stirnulatory GTP-Binding Regulatory Protein: G/F G/F is by far the best studied and understood protein of the adenylate cyclase system, largely because of technical reasons. It was noted soon after its discovery that it was the most stable protein (Ross, et al., 1978) and that it behaved as a monodisperse species in solutions of several detergents (Howlett and Gilman,
122
ELLIOlT M. ROSS ET AL.
1980; Kaslow et al., 1980; Sternweis et al., 1981). It can be [3ZP]ADP-ribosylated on one of its subunits, facilitating its identification in crude mixtures (Gill and Meren, 1978; Cassel and Pfeuffer, 1978). It can also be identified by its binding of a 32P-labeled photoaffinity analog of GTP and by its binding to a GTP-agarose affinity matrix (Pfeuffer, 1977). These properties motivated the initial purification of G/F from rabbit liver by Northup er al. (1980). This preparative procedure has been markedly improved and applied to G/F from turkey and human erythrocytes (Sternweis et al.. 1981; Hanski et al., 1981). A more detailed description of G/F and its properties is available in these references and in a discussion by Smigel et al. (1982). Purified preparations of G/F contain two or three polypeptides of molecular weights 35,000, 42,000-45,000, and 52,000, as determined by electrophoresis of the dodecyl sulfate-denatured protein (Northup et al., 1980). The purified native protein behaves hydrodynamically as a particle with a molecular weight of about 80,000 (Sternweis et al., 1981; Hanski et af.,1981). The discrepancy between the native molecular weight and the sum of the molecular weights of the three subunits, combined with other data, suggests strongly that a molecule of G/F is a dimer composed of one 35,000-dalton subunit and one subunit of either 45,000 or 52,000 daltons (Sternweis et al., 1980). It is likely that the 45,000dalton protein is a proteolytic product of the 52,000-dalton protein, since the two are similar functionally (see below) and yield similar peptide maps (Hudson and Johnson, 1980). The 52,000-dalton form is entirely lacking in avian erythrocytes (Gill and Meren, 1978; Cassel and Pfeuffer, 1978; Pfeuffer, 1977; Hanski et al., 1981), and Larner and Ross (1981) have shown that it is preferentially lost as rat erythrocytes mature from the reticulocyte stage. Purified hepatic G/F has been shown to bind at least 1 mole/mole of [35S]GTPySwith a concentration dependence similar to that observed for activation, and other nucleotides compete with [3sSS]GTPySfor binding to this site with appropriate potency (Smigel et al.. 1982). It is uncertain whether binding sites of lower affinity exist, but it is likely that this high-affinity site resides on the 52,000 (45,000) -dalton polypeptide. This conclusion derives from the labeling of this protein with [32P]GTP-y-azidoanilide(Pfeuffer, 1977). This polypeptide also contains the site at which G/F is specifically ADP-ribosylated by cholera toxin, thus suggesting that it also contains the active site for the hydrolysis of GTP (Cassel and Selinger, 1977b). All data so far available suggest that this larger polypeptide is the proximal activator of C. The key question in the study of G/F and its function is the process whereby it is activated. Unliganded G/F does not appreciably alter the activity of C. It is its activation, either by fluoride or a nonhydrolyzable analog of GTP (or, transiently, by GTP), that converts G/F into a potent stimulator of the adenylate cyclase activity of C. Howlett et al. (1979) first showed that G/F, in the absence of C, could be stably activated by fluoride or Gpp(NH)p in detergent solution.
123
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
Activated G/F was assayed subsequently according to its ability to stimulate C’s catalytic activity. Activation of G/F, i.e., its conversion to a form which can stimulate C , is not synonymous or necessarily coincident with binding of the activating ligand. Activation and deactivation of G/F are both first-order processes, and their rates are dependent upon both the concentration of ligand and the concentration of the 35,000-dalton subunit (Smigel et af., 1982). Activation by nucleotide is also dependent on rather high concentrations of Mgz+ (Sternweis er al., 1981; Hanski et al., 1981), and activation by fluoride has recently been shown to be dependent on the presence of A13 or Be2 (Sternweis and Gilman, 1982). The accurate measurement of activation and inactivation is complex, and the molecular mechanism whereby it is regulated is still speculative. Interested readers are directed to an excellent discussion of the problem by Smigel and co-workers ( I 982). G/F is best assayed reconstitutively by virtue of its ability, when activated by fluoride or a guanine nucleotide, to stimulate the catalytic activity of C (Ross and Gilman, 1977b; Ross et al., 1978; Sternweis et af., 1981). The sample to be assayed is mixed in an appropriate medium with an excess of C and the mixture is allowed to anneal in the presence of a stirnulatory ligand of G/F (fluoride or GTPyS). Adenylate cyclase activity is then measured in the presence of Mg2+ (Northup et af.. 1980; Sternweis et af., 1981). This assay is sensitive, easy, and reliable. It is strictly quantitative, however, only when certain conditions are met (Sternweis er al., 1981). First, the amount of C used in the assay must truly be saturating with respect to the amount of G/F that is to be assayed (Fig. 7), reflecting the bimolecular nature of the G/F-C interaction. It is clearly not sufficient to use an amount of G/F small enough so that reconstituted activity is linear with added G/F (Sternweis er al., 1981; Lamer and Ross, 1981). The second major problem encountered in assaying G/F is that detergents used to solubilize G/F, primarily cholate, can inhibit or denature the C that is used in the assay, necessitating the dilution of G/F into a standard medium prior to assay. These two limitations have detracted from the quantitative accuracy and reproducibility of several published measurements of G/F, including the early work of Ross et al. (1978) (see Lamer and Ross, 1981, for more discussion). G/F has been recognized recently as one of the prominent loci at which variation in the regulation of adenylate cyclase systems among different cells is expressed. Kaslow et al. (1979) first suggested that differences in the quantitative responses to hormones and guanine nucleotides that exist between adenylate cyclases from different tissues might be due to differences in the G/F protein. These authors compared G/F from wild-type S49 lymphoma cells with G/F from turkey erythrocytes. The former is a typical mammalian system. The latter is relatively unresponsive either to hormone in the presence of GTP [as compared with Gpp(NH)p or GTPyS] or to Gpp(NH)p alone in the absence of hormone. Allowing for inefficient reconstitution and low activities, these properties were +
+
124
ELLlOlT M. ROSS ET AL.
FIG.7. Assay of G/F in rat erythrocyte plasma membranes. G/F activity was solubilized from rat erythrocyte membranes using cholate at high ionic strength. Aliquots of extract, at varying concentrations of protein, were mixed with aliquots of cyc- plasma membranes (as the source of C). Several different concentrations of cyc- membrane were used, and contributed to each assay the amount of protein shown at the right. The reconstituted mixtures were then activated with GTPyS, and adenylate cyclase was assayed in the presence of MgC12. Note that activity is a relatively linear function of the amount of G/F added to each assay, even at low concentrations of C. Only with greater amounts of C. however, is the measured specific activity of GIF (i.e., the slope of the line) both maximal and constant. (From Larner and Ross, 1981.)
retained when G/F from each of the two different cell types was reconstituted into plasma membranes from cyc- S49 cells. This work has now been confirmed with purified G/F (Hanski et al., 1981). These data argue that G/F itself determines qualitative aspects of the regulation of adenylate cyclase in plasma membranes. This finding is perhaps more interesting when combined with the observation that G/F from avian erythrocytes lacks the 52,000-dalton form of the GTP-binding subunit of G/F-only the 45,000-dalton form is detectable. When Sternweis et al. (1981) compared fractions of rabbit hepatic G/F that were enriched in either one form or the other, they found that the fraction rich in the 45,000-dalton form behaved similarly to turkey erythrocyte GIF with respect to kinetics of activation and Mg2+ dependence. Lamer and Ross (1981) made a similar finding in a study of the maturing rat reticulocyte. They found that the loss of the 52,000-dalton subunit of G/F during maturation correlated well with the increased preference for GTPyS over GTP as cofactor for hormonal stimulation of adenylate cyclase. Changes in Mg2+ dependence for the activation of G/F were also noted by these authors. These observations in several different systems all lead to the speculation that the proteolysis of the 52,000-dalton subunit of G/F to the 45,000-dalton form leads to qualitative changes in the
PROTEIN COMPONENTS OF ADENYLATE CYCIASE
125
regulation of adenylate cyclase. It will be fascinating to determine if this proteolysis is regulated by endocrine or developmental factors. A second modification of G/F which may be of importance in the physiological regulation of adenylate cyclase is suggested by the phenotype of another variant S49 lymphoma cell. Haga et al. (1977b) described a stable variant clone in which hormones (P-adrenergic amines or prostaglandin E,) failed to stimulate adenylate cyclase activity. These cells retain adenylate cyclase activity, which can be stimulated by fluoride or Gpp(NH)p, and also retain P-adrenergic recepThe lesion tors, as measured by the binding of [ ‘251]iodohydroxybenzylpindolol. appears as an uncoupling of receptors from enzyme, leading to the name “UNC,” for uncoupled. The UNC lesion and the cyc- lesion are not complementary with regard to hormonal stimulation of the enzyme, as assayed either by reconstitution protocols (Ross and Gilman, 1977a; Sternweis and Gilman, 1979; Schwarzmeier and Gilman, 1977) or in somatic cell hybrids (Naya-Vigne et al., 1978). It can be inferred, therefore, either that cyc- cells are deficient both in G/F and a putative “UNC factor” or that G/F in UNC cells is somehow defective. Sternweis and Gilman (1979) argued for the presence of an altered GIF in UNC plasma membranes by demonstrating that a crude preparation of G/F from wild-type S49 cells or rabbit liver can restore hormonal responsiveness to UNC plasma membranes. This ability to reconstitute hormone responses in UNC membranes cofractionates several thousand-fold with G/F, When UNC G/F is [”PJADP-ribosylated using cholera toxin, it is also observed that the 45,000dalton subunit of G/F is shifted to a more acidic isoelectric point (Schleifer e t a l . , 1980). The UNC lesion also abolishes control by guanine nucleotides of the affinity of hormone binding to receptors (Haga et al., 1977b), and this loss is reversed by reconstitution with crude G/F (Sternweis and Gilman, 1979). These results, taken together, provide a strong argument that the UNC lesion represents a modification (or lack of required modification) of the G/F protein such that it can no longer fulfill its role as a coupling factor between receptor and C. Since several physiological states have been described in which G/F appears to be uncoupled (see Ross and Gilman, 1980), it is tempting to speculate that the UNC lesion represents the uncontrolled expression of a normal physiological regulatory function. Understanding the chemical nature of the lesion should allow us to explore the basis of this regulation.
E. Cell Surface Receptors That Stimulate Adenylate Cyclase The ligand-binding properties of a wide variety of hormone receptors that act via adenylate cyclase have been studied in the membranes of target cells by the use of appropriate radioactive ligands; a rather large number of receptors have
126
ELLIOlT M. ROSS ET AL.
also been characterized after detergent solubilization. (see Ross and Gilman, 1980, for a brief review). Several receptors have also been affinity labeled, allowing the identification of their subunits by dodecyl sulfate-polyacrylamide gel electrophoresis (Johnson et al., 1981; Ji and Ji, 1980; Rebois et al., 1981; Atlas and Levitzki, 1978; Rashidbaigi and Ruoho, 1981; Lavin et al., 1981), and the P-adrenergic receptor has been substantially purified (Vauquelin et al., 1979; Durieu-Trautmann et al., 1980; Shorr et al., 1981). Ligand-binding characteristics of soluble receptors are generally unchanged from those of the membranebound protein, although some hormone-binding activities are altered by the receptor's environment. In the case of the glucagon receptor, it has not been possible to bind hormone to the solubilized protein, although a receptor-ligand complex has been solubilized from hepatic membranes that were first incubated with [ 1251]iodoglucagon(Welton et al., 1977). This may reflect a stabilizing effect of ligand upon the receptor, implying a relative instability of the unliganded, detergent-solubilized protein. This effect has been noted to a lesser extent with other receptors. Similarly, alkyl polyethylene oxide detergents (Lubrol, Brij) have permitted only solubilization of preformed complexes of (3-adrenergic receptors and their radioactive ligands (Haga et al., I977a), although Caron and Lefkowitz (1976) showed that digitonin could be used to solubilize P-adrenergic adaptors in a form that could bind the antagonist ligand [3H]dihydroalprenoloI (DHA). They further demonstrated the binding of unlabeled agonist and antagonist ligands to soluble receptors by using competitive binding assays that employed [3H]DHA. Similar results have been reported briefly by Witkin and Harden (198 I ) , who used [ 12sI]iodopindolol, also an antagonist, as their probe. Another specific P-adrenergic antagonist ligand, [ '251]iodohydroxybenzylpindolol (IHYP), seems to be most sensitive to small effects of membrane environment on receptor structure. It has not been possible to detect binding to digitonin-solubilized receptors using this ligand-binding activity is lost upon the addition of detergent (Haga et al., 1977a; Fleming and Ross, 1980; Witkin and Harden, 1981). Fleming and Ross (1980) demonstrated that the ability of digitonin-solubilized P-adrenergic receptors to bind [ 1251]IHYP can be restored by their reconstitution into phospholipid vesicles. Starting with a mixture of soluble receptors and dimyristoylphosphatidylcholine,they used gel filtration (and, originally, sucrose density gradient centrifugation), to remove digitonin and to incorporate receptors into unilamellar vesicles of 500- 1000 A diameter. The reconstitution had little effect on the number of receptors that could be assayed using ["IDHA or on the K , for this ligand but was efficient in restoring the binding of [ 1251]IHYPto expected levels. Since this reconstitution of [1251]IHYP-bindingactivity utilizes receptors that were solubilized in digitonin, it should be equally applicable to receptors that are purified in that detergent by the techniques cited above. The procedure has now been applied to receptors derived from rat and turkey erythrocytes and from murine S49 lympho-
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
127
ma cells. [12sI]IHYPbinding can be similarly restored to P-adrenergic receptor that have been solubilized with deoxycholate by removing detergent and incorporating the receptors into phosphatidylcholine vesicles (Citri and Schramm, 1980; Pedersen and Ross, 1982a,b). In the case of deoxycholate, the solubilized receptors were unable to bind either [3H]DHA or ['251]IHYP, and reconstitution restored the ability to bind both ligands (Pedersen, and Ross, 1982b). The molecular events that cause the reversible inhibition of IHYP binding by digitonin and the inhibition of either IHYP or DHA binding by deoxycholate are unknown. However, these effects may suggest that the hydrophobic environment of the receptor is important in determining its ability to bind P-adrenergic ligands, presumably because of effects on its conformation. The distinct behavior of IHYP and DHA with digitonin-solubilized receptors suggests that detergents do not merely occlude a binding site. This role of lipids on a receptor's ligand-binding activity is apparently distinct from the requirements for a phospholipid membrane for the interaction of receptor with GIF. However, both of these effects may indicate an exquisite sensitivity of the conformation of hormone receptors to their surroundings in the plasma membrane bilayer.
111.
PROTEIN-PROTEIN INTERACTIONS AND THE REGULATION OFADENYLATECYCLASE
A. General Considerations The hormonal regulation of adenylate cyclase activity can be thought of most simply as a sequence of two distinct regulatory interactions between two different pairs of proteins. First, receptor binds hormone, undergoes some conformational change, and acts allosterically on G/F to promote the activation of G/F by guanine nucleotide. Second, activated G/F binds to C and stimulates its catalytic activity. Since this scheme involves two small and two macromolecular allostenc ligands, it is clearly not all that simple. There is also good reason to believe that, in native membranes, several of these reactions may be concerted or that stable assemblies of these proteins may exist (see Tolkovsky et al., 1982; Braun and Levitzki, 1979; Tolkovsky, this volume; and references therein for some of the best arguments in this direction). Such complexes may be further stabilized when inserted correctly in a membrane bilayer or they may be stabilized by cytoskeletal elements (see, for example, Rudolph et af., 1977; Insel and Kennedy, 1978; Rasenick et a!. , 1981 ; Sahyoun et al., 1977, I98 1). Regardless, considering the total system as the sum of two ligand-mediated protein-protein interactions has the virtue of relative conceptual simplicity and seems to be a valid experimental approach. Each interaction has been studied separately. The interaction of receptors with G/F has been studied by measuring
128
ELLIOlT M. ROSS ET AL.
the stimulatory effects of hormone binding on the rate of activation of G/F by guanine nucleotides in reconstituted systems (Citri and Schramm, 1980; Pedersen and Ross, 1982b) as well as in membranes of HC-I hepatoma cells, which are essentially deficient in C (E. M. Ross, unpublished; see also Ross et al., 1978). The negatively cooperative binding interactions of guanine nucleotides and hormones, which also reflect the coupling of receptors to G/F, are also observable in HC-1 membranes or in wild-type S49 cell membranes in which C has been chemically or thermally denatured (Ross et al., 1978). Preliminary data from our laboratory indicate that they may also be observed in phospholipid vesicles that contain G/F and P-adrenergic receptors. The stimulation of C by activated G/F has been studied in relatively well resolved systems (see, for example, Sternweis et a!., 1981; Smigel e t a l . , 1982; Ross, 1981, 1982). Reconstituted systems containing only two interacting proteins have the advantage that one can independently manipulate the relative concentrations of each protein in order to estimate their affinities for each other, their rates of interaction and dissociation, and the modulation of these rates by regulatory ligands and by the lipid composition and structure of the membrane. The discussion below will concentrate primarily on work that has utilized this approach.
B. Activation of C by G/F Until 1977, C and G/F together were generally considered to compose a single enzyme, adenylate cyclase, that was variably stimulated in different plasma membranes by Mg2 , Mn2 , F- , and guanine nucleotides. In many cases, this assumption is at least practically useful, in that the binding of C and G/F can be quite stable, such that they apparently do not dissociate over several days. Hydrodynamic studies of the detergent-solubilized C-G/F complex, usually stabilized by an activating ligand such as Gpp(NH)p, have yielded profiles of monodisperse species with properties consistent with a molecular weight of about 2 X lo5 (see Ross and Gilman, 1980, for a review). Tolkovsky et al. (1982) and Tolkovsky and Levitzki (1981) proposed that a stable C-G/F complex, rather than free G/F, is the principal species that is activated by hormone receptors in native membranes. They found that in turkey erythrocytes, the formation of the C-G/F complex was not rate limiting for the hormonal activation of adenylate cyclase and concluded that the C-GIF complex was therefore relatively stable prior to activation. This conclusion may not be general, however. In plasma membranes from S49 lymphoma cells, for example, there appears to be a molar concentration of G/F greater than or equal to the concentration of C plus P-adrenergic receptors. Thus, when adenylate cyclase activity is maximally stimulated by Gpp(NH)p, there is still enough G/F to interact reversibly with receptors (Ross et ai., 1977). This is also true in hepatic plasma membranes and +
+
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
129
detergent extracts therefrom (Welton et al., 1977; Lad et al., 1977). If a C-GF complex does exist, it must be relatively transient and allow for the fairly rapid exchange of G/F and C molecules, since excess exogenous G/F molecules added to a native membrane have been shown to interact with endogenous C. Thus, G/F from wild-type 549 lymphoma cells can, when added to plasma membranes from UNC cells, compete for C with endogenous UNC G/F; added ADP-ribosyl-G/F can compete with endogenous unmodified G/F (Sternweis et al., 1979); and G/F that has been preactivated with GTPyS competes with native G/F in membranes to which it has been added (Larner and Ross, 1981). These studies argue for the potential of rapid and regulated exchange of C and G/F molecules in membrane, but it is difficult to assess how rapidly exchange occurs in an unperturbed membrane.. It is also frustrating that it has been impossible to count molecules of C in a membrane, so that there is no real way to determine the molar stoichiometric relationship of C and G/F. Such measurements will probably demand reconstitution of purified preparations of each protein in a suitable medium. In crude reconstituted systems and in detergent solution, it has been somewhat easier to deal with the interaction of C and G/F in at least semiquantitative terms. Sternweis et al. (1981) showed that the formation of the activated C-G/F complex could be treated as the simple binding equilibrium, C + G/F e C-G/F. They used the activation of C to measure the amount of complex that was formed and used purified, GTPyS-activated hepatic G/F and cyc- plasma membranes as their sources of each component. The concentration of G/F was manipulated directly. The concentration of C was altered either by changing the concentration of cyc - membranes or by mixing varying proportions of native membranes and membranes in which C had been inactivated. These findings are consistent with the less extensive data of Larner and Ross (198 l), which were obtained using crude rat erythrocyte G/F. Pfeuffer (1979) used sucrose density gradient ultracentrifugation in Lubrol solution to study the interaction of Lubrol-solubilized C and G/F that had been separated by affinity chromatography. He found that in the presence of active C, G/F could be caused to sediment as a larger particle by the addition of GTPyS. Centrifugation of G/F alone, of G/F plus C but in the absence of nucleotide, or of G/F plus C in the presence of GDPpS gave similarly smaller sedimentation rates for G/F. These data were interpreted to indicate that only nucleotide-activated G/F could form a stable C-G/F complex in Lubrol solution. This conclusion is consistent with numerous observations from other laboratories on the stabilization of detergent-soluble adenylate cyclase activity by fluoride or by analogs of GTP. A major uncertainty about the interaction of membrane-bound C with added soluble G/F is the extent to which G/F can reassociate with the membrane. Ross and Gilman (1977a) initially found that Lubrol-solubilized G/F could stimulate C on cyc - membranes and mediate hormonal stimulation of adenylate cyclase
130
ELLIOlT M. ROSS ET AL.
activities under conditions where it was not stably bound to the membrane. Howlett et al. (1979) confirmed these results and showed that such a preparation of G/F became tightly associated with cyc- membranes only when it had been stably activated either by fluoride or by Gpp(NH)p or GTPyS. GTP alone or in the presence of hormone did not promote binding. Sternweis and Gilman (1978) showed that this is not the case when G/F is solubilized in cholate. Cholatesolubilized G/F bound to the membrane after dilution of detergent. Binding required warming and was promoted by the addition of GTP alone, but did not require stable activation of G/F. These data, data on the solubilization of G/F (Ross et al., 1978), and data on the protease susceptibility of G/F (Hudson et al., 1981) have suggested that G/F may interact with membranes through a small hydrophobic domain and may not be a typical, globular, integral membrane protein. As such, it may interact with receptor or C without binding to the lipid bilayer. Recent studies in our laboratory have pointed out a probable role for phospholipids in the stabilization of the C-GIF complex. Rabbit hepatic C, prepared by gel filtration in cholate solution (Ross, 1981), is essentially free of endogenous phospholipids. Using this preparation, we showed that phosphatidylcholine markedly potentiates the ability of activated G/F to stimulate the catalytic activity of C (Ross, 1982). A lipid-free mixture of C and G/F is only slightly stimulated
0
0.4
0.8
1.2
1.4
[DMPC] (mM) FIG. 8. Effect of lecithin on the interaction of C and G/F. Lipid-free preparations cf hepatic C and excess purified hepatic GIF were combined and diluted in the presence of increasing amounts of dimyristoylphosphatidylcholine (DMPC). Adenylate cyclase activity was measured either with Mn2+ (0) or with GTPyS plus Mg2+ (0). Specific activities shown are relative to the amount of protein in the preparation of C. (See Ross, 1982. for experimental details.)
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
131
by GTPyS (Fig. 8) or by fluoride, and attains a maximal activity less than that elicited by Mn2+ alone. The addition of an optimal concentration of dimyristoylphosphatidy Icholine increases G/F-mediated stimulation by fluoride or GTPyS as much as %fold, to yield the 10- to 20-fold stimulation over basal activity that is typical of native membranes. In contrast, phosphatidylcholine has little effect on activity stimulated by Mn2+ or by forskolin, neither of which acts via GIF. The small increase in Mn2 -stimulated activity that is observed is due primarily to stabilization of C by the lipid during the assay. Phosphatidylcholine also does not potentiate the initial activation of G/F by GTPyS or by fluoride. Thus, by elimination, these results are interpreted as indicating that phosphatidylcholine promotes or is required for the productive interaction of activated G/F with C. Various synthetic and natural phosphatidylcholines display this stimulatory effect, whereas detergents (cholate, digitonin, Lubrol 12A9, lysophosphatidylcholine), cholesterol, and several other phospholipids do not (Ross, 1982). The mechanism whereby phosphatidylcholine acts in this system is unclear, as is its possible physiological significance. These results do, however, point to the sensitivity of the interaction of C and G/F to the nature of their hydrophobic environment. Studies on the role of phospholipids on the interaction of C and G/F have also suggested a novel experimental system in which to pursue these effects. After the addition of phosphatidylcholine to cholate-solubilized C, cholate can be removed by dialysis and centrifugation to yield a preparation of large, unilamellar phospholipid vesicles to which C is bound. In a second stage of reconstitution, G/F can be added to these C vesicles in varying amounts to yield a vesicle-bound adenylate cyclase that can be regulated by fluoride or guanine nucleotides (Ross, 1982). The extent of stimulation by fluoride or by GTPyS is related to the amount of G/F added to the vesicles, and displays saturation with respect to G/F at appropriate concentrations. This system, in which the concentration of C and G/F and their molar ratios can be varied in a membrane of known lipid composition, promises to be useful for further mechanistic studies of the interaction of the two proteins. +
C. Regulation of the Activation of G/F by Receptor and Hormone Several basic properties of hormonal stimulation of adenylate cyclase indicate that the interaction of receptor with G/F is the key to the action of hormone in this system. First, hormonal stimulation of adenylate cyclase activity requires the presence of a guanine nucleotide, as first proposed by Rodbell and co-workers (197 I ) . If the nucleotide is GTP, hormonal activation is rapidly reversible and the steady-state level of adenylate cyclase activity reflects the number of recep-
132
ELLIOTT M. ROSS ET AL.
tors and their fractional saturation by hormone. If a p r l y hydrolyzable analog of GTP is present or if G/F has been treated with cholera toxin, activation is slowly reversible or irreversible, and the hormone acts merely to increase the rate at which enzyme is activated (see Maguire er al., 1977; or Ross and Gilman, 1980, for review). More detailed kinetic studies, primarily in the laboratories of Selinger, Levitzki, and Birnbaumer, have led to the notion that the receptor-hormone complex essentially acts catalytically to increase the rate at which adenylate cyclase, or G/F, is activated. The rate of deactivation reflects the cellular source of the G/F and the identity of the guanine nucleotide, but is generally not regulated by hormone (reviewed in Ross and Gilman, 1980; see also Tolkovsky, this volume). Thus, in the presence of GTP, the steady-state level of hormone-stimulated activity reflects the balance of a hormone-catalyzed activation rate and a tonic rate of deactivation. Under special circumstances, the liganded receptor may also catalyze deactivation (Cassel and Selinger, 1977a; Sevilla and Levitzki, 1977; Arad el a / . , 1981). but this is probably not of physiological significance. The nature of the rate-limiting step in the activation of adenylate cyclase by hormone is uncertain. The simplest model for the mechanism of hormone action holds that ( I ) G/F is a GTPase, (2) only the GTPliganded form of G/F is active, and (3) hormone merely facilitates the displacement of GDP, the GTPase product, so that a new molecule of GTP can bind (Cassel et a f . , 1977; review by Ross and Gilman, 1980). More commonly, the conformational change of GTP-liganded G/F from inactive to active states may be rate limiting. Regardless, the physiological stimulation by hormone derives from the ability of H.R to catalyze the activation. In numerous systems, it is the formation of the H - R G / F catalytic intermediate that is the key step in activation by hormone. The questions to be asked now concern the detailed mechanism of the formation of this complex and the molecular nature of the catalytic event. The productive interaction of G/F with liganded receptor and, hence, the hormonal stimulation of adenylate cyclase require that both G/F and receptor be incorporated into a suitably unperturbed lipid bilayer. Furthermore, the chemical composition of the bilayer and its physical structural organization can have profound effects upon the efficiency and extent of hormonal control (for review see Ross and Gilman, 1980; Henis et a f . , 1982; Houslay and Gordon, this volume). Studies of native biological membranes are inherently limited, however, because one cannot independently or specifically alter the concentration of individual proteins or the phospholipid composition of the bilayer. It is also difficult to identify the details of the interaction of receptor and G/F in the presence of C. We therefore have attempted to develop reconstituted experimental systems in which receptor and G/F can interact in a bilayer of known composition and structure. Two such systems have yielded results so far. As discussed above, Fleming and Ross (1980) succeeded in introducing digitonin-solubilized P-adrenergic receptors into phospholipid vesicles by a two-step
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
133
procedure involving gel filtration and sucrose density gradient centrifugation. Reconstitution into vesicles composed primarily of dimyristoylphosphatidylcholine restored both the stability and the ligand-binding capabilities that are characteristic of the native, membrane-bound receptor, and should provide a population of receptors that are capable of catalyzing the activation of G/F. A second reconstituted system has now been exploited further in our laboratory to study the receptor-G/F interaction. The procedure is an extensive modification of that developed by Citri and Schramm (1980), in which P-adrenergic receptors are first solubilized using deoxycholate, essentially according to Eimerl et al. (1980). The ability to bind either [3H]DHA or [1251]IHYPis lost upon solubilization. Addition of dimyristoylphosphatidylcholine to this extract, followed by gel filtration, yields P-adrenergic binding activity eluted in a turbid fraction at the void volume. Of the binding activity originally in the membranes, greater than 60% is recovered in these fractions, while more than 99% of the deoxycholate is removed. Receptors in this preparation bind both [ *251]IHYPand [3H]DHA with a specific activity greater than I pmole mg - I protein and display appropriate P-adrenergic specificity for unlabeled agonist and antagonist ligands in competitive binding assays. While this preparation has not yet been extensively characterized either hydrodynamically or by electron microscopy, the binding activity is in a particulate fraction containing about 3 mg phospholipid/ mg protein that has displayed, in preliminary experiments, properties expected of vesicles in the 500-2000 A size range. Purified rabbit hepatic G/F added to these P-adrenergic receptor vesicles in a second reconstitution step can be stably activated by GTPyS in a time-dependent reaction. (Activated G/F is subsequently assayed by the addition of Lubrol 12A9 and reconstitution into cyc membranes.) At relatively low concentrations of Mg2+, the rate of activation of G/F in these vesicles is low, as predicted by studies of soluble G/F (Sternweis et al. 1981). However, the addition of (-)isoproterenol stimulates the rate of activation of G/F up to 4-fold (Fig. 9). These results do not compare unfavorably with those of Citri and Schramm (1980), who reported up to 12-fold stimulation in the presence of receptor and hormone. These authors, however, used crude turkey erythrocyte G/F, which has a far lower basal rate of activation than does the rabbit hepatic protein (compare Hanski et al., 1981; and Sternweis ef al., 1981). The increase in rate caused by isoproterenol displays characteristic P-adrenergic chemical and stereochemical selectivity both for stimulation by agonists (Fig. 10) and blockade by antagonists, and is also abolished if P-adrenergic binding sites are destroyed by N,N'dicyclohexylcarbodiimide. This latter procedure does not inactivate G/F. This system thus appears to represent the restoration of the hormone-specific, catalytic interaction of liganded receptors with nucleotide-liganded G/F. Our reconstitution of P-adrenergic stimulation of the activation of G/F is still in its preliminary stages of development. First, only G/F and phospholipid are ~
134
ELLIOlT M. ROSS ET AL.
). I
I
0
2
4
6
8
1015
Time (min)
FIG. 9. Activation of GIF by GTPyS is catalyzed by P-adrenergic receptors in reconstituted phospholipid vesicles. P-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes essentially according to Eimerl et a / . ( 1980), mixed with dimyristoylphosphatidylcholine, and incorporated into phospholipid vesicles by gel filtration in the absence of detergent. Concentrated vesicles were mixed with purified rabbit hepatic G/F (Sternweis et al., 1981)such that the final concentration of Lubrol (which was added with the G/F) war about 20 pg/ml. This mixture was incubated at 3OoC in the presence of 10 @f GTPyS and I @f (-)isoproterenol ( 0 )or of GTPyS, isoproterenol, and 10 p M (-)propranolol The activation reaction was quenched at the indicated times by diluting aliquots of these mixtures with buffer containing 1 0 - 5 M (-)propranolol, 5 mM EDTA, and 0.1 mM GDPPS. The samples were then assayed for activated G/F essentially as described by Sternweis er al. (1981). Each G/F assay tube contained a volume of activation reaction mixture equivalent to 0.46 fmole of P-adrenergic receptors, as assayed using [ '2sI]IHYP. Thus, if the reconstitutive activity of activated G/F is about lo4 U/mg = 160 Ulnmole (Sternweis et ot., 1981), the hormone-catalyzed activation of 1.9 X 1 0 - 3 Uiminute (2.45 x total - 0.55 x 1 0 - 3 basal) represents a turnover rate of more than 5 moles G/F/rnole receptodminute over the first I minute of the activation reaction.
(c).
added as pure components. A purer preparation of receptors must be used in order to accommodate a wider range of receptor:G/F molar ratios in the vesicles and to provide the general security of working with pure proteins. The kinetics of the reaction must also be studied in greater detail. The activation of G/F by receptor-hormone complex is characterized by a burst that is nearly complete in 1 minute. This behavior is not understood, but does not simply reflect denaturation of G/F or receptors during the activation reaction. It is also significant that while the hormone-stimulated rate falls after the burst, it remains higher than the basal rate as both approach linearity. Hypothetically, this nonlinear time course may reflect a rate-limiting exchange of G/F among vesicles. It might also represent the reconstitution of a rapid down-regulation of receptor-G/F coupling (Su et af., 1980). It should be pointed out, however, that initial rates do indicate a catalytic event-we calculate that the initial rate of activation reflects the activation of more than five molecules of G/F per minute per receptor molecule in the experiment shown here, and higher rates have been observed. This calculation does not make allowances for the possible segregation or occlusion of some receptors, so it is probably an underestimate of the catalytic capacity of the active, vesicle-bound receptor molecules. Another estimation of the efficiency of
135
PROTEIN COMPONENTS OF ADENYLATE CYCLASE
A L
0
10-'0
10-0
10-0
10-4
ISOPROTERENOL (M)
2.5
2 .o 1.5 1.o
0.5
t
04.
I
I
I
I
B
Fic. 10. P-Adrenergic specificity of reconstituted, hormone-stimulated activation of C/F. GIF was activated in 0-adrenergic receptor-containing vesicles as described in the legend to Fig. 9, but in the presence of increasing concentrations of each stereoisomer of isoproterenol (A) or in the presence of I phf (-)isoproterenol and increasing concentrations of each stereoisomer of propranolol (B). In each figure, the open symbol is the ( + ) isomer. In (A), the activation was carried out for I minute. In (B). the activation was carried out for 5 minutes, causing the relatively high basal rate at saturating concentrations of propranolol. Also shown in (A) is the effect of added 5 phf (-)propranolol at two concentrations of (-)isoproterenol
(A).
the reconstitution is the comparison of the hormone-stimulated activation rate with the rate stimulated by high concentrations of M g 2 + . This parameter, evaluated at 1 minute (Table II), suggests a process about 30-40% efficient, but this fraction falls to 15% at longer activation times. Again, this parameter does not account for the possible spatial segregation of some GIF and receptor molecules, since all G/F is presumably accessible to Mg2 , but probably not to receptors. + +
136
ELLIOT M. ROSS ET AL.
TABLE I1 ACTIVATIONOF G/F AI-TITERRECONSTITUTION INTO PHOSPHOLIPID VESICLES CONTAINING P-ADRENERGIC RECEFTORS"
Additions
Rate of activation of G/F (units rnin-1 x 103)
None ( - )Isoproterenol ( 10- M ) ( - )Propranolol (10 - 5 M ) lsoproterenol plus propranolol M) Terbutaline Terbutaline plus propranolol Phentolamine ( 1 0 - 5 M ) Isoproterenol plus phentolamine MgClz (0.05 M )
0.7 2.9 0.7 0.8 2.1 0.7 0.8 2.7 8.7
" Preparation of vesicles and the addition of purified G/F were performed as described in the legend to Fig. 9. Activation was carried out for I minute at 30°C in the presence of the indicated compounds.
Thus, this level of efficiency is not a bad place to begin. A more fundamental test of the efficiency of receptor-G/F coupling in a reconstituted system may be the negatively cooperative binding interactions of guanine nucleotides and agonist ligands (see Maguire et a l . , 1977; or Ross and Gilman, 1980, for a review). The extent to which the addition of GIF increases the affinity with which agonists bind to the receptor and the ability of GTP to block this interaction should provide at least a thermodynamic estimate of the association of the two proteins. In preliminary experiments, we were able to observe a small but definite increase in the affinity with which isoproterenol bound to receptor vesicles when purified G/F was present. We thus foresee multiple ways in which we can study the coupling of the two proteins after reconstitution. The methods described here should ultimately allow independent and welldefined variation of lipid composition, j3-receptor concentration, and G/F concentration during reconstitution. The concentrations of receptors and of G/F can now be varied only within limited ranges. The concentration of receptors within a vesicle is constrained by currently low specific activity in our preparations. Consequently, we are attempting to apply the reconstitutive procedure to purer preparations of receptors. Manipulation of G/F is limited only by the detergent that is introduced into the system with the purified protein, a problem that may be circumvented by the use of more concentrated preparations or a detergent removal step. Those developments, along with physical studies of purified, functional j3-adrenergic receptors, should allow the detailed molecular study of the receptor-G/F interaction.
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IV. ASSESSMENT OF PROGRESS The last 5 years of research on hormone-sensitive adenylate cyclase have focused on the questions of how many proteins are involved in an observed activity or regulatory events, what the role of each protein is, and the sequence in which individual events occur. Identifying proteins has been a slow process, but is a prerequisite to obtaining the proteins in a pure form, suitable for study. We are now at the point at which the individual protein components must be analyzed in detail by essentially classic physical and enzymological approaches, and we can begin to construct reconstituted model systems in which to test our hypotheses. Many of the outstanding questions are obvious. Direct information on the conformational alteration of a receptor by a hormone requires the study of workable quantities of purified receptors. Questions of how receptors catalyze the activation of G/F demand both kinetic and structural approaches, particularly with regard to the effects of the membrane bilayer on the ability of the two proteins to interact. Whether C is one or several proteins awaits better purification, and the study of the regulation of C is virtually dependent on purer preparations. Ideally, these biochemical problems can be pursued in parallel with the questions of long-term endocrinologic regulation of these proteins and of the control and coordination of their synthesis. ACKNOWLEDGMENTS Studies from this laboratory cited here have been supported by USPHS grant GM30355. E. M. R. is an Established Investigator of the American Heart Association and V. A. F. is the recipient of a graduate fellowship from the Pharmaceutical Manufacturers‘ Association Foundation. REFERENCES Arad, H., Rimon. G . , and Levitzki, A. (1981). The reversal of the Gpp(NH)p-activated state of adenylate cyclase by GTP and hormone is by the ”collision coupling” mechanism. J. B i d . Chem. 256, 1593-1597. Atlas, D., and Levitzki, A. (1978). Tentative identification of P-adrenoceptor subunits. Nature (London) 272, 370-371. Bourne, H. R., Coffino, P., and Tomkins. G. M. (1975). Selection of a variant lymphoma cell deficient in adenylate cyclase. Science 187, 750-752. Braun, S., and Levitzki, A. (1979). Adenosine receptor permanently coupled to turkey erythrocyte adenylate cyclase. Biochemistry 18, 2134-2138. Caron, M. G., and Lefkowitz, R. J . (1976). Solubilization and characterization of the P-adrenergic receptor binding sites of frog erythrocytes. J . Biol. Chem. 251, 2374-2384. Cassel, D., and Pfeuffer, T. (1978). Mechanism of cholera toxin action: Covalent modification of the guanyl nucleotide-binding protein of adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 75, 2669- 2673. Cassel, D., and Selinger, 2. (1976). Catecholamine-stimulated GTPase activity in turkey erythrocytes. Biochrm. Biophys. Acta 452, 538-55 I . Cassel, D., and Selinger, Z. ( l977a). Catecholamine-induced release of [‘H]Gpp(NH)p from turkey erythrocyte adcnylate cyclase. J . Cvclic Nucleotide Res. 3, 11-22.
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Cassel. D., and Selinger, Z. (1977b). Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site. Proc. Narl. Acad. Sci. U.S.A. 74, 3307-33 1 1 . Cassel. D., Levkovitz. H.. and Selinger. Z . (1977). The regulatory GTPase cycle of turkey erythrocyte adenylate cyclase. J. Cyclic Nuclrotide Res. 3 , 393-406. Cheung. W. Y . . Lynch, T. J., and Wallace, R. W. (1978). An endogenous Caz+-dependent activator protein of brain adenylate cyclase and cyclic nucleotide phosphodiesterase. Adv. Cyclic Nucleotide Res. 9, 233-251. Citri. Y . , and Schramm, M. (1980). Resolution, reconstitution, and kinetics of the primary action of a hormone receptor. Nature (London) 287, 297-300. Clarke, S . (!975). The size and detergent binding of membrane proteins in detergent solution. J . Biol. Chem. 250, 5459-5469. Durieu-Trautmann. O., Delavier-Klutchko, C.. Vauquelin, G . , and Strosberg. A. D. (1980). Visualization of the turkey erythrocyte P-adrenergic receptor. J . Suprumol. Strucf. 13, 41 1-419. Eirnerl. S., Neufeld. G., Korner. M., and Schramm. M. (1980). Functional implantation of a solubilized P-adrenergic receptor in the membrane of a cell. Proc. Natl. Acad. Sci. U . S . A . 77, 760-764. Fleming, J. W., and Ross, E. M. (1980). Reconstitution of beta-adrenergic receptors into phosbinding to digitonin-solpholipid vesicles: Restoration of [ ~~~I]iodohydroxybenzylpindolol ubilized receptors. J. Cyclic Nudeotide Res. 6, 407-4 19. Florio, V. A,. and Ross, E. M. (1982a). Direct inhibition of the catalytic protein of adenylate cyclase at the adenosine P site. Fed. Proc. Fed. Am. Soc. Erp. Eiol. 41, 1408. Florio, V. A , , and Ross, E. M. (l982b). Regulation of the catalytic component of adenylate cyclase: Cooperative interaction of stimulatory ligands and ribose-modified adenosine analogs. Mol. Pharmacol., in press. Gill. D. M., and Meren, R. (1978). ADP-ribosylation of membrane proteins catalysed by cholera toxin: Basis of the activation of adenylate cyclase. Proc. Nut/. Acud. Sci. U . S . A . 75, 30503054. Haga, T., Haga, K.. and Gilman. A. G. (1977a). Hydrodynamic properties of the P-adrenergic receptor and adenylate cyclase from wild type and variant S49 lymphoma cells. J. Biol. Chem. 252, 5776-5782. Haga, T., Ross, E. M., Anderson, H. J . , and Gilman, A. G. (1977b). Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells. Proc. Natl. Acad. Sci. U . S . A . 74, 2016-2020. Hanski, E., Sternweis, P. C., Northup, J . K.. Dromerick, A. W., and Gilman, A. G. (1981). The regulatory component of adenylate cyclase. Purification and properties of the turkey erythrocyte protein. J. BkJl. Chem. 256, 1291 1-12919. Hazeki, 0.. and Ui. M. (1981). Modification by islet-activating protein of receptor-mediated regulation of cyclic AMP accumulation in isolated rat heart cells. J. Biol. Chrm. 256, 2856-2862. Henis, Y . I . , Rimon, G., and Felder, S. (1982). Lateral mobility of phospholipids in turkey erythrocytes. Implications for adenylate cyclase activation. J. B i d . Chem. 257, 1407- 141 I. Howlett, A. C., and Gilman, A. 0. (1980). Hydrodynamic properties of the regulatory component of adenylate cyclase. J. Biol. C h r m . 255, 2861-2866. Howlett, A. C., Sternweis, P. C., Macik. B. A,, Van Arsdale, P. M . , and Gilman, A. G . (1979). Reconstitution of catecholamine-sensitive adenylate cyclase. Association of a regulatory component of the enzyme with membranes containing the catalytic protein and P-adrenergic receptors. J. Biol. Chem. 254, 2287-2295. Hudson, T. H., and Johnson, G. L. (1980). Peptide mapping of adenylate cyclase regulatory proteins that are cholera toxin substrates. J. B i d . Chem. 255, 7480-7486. Hudson, T. H., Roeber, J. F.. and Johnson, G. L. (1981). Conformational changes of adenylate cyclase regulatory proteins mediated by guanine nucleotides. J . B i d . Chem. 256, 1459- 1465.
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Insel. P. A., and Kennedy, M. S. ( 1978). Colchicine potentiates P-adrenoceptor-stimulated cyclic AMP in lymphoma cells by an action distal to the receptor. Nature (London) 273, 471473. Insel, P. A.. Maguire, M. E.. Gilman, A. G., Bourne, H. R.. Coffino. P., and Melmon. K . L. ( 1976). Beta adrenergic receptors and adenylate cyclase: Products of different genes'? Mol. Pharmacol. 12. 1062- 1069. Ji. I . , and Ji. T. H. (1980). Macromolecular photoaffinity labeling of the lutropin receptor on granulosa cells. Proc. Narl. Acad. Sci. U.S.A. 77, 7167-7170. Johnson, G . L., MacAndrew, V. I.. and Pilch, P. F. (1981 1. Identification of the glucagon receptor in rat liver membranes by photoaffinity cross-linking. Pruc. Narl. Acud. Sci. U.S.A. 78, 875-878. Kaslow. H . R.. Farfel, Z . , Johnson, G. L.. and Bourne, H. R. (1979). Adenylate cyclase assenibled in virrot Cholera toxin substrates determine different patterns of regulation by isoproterenol and guanosine S'-triphosphdte. Mol. Pharmacol. 15, 472-483. Kaslow, H. R., Johnson. G . L., Brothers. V. M., and Bourne, H. R. ( 1980). A regulatory component of adenylate cyclase from human erythrocyte membranes. J . B i d . Chem. 255, 3736-3741. Katada. T.. and Ui. M. (1981). Islet activating protein. J . Biol. Chem. 256, 8310-8316. Katada. T.. and Ui, M. (1982). Direct modification of the membrane adenylate cyclase system by islet-activating protein due to ADP-rihosylation of a meinbrane protein. Proc. Nut/. Acad. Sci. U.S.A. 79, 3129-3133. Lad. P. M.. Welton. A. F.. and Rodbell, M. (1977). Evidence fordistinct guanine nucleotide sites in the regulation of the glucagon receptor and of adenylate cyclase activity. 1. Biol. Chem. 252, 5942-5946. Lamer, A. C.. and Ross, E. M. (1981). Alteration in the protein components of catecholaminesensitive adenylate cyclase during maturation of rat reticulocytes. J . Biol. Chcm. 256, 9551-9557. Lavin. T. N., Heald. S. L.. Jeffs. P. W . . Shorr, R . G . L.. Letkowitz, R. J.. and Caron, M. G . (19x1). Photoaffinity labeling of the P-adrenergic receptor. J. B i d . Chrtn. 256, 11944-1 1950. Limbird. L. E. (1981). Activation and attenuation of adenylate cyclase: GTP-binding proteins as molecular messengers in receptor-cyclase coupling. Biorhrm. J. 195, I- 13. Limbird. L. E.. and Letkowitz, R. J. (1977). Resolution of P-adrenergic receptor binding and adenylate cyclase activity by gel exclusion chromatography. J. B i d . Chem. 252, 799-801. Limbird. L. E.. McMillan, S . T.. and Smith. S. K. (1981). Solubilization of human platelet aadrenergic receptors: Evidence that agonist occupancy of the receptors stabilizes receptoreffector interactions. Proc. Narl. Acad. Sei. U.S.A. 78, 4026-4030. Lindner. E.. Dohadwalla, A. N.. and Bhdttdcharya, B. K . (1978). Positive inotropic and blood pressure lowering activity of a diterpene derivative isolated from Colrit.s,~orsX.ohlii:Forskolin. Armeim. Forsch. 28, 284-289. Londos, C.. and Wolff. J. ( 1977). Two distinct adenosine-sensitive sites on adenylate cyclase. Pro(,. Nutl. Acad. Sci. U.S.A. 74, 5482-5486. Londos. C., Wolff. J.. and Cooper, D. M. F. (1979). Action of adenosine on adenylate cyclase. In "Physiological and Regulatory Functions of Adenosine and Adenine Nucleotides" (H. P. Baer and 0.I . Drummond. eds.), pp. 271-281. Raven. New York. Maguire. M. E.. Ross, E. M . . and Cilman. A.G. (1977). 0-Adrenergic receptor: Ligand binding properties and the interaction with adenylyl cyclase. Adv. Cyclic Nircleotide Res. 8, 1-83. Murad. F.. Chi. Y . M., Rall, T. W.. and Sutherland. E. W. (1962). The effect of catecholamines and choline esters on the formation of adenosine 3',5'-phosphate by preparations from cardiac muscle and liver. J . B i d . Chem. 237, 1233-1238. Naya-Vigne. J . , Johnson, G . L., Bourne. H. R.. and Coffino. P. (1978). Complementation analysis of hormone-sensitive adenylate cyclase. Nature (London) 272, 720-722.
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Neer. E. J. (1979). Interaction of soluble brain adenylate cyclase with manganese. J . Eiol. Chem. 254, 2089-2096. Neer. E. J . , and Salter, R. S. (1981). Reconstituted adenylate cyclase from bovine brain. J . Biol. Chem. 256, 12102-12107. Northup. J . K.. Sternweis, P. C., Smigel, M. D.. Schleifer, L. S., Ross, E. M.. and Gilman, A. G . (1980). Purification of the regulatory component of adenylate cyclase. Proc. Nut/. Acud. Sci. U.S.A. 77, 6516-6520. Pedersen. S. E., and Ross. E. M. ( 1982a). Functional reconstitution of P-adrenergic receptors and the guanine nucleotide-binding regulatory protein of adenylate cyclase. Fed. Proc. Fed. Am. Sor. Exp. B i d . 41, 141I . Pedersen. S. E.. and Ross. E. M. (1982b). Functional reconstitution of 0-adrenergic receptors and the stimulatory GTP-binding protein of adenylate cyclase. Proc. Nut/. Acrid. Sci. U.S.A. In press. Perkins. J . P. (1973). Adenyl cyclase. Adv. Cyclic Nucleotide Res. 3, 1-64. Pfeuffer, T . (1977). GTP-binding proteins in membranes and the control of adenylate cyclase activity. J . Biol. C h m . 252, 7224-7234. Pfeuffer, T . ( 1979). Guanine nucleotide controlled interactions between components of adenylate cyclase. FEES Lett. 101, 85-89. Premont. J . , Cuillon. G.. and Bockaert. J . (1979). Specific Mg’+ and adenosine sites involved in a bireactant mechanism for adenylate cyclase inhibition and their probable localization on this enzyme’s catalytic component. Eiochein. Eiopphys. Res. Coinmun. 90, 5 13-5 19. Rasenick, M. M.. Stein. P. I . , and Bitensky, M. W. (1981). The regulatory subunit of adenylate cyclase interacts with cytoskeletal components. Nature (London)294, 560-562. Rashidbaigi, A.. and Ruoho, A. E. (1981). Iodoazidopindolol. a photoaffinity probe for the P-adrenergic receptor. Proc. Nut/. Acad. Sci. U.S.A. 78, 1609-1613. Rebois, R. V . , Omedeo-Sale, F.. Brady. R. 0..and Fishman. P. F. (1981). Covalent crosslinking of human chorionic gonadotropin to its receptor in rat tcstes. Proe. NmI. Acad. Sri. U.S.A. 78, 2086-2089. Rodbell. M. (1972). Regulation of glucagon action at its receptor. I n “Clucagon: Molecular Physiology, Clinical Therapeutic Implications” (P. S. Lefebvre and R. H. Unger, eds.), pp. 61-75. Pergamon, Oxford. Rodbell. M., Birnbaumer, L., Pohl, S. L., and Krans, H. M. J . (1971). The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. V. An obligatory role of guanyl nucleotides in glucagon action. J . Eiol. Chein. 246, 1877-1882. Ross, E. M. (1981). Physical separation of the catalytic and regulatory proteins of hepatic adenylate cyclase. J. Eiol. Chein. 256, 1949-1953. Ross, E. M. ( 1982). Phosphatidylcholine-promoted interaction of the catalytic and guanine nucleotide-binding proteins of adenylate cyclase. J . B i d . Chem., in press. Ross, E. M.. and Gilman. A. G. (l977a). Reconstitution of catecholamine-sensitive adenylate cyclase activity: Interaction of solubilized components with receptor-replete membranes. Proc. Nut/. Arad. Sci. U.S.A. 14, 3715-3719. Ross. E. M.. and Gilman. A. G. (1977b). Resolution of some components of adenylate cyclase necessary for catalytic activity. J . Eiol. Chem 252, 6966-6970. Ross. E. M., and Gilman. A. G. (1980). Biochemical properties of hormone-sensitive adenylate cyclase. Anriu. Rev. Biot,hem. 49, 533-564. Ross, E. M., Maguire, M. E., Sturgill, T. W . . Biltonen. R. L.. and Gilman, A. G. (1977). Relationship between the P-adrenergic receptor and adenylate cyclase. Studies of ligand binding and enzyme activity in purified membranes of S49 lymphoma cells. J . B i d . Chem. 252, 5761-5775. Ross, E. M.. Howlett, A. C . , Ferguson, K. M., and Gilman. A. G . (1978). Reconstitution of
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hormone-sensitive adenylate cyclase activity with resolved components of the enyzme. J . Biol. Chem. 253, 6401-6412. Rudolph, S . A.. Greengard, P., and Malawista, S . E. (1977). Effects of colchicine on cyclic AMP levels in human leukocytes. Proc. Natl. Acad. Sci. U . S . A . 74, 3404-3408. Sahyoun. N., Hollenberg. M. D.. Bennet. V . . and Cuatrecasas. P. (1977). Topographic separation of adenylate cyclase and hormone receptors in the plasma membrane of toad erythrocyte ghosts. Proc.. Natl. Acad. Sci. U.S.A. 74, 2860-2864. Sahyoun. N. E.. LeVine, H., 111, Hebdon, G. M., Hemadah, R., and Cuatrecasas, P. (1981). Specific binding of solubilized adenylate cyclase to the erythrocyte cytoskeleton. Proc. Nail. A m d . Sci. U . S . A . 78, 2359-2362. Salter. R. S . . Krinks. M . H.. Klee, C. B.. and Neer, E. J. (1981). Calmodulin activates the catalytic unit of brain adenylate cyclase. J. Biol. Chem. 256, 9830-9833. Sano. M . . and Drummond, G. I. (1981). Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein. J . Neurochem. 37, 558-566. Schleifer. L. S . , Garrison. J . C.. Sternweis, P. C . . Northup. J. K., and Gilman, A. G.(1980). The regulatory component of adenylate cyclase from uncoupled S49 lymphoma cells differs in charge from the wild type protein. J . Biol. Chem. 255, 2641-2644. Schleifer. L. S . . Kahn. R. A,, Hanski, E., Northup, J . K., Sternweis, P. C.. and Gilman, A. G. ( 1982). Requirements for cholera toxin-dependent ADP-ribosylation of the purified regulatory component of adenylate cyclase. J . Biol. Chem. 257, 20-23. Schwarzmeier, J. D.. and Gilman, A. G. ( 1977). Reconstitution of catecholamine-sensitive adenylate cyclase activity: Interaction of components following cell-cell and membrane-cell fusion. J . Cvclic Nideotide Res. 3, 227-238. Seamon. K., and Daly, J . W. (1981). Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J . Biol. Chem. 256, 9799-9801. Seamon. K. B., Padgett. W., and Daly, J . W. (1981). Forskolin: Unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. U.S.A. 78, 3363-3367. Sevilla. N., and Levitzki. A. (1977). The activation of adenylate cyclase by 1-epinephrine and guanylimidodiphosphate and its reversal by I-epinephrine and GTP. FEBS Lett. 76, 129- 134. Shorn. R. G . L.. Lefkowitz, R. J . , and Caron. M. G. (1981). Purification of the P-adrenergic receptor. Identification of the hormone-binding subunit. J . Biol. Chem. 256, 5820-5826. Sniigel. M. D., Northup. J . K., and Gilman, A. G . (1982). Characteristicsoftheguanine nucleotidebinding regulatory component of adenylate cyclase. Recent Prog. Horm. R e x . 38, 601-622. Skmweis, P. C.. and Gilman, A. G. ( 1979). Reconstitution of catecholamine-sensitive adenylate cyclase. Reconstitution of the uncoupled variani of the S49 lymphoma cell. J. Biol. Chem. 254, 3333-3340. Sternweis, P. C . , and Gilman, A. G . (1982). Aluminum: A requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proc. Natl. Acad. Sci. U.S.A. 79, 4888-489 I , Sternweis, P. C . . Northup. J . K . . Smigel, M. D., and Gilman, A. G . (1981). The regulatory component of adenylate cyclase. Purification and properties. J . Biol. Chem. 256, 1151711526. Strittmatter, S . , and Neer. E. J . (1980). Properties of the separated catalytic and regulatory units of brain adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 77, 6344-6348. Su. Y . - S . , Harden. T. K . , and Perkins, J . P. (1980). Catecholamine-specific desensitization of adenylate cyclase. Evidence for a multistep process. J . Biol. Chem. 255, 7410-7419. Tolkovsky. A. M . . and Levitzki, A. (1981). Theories and predictions of models describing sequential interactions between the receptor, the GTP regulatory unit. and the catalytic unit of hormone dependent adenylate cyclase. J . Cvclic Nucleotide Res. 7, 139-150.
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Tolkovsky, A. M.. Braun, S., and Levitzki, A. (1982). Kinetics of interaction between P-receptors, GTP protein. and the catalytic unit of turkey erythrocyte adenylate cyclase. Proc. Narl. Acad. Sci. U.S.A. 79, 213-217. Toscano, W. A,, Jr., Westcott, K. R., LaPorte, D. C.. and Storm, D.R. (1979). Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase. Proc. Nafl. Acad. Sci. V.S.A. 76, 5582-5586. Vauquelin, G.,Geynet, P., Hanoune, J . , and Strosberg, A. D. (1979). Affinity chromatography of the P-adrenergic receptor from turkey erythrocytes. Eur. J. Biochem. 98, 543-556. Weber, G . (1975). Energetics of ligand binding to proteins. Adv. Protein Chem. 29, 1-83. Welton, A. F., Lad, P. M., Newby, A. C., Yamamura, H., Nicosia, S., and Rodbell, M. (1977). Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states. J. Biol. Chem. 252, 5947-5950. Westcott, K. R., LaPorte, D. C., and Storm, D. R. (1979). Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-Sepharose affinity chromatography. Proc. Nafl. Acad. Sci. U.S.A. 76, 204-208. Witkin, K. M., and Harden, T. K. (1981). A sensitive equilibrium binding assay for soluble P-adrenergic receptors. J. Cyclic Nucleotide Res. 7 , 235-246. Wolff, D. J., and Brostrom, C. 0. (1979). Properties and functions of the calcium-dependent regulatory protein. Adv. Cyclic Nucleotide Res. 11, 27-88. Wolff, J., Londos, C., and Cook, G. H. (1978). Adenosine interactions with thyroid adenylate cyclase. Arch. Biochem. Eiophys. 191, 161-168.
CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I8
The Regulation of Adenylate Cyclase by Glycoprotein Hormones BRIAN A . COOKE Department of Biochemistry Roval Free Hospital School of Medicine University of’ London London. England
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nature of the Hormones., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nature of the Receptors . ................... IV. Involvement of Cyclic AMP in Hormone Action.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Important Features of the Hormone Receptor-Adenylate Cyclase System. . . . . . . . . . Effect of Guanine Nucleotides on Binding of the Hormone to Its Receptor. . . . . v1. Desensitization and Down-Regulation by Homologous Hormone . . . . . . . . . . . . . . . . . A. Desensitization and Down-Regulation of a LH-Responsive Leydig Cell Tumor . B. The Desensitizing Effect of GTP on Isolated Plasma Membranes. . . . . . . . . . . . . C. Determination of the Site of Lesion in LH-Desensitized Leydig Tumor Cells. . . D. Possible Mechanisms Involved in Desensitization . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.
11. 111.
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INTRODUCTION
Most mammalian cells controlled by hydrophilic hormones contain plasma membrane bound adenylate cyclase and specific hormone receptors. The target cells controlled by the glycoprotein hormones, lutropin (LH), follitropin (FSH), and thyrotropin (TSH), are no exception. What distinguishes these hormones from other hormones working through the same mechanism is their structure and binding characteristics of the hormone receptor interaction. They each contain two essential subunits with different but not well-defined functions. These hormones bind to their receptors in a manner which is not readily reversible and are unaffected by guanine nucleotides in terms of their binding characteristics. As 143 Copyright D 19x3 by Academic Press. Inc. All rights of reproduclion In any lbml reserved. ISBN 0-12-153318-2
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pointed out by Abramowitz et (11. (1981) this appears to make the glycoprotein hormone receptors unique, because all other receptor systems that affect adenylate cyclase activity bind to hormones in a manner that is readily reversible and are affected by guanine nucleotides; in the presence of the latter the affinity of the nonglycoprotein hormone receptor interactions is lowered by as much as 10-fold. The glycoprotein hormone-adenylate cyclase systems have, in common with other systems, a protein (or proteins) which couple their receptors to the adenylate cyclase catalytic protein. This coupling protein contains a GDP/GTP binding site. The interaction of the hormone, receptor, and guanine nucleotide binding protein (referred to as G or N or GIF protein) to the adenylate cyclase results in the formation of cyclic AMP from ATP and requires the presence of Mg2+. In addition to the stimulatory effect on cell function it has been shown that the glycoproteins desensitize their target cells, resulting in a loss of response to further stimulation. Initially this involves a decrease in adenylate cyclase activity, which is followed by loss of hormone receptors (down-regulation). The mechanisms involved have not been fully elucidated, but probably an uncoupling of the hormone receptor complex from the G-protein-adenylate cyclase occurs followed by internalization of the hormone receptor. In some systems these processes have been found to depend on protein synthesis. The purpose of this article is to highlight some of the more important aspects of the recent advances in our understanding of the above-mentioned mechanisms. Because of the current state of knowledge and the interests of this author emphasis will be placed on the mechanism of action of one of the glycoproteins-lutropin-and its action on the Leydig cell. For more detailed reviews on other aspects of the regulation of receptors and adenylate cyclase the reader is referred to Abramowitz et ul. (1979), Jacobs ( 1979), Ross and Gilman (1980), Schulster and Livitzky (l980), Limbird (1981), Spiegel e t a l . (1981), and Cooke (1982).
II. NATURE OF THE HORMONES The glycoprotein hormones follitropin (follicle-stimulating hormone, FSH), lutropin (luteinizing hormone, LH), thyrotropin (thyroid-stimulating hormone, TSH), and choriotropin (human choriogonadotropin, hCG) are structurally related. Each molecule is composed of two nonidentical polypeptide chains (a and p). The carbohydrate moiety represents 7-30% of the total molecular mass. The carbohydrate residues are linked to the polypeptide chains through N-glycosidic linkages to either the amide group of asparagine or the hydroxyl group of serine or threonine residues. Qualititative and quantitative variations occur in the carbohydrate contents of the glycoproteins from different species. Sequence analysis of the amino acid residues has shown that the a-chains of
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the glycoproteins are remarkably similar and the P-chains are quite different. Dissociation of the hormone into the a and P subunits can be achieved under nondenaturing conditions indicating that they are linked by noncovalent bonds. The dissociated units are biologically inactive. Reassociation at 4°C can be achieved with restoration of most of the original activity. It has been suggested that the p subunit of the glycoprotein hormone determines the biological specificity of the molecule, but that this can be achieved only in the presence of the (Y subunit. Specific gangliosides may be involved in the binding of the glycoproteins to their receptor sites (see review by Kohn, 1978). The role of the carbohydrate moiety in the glycoprotein hormones is not clear, although it has been shown that removal of the sialic acid residues from hCG makes this hormone more susceptible to metabolism in vivo but has little effect on its binding to testicular plasma membranes (Van Hall et a l . , 1971). In the female both FSH and LH are involved in the maturation of the ovarian follicle. FSH, together with estradiol- 17p, prepares the follicle for ovulation, and LH (and prolactin) is mainly concerned with the maintenance of the corpus luteum and steroid production (Richards, 1979). In the male, FSH specifically controls the activity of the Sertoli cells in the seminiferous tubules (see review, Dorrington and Armstrong, 1979), and LH the Leydig cells where the male androgen testosterone is formed (see review by Cooke et af., 1981b). hCG is secreted by the placenta and, unlike the other trophic hormones, is independent of the hypothalmus or pituitary gland. This hormone, together with human placental lactogen, is secreted in large amounts during the first few weeks of pregnancy and during this time may well play an important luteotrophic role. The roles of these hormones after this time, however, are still unclear. The biological properties of hCG are very similar to those of LH, but they differ in chemical structure and in their metabolic half-life (hCG has a much longer halflife than LH). Because of the stability of hCG and its similarity to LH and availability in a highly purified form, it is often used in place of LH in in vivo and in v i m studies on the testes and ovaries. TSH has been shown to be capable of stimulating almost every metabolic process in the thyroid. This includes iodide trapping, formation and release of the thyroid hormones, and general metabolic effects on glucose oxidation, ribonucleic acid synthesis, and phospholipid formation. 111.
NATURE OF THE RECEPTORS
A11 of the hormones listed in Table 1 have been shown to have plasma membrane receptors in their target cells with the characteristics necessary for fine control of their functions, i.e., high specificity, sensitivity, and affinity. The affinity constants obtained are of the order of 0.1 nM. These and other physical
TABLE I PROPERTIES OF THE GLYCOPROTEIN HORMONES
Molecular weight
Number of amino acid residues
Origin
Hormone
Molecular nature
Pituitary
Follitropin (follicle-stimulating hormone, FSH)
GI ycoprotein 2 subunits (a,P)
36,000
204 89 (a)
Lutropin (luteinizing hormone, LH)
GIycoprotein
Thyrotropin (thyroid-stimulating hormone, TSH)
Glycoprotein 2 subunits (a,p)
28,850 15,750 (a) 15.350 (P) 25,000
Choriotropin (human choriogonadotropin, hCG)
Glycoprotein 2 subunits (a,P)
Placenta
36,000 14.500 (a) 22,200 (P)
Site of action
26
Ovary (follicle), testis (Sertoli cells)
218 89 (a) 129 (P) 215
23 16 (a) 7 (P) 20
Ovary (follicle), testis (Leydig cells)
90 (a) 125 ($1
14 (a)
23 I 92 (a) 139 (p)
45
115
2 subunits (a,f3)
Carbohydrate content (res mole-1)
(P)
6
(PI
16 (a) 19 (P)
Thyroid, adipose tissue
REGULATION BY GLYCOPROTEIN HORMONES
147
properties and the data obtained on the purification of hormone receptors are summarized in Table 11. In order to characterize the hormone binding sites it is necessary to prepare radioactively labeled hormones of high specific radioactivity. For the protein hormones, usually the i2sl-labeled derivatives are used. The development of mild labeling techniques (e.g., with lactoperoxidase, Thorell and Johansson, 1971) has enabled the preparation of '2s1-labeled hormone usually without a loss in biological activity. Using these labeled hormones it has been possible to demonstrate specific binding in target issues. High-affinity capacity receptors have been reported in target cells. However, in view of the virtual irreversibility of the binding (see p. 152 for references), especially with lutropin, the theoretical assumptions on which many of the binding data are based (e.g., reversible binding, equilibrium conditions) may not be valid. Nonionic detergents have been extensively used to solubilize hormone receptors, and this has been achieved for LH/hCG (see Dufau et al., 1975, for references) and FSH (Dufau et al., 1977a). In the male, the plasma membrane receptor for LH is located in the testis Leydig cell, and in the female the ovarian granulosa cell. The LH receptor has been solubilized and purified from both the testis and ovary (Dufau and Catt, 19761, and various physicochemical parameters have been measured (see Table 11). The results obtained suggest that a common macromolecular configuration is shared by the gonadotropin receptors extracted from testis and ovary (Dufau et a / . . 1974). The LH receptor was extracted from rat testis interstitial cells by treatment with Triton X- 100. The solubilized receptors retained their hormonal specificity but the association constant at 24°C was lower (0.5-1 X loioM - '1 than that of the original particulate receptor for hCG (2.4 x I O ' O M - I). Exposure of particulate and soluble receptors to trypsin caused loss of gonadotropin binding activity, indicating the protein nature of an essential component of the receptor site. In addition, a significant role of phospholipid in the receptor was suggested by the reduced binding activity observed after treatments of particulate and soluble receptors with phospholipase A. By reference to the behaviors of standard proteins during filtration on Sepharose 6B, the hydrodynamic radius of the receptor was calculated to be 64 A. The sedimentation constant of the free receptor was 6.5 S, and that of the hormone-receptor complex was 7.5 S. Based on these physical analyses, on gel filtration and density gradient centrifugation, it was concluded that both the free and hormone-bound receptor exist in solution as elongated molecules with molecular weights of 194,000 and 224,000, respectively (Dufau et af.. 1973). The solubilized receptors were purified 15,000-fold by affinity chromatography on agarose coupled to hCG columns (Dufau et al., 1975). A water-soluble hCG binding protein has also been isolated from Leydig
TABLE I1 PROPERTIES OF HORMONE RECEFTORS Affinity consiani
Hormone Luvopin
Folh~ropin Thyroid-stimulating hormone
Tissue Ovary
Solvent used for solubilizaiion of receptor Tnton X-I00
Changes in specificity after solubilirarion
Before roiubiiirauon (M- '1
None
After solubilization (M-1) 0.5-1
Testii
Tnton X-100
None
24 x
Testis Thyroid
Tnton X-I00 Lithium diiodooxalicylate
None None
8.5 x 10'0
10'0
Y
10"'
Naiure of receptor Protein,
phospholipid 0.5-1 x 10i" Protein1 phospholipid 1.6 X lo9 Protein
Purification Estimdicd achieved molecular I +fold1 weight
250
wdimenlation constant 6.0. 6 75 Stokec radius 6 4 nm (61A,. red,mentation constant 7.55
Slokei radius 6.0 nrn (60 Al.
15.ooO
15,oOO
Other propenies
194.000
15.-
270.000
References Dufau er 01. (19741;
Haour and Saxena (19741 Duf~ucr a/ (1973) Dufau er a / . ( 1 9 7 7 ~ ) Tate cr 01. (1975a.b)
REGULATION BY GLYCOPROTEIN HORMONES
149
cell membrane fractions (Pahnke and Leidenberger, 1978). The molecular weight, sedimentation coefficients, and K, were found to be 71,500,4.35 S , and 0.75-1 .O X 1O1() M - ’ , respectively. The evidence obtained suggested that the hCG water-soluble binding protein was derived from membrane-bound LH receptors possibly cleaved by lysosomal or other proteolytic enzymes or, alternatively, they might be newly formed receptor material that had not been inserted into the membrane. An antiserum to soluble rat luteal LH receptors has been raised in rabbits (Lubrosky and Behrman, 1979). LH-dependent progesterone secretion from isolated rat ovarian cells was reduced by the antiserum, although there was no change in the LH binding, indicating that the antiserum was bound at a site different from the LH binding site. FSH receptors are present in the Sertoli cell plasma membranes in the rat testes seminiferous tubules (see review by Dorrington and Armstrong, 1979). These receptors have been solubilized by extracting 20-day-old male rat testes preparations with 1% Triton X-100 (Dufau ef al., 1977b). The association constant of the detergent-solubilized FSH receptors was higher than that of the particulate receptors (8.5 x lo”, I .6 X lo9 M - I , respectively), which is in contrast to the fall in binding affinity observed during solubilization of testicular LH receptors (Dufau et al.. 1973, 1975). Water-soluble binding sites with a high affinity for 12sI-labeledFSH ( K , I . 17 X 10yM- I ) were also detected. These water-soluble receptors represented about 20% of the FSH receptors in the testis. Bovine TSH receptors from thyroid tissue have been solubilized with lithium diiodooxalicylate and were found to be heterogeneous in size, in that they had binding components with molecular weights of 268,000, 160,000, 75,000, and 15,000-30,000 (Tate et a/., 1975a,b). Tryptic digestion converted all three higher molecular weight components to the same 15,000- to 30,000-dalton species. The latter had all the binding properties of the higher molecular weight forms including nonlinear Scatchard plots. The tryptic fragment of the solubilized receptor was purified approximately 250-fold by affinity chromatography on TSH-Sepharose columns.
IV. INVOLVEMENT OF CYCLIC AMP IN HORMONE ACTION It has been clearly shown that cyclic AMP can mimic the action of FSH, LH, and TSH, and there is good evidence that it plays an obligatory role in the action of these hormones. The early investigations revealed that cyclic AMP (or more active derivatives) mimicked the effects of the hormone on the cell response (e.g., steroidogenesis in ovarian and testicular cells) and that the effect of cyclic AMP was not additive to the maximum stimulating level of the hormone. The
150
BRIAN A. COOKE
hormone-induced increase in cyclic AMP preceded the effect on cell response and phosphodiesterase inhibitors potentiated the hormonal effect on cyclic AMP production and the cellular response. These data satisfied the criteria proposed by Sutherland and co-workers (Robison et al., 1971) for the mediation of cyclic AMP as the second messenger. However, in some systems cyclic AMP production was observed to be undetectable at levels of the hormone that gave a submaximal response of the cell; for example, in ovarian and testicular cells it was demonstrated that increased steroidogenesis could be detected with hormone concentrations 10 times lower than those required to detect changes in cyclic AMP levels (Marsh 1966; Catt and Dufau, 1973; Moyle and Ramachandran, 1973; Rommerts et al., 1973). This led to the conclusion that the role of cyclic AMP at physiological concentrations of hCG or LH may not be obligatory. However, it was argued that small changes in cyclic AMP could occur within the cells which were not detectable by the methods used; that the latter was true was indicated by the stimulatory effects of phosphodiesterase inhibitors on testosterone release in testis cells, but not on detectable cyclic AMP production, by low amounts of hCG (Catt and Dufau, 1973). LH was known to stimulate cyclic AMP-dependent protein kinases in Leydig cells (Cooke and van der Kemp, 1976) (the only known mechanism of cyclic AMP action), and therefore it was investigated whether at low concentrations of LH cyclic AMP-dependent protein kinase activation in testis Leydig cells was a more sensitive parameter than the cyclic AMP concentration itself (Cooke et al., 1976). It was clearly demonstrated that all concentrations of LH which stimulated testosterone production also stimulated protein kinase activation. Again with the lower stimulating amounts of LH (<1 ng/ml) there were no detectable changes in cyclic AMP production. Similarly, a close correlation was obtained between the phosphorylation of endogenous proteins and testosterone production in rat Leydig cells (Cooke er al., 1977). Ling and Marsh (1977) also reported a positive correlation between the activation of cyclic AMP-dependent protein kinase and the stimulation of steroidogenesis in the bovine corpus luteum. Another approach utilized by Dufau et al. (1977b) was the measurement of free and occupied cyclic AMP binding sites of the regulatory subunit of Leydig cell protein kinase in basal and hCG-stimulated cells. These studies demonstrated a close correlation between hCG-stimulated testosterone production and the occupation by endogenous cyclic AMP of the intracellular cyclic AMP receptor binding protein. Thus, all the studies involving indirect measurements of cyclic AMP production by measuring protein kinase activation or by occupation of the cyclic AMP binding protein are consistent with an obligatory role for cyclic AMP in the action of trophic hormones on ovarian and testicular tissues. Direct measurements of cyclic AMP, even using very sensitive radioimmunoassay methods, are not consistent with this role. The results from a recent study on direct cyclic AMP measurements in mouse Leydig cells in culture also argue against a role for
REGULATION BY GLYCOPROTEIN HORMONES
151
cyclic AMP at low levels of lutropin (Cooke et af., 1982). In these studies isoproterenol-stimulated cyclic AMP and testosterone production were measured. It was found that isoproterenol gave a fivefold stimulation of cyclic AMP production and the same stimulation of testosterone production as 0.1 ng/ml of lutropin. With this amount of lutropin, however, no change in cyclic AMP production could be detected.
V. IMPORTANT FEATURES OF THE HORMONE RECEPTOR-ADENYLATE CYCLASE SYSTEM Using plasma membrane preparations and sensitive methods of measuring cyclic AMP, the kinetics and substrate requirements for activation of adenylate cyclase have been investigated (Birnbaumer, 1973; Birnbaumer and Yang, 1974). In addition to the appropriate hormone, it has been demonstrated that there is an absolute requirement for guanine nucleotides and divalent cations (see Rodbell et a / . , 1975). There is also convincing evidence indicating that the hormone receptor and adenylate cyclase are distinct and separable molecules linked during activation via a guanosine nucleotide binding protein (see Fig. 11). The latter is, or is associated with, a GTPase. The “floating receptor” model of Cuatrecasas (1974) indicates that the receptor and the catalytic unit of adenylate cyclase systems exist as independent molecules which after binding of the hormone to the receptor interact by lateral diffusion within the membrane. There is now good evidence to support this concept. Using the technique of cell fusion the transfer of receptors from one cell type to another and subsequent coupling to the adenylate cyclase has been demonstrated (Orly and Schramm, 1976). Using solubilized membrane preparations the chromatographic separation of the receptor, guanine nucleotide binding site, and adenylate cyclase has been achieved (Limbird and Lefkowitz, 1977; Pfeuffer, 1977). Mutants (especially of S49 cells) have been produced which lack either guanine nucleotide binding protein or the receptor or both (Bourne et al., 1975, Haga et al., 1977; Ross and Gilman, 1980). This evidence obtained with different hormones and cells suggests that there is a general mechanism of hormone receptor-adenylate cyclase interaction. The important features of the roles played by guanine nucleotides with this mechanism can be summarized as follows: I . GTP can stimulate adenylate cyclase in the absence of hormone except in turkey erythrocytes, where its presence is required. 2. Nonhydrolyzable analogs of GTP as p(NH)ppG’ and GTPyS are more active than GTP and convert the adenylate cyclase to a persistently active state. ‘Abbreviations used: p(NH)ppG, guanosine 5’-[P,y-imido]triphosphate;GTPyS (= [slppG), guanosine 5‘-[y-thio]triphosphate.
152
BRIAN A. COOKE
3. GTP activates adenylate cyclase via a regulatory protein (G-protein). 4. The GTP regulatory protein is associated with or is a GTPase. 5 . The GTPase “turns off” the activity of the adenylate cyclase by hydrolysis of GTP to GDP. 6 . The hormone increases GTPase activity (in turkey erythrocytes). 7. GTP increases the rate of dissociation of the hormone from its receptor (except the glycoprotein hormones). Effect of Guanine Nucleotides on Binding of the Hormone to Its Receptor The early work of Rodbell (1975) clearly showed that GTP and p(NH)ppG added to liver plasma membranes increased the rate of dissociation of 1251labeled glucagon, thus indicating that glucagon has a decreased affinity for its receptor in the presence of these guanine nucleotides. This has now also been shown for P-adrenergic hormones and their receptors in rat glioma cells, frog erythrocytes, and other cells (see Simpson and Pfeuffer, 1980, for references). Binding of glycoprotein hormones to their receptors, however, is unaffected by guanine nucleotides (Lee and Ryan 1972, 1973; Labarbera et al., 1980; AminZaltaman and Salomon, 1980). Considerable progress is now being made on the further characterization of the G-protein from rabbit liver and turkey erythrocyte plasma membranes (Northup et al., 1980; Sternweiss et al., 1981a,b). Again the G-protein has been assayed using the cyc- S49 lymphoma cells. The G-protein complex isolated from rabbit liver membranes consists of three nonidentical subunits of molecular weights of 52,000, 45,000, and 35,000, whereas the G-protein from turkey erythrocytes contains only the 45,000- and 35,00044, species. The 45,000-M, subunit has an M , value similar to that identified by Pfeuffer (1977) (42,000) using the 3Hlabeled oxidoanilide analog of GTP and may be responsible, at least in part, for restoring the p(NH)ppG-stimulated and NaF-stimulated activity to adenylate cyclase preparations following exposure to GTP-Sepharose resin. The same subunit is most probably involved in the ADP-ribosylation induced by cholera toxin. However, the purified G-protein isolated by Sternweiss er al. (1981a) is not a substrate for this reaction, since this reaction takes place only after the addition of membrane fractions. GTPase activity is also absent from the purified protein.
VI.
DESENSITIZATION AND DOWN-REGULATION BY HOMOLOGOUS HORMONE
In addition to having a stimulatory effect, it is well established that certain hormones cause desensitization of their target cells, resulting in a loss of response to further stimulation; this can be caused by over- or underexposure to the
REGULATION BY GLYCOPROTEIN HORMONES
153
hormone. In general, this phenomenon is characterized by loss of adenylate cyclase activity (desensitization) and may also include loss of hormone receptors (down-regulation). The various hormones differ in the degree to which they conform to this general pattern. Recent studies have concentrated on elucidating the mechanism of desensitization and determining whether the modification in function of the cell is due only to uncoupling of the adenylate cyclase or to loss of receptors or both. Roth and colleagues (Kahn et al., 1973; Sell et al., 1975; Gavin et al., 1974) made the initial observations on the down-regulation of insulin receptors in lymphocytes and liver tissue. Since that time, other hormones, for example, growth hormone (Lesniak and Roth, 1976) and P-adrenergic hormones (Perkins et al., 1978; Kebabian et al., 1975; Mukherjee and Caron, 1975; Su et al., 19801, have been shown to cause loss of adenylate cyclase and their respective receptors. Lutropin especially has been shown to have a dramatic effect on the levels of its receptor and adenylate cyclase activity in both the rat luteal and testis Leydig cell (see review by Catt et al., 1979). Hormone-induced receptor loss in some systems has been found to be dependent on protein synthesis (Hinkle and Tashjian, 1976; DeMeyts et al., 1976; Saez et al., 1978; Sharpe, 1977; Dix and Cooke. 1981), whereas it is apparently independent of protein synthesis in other systems (Lesniak and Roth, 1976; Mukherjee and Caron, 1975). There is relatively little information on the possible negative effects of TSH on target cells. Prolactin has been shown to decrease its own receptor levels in rat mammary gland and liver (Djiane et al., 1979) but increase LH receptor levels in Leydig cells (Zipf et al., 1978). In order to illustrate the work carried out on desensitization, the data on LH effects on ovarian cells and testis Leydig cells will be mainly referred to [for discussions of other systems, the reader is referred to Catt et al. (1979) and Perkins et al. ( 1978)]. In the Leydig cells of the testis and luteal cells of the ovary, LH binds specifically to its receptor with high affinity (K, = 0. I mM) and increases cyclic AMP production. This is followed by activation of cyclic AMP-dependent protein kinases and subsequently steroidogenesis (testosterone production in the Leydig cells and progesterone in the luteal cells). The responsiveness of these cells to LH can be conveniently measured in vitro, in purified cell suspensions (Janszen et al., 1976; Conn el al., 1977; Schumacher ef al., 1978; Cooke et al., 198 I a, b). The initial occupancy of the LH receptors in the Leydig and luteal cells by exogenously administered hormone lasts up to 24 hours and is followed by a prolonged loss of LH receptors which is maximal after 1 to 2 days and lasts up to 8 days in both the testis and ovary. The extent of receptor loss is much greater than the initial occupancy by administered hormone (Conti et al., 1977). In both the testis and ovary, the period of net receptor loss is followed by restoration of receptor numbers to normal levels over the next 6 to 10 days. These in vivo
154
BRIAN A. COOKE
studies also indicate that there is an uncoupling of adenylate cyclase from the receptor during the initial or partial occupation of receptors in the ovary and testis, i.e., the adenylate cyclase becomes unresponsive to further stimulation even in the presence of unoccupied receptors. In both the luteal and Leydig cells there is a correlation between the loss of LH receptors, adenylate cyclase activity, and steroid production. However, in the Leydig cell it has been demonstrated that loss of testosterone production could not be fully accounted for by loss of LH receptors and that there was an additional block in the steroid biosynthetic pathway between pregnenolone and testosterone. This has been found to be caused by inhibition of the 17,20-desmolase which becomes rate limiting in the desensitized state with accumulation of 17-hydroxylated precursors, The mechanisms involved have not been fully elucidated, but they may involve estrogen production in the Leydig cell which has been shown to inhibit the 17,20-lyase (Cigorrago et al., 1978). It has also been demonstrated that LH-releasing hormone (LHRH) analogs can cause desensitization of ovarian and luteal cells when administered in vivo to hypophysectomized rats (see review by Catt et al., 1979). Furthermore, LHRHlike compounds are actually secreted by the rat testes and ovaries (Sharpe and Fraser, 1980), probably by the Sertoli and granulosa cells, respectively. Their secretion is controlled by gonadotropins (Sharpe and Fraser, 1980). Furthermore, it has now been found that LHRH analogs initially stimulate rat testis Leydig cell steroidogenesis in vitro (Hunter et al., 1982; Sharpe and Cooper, 1982) followed at later times (after approximately 30 hours) by inhibition (Hunter er al., 1982). In addition, these effects of LHRH analogs may be species specific, because no effects were detected with mouse Leydig cells (Hunter et al., 1982).
A. Desensitization and Down-Regulation of a LH-Responsive Leydig Cell Tumor 1. INTACT CELLS In order to investigate these mechanisms in detail, a model for studying desensitization of Leydig cells in virro has been developed (Dix and Cooke, 1981). This utilizes a LH-responsive Leydig cell tumor which has many of the characteristics of normal Leydig cells (Cooke et al., 1979). Initial studies with this model have shown that desensitization of the tumor cells occurs after exposure to LH in virro; a dose-dependent loss of both LH receptors and LHstimulated cyclic AMP production occurred over a period of 12 hours (Dix and Cooke, 1981). Typical results from these experiments are given in Figs. 1 and 2. In Fig. 1, the control cells not treated with LH showed an initial increase in the apparent number of LH receptors over the first 2 hours, which then remained constant during the next 8 hours. This initial rise in the number of receptors was
REGULATION BY GLYCOPROTEIN HORMONES
155
Time after preincubation ( h )
FIG. I . Effect of pretreatment with saline on tumor Leydig cells. Suspensions of cells from a Leydig cell tumor were prepared by incubation of the tumor with collagenase followed by purification of the cells on Percoll density gradients. Portions of the cells were incubated with saline for 30 minutes, washed, and then incubated in suspension cultures. At various times the cells were removed and cyclic from the cultures and binding of ’2sI-labeled hCG ( O ) ,basal cyclic AMP production (0). AMP production (A)during incubation with LH for 1 hour was measured. (From Dix and Cooke, 1981.)
Time after pretreatment ( h 1 FIG. 2. The effect of pretreatment with lutropin on tumor Leydig cells. The same conditions as those given in Fig. I were used except that the cells were pretreated in medium containing LH (100 ngiml. NIH S20: I .9 IU SIlmg). (From Dix and Cooke, 1981.)
156
BRIAN A. COOKE
accompanied by an initial increase in basal cyclic AMP production, whereas the LH-stimulated cyclic AMP production remained constant throughout the incubation. Preincubation of the cells with LH also resulted in an apparent LH receptor number followed by a dramatic decrease. There was a marked decrease in the ability of these cells to respond to LH in terms of cyclic AMP production even when the LH receptors were increasing, indicating that uncoupling of the receptors from the adenylate cyclase had occurred (Fig. 2). The LH-dependent decrease in cyclic AMP production and the loss of LH receptors were completely prevented by inclusion of cycloheximide in the incubation media, thus indicating that this desensitization process is dependent on protein synthesis (Dix and Cooke, 1981). This agrees with studies in vivo (Saez et al., 1978; Sharpe, 1977). Also in agreement with studies in vivo (Huhtanierni er uf.. 1978) was the observation that the initial rise in the number of LH receptors was not prevented by cycloheximide and is, therefore, probably not dependent on protein synthesis de novo. 2. CHARACTERIZATION A N D DESENSITIZATION OF ISOLATED PLASMAMEMBRANES Further progress on the elucidation of the molecular events taking place in the plasma membranes of Leydig cells from normal and desensitized cells has been hampered by the poor responsiveness of the isolated membranes to LH in vitro (Dufau et al., 1977). Recently there have been reports of the successful isolation of lutropin and follitropin-sensitive adenylate cyclase from rat testis preparations (Dufau et af., 1980; Jahnsen et al., 198I ) . However, these studies were carried out with mixed testis cell populations, making it difficult to study directly the Leydig cell adenylate cyclase. Lutropin-sensitive plasma membranes from Leydid cell tumors have been successfully isolated and characterized (Levi er ul., 1982a). The plasma membranes were prepared in a hypotonic buffer using a two-phase (dextran-polyethylene glycol) method for separation from other subcellular constituents. Some of the characteristics of these membranes are as follows. a. EfJect of Magnesium Ion Concentration. In agreement with results from other systems, it was found that stimulation of adenylate cyclase was highly dependent on Mg2+ concentrations (Fig. 3). In the presence of fluoride ions, activity of the adenylate cyclase was not much greater than basal activity at concentrations of Mg2+ less than 2 mM. At 1 mM M g 2 + , fluoride-stimulated activity was threefold greater than basal activity, and it was maximal at 6 mM Mg2 . Thereafter there was a small decrease in activity as the concentration of Mg2 was increased. Stimulation of adenylate cyclase by lutropin plus p(NH)ppG was again evident only at concentrations of Mg2+ greater that 2 mM, and was maximum at 4-6 mM. Basal activity followed a similar bimodel pattern, being highest at concentrations of 4-8 mM Mg2 + . +
+
157
REGULATION BY GLYCOPROTEIN HORMONES
M Q ~(+ m ~ )
FIG. 3 . Effect of varying magnesium ion concentrations on adenylate cyclase activity in tumor Leydig cell plasma membranes. Plasma membranes were incubated for 30 minutes at 30°C with p(NH)ppG (0.I mM) increasing concentrations of MgClz in the presence of sodium fluoride (O), plus lutropin ( I kg/ml) o r assay mixture alone ( 0 )Results . represent means 2 SEM (ti = 3 ) . (From Levi ef a/..1982a.)
(A),
In addition to being necessary during assay of adenylate cyclase, it was found that the activity of this enzyme was dependent on the concentration of Mg2+ present during preparation of the plasma membranes (Levi et al., 1982a). Therefore, it may also stabilize some part of the receptor-guanine nucleotide regulatory protein-adenylate cyclase system. lyengar and Bimbaumer (198 I ) have shown that the guanine nucleotide regulatory protein has a binding site not only for guanine nucleotides but also for Mg2 . Their work indicates that Mg2 may be an essential activator of the regulatory component. The other essential role of Mg2+ in its complex with ATP is well established as the natural substrate of adenylate cyclase. It was observed that whereas at 2 mM MgCI, the response to p(NH)ppG plus lutropin was 64% of its maximum, fluoride-stimulated activity was barely greater than basal activity (Fig. 3). In this experiment ATP and EDTA concentrations were both 1 .O mM. Therefore, the fluoride-stimulated response is clearly consistent with a role of magnesium ion in complexing ATP, stimulation becoming manifest when this “nucleotide” or “substrate” requirement is met. The observation that in the presence of p(NH)ppG plus hormone the +
+
158
BRIAN A. COOKE
enzyme was stimulated at a lower magnesium concentration is consistent with the findings of other workers. For example, GTP and corticotropin appear to act synergistically to reduce the requirement of adrenocortical adenylate cyclase activity for Mg2+ (Glynn et af., 1977), possibly by raising the affinity of the system for this cation. 6. Effect ofp(NH)ppG and GTP. The effects of increasing the concentrations of p(NH)ppG and GTP in the presence and absence of lutropin and fluoride ions were investigated. With p(NH)ppG alone, stimulation was detectable at lop8M , half-maximal at 2.5 X IOW’M, and maximal at M p(NH)ppG (Levi e t a / . , 1982a) (Fig. 4). Lutropin ( I pg/ml) stimulation of adenylate cyclase was greatly increased in the presence of p(NH)ppG. This effect was detectable at lo-* M M, representing a 55-fold increase p(NH)ppG and reached its maximum at over adenylate cyclase activity in the presence of lutropin alone. This activity was 86% of that in the presence of sodium fluoride alone. The effect of GTP upon basal adenylate cyclase activity showed an initial increase at M GTP with a maximum at l o p 5 M , followed by a significant decline at higher concentrations (Levi et a/., 1982a) (Fig. 5). Lutropin-stimulated activity was also considerably enhanced by the nucleotide (Fig. 5 ) , with
/r
.m
c .-
I
600
4
P
8
J
BASAL
400
200
,$ p(NH)ppG ( M I
FIG. 4. Effect of varying p(NH)ppG concentrations on adenylate cyclase activity in tumor Leydig cell plasma membranes. Plasma membranes were incubated for 30 minutes at 30°C with increasing concentrations of p(NH)ppG in the presence of sodium fluoride (O), LH (1 p,g/ml) (0). and assay mixture alone (A), Results represent means 2 SEM (n = 3).
159
REGULATION BY GLYCOPROTEIN HORMONES
T
I
1501
/I
T
\
GTP ( M I
FIG.5. Effect of varying GTP concentrations on adenylate cyclase activity in tumor Leydig cell plasma membranes. Plasma membranes were incubated for 30 minutes at 30°C with increasing and assay mixture concentrations of GTP in the presence of sodium fluoride ( O ) ,LH ( I pgiml) (0). above (A). Results represent means ? SEM ( n = 3 ) .
maximal activity occurring at M GTP; this was followed by a significant decline in hormone-stimulated activity at l o p 3 M ,and GTP was 11% of that in the presence of sodium fluoride alone. The maximal enhancement of hormonestimulated adenylate cyclase activity by GTP was only 13% of that elicited by p(NH)ppG, and no decrease in activity was observed with increasing concentrations of p(NH)ppG. Fluoride-stimulated activity was not affected by either nucleotide (Figs. 4 and 5 ) . The dependence of tumor Leydig cell adenylate cyclase on guanine nucleotides was therefore clearly indicated by the ability of GTP and p(NH)ppG to amplify the hormonal stimulation of the enzymes by lutropin. Moreover, the variation in cyclase activity at the concentrations of GTP and p(NH)ppG studied is very similar to the pattern exhibited by homogenates from testis membranes (Dufau et al., 1980). This indicates that the adenylate cyclase system of tumor Leydig cells and testis cells is fundamentally similar. The inhibitory effect of high GTP concentrations was in contrast to the progressively greater effects of increasing concentrations of the nonhydrolyzable analog, p(NH)ppG. This inhibitory effect of GTP has also been observed in the ovary (Ezra and Salomon, 1980) and the testis (Dufau et a l . , 1980). L'. Kinetics of Adenylate Cyclase Stimulation. The time course of the response of adenylate cyclase to the various stimulants was also investigated (Levi et al., 1982a). Basal activity was increased twofold by lutropin alone, and the time course of cyclic AMP production was linear (Fig. 6). With GTP the mean
160
BRIAN A. COOKE T
320
-
260
-
:. : E" c
240-
200-
\
e 5
-aE 5
0 .-
3
160-
12060-
0 D .
40
-
0
5
lo
b
25
20 TIME
30
35
40
45
(mid
FIG. 6. Time course of adenylate cyclase activation in tumor Leydig cell plasma membranes. Pldsma membranes prepared were incubated for varying times at 3OoC in the presence of GTP (0.02 mM) plus lutropin ( 1 pgirnl) (A),GTP (0.02 mM) lutropin ( I Fg/ml) ( O ) ,or assay mixture alone ( 0 ) Results . represent means k SEM (n = 3). (From Levi et a / . , 1982a.)
(A),
enzyme activity was increased threefold; however, there was a pronounced lag during the first 10 minutes of the response to GTP (Fig. 6); enzyme activity increased between 10 and 20 minutes and then progressively decreased. In the presence of lutropin no lag was observed in the response to GTP. Stimulation was initially sixfold greater than basal, but this rate was maintained for only 20 minutes; thereafter, cyclase activity declined (Fig. 6). Figure 7 shows the response of adenylate cyclase in the presence of p(NH)ppG; again there was a lag phase of about 10 minutes before maximal rate of activation was reached. The presence of lutropin abolished the lag in the p(NH)ppG-stimulated response. The decrease in enzyme activity observed with GTP was not evident in the response to p(NH)ppG in the absence or presence of lutropin. The fluoride-stimulated activity was essentially linear with a minor lag phase between 0 and 10 minutes and a slight decline in enzyme activity by 45 minutes (Fig. 7). Therefore, for both GTP- and p(NH)ppG-dependent activities there was a 10to 15-minute lag phase in the response of the cyclase. Since the lag period is a measure KoN, the constant which characterizes the rate of adenylate cyclase activation by guanine nucleotides (Levitzki, 1980), the inference is that both GTP and p(NH)ppG may be acting at the same regulatory site with similar
161
REGULATION BY GLYCOPROTEIN HORMONES
I400
1000
Time ( m i n ) F I G . 7. Time courses of adenylate cyclase activation in tumor Leydig cell plasma membranes. Plasma mcmbranes were incubated for varying times at 30°C in the presence of sodium fluoride (10 mM) (0). p(NH)ppC (0.1 mM) plus lutropin ( I Kgiml) or p(NH)ppG (0. I mM) (0).Results represent means ? SEM ( n = 3 ) . (From Levi et a l . , 1981-a.)
(A),
binding characteristics. The effect of lutropin on GTP- and p(NH)ppG-stimulated activities was to decrease the lag period in the response to these nucleotides. In addition, lutropin enhanced 1.4-fold the steady-state rate given by p(NH)ppG alone, and increased maximal activity in the presence of GTP by 25%. These hormone-mediated effects have also been observed in the epinephrine-stimulated fat cell cyclase (Rodbell, 1975) and the glucagon-stimulated liver cyclase (Salomon et a l . , 1975) systems. However, with rat ovarian adenylate cyclase lutropin enhanced GTP- and p(NH)ppG-stimulated activities but there was no time lag with guanine nucleotides alone (Ezra and Salomon, 1980). Dufau ei a/. ( 1980)also did not observe a time lag with guanine nucleotide when added to testis membranes.
B. The Desensitizing Effect of GTP on Isolated Plasma Membranes Further experiments were carried out with GTP and related guanine nucleotides in which the ATP and the ATP-regenerating system were omitted from
162
BRIAN A. COOKE
4001 350
Time (min ) FIG. 8. Time courses of p(NH)ppG plus lutropin-stimulated cyclic AMP production in guanine nucleotide/lutropin pretreated tumor Leydig cell plasma membranes. Plasma membranes were preincubated for 40 minutes at 30°C in the presence of GTP ( 10 fl)plus lutropin ( I pg/ml) (a), GDP ( 10 pM) plus lutropin ( I pg/ml) (0). or buffer alone (A). and the subsequent time course of lutropin- ( I pg/rnl) plus p(NH)ppG- (0.I mM) stimulated cyclic AMP production was determined. Results represent means ? SEM ( n = 3). (From Levi t r a/.. 1982b.)
the preincubation medium (Levi et a/.,1982b). Pretreatment wirh GTP and lutropin was found to impose a 5- to 10-minute lag on p(NH)ppG/lutropinstimulated cyclase activity which, even in the steady state, was 50% lower than control (Fig. 8). Thus the desensitizing effect of GTP was only partly reversible by incubation with p(NH)ppG plus lutropin. GDP caused a similar lag in cyclase activity, and steady-state activity remained 20% below control. GMP and guanosine imposed a 10-minute lag in cyclase activity, bur in each case steadystate activity was the same as that of the control enzyme (Fig. 9). Thus, the effects of GMP and guanosine on adenylate cyclase were entirely reversed by incubation with p(NH)ppG plus lutropin. It was concluded from these studies that pretreatment of tumor Leydig cell plasma membranes with GTP, in the absence of ATP, desensitizes adenylate cyclase to further stimulation by several agents, including sodium fluoride. While lutropin alone was not able to induce similar desensitization, this hormone was able to enhance the desensitizing effect of GTP. These effects on cell-free preparations had not been reported for Leydig cells, but there are similarities and differences in those reported for desensitization of ovarian plasma membrane
REGULATION BY GLYCOPROTEIN HORMONES
163 T
400i 350
; 300.8
f! . 2
-E
250-
200-
a
Guonosine
L
+ LH
u ._ -
K 50
5
10
15
20
25
30
35
40 45
Time ( min )
FIG.9. Time courses of p(NH)ppG plus lutropin-stimulated cyclic AMP production in guanine nucleotideilutropin-pretreated tumor Leydig cell plasma membranes. Plasma membranes were preincubatcd for 40 minutes at 30°C in the presence of G M P (10 p M ) plus lutropin ( I @g/ml) (H). guanosine (10 plus lutropin ( I pgiml) (A), or buffer alone (A), and the subsequent time course of lutropin- ( I pgirnl) plus p(NH)ppG- (0. I iM stimulated cyclic AMP production was determined. Resulta represent means SEM ( n = 3). (From Levi rr u l . . 1982b.3
a)
*
adenylate cyclase by GTP (Bockaert et d..1976; Ezra and Salomon et a / . , 1980). Each of the guanine nucleotides tested imposed a lag phase ( 5 - 10 minutes) on subsequent enzyme activity assayed in the presence of lutropin plus p(NH)ppG. This may represent the time required for the displacement of the nucleotide from the regulatory site. It has been shown that treatment of turkey erythrocyte membranes with hormone plus GMP removes endogenous GDP (Abramowitz et ml., 1980). Alternatively, the guanine nucleotide may decrease the affinity of the hormone receptor for lutropin, a well-recognized phenomenon in other tissues (Rodbell et a / . , 1971; Simpson and Pfeuffer, 1980; Maguire et a / . , 1977). This would impair the ability of lutropin to reduce the lag phase previously observed in p(NH)ppG activation (Levi et al., 1982a). The effects of GMP and guanosine were entirely reversed during the assay with lutropin plus p(NH)ppG since, once the lag phase was overcome, steady-state activity of the GTP-treated enzyme was only 50% lower than control, indicating that the desensitization induced by GTP was only partly reversed. The desensitizing effects of GDP, too, were not fully reversed, since steady-state activity remained 20% below control activity. Pre-
164
BRIAN A. COOKE
treatment with hormone plus p(NH)ppG achieved quite the reverse-the enzyme was persistently activated, basal activity being increased 40-fold. Since the effect of the guanine nucleotides appears to be related to the phosphate content of the desensitizing nucleotide, it may be mediated by a guanine nucleotide-dependent phosphorylation reaction. If this is true, the difference in the abilities of GTP and p(NH)ppG to desensitize the cyclase would result from the difference in the abilities of the two nucleotides to participate in phosphotransferase reactions. A GTP-mediated phosphorylation was also proposed as a mechanism for the desensitization of ovarian plasma membrane adenylate cyclase (Ezra and Salomon, 1980). Bockaert et ul. (1976), using pig follicular membranes, first demonstrated that cell-free desensitization occurred under comparable incubation conditions. In these experiments desensitization was dependent on the presence of M g 2 + , ATP, and lutropin. Incubation of the desensitized membranes with fractions enriched in phosphoprotein phosphatase and alkaline phosphatase activities resulted in resensitization of the lutropin response. More recently, Iyengar et ul. (1980) observed that treatment of rat liver plasma membrane with Mg2 in the presence of ATP led to a loss of glucagon stimulation. In this system desensitization stimulated by glucagon was dependent upon coaddition of GTP. Phosphorylation reactions have been implicated, but not proved, in the desensitization of a large number of other adenylate cyclase systems. In contrast to the studies of Bochaert et al. (1976), desensitization in Leydig cells was shown to be independent of ATP. Ezra and Salomon (1980) also had ATP in their incubations, although a more recent study demonstrated that GTP-induced desensitization of ovarian plasma was independent of ATP (Ezra and Salomon, 1981). In the experiments in which the plasma membranes were preincubated with lutropin plus p(NH)ppG, there was a resulting elevated basal activity due to the binding of p(NH)ppG at the regulatory site, and 30 minutes of incubation under the conditions used has been shown to be adequate for maximal cyclase activation. Lutropin was included since it has been shown to increase the guanine nucleotide binding capacity of the enzyme (Dufau et a / ., 1980). This could be attributed to exposure of hindered sites or, as recently proposed, to hormoneinduced exchange of guanine nucleotides with the species (probably GDP) that occupies the regulatory site. It is interesting to compare the GTP-induced desensitization of Leydig cell membranes with that of ovarian membranes (Ezra and Salomon, 1980). The latter authors found that lutropin plus GTP, NaF, and lutropin plus p(NH)ppG initially all gave similar stimulations of adenylate cyclase activity. After 15 minutes the rate with lutropin and GTP declined; not only was this prevented by NaF, but the combination of all three substances gave a much higher stimulation than NaF alone. The GTP-induced desensitization was reversed by p(NH)ppG. In the Leydig cell membranes, lutropin and p(NH)ppG or GTP did not enhance +
REGULATION BY GLYCOPROTEIN HORMONES
165
the NaF effects. The GTP-induced desensitization was not easily reversed; neither NaF nor p(NH)ppG completely prevented or reversed the GTP-mediated desensitization.
C. Determination of the State of Lesion in LH-Desensitized Leydig Tumor Cells Having established a method for the preparation of LH-sensitive plasma membranes it now became possible to prepare plasma membranes from desensitized cells so that the site of desensitization in the hormone receptor-adenylate cyclase system could be determined. In these studies it was also important to determine whether the loss of receptors and desensitization were separate processes and to define more clearly the kinetics of cyclic AMP formation in the desensitized systems (Dix et a / . , 1982). It was found that maximum cyclic AMP production was achieved in control and lutropin-pretreated cells with 1 p g of lutropin; however, the maximum stimulated level of pretreated cells was only 30% of that of control cells. The sensitivity to lutropin was also decreased in lutropin-pretreated cells with an ED,, value of 60.0 ng/ml for pretreated cells compared with 8.4 ng/ml for control cells. Consistent with a decreased maximum cyclic AMP production in lutropin-pretreated cells was a decreased rate of cyclic AMP production (0.58 compared with 1.89 pmolesl lo6 cells/minute). Although the initial basal levels of cyclic AMP were higher in the pretreated cells, the subsequent cyclic AMP production was much reduced compared with control cells (0.08 and 0.38 pmoles/ 1 O6 cells/minute, respectively). The decreased lutropin-stimulated cyclic AMP production was evident 6 hours after the preincubation, which was in agreement with previous observations. However, cholera toxin-stimulated cyclic AMP production was not decreased by lutropin preincubation and a significant potentiation of the cholera toxin-stimulated cyclic AMP production could be seen at all time points studied. In further experiments, purified tumor Leydig cells were preincubated with lutropin or dibutyryl cyclic AMP followed by preparation of the plasma membranes. The adenylate cyclase activity of the plasma membranes in the presence of lutropin, p(NH)ppG, lutropin plus p(NH)ppG, and sodium fluoride plus lutropin was determined. In addition, the lutropin- and cholera toxin-stimulated cyclic AMP production of the intact cells with and without acid washing was determined. In these experiments the cells were preincubated for 2 hours with a higher concentration of lutropin (10 pg/ml) than in previous experiments. Under these conditions, although the mean cholera toxin-stimulated cyclic AMP production was consistently higher in the lutropin-pretreated cells, these values were not significantly different from control values (Tables I11 and IV, p > 0.05). In
THEE p t E m
Pretreatment Saline Saline Lutropin Lutropin
Ok
TABLE 111 LUTROPIN O N THt LUTROPIN RtCtPrOR CON I t N T A N U T H t CYCLIC AMP PRODUCTION O F LLYUlC TUMOR CtLLS” ”
Washing conditions (pH)
Specific 12s1-labeledhCG binding (fmole/106 cells)
7.4 3.0 7.4 3 .O
7.3 ? 0.7 5.9 2 0.9 0.4 2 0.1 8.4 2 0.9
Basal cyclic AMP production (pmole/106 cellsl2 hours) 4.5 3.3 24.1 9.0
Lutropin-stimulated cyclic AMP production (pmo1e/lO6cells/7 hours)
0.5 0.5 2 0.7 5 0.7
193.9 f 190.6 i71.1 f 70.3 It
2 -t
~
3.0 12.7 7.2 14.7
________________
Cholera toxin-stimulated cyclic AMP production (pmole!lO6 c e W 2 hours) 178.4 2 1.6 199.8 2 18.9 217.5 k 6.8 169.1 2 14.2 _
_
_
_
~
From Dix e r a / . (1982). Leydig tumor cells were prepared and then preincubdted with or without lutropin (10.0 Kgiml) for 2 hours at 32°C. Cells were then centrifuged (200 g for 10 minutes) and incubated in 0.98 saline. 50 mM glycine at M 0 Cfor Z minutes at pH 3.0 or 7.4. The cells were then washed in phosphatebuffered saline (pH 7.4), and specific 12s1-laheledhuman choriogonadotropin binding and basal. lutropin- ( 10 +g/ml) stimulated, and cholera toxin(4 &g/ml) stimulated cyclic AMP production was determined. Results rcpresent means f SEM ( u = 3 ) . ‘I
167
REGULATION BY GLYCOPROTEIN HORMONES
Pretreatment
Bahal
Lutropin-~tiniulated
Cholera toxin-stiiiiulatcd
Control Dibutyryl cyclic AMP Lutropin
2.1 -c 0.2 14.5 2 I .5 43.5 2 4 . 3
253.1 34.3 214.4 t 8.5 65.9 2 0.5
260.3 2 24.8 248.9 2 22.6 231.5 2 14.0
From Dix
el
_f
ol. ( 1982).
'' Leydig cells were prepared
and then preincubated with lutropin ( I 0 pglml). dibutyryl cyclic AMP ( 2 mM), or saline for 2 hours at 32°C. Cells were then washed twice in suspension media and finally resuspended in subpension media containing isohutylmethylxanthine (0.5 mM) and the basal. lutropin- ( 10 Fgiml) stimulated, and cholera toxin- (4 pg/ml) stimulated cyclic AMP production was determined. Results represent means 2 SEM 01 = 3 ) .
agreement with the previous experiments, with lutropin pretreatment there was no decrease in the cholera toxin-stimulated cyclic AMP production, whereas there was a decrease in the subsequent lutropin-stimulated cyclic AMP production. Preincubation with dibutyryl cyclic AMP ( 2 mM) had no effect on either lutropin or cholera toxin-stimulated cyclic AMP production. The specific 12sIlabeled choriogonadotropin binding was decreased to approximately 5% of that of control cells by preincubation with lutropin (Table Ill). This decrease was aboli4iLd if the cells were washed under acidic conditions (pH 3.0 for 2 minutes ; I I 1 ) -4°C). Although this acid-washing procedure reversed the lutropin-induced decrease in specific ZsI-labeled human choriogonadotropin binding it had no effect on the lutropin-induced decrease in lutropin-stimulated cyclic AMP production of these cells (Table Ill). However, the slight potentiation in cholera toxin cyclic AMP production seen in lutropin-pretreated cells was reversed by this acid-washing procedure (Table 111). The results with purified plasma membranes from lutropin and dibutyryl cyclic AMP-pretreated cells were consistent with the results from intact cells (Dix et al., 1982). In plasma membranes from control cells both lutropin plus p(NH)ppG and sodium fluoride plus lutropin caused a 50- to 60-fold linear increase in cyclic AMP production over 40 minutes compared with 15-fold with p(NH)ppG and 6-fold with lutropin alone (Fig. 10). In the plasma membranes isolated from lutropin-pretreated cells the p(NH)ppG and sodium fluoride-stimulated cyclic AMP production rates were not significantly different from those of control plasma membranes, but no effect of lutropin could be demonstrated with or without the addition of p(NH)ppG (Fig. 10). The plasma membranes from the
168
BRIAN A. COOKE
1600- A
I400-
I400-
c .c
ea
. W 0
E
c .-
1200-
0) c
g
1000-
/
aoo -
I
a
n
2
a
I
5
a
0 ._
0 21
20
40
30
Incubation time ( m i n
10
20
30
40
Incubation time ( m i n )
)
t -e a
W
0
0
20
30
40
Incubation time ( m i n )
FIG. 10. Effect of pretreatment of tumor Leydig cells on the plasma membrane adenylate cyclase activity. Purified rat tumor Leydig cells were purified and incubated for 2 hours at 32°C in the presence of ( A ) suspension media, (B) lutropin (I0 pg/ml). and (C) dibutyryl cyclic AMP (2 mM). Cells were then washed and plasma membranes prepared as previously described (Levi er al.. 1982a), and the basal- (a),lutropin- ( I Fgiml) (0). p(NH)ppC- (0. I mM) lutropin ( I pgiml) plus p(NH)ppC- (0. I mM) (A), and fluoride (10 mM) plus lutropin- ( 1 pg/ml)) . ( stimulated cyclic AMP production was determined by radioimmunoassay. Results represent means 2 SEM for triplicate determinations. (From Dix e t a / . , 1982.)
(A),
REGULATION BY GLYCOPROTEIN HORMONES
169
dibutyryl cyclic AMP-pretreated cells had cyclic AMP production rates similar to those of the control cells with all the stimulants studied (Fig. 10). Evidence obtained from in vivo studies suggested that a lesion between the lutropin receptor and the adenylate cyclase moiety develops in the rat testis Leydig cell after administration of human choriogonadotropin (Tsurahara rt a/., 1977; Saez et ( I / . , 1978; Jahnsen et ul., 198I ) . Similar conclusions were reached with ovarian cells (Hunzicker-Dunn and Birnbaumer, 1976; Lamprecht et a / ., 1977). The work just described defined more clearly the site of this lesion as being between the guanine nucleotide binding protein and the lutropin receptor under conditions where no loss in lutropin receptor sites occurred. In the work of Tsurahara et a / . (1977) it was observed that cyclic AMP responses to cholera toxin were retained in testis interstitial cells from human choriogonadotropin-treated rats. In these studies the response to cholera toxin was measured 2 days after human choriogonadotropin treatment at a time when there had been a decrease in lutropin receptor levels. However, with the lowest dose of human choriogonadotropin used (0.2 p.g) causing a loss in lutropin receptors, there had been no decrease in the response to human choriogonadotropin with respect to cyclic AMP production, indicating that the hormone receptors were still able to couple to the guanine nucleotide regulatory protein. In contrast, with the Leydig cells it has been shown that, under conditions in which the number of hormone binding sites is unaltered, lutropin produced a decrease in lutropin stimulation of adenylate cyclase in both intact cells and purified plasma membranes, whereas the cholera toxin response of intact cells and the fluoride and p(NH)ppG responses of purified plasma membranes were not decreased. Since it has been shown that cholera toxin acts through ADP-ribosylation of the G-protein (Cassell and Pfeuffer, 1978) and p(NH)ppG activates the adenylate cyclase through this regulatory protein, the above results clearly demonstrate that the coupling between the G-protein and the adenylate cyclase moiety is intact in lutropin-desensitized cells. They furthermore suggest that the lesion lies at, or proximal to, the coupling of the guanine nucleotide regulatory protein to the lutropin receptor. Such a conclusion is in agreement with those of other workers investigating the isoproterenol-induced desensitization of S49 lymphoma cells (Iyengar et al., 1980). This lesion may be necessary before the internalization of the hormone receptor complex can occur (Conn et a / . . 1978). A further point of interest is that lutropin-desensitized cells show an increased cholera toxin-stimulated cyclic AMP production (Tsurahara et a/., 1977; Dix et al., 1982). Similar potentiation of isoproterenol-stimulated cyclic AMP production in S49 lymphoma cells by cholera toxin has been reported (Insel er a/., 1981). Dufau et d.(1980) have shown that lutropin causes an increase in the availability of the binding sites for GTP on the regulatory protein in rat testicular cell membranes. Such an increase in GTP binding sites may explain the potentia-
170
BRIAN A. COOKE
tion of the action of cholera toxin in lutropin-desensitized tumor Leydig cells and the loss of this potentiation when the bound lutropin is removed. In conclusion, this study clearly indicates that the initial events of lutropininduced desensitization involves an uncoupling of the lutropin receptor from the guanine nucleotide-dependent regulatory protein before any loss of lutropin receptors occur. Although molecular events of this process are still not understood, the ability to prepare large numbers of pure cells and pure plasma membranes from the tumor cells used should facilitate the elucidation of these molecular events.
D. Possible Mechanisms Involved in Desensitization Although little is known if the molecular events which cause desensitization, Hunzicker-Dunn et al. (1979) and Ezra and Salomon (1980) have suggested that desensitization of the ovarian adenylate cyclase system by lutropin involves a phosphorylation reaction. It is of interest to speculate that lutropin-induced desensitization of rat tumor Leydig cells involves phosphorylation, either of the lutropin receptor or of the guanine nucleotide-dependent regulatory protein, and that this phosphorylation impairs the coupling between the hormone receptors and the regulatory protein. However, dibutyryl cyclic AMP did not cause desensitization of the tumor Leydig cells, and therefore if a phosphorylation step is involved it appears not to be cyclic AMP mediated. Experiments carried out with isolated Leydig cell tumor plasma membranes suggested that it may be GTP mediated (Levi et ul., 1982b). It is not known, however, if the desensitized states induced by lutropin in intact cells and by lutropin/GTP in plasma membranes are regulated by the same mechanisms. One main difference that requires investigation is that desensitization in the intact cell is dependent on protein synthesis (Dix and Cooke, 198 I), whereas obviously in the isolated plasma membrane the same mechanism cannot operate. Desensitization could be due to a modification of the receptor and/or the G-protein. Harden et af. ( 1980) found that plasma membrane-receptor preparations devoid of adenylate cyclase migrated at a different rate on concanavalin/ sucrose columns when isolated from desensitized cells compared with controls, suggesting that the receptor had been modified. The same conclusion was reached by lyengar et al. (1980) because they found that the G-protein was not modified in isoproterenol-desensitized S49 cells. Similarly, in the desensitized Leydig cell work described above it was shown that cholera toxin, p(NH)ppG, and fluoride-stimulated cyclic AMP production was not impaired, suggesting that the G-protein was not altered. However, in view of recent results on the purified G-protein it is possible to interpret these results in another way. As described earlier (p. 152) the G-protein contains two subunits (see Fig. 1 I): the
REGULATION BY GLYCOPROTEIN HORMONES
171
Fic;. I 1 , Hypothetical scheme for the hormone-induced desensitization of adenylate cyclase. Binding of the hormone ( H ) with its receptor ( R ) causes activation of adenylate cyclase via the GTP binding protein. The latter is composed of several subunits (CY and p). The p subunit is thought to be the site of interaction of CTP. p(NH)ppG. tluoride ions ( F - ). and cholera toxin. Desensitization. i.e.. decrease o r loss of ability of the hormone to activatc adenylate cyclase, may be caused by a change in the conformation of the G T P binding protein (a subunit?) (involving GTP-mediated phosphorylation?) so that i t can no longer couple to thc receptor. However. the p subunit can still interact with F , cholera toxin (CT). GTP, or p(NH)ppC with subsequent formation of cyclic AMP (CAMP). -
45K/52K (p) unit is the probable site of interaction of F - , cholera toxin, and GTP binding, whereas the 35K subunit (a)may bind to the hormone receptor. It is possible, therefore, that during desensitization the a subunit is modified (via GTP-mediated phosphorylation'?) so that it is no longer able to couple to the receptor, whereas the p site is unmodified and is still able to interact with F - , cholera toxin, and GTP [or p(NH)ppG]. The availability of the purified Gprotein subunits should in the near future make it possible to test this hypothesis. Reversal of desensitization and down-regulation have been shown to occur slowly in virw in both the ovary (Harwood et a l . , 1978) and testis (Hsueh et at., 1976, 1977). In the ovary, for example, the number of receptors for hCG and adenylate cyclase activity were restored to control values after 7 days after injection of amounts of hCG which caused 50-90% loss of receptors. The progesterone production was fully recovered after 5 days (Conti et ul., 1977). In these studies there was a transient loss of the fluoride effect on adenylate cyclase
172
BRIAN A. COOKE
with recovery in 24-36 hours. Desensitization of the desensitized follicular adenylate cyclase to lutropin has also been demonstrated in cell-free preparations (Hunzicker-Dunn rt al., 1979). This was achieved by addition of exogenous phosphoprotein phosphatase partially purified from porcine follicular cytosol. Further experiments with lutropin-desensitized Leydig cells have shown that this can also be reversed (Dix and Cooke. 1982). This was achieved by incubating the plasma membranes prepared from the desensitized Leydig cells with a 10-fold excess of plasma membranes from human erythrocytes. Incubation of the human erythrocyte membranes (which contain the G-protein) (in the absence of chemical fusogens) has been reported (Nielson et al., 1980) to restore adenylate cyclase activity to cyc- cells (which lack the G-protein). Whether the G-protein is actually transferred from the human erythrocyte membranes or somehow acts across the two membranes is not clear. The results with the Leydig cell membrane resensitization are not contrary to the hypothesis depicted in Fig. 11; the a and p subunits may be “transferred” from the human erythrocyte membranes. Alternatively, the processes involved may not be via transfer of the G-protein; they could be a simple human erythrocyte-mediated dephosphorylation of the Leydig cell plasma membranes. ACKNOWLEDGMENTS 1 would like to thank the Wellcome Trust for financial support and Miss B. E. Macey for typing the manuscript.
REFERENCES Abramowitz, I.. lyengar. R., and Birnbdumer, L. (1979). Review: Guanyl nucleotide regulation of hormonally-responsive adenylyl cyclase. M o l . Cell. Endrocrinol. 16, 129- 146. Abramowitz, J . . lyengar. R.. and Birnbaumer. L. (1980). On the mode of action of catecholamines and turkey erythrocyte adenylyl cyclase. J . B i d . Chem. 255, 8259-8265. Abramowitz. J . , Iyengar. R., and Birnbaumer, L. (1981). Receptor regulation and receptor coupling in the gonadotropin-sensitive corpus luteuni. Do gonadotropin and catecholamine receptors activate adenylyl cyclase by different mechanisms? In “Functional Correlates of Hormone Receptors in Reproduction” ( V . B. Mahesh, T. G . Muldon, B. B. Saxena, and W. A. Sadler, eds.), pp. 335-365. Elsevier, Amsterdam. Amir-Zaltsman, Y.. and Salomon, Y . (1980). Studies on the receptor for luteinizing hormone in a purified plasma membrane preparation from rat ovary. Endocrinology 106, 1166- 1172. Birnbdumer, L. ( 1973). Hormone-sensitive adenylyl cyclases useful models for studying hormone receptor functions in cell free systems. Biochim. Biophys. Acfa 300, 129- 158. Birnbdumer. L., and Yang, P. C . (1974). Studies on receptor-mediated activation of adenylyl cyclases 111. J . B i d . Chem. 249, 7867-7873. Bockaert, J . Hunzicker-Dunn. M., and Birnbaumer, L. ( 1976). Hormone-stimulated desensitization of hormone-dependent adenylyl cyclase. J . B i d . Chem. 251, 2653-2663. Bourne, H. R., Coffins, P., and Tomkins. C . (1975). Selection o f a variant lymphoma cell deficient in adenylate cyclase. Srietwe 187, 750-752. Cassel, D.,and Pfeuffer, T. (1978). Mechanism of cholera toxin actions covalent modification of the guanyl nucleotide binding protein of the adenylatc cyclase system. Proc. N a t l . Acud. Sci. U.S.A. 75, 2669-2673.
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME i n
The Activity of Adenylate Cyclase Is Regulated by the Nature of Its Lipid Environment MILES D.HOUSLAY**tAND LARRY M. GORDONf-$ *Department of Biochemistry University of Manchester Institute of Science and Technology Manchesier, England 'California Metabolic Research Foundation La Jolla, California and fRees Stea1.v Research Foundation San Diego, California
Structure of Biological Membranes ................................ Structural Aspects of Hormone Receptor-Adenylate Cyclase Interaction Membrane Fluidity as a Regulator of Adenyl ity ................ A. Benzyl Alcohol.. . . . . . . . . . . . . . . . . . . .................... B. Fatty Acids as Modulators of Adenylate ........... C. Temperature Effects on Adenylate Cyclase Activity and HQWThey Ca Used to Determine a Sensitivity to an Asymmetric Lipid Environment . . . . . . . . D. Manipulation of Phospholipid Species. . . .................... E. Cholesterol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......... F. Hormone-Mediated Alterations in Lipid Fluidity .......................... IV. Selective Modulation te Cyclase by Asymmetric Perturbations of the ....................... Membrane Bilayer.. . A. Positively and Ne harged Local Anesthetics . . B. Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mitogenic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Phospholipid Headgroup Composition and Adenylate Cyclas VI. Disease States., .................................... ........ 1. 11. 111.
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I. STRUCTURE OF BIOLOGICAL MEMBRANES Membrane proteins are noncovalently associated with the lipid bilayer that forms the matrix of the membrane. Such proteins may be divided into two categories: ( 1 ) integral proteins which are firmly embedded in the bilayer; and (2) peripheral proteins which are associated with the membrane through electrostatic interactions. Each copy of a particular integral protein exhibits an absolute asymmetry that is set up during biosynthesis, and the extraction of these proteins from the membrane necessitates the use of detergents. Peripheral proteins, however, can be liberated by merely adding chelating agents or by changing the pH or ionic strength. Recent studies have demonstrated that integral membrane proteins can be variously classified based upon the degree of penetration of their globular portions into the lipid bilayer. Peripheral proteins, on the other hand, can be divided into two categories: those that interact predominantly with phospholipids and those that bind specifically to integral proteins (see Fig. 1). An important feature of the fluid mosaic model is that lipids and proteins, unless specifically restrained, exhibit rapid lateral movement in the bilayer plane (i.e., translational diffusion) or rotational motion about an axis perpendicular to the bilayer plane. The inability of both proteins and lipids, except during biosynthesis, to pass across membranes means that biological membranes are asymmetric with respect to both their protein and lipid constituents (see Warren and Houslay, 1980, and Houslay and Stanley, 1982, for reviews). That a significant fraction of lipid present in biomembranes is in the fluid state has been indicated by the use of electron spin resonance (ESR) probes. Most often these are either fatty acids or phospholipids with a nitroxide reporter group attached to the acyl chain. These labels intercalate into the membrane so that their long molecular axes are perpendicular to the plane of the bilayer where they execute rapid axial rotation and translational diffusion at physiologic temperatures. The fatty acyl chain containing the nitroxide reporter group will concurrently undergo segmental flexing or bending motions, and this may be quantitated by calculating either a polarity-corrected order parameter S or polarityuncorrected order parameters S(T ) and S(T,) from the ESR spectra (Gordon el ul., 1978). These parameters may each range between 0 and I , with the extreme values indicating that the probe samples fluid or immobilized environments, respectively. Since the ESR spectra indicate that such probes primarily sample the fluid lipid phase of biological membranes, the order parameter may be viewed as a measure of the bulk lipid fluidity. Thus the term “lipid fluidity,” as used so frequently, merely denotes the relative motional freedom of reporter groups, be they fluorescent or spin probes, in the membrane. Biological membranes contain a complex mixture of lipids and so do not undergo a well-defined lipid phase transition, as does a pure lipid species, where all of the lipid passes from a fluid to a solid state at a well-defined temperature
181
THE LIPID ENVIRONMENl
. . . . . .
FIG. I . Classes of integral and peripheral proteins. A schematic representation of the various relationships of integral (I-IV) and peripheral (V-VI) proteins with the bilayer. 1. transmembrane protein with globular functioning region inserted into the bilayer: 11, transmembrane fibrous integral protein with globular mass in aqueous compartment: 111, as 11 but with hydrophobic pellicle that does not span the bilayer; IV, globular integral protein associated essentially with one or the other half of the bilayer. A fibrous transmembrane tail may or may not be associated; V . peripheral protein associated with phospholipid headgroups; and VI, periphcral protein associated with integral proteins. (From Houslay, 1981b.)
(see Warren and Houslay, 1980). Instead, lipid phase separations occur where upon lowering the temperature “quasi-crystalline clusters” (QCC) of lipid form domains that coexist with fluid lipid (see Fig. 2 ) . In this instance the QCC are more ordered than the fluid lipid (L) matrix but less ordered than solid lipid (S) which would pack in a quasi-hexagonal assay. Thus as the lipids with the higher lipid phase transition temperatures tend to preferentially form QCC in biomembranes, then changes in both the composition and the fluidity of the bulk fluid lipid pool will result at temperatures below the lipid phase transition. As integral proteins are excluded from ordered lipid domains, then at temperatures below the lipid phase separation we might well expect to see changes in their activity due to both alterations in the composition of the fluid lipid environment
MILES D. HOUSLAY AND LARRY M. GORDON
182 Above lipid phase separation To Ts
>
f I
*.
'
o..;;.:.
<
;0.
fluid lipid wol of compo&ion A
dispersed membrane proteins
Below lipid phase separation To Ts
protein clusters fluid lipid pool Of composition B quasi-crystalline lipid excluding proteins
FIG. 2. The effect of a lipid phase separation on a biological membrane. Lowering the temperature below the onset temperature (T,) of the lipid phase separation causes a preferential segregation of certain phospholipids to form domains of "quasi-crystalline" lipid. Proteins are excluded from this phase and so are left in a fluid lipid domain of diminished size and altered composition. This can lead to changes in activity.
and its reduced size. Effects on lipid composition could affect activity by virtue of either any alterations in lipid fluidity or the availability of specific phospholipids, and diminution in the fluid lipid pool size could promote protein association and hence altered activities (see Fig. 2). These occurrences account for the appearance of break points in the Arrhenius plots of the activities of many integral membrane enzymes and transport proteins (see Kimelberg, 1977). Here, the break point usually coincides with a lipid phase separation, indicating that the altered lipid environment affects the activity of the biological process. Recent studies, however, report that at physiologic temperatures, the lipids of many biological membranes are not homogeneously distributed. Instead lipid domains of differing fluidity and/or structure may coexist in biomembranes (see Marinetti and Crain, 1978; Malchior and Steim, 1979). Indeed, there is evidence to suggest that cholesterol-rich and -poor domains can coexist in plasma membranes. This would explain the occurrence of thermotropic lipid phase separations in membranes with cholesterol contents (cholesteroliphospholipid molar ratio 30.7)sufficient to abolish any such event if it were homogeneously distributed (see Montesano et al., 1979; Houslay and Palmer, 1978).
THE LIPID ENVIRONMENT
183
That the formation of relatively solid lipid domains in a liquid lipid matrix dramatically influences the activity of penetrant enzymes is not unexpected, in view of the effects that lowering the temperature below T, has on the distribution of membrane proteins and the lipid environment of the sequestered proteins (Fig. 2 ) . Nevertheless, the mere fact that Arrhenius plots of an enzyme activity exhibit “breaks” at temperatures similar to those detected by extrinsic probes provides only circumstantial evidence that such a relationship exists. Additional studies are required to rule out the possibility that any breaks in Arrhenius plots of activity are simply due to protein-mediated events that are independent of the lipid environment of the enzyme. One approach to this problem is to manipulate the lipid environmenVfluidity of the membrane with various agents and then concurrently determine Arrhenius plots of the enzyme activity and the mobility of an incorporated reporter group. Perturbants that have been used successfully in such experiments include ( I ) addition of the neutral local anesthetic benzyl alcohol or charged local anesthetics; ( 2 )incorporation of exogenous lipid through lipid substitution or fusion techniques; (3) alterations in ambient pressure; and (4) dietary manipulation, which can take the form of the addition of supplements or inhibitors of the biosynthesis of specific lipid to either the culture media of cells or to the diet of animals. By simultaneously examining the action of these interventions on the lipid phase separation sensed by extrinsic probes and the breaks in Arrhenius plots of enzyme activity, it should be possible to assess unequivocally any relationship that may exist between the lipid phase separation and the enzyme activity (Houslay and Stanley, 1982). A second approach involves the solubilization of the membrane enzyme with an appropriate detergent and then the construction of Arrhenius plots of the solubilized activity. If the breaks observed in the Arrhenius plots of the membrane-bound activity are indeed a consequence of the thermotropic lipid phase separation, such treatment should profoundly alter the form of the Arrhenius plots and may even act to abolish the break point (Dipple and Houslay, 1979a). Hormone-stimulated adenylate cyclase presents us with a complex system of asymmetrically oriented integral membrane proteins. This article examines the role that the membrane environment has in influencing its activity. II. STRUCTURAL ASPECTS OF HORMONE RECEPTOR-ADENYLATE CYCLASE INTERACTION Detailed kinetic and molecular descriptions of the interactions among hormone receptors, the guanine nucleotide coupling protein, and adenylate cyclase will be found elsewhere in this volume (see articles by Tolkovsky and Martin). However, it is of some importance to describe the organizational aspects of the interactions among these components as they pertain to our interpretation of lipidmediated effects.
184
MILES D. HOUSLAY AND LARRY M. GORDON
Receptors specific for certain ligands, the guanine nucleotide coupling protein, and the catalytic unit of adenylate cyclase are known to be distinct proteins under separate genetic control (see Ross and Gilman, 1980). A number of independent approaches support the concept that these three entities are able to undergo independent free lateral diffusion within the bilayer, interacting functionally and structurally only in the presence of appropriate stimulating ligands (see Houslay et al., 1977, 1980a; Houslay, 1981a; Schramm et al., 1977; Tolkovsky and Levitzki, 1978; Hanski et al., 1979; Martin et al., 1979). In general it would seem that hormones activate adenylate cyclase in the presence of GTP by a collision coupling mechanism. The following equilibria may thus exist between hormone-occupied receptor (HR), guanine nucleotide coupling protein ( G ) ,and the catalytic unit of adenylate cyclase in its basal (C) and activated (C*) activity states: HR G*
+G +C
-
HRG* GC*-t G
+ C*
c* + c
In this instance the steady-state activity that is measured reflects the active states of GC* and perhaps C*. However, in rat liver plasma membranes at suboptimal GTP concentrations (10-7-10-8 M ;see Kimura and Nagata, 1977) a mobile receptor model is obeyed (see Houslay, 1981a; Houslay et al., 1980a) such that the active species that accumulates is a transmembrane complex formed from HR, G, and C*. This is in contrast to the collision coupling model which is observed when optimal GTP concentrations (lopsM )are used (Houslay, 1981a). All of the components of this system are asymmetrically disposed within the bilayer such that R has its binding site for hormone at the external surface of the cell, whereas C and G have their substrate binding sites at the internal surface of the membrane (Houslay et al., 1980a). Although all three entities are integral proteins, G and C bind little detergent (see Neer, 1976; Ross and Gilman, 1980), indicating that only a relatively small fraction of their mass is actually interpolated into the bilayer. Indeed, as will be developed in Section 111, it would seem that the functional globular part of both G and C penetrates only into the cytosolfacing half of the bilayer. Thus the steady-state, basal, and guanine nucleotide/ fluoride activities are influenced only by the lipid environment of the internal half of the bilayer. On the basis of such considerations it has been suggested that all receptors should be transmembrane entities in order that they may interact appropriately with the guanine nucleotide (G) unit localized at the cytosol surface of the membrane (Houslay, 1981a). This is because reactions among proteins are likely to exhibit greater specificity if they occur in a polar environment. On the basis of such structural interactions we would expect only the steady-state activity of the catalytic unit to be sensitive to the physical properties of the external
185
THE LIPID ENVIRONMENT
half of the bilayer when a mobile receptor model is obeyed, yielding a complex of HR-G-C*. The steady-state activity during a collision coupling mechanism would, on the other hand, merely exhibit a sensitivity to the physical properties of the inner half of the bilayer. However the rate constant for activation of G by HR would undoubtedly be affected by the properties of the lipids of the external half of the bilayer as well as the internal half (see Rimon et al., 1978, 1980). Such results have been clearly demonstrated for the rat liver and hamster liver enzymes (Houslay et al., 1980a). As will be seen in Section 111, lipid fluidity has a marked effect on the steadystate activity of adenylate cyclase, presumably by altering the physical constraints imposed upon the enzyme by the bilayer. However, it has also been demonstrated (Rimon et al., 1978) that the fluidity of the bilayer can influence the rate of activation of turkey erythrocyte adenylate cyclase by P-agonists. This would be consistent with the need for the components to undergo independent lateral diffusion within the plane of the bilayer before functional collisions occurred. However, as the full activation of adenylate cyclase by hormones occurs within seconds, then the metabolically relevant effects of fluidity are going to be predominantly on the steady-state activity of the enzymes and not on the rates of activation of the enzyme. These are discussed in the following sections.
111.
MEMBRANE FLUIDITY AS A REGULATOR OF ADENYLATE CYCLASE ACTIVITY
The ring of lipids that immediately surrounds an integral membrane protein has been termed annular lipids (see Warren et al., 1975). Lipids in the annular domain act to solvate integral proteins into the membrane bilayer. These lipids, which are able to exchange with lipids in the bulk lipid pool, albeit at a slower rate, interact with the integral proteins in such a way as to minimize nonspecific leakage at the lipid-protein interface and to provide a suitable environment for the protein to function. There are two basic ways in which the lipid bilayer can modulate the activity of integral proteins. First, the chemical nature of the lipid may exert specific effects, and second, the physical properties of the bilayer, in particular its fluidity or rigidity, can influence not only the lateral migration of the protein, but also the internal motions of groups or peptide chains within the protein connected with its function (Houslay and Stanley, 1982). The lipid fluidity can be perturbed in a number of ways. One of the simplest means of achieving this is to change the temperature, where increased temperature relates directly to increased fluidity. However, lipid fluidity can be altered at physiologic temperatures in in vitru experiments using a wide variety of agents such as local and general anesthetics, fatty acids and fatty alcohols, and cations such as Ca2 , Mg2 , and La3 . Such perturbants can also be expected to have +
+
+
186
MILES 0.HOUSLAY AND LARRY M. GORDON
direct effects on the protein that are independent of any changes in bilayer fluidity or lipid domain organization that may be effected by the agents. In this section we attempt to portray the important role that bilayer fluidity plays in determining the activity of adenylate cyclase in its membrane environment.
A. Benzyl Alcohol This is a small, water-soluble molecule that readily partitions into lipid bilayers where it is known to orient itself with its hydroxyl group in the headgroup (polar) region and its aromatic nucleus directed into the apolar core of the bilayer (Colley and Metcalfe, 1972). Benzyl alcohol, when inserted into a lipid bilayer, increases the fluidity and decreases the lipid base transition (Ipt)/separation (Ips) temperature by some 6"-8"C at a concentration of 40 mM. This depression is simply analogous to an impurity's depressing the melting point and reflects the extra crystal lattice energy required to expel benzyl alcohol from solid (crystalline) phase lipid. Benzyl alcohol is a neutral molecule; this precludes any selective charge interactions with the protein or bilayer lipids, and,
FIG. 3. Benzyl alcohol increases the fluidity of rat liver plasma membranes detected with 5-nitroxide stearate. This demonstrates the decreases in order parameters, which signify an enhanced bilayer tluidity. with increasing concentrations of benzyl alcohol. Rat liver plasma membranes at 37°C were the membrane source. (Adapted from Gordon ei al., 1980a.)
187
THE LIPID ENVIRONMENT
because it is water soluble, it can simply be removed by washing or diluting the membranes. As such, it is an ideal modulator of bilayer fluidity and has been used successfully to probe the effects of increasing fluidity, as detected by ESR and fluorescence studies, on adenylate cyclase from a number of sources (Dipple and Houslay, 1978; Gordon er al., 1980a; Brasitus and Schachter, 1980; Krall et af.. 1981; Amir et al., 1981). In rat liver plasma membranes, benzyl alcohol achieves a marked increase in bilayer fluidity (Fig. 3) and at 40 mM benzyl alcohol this effect is equivalent to that elicited by a 6°C rise in temperature (Fig. 4). The action of benzyl alcohol on the adenylate cyclase activity associated with these membranes is really quite remarkable. The hormone response is activated some twofold and considerable effects are seen on the fluoride-, guanine nucleotide-, and glucagon-stimulated activities, although the effect on the basal activity is quite small (Fig. 5). Indeed,
3.5 -
3.040 I
30 l
T ("c)
20
10 I
l
0.70 L A
I
-
. 0.65
2.5-
W
OI
-
I
c
t
2.0 -
Glucagon Native
1.5-
I
3.20
I
I
I
I
3.30
llrrPKll
3.40 x
I
I
3.50
3.2
3.3
3.4
3.5
3.6
IO~IT~K)
FIG. 4. Benzyl alcohol depresses the lipid phase separation temperature in rat liver plasma membranes. A decrease in the high-temperature onset of the lipid phase separation from 28" to 22°C by 40 mM benzyl alcohol (A) is detected by ESR studies using a fatty acid spin label, and also in Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase (B). (Adapted from Dipple and Houslay. 1978, and Gordon et a/.. 1980a.)
3.7
MILES D. HOUSLAY AND LARRY M. GORDON
188
GLUCAGON
GLUCAGON
im
+ GTP
U
u
OO
10
Y)
110
S o i m
[BENZYL ALCOHOL] mM Fia. 5 . Effect of the neutral, local anesthetic benzyl alcohol on ligand-stimulated adenylate cyclase activity from rat liver plasma membranes at 30°C. p(NH)ppG, Guanosine 5’-(pwimidoltriphosphdte. (Adapted from Dipple and Houslay, 1978. and Houslay ei u/.r 1980a.)
the relative insensitivity of basal activity, compared with the ligand-stimulated activities, to changes in bilayer fluidity means that the physical state of rnembrane lipid can have a strong influence on the fold stimulation achieved by these ligands. The change in fluidity elicited by 40 mM benzyl alcohol can double the activating potency of glucagon on this system (Fig. 6), whereupon it can achieve a 44-fold, rather than a 22-foId, stimulation (Houslay et al., 1980a). How do we know whether benzyl alcohol is exerting its effects on adenylate cyclase by increasing membrane fluidity? In the first instance, low concentra-
THE LIPID ENVIRONMENT
189
1.8
-
1.6 -
1.4 -
ACTIVATION RATIO o / a c
%BAC
-
1.2
1.0
c--
.\
0.8 0
20
4
0
6
0
8
0
[BENZYL ALCOHOL] mM
FIG. 6 . Benzyl alcohol increases the net activation of adenylate cyclase by glucagon. Glucagon normally activates adenylate cyclase about 20-fold over basal (see Houslay et d.,1980a). However, increases in bilayer fluidity. achieved by benzyl alcohol, can almost double this effect (data adapted from Fig. 5). GSAC, Glucagon-stimulated adenylate cyclase: BAC, basal adenylate cyclase activity.
tions of benzyl alcohol increase in parallel both enzyme activity and the membrane fluidity detected with a fatty acid spin label (see Fig. 7). Furthermore, if a detergent-solubilized enzyme preparation is treated with benzyl alcohol, then no activation of the enzyme occurs over the range of concentrations tested here (see Dipple and Houslay, 1978; Gordon et af., 1980a). Finally, Arrhenius plots of all ligand-stimulated activities exhibit decreased activation energies and, in the case of the glucagon-stimulated activity in the presence of 40 mM benzyl alcohol, a depression of the break point from 28" to 22°C (Dipple and Houslay, 1978). This is in accord with the depression of the high-temperature onset of the lipid phase separation occurring in the external half of the bilayer of these membranes (Dipple and Houslay, 1978; Gordon et al., 1980a; see Fig. 4). It is likely that low concentrations of benzyl alcohol, by increasing the fluidity of the membranes, relieve the constraint imposed upon the enzyme by its surrounding lipids; hence its conformational flexibility, and thus its activity, is increased. Two other facets of these dose-response curves require comment. These are the inhibition at high benzyl alcohol concentrations, which occurs even though the lipid fluidity is increasing, and the inhibition of fluoride-stimulated activity manifest at low benzyl alcohol concentrations. The high-concentration inhibition seen with a variety of neutral and charged anesthetics for adenylate cyclase and some other integral proteins (see Gordon el al., 1980a,b) is not observed using detergent-solubilized membrane preparations and so is not due to the direct action of these drugs at the hydrophilic surfaces of the protein. instead this effect
190
MILES D. HOUSLAY AND LARRY M. GORDON
s (%)
GLUCAGON- (C STIMULATED ADENYLATE CYCLASE
1
160
-A order parameter (%)
"t
b
10
144
120
0
2 0 4 0 0 8 0
[BENZYL ALCOHOL] mM
GLUCAQON-STIMULATED ADENYLATE CYCLASE (YO)
FIG. 7 . Benzyl alcohol activates adenylate cyclase by increasing bilayer fluidity at 37°C. The concentration dependence of benzyl alcohol's activation of adenylate cyclase (a) parallels its ability to increase the fluidity of the lipid bilayer as measured by the changes in order parameter (AS, A.S(Tll))for a fatty acid spin probe inserted in the membrane (b).(Data adapted from Gordon el a / . . I980a.)
is believed (see Gordon er al., 1980a) to be due to benzyl alcohol competing for domains on the protein that are normally occupied by annular lipid. Differences in sensitivity to inhibition by high benzyl alcohol concentrations [e.g., the reduced sensitivity seen with guanine nucleotides (Fig. 5 ) ] imply a changed conformation of the enzyme and perhaps an altered interaction of the enzyme with bilayer lipids. Thus when the anesthetic concentration is sufficiently high it can displace the-annular lipid, leading to a loss of function, either because lipid is essential for activity or because benzyl alcohol can itself inhibit the reaction when it occupies this site. Such an effect has been termed the annular displacement or disruption model (Gordon et d . , 1980a; Katz and Messineo, 1981) and has been proposed as a mechanism for local anesthesia where the target protein would be the Na+ channel. The inhibition of fluoride-stimulated activity seen at low benzyl alcohol concentrations (Fig. 5 ) is believed to be due to a disruption in the headgroup region of the bilayer elicited by the aromatic nucleus of the alcohol (see Dipple and Houslay, 1978). This effect is not observed by using homologs of benzyl alcohol where the aromatic ring is embedded deeper into the bilayer core away from the headgroup region. Furthermore, if stimulation is achieved using other ligands (e.g., glucagon or guanine nucleotides) then no inhibition is seen either because the perturbation of this area of the bilayer is insignificant or because the proteins now adopt a very different vertical position (e.g., by receptor-catalytic unit coupling). There is one caution about the use of benzyl alcohol in membrane studies, namely, that at high concentrations (ca. 100 mM) it has been shown to induce
191
THE LIPID ENVIRONMENT
fusion and to activate a Ca2 +-dependent membrane proteinase in erythrocytes (Ahkong et al.. 1980). In the experiments described above, all of the effects could be readily reversed by washing to remove the alcohol (Dipple and Houslay, 1978). Furthermore, the addition of N-a-tosyl-L-lysine chloromethylketone, a specific inhibitor of the proteinase, did not influence the results (L. Needham and M. D. Houslay, unpublished results). Are such effects on purified plasma membranes of any physiological relevance? It is significant that if isolated whole rat hepatocytes are exposed to benzyl alcohol, both the glucagon-mediated rate and degree of increase of intracellular cyclic AMP concentration are augmented, presumably by the increase in bilayer fluidity achieved (Fig. 8). The effect of benzyl alcohol in activating adenylate cyclase by increasing bilayer fluidity is apparently a general phenomenon. It has now been observed in liver plasma membranes (Dipple and Houslay, 1978; Gordon ef al., 1980a), intestinal basolateral membranes (Brasitus and Schachter, 1980), uterine smooth muscle (Krall et al., 1981), human thyroid membranes (Amir ef al., 1981), and brain caudate nucleus (L. Needham and M . D. Houslay, unpublished results). The activations observed at physiologic temperatures are really quite dramatic, varying from 2- to 10-fold at 50 mM benzyle alcohol, depending upon the enzyme source and the ligand used for stimulation. Such results imply that
50-
glucagon
+ 10 rnM benzyl alcohol
a--. a.------
10 -
OV 0
I
I
I
1
I
2
4
6
0
10
TIME (rnin) FIG. 8. Benzyl alcohol augments the glucagon-triggered rise in intracellular cyclic AMP in hepatocytes. Hepatocytes were incubated under conditions described in detail previously (Houslay et 01.. 1980a). (Unpublished data of K . R . F. Elliott and G . A. Turner.)
192
MILES D. HOUSLAY AND LARRY M. GORDON
adenylate cyclase activity is exquisitely sensitive to alterations in membrane fluidity. Thus an appreciation of the bilayer fluidity and how it can be perturbed by xenobiotics or disease is of considerable interest to our understanding of the regulation of adenylate cyclase.
B. Fatty Acids as Modulators of Adenylate Cyclase Activity Fatty acids of various chain lengths and degree of unsaturation have been used to probe adenylate cyclase activity in turkey erythrocytes (Orly and Schramm, 1975). Certain of these led to a 2- to 3-fold activation of catecholamine-stimulated adenylate cyclase activity at 37"C, and as much as a 16-fold activation at 20"C, whereupon it was suggested that they could be exerting their effect by increasing membrane fluidity. The most potent agent, cis-vaccenic acid, does indeed increase membrane fluidity as detected by a fluorescent probe (Hanski et al., 1979). However, it would seem, from the available evidence, that the activating effects are extremely sensitive to the structure of the fatty acid, suggesting that they act through direct interactions on components of the adenylate cyclase system as well as by perturbing the membrane fluidity. Consistent with such an interpretation is the observation that there is no close correlation between the increase in membrane fluidity achieved by cis-vaccenic acid and the activation of adenylate cyclase (Fig. 9). Moreover, the lower the melting point of the fatty acid, the more potent a fluidizing agent it should be, and hence one might
[cis
- Vaccenic Acidj
mole mg'' protein
FIG. 9. Stimulation of adenylate cyclase by cis-vaccenic acid in turkey erythrocytes. The increase at 25"C, in both the steady-state L-epinephrine + GppNHp adenylate cyclase activity and membrane fluidity monitored using DPH microviscosity is shown as a function of cis-vaccenic acid concentration for turkey erythrocyte plasma membranes. (Data adapted from Hanski et a / .,
1979.)
193
THE LIPID ENVIRONMENT
ACTIVATION RATIO
t
-10
0
10
20
30
40
50
€4 70
Melting point ("C)
FIG. 10. The dependence of the activation of turkey erythrocyte adenylate cyclase activity on the melting point of fatty acids at 30°C. The activation ratio (activity plus fatty acidiactivity without fatty acid) for the isoproterenol-activated state of the enzyme as a function of the melting point of a selection of fatty acids having different chain lengths and varying in their degree of saturation.
expect the more potent an activating agent. Figure 10 shows that this does not appear to be the case. The fatty acids that are the most vigorous activators are unsaturated and it may well be that the specificity for a direct interaction lies at the position of the double bond (Fig. 1I ) . Here it can be seen that, irrespective of chain lengths between 16 and 20 carbons, optimum activating effects are observed with fatty acids having the double bond in the C-9-C-10 or C-11-C-12 position even though they exhibit disparate melting points. Indeed disruption of this region with two double bonds, as occurs in linoleic acid (A9,12-C18:2c), dramatically reduces the activation effect to only 4-fold, even though the melting point of this fatty acid (-5°C) is lower than those fatty acids eliciting 10- to 16-fold activations. Furthermore, the enzyme, after full preactivation by both guanyl nucleotides and hormone, is essentially insensitive to the action of fatty acids. Although changes in conformation induced by the state of activation can modulate the response to fluidity (see Fig. 5; Dipple and Houslay, 1978; Houslay, 1981a), the dramatic nature of this effect is more likely to be due to the inability of the complex to interact directly with the fatty acid. Clearly it would be of interest to examine the effect of fatty acids on the solubilized enzyme and also to look at their dose-response curves. To discriminate unequivocally between direct effects on the enzyme and those transmitted through the lipid bilayer, the action of fatty acids on the fluidity of membranes labeled with extrinsic reporter groups (e.g., fluorescent or spin labels) must also be assessed. Studies on fatty acid effects are of importance in their own right, as
194
MILES D. HOUSLAY AND LARRY M. GORDON
16 14
0
s I-
13OC
a
-
12 10-
z 0
l-
8-
a
2
iV
a
6443oc
0
6-7 7-8 8.9 9-10 10.11 11-1212-1313-14 14-15 15-16
POSITION OF CIS DOUBLE BOND FIG. 1 I . The influence of double bond position on the activation of turkey erythrocyte adenylate cyclase by unsaturated fatty acids. The activation ratio of the isoproterenol-activated enzyme as a function of the position of the cis double bond in the applied fatty acid. Melting point and chain length are denoted for each fatty acid. All fatty acids were I mM and experiments were carried out at 30°C.(Data adapted from Orly and Schramm, 1975.)
fatty acids are released in membranes through the action of phospholipases and as high levels of circulating fatty acids have been observed in patients after myocardial infarction. Fatty acids may be involved in cell fusion events and have also been implicated in the release of cytosol enzymes from ischemic myocardial cells (Katz and Messineo, 1981). However, they do not appear to be particularly useful tools for investigating the relationship between membrane fluidity and adenylate cyclase, as they undoubtedly perturb adenylate cyclase directly as well as indirectly by modulating bilayer fluidity.
C. Temperhture Effects on Adenylate Cyclase Activity and How They Can Be Used to Determine a Sensitivity to an Asymmetric Lipid Environment Arrhenius plots of enzyme activity can be useful indicators of a sensitivity of the function to its lipid environment, providing that adequate data points are collected, certain controls are made, and any breaks observed are rigorously shown to be due to the lipid environment (see Section 111,A). Lipid phase separations occur at 26" and 13°C over a temperature range of 0"-45"C in plasma membranes from hamsters (Mesocricetus aureus). Both of these thermotropic lipid phase separations are detected by the activity of glu-
195
THE LIPID ENVIRONMENT
cagon-stimulated adenylate cyclase (Houslay and Palmer, 1978) under conditions in which it undergoes a mobile receptor mechanism, forming a transmembrane complex. However the fluoride- and guanine nucleotide-stimulated activities sense only the lipid phase separation occurring at 26°C. Due to the known asymmetry of the adenylate cyclase system and that of other integral plasma membrane enzymes which sense both or either one or the other of these lipid phase separations dependent upon their penetration into the bilayer, the lipid phase separation at 26°C has been assigned to the inner (cytosol-facing) half of the bilayer and that at 13°C to the outer half [ i .e., that originally facing the cell exterior (Fig. I2)]. Interestingly, hamsters, which normally have a body temperature of ca. 40°C when housed at 20"C, can be induced to hibernate in the dark at 4°C. Under such conditions their body temperature drops to ca. 6°C. This is accompanied by an adaptive response in their plasma membranes whereupon an asymmetric change leads to the selective depression of the lipid phase separation, occurring in the outer half of the bilayer, to 4°C (Houslay and Palmer, 1978).Indeed many organisms exhibit such adaptive responses in an attempt to maintain a constant mem-
UCTERNAL SURFACE
Conl.
Hib.
13
4
26
26
INTERNAL SURFACE
FIG. 12. Sensitivity of integral enzymes in hamster liver plasma membranes to lipid phase separations. Distinct lipid phase separations occur in both halves of the bilayer of hamster liver plasma membranes. Dependent upon whether integral enzymes are transmembrane o r their functional globular region lies in one or the other half of the bilayer. the activity of these enzymes is modulated by one or both lipid phase separations. When adenylate cyclase is in its basal state or coupled to the guanine nucleotidc regulatory component (which would appear to reside exclusively in the inner half of the bilayer). i t only senses lipid phase separations occuring in the inner half. When a transmembrane complex of receptor. G-protein, and C-unit form, then the activity senses both lipid phase scparations. A change in membrane asymmetry occurs upon hibernation (Hib.) of control (Cont.) animals. Mg*+ ATPase. Mg'+ -dependent ATPase; S'Nu, S'huclcotidase; AC. adcnylate cyclasc complex of catalytic unit and G-protein; GR, glucagon receptor; C. glucagon: CAMP PD. cyclic AMP phosphodicstcrasc; PD I . phosphodicstcrasc I '. (From Houslay and Palmer. 1978.)
MILES D. HOUSLAY AND LARRY M. GORDON
196
brane fluidity, and this phenomenon has been termed homeoviscous adaptation (Kimelberg, 1977; Houslay and Stanley, 1982). This effect appears to facilitate activation of adenylate cyclase by hormone at the decreased body temperature, as can be seen from Arrhenius plots of the fold activation of adenylate cyclase by glucagon (Fig. 13). In normal animals the activation energy for this process is 32 kJ mole- I at 40"C, and even higher at 42 kJ mole- if this process were to occur at ca. 6°C. However, in membranes from hibernating animals the activation energy is reduced to only 6 kJ mole- at 6°C; the activation energy is now only some 18% of that seen with normal animals at
CONTROL ANIMALS
0.9 -
P
3
0.8 -
z
0.7 -
7
'5 ;
4
.
13.5 "C
'.
't
0.6-
0.5 -
8 0 3.15 -1
3.25
3.35
3.45
3.55
3.65
[I/T°K] x lo00
HIBERNATING ANIMALS
a
5
5 5
-1
0.8 0.7 0.6
I
0.3 3.15
4°C'
3.25
3.35
u 3.45 3.55 3.65
[I I T lq x lo00
FIG. 13. Arrhenius plots of the net stimulation by glucagon of liver plasma membrane adenylate cyclase activity from control and hibernating hamsters. The fold stimulation by glucagon (glucagon activity/basal activity) is shown as a function of temperature. thus yielding the activation energy for the activation process. (Data adapted from Houslay and Palmer, 1978.)
THE LIPID ENVIRONMENT
197
37°C or only 14% of that of normals at 6°C. Thus changes in the lipid environment can have a marked effect on adenylate cyclase function, showing, in this instance, a considerable facilitation of the activation process by a mechanism which presumably increases the fluidity of the external half of the bilayer (Houslay and Palmer, 1978). In rat liver plasma membranes there is a thermotropic lipid phase separation occurring at 28°C which, by the criteria discussed above (see also Section lll,B), is apparently localized to the external half of the bilayer (see Houslay el af., 1976a,b,c). N o corresponding lipid phase separation occurs in the inner half of the bilayer over a temperature range of 0"-45°C. The break occurring at 28°C in Arrhenius plots of activity for enzymes connected in some way with the external half of the bilayer (see Fig. 12) reflects the sensitivity of these proteins to the high-temperature onset of the lipid phase separation. This is believed to be due to the formation of "quasi-crystalline" clusters (QCC), having a packing density between that of solid-phase (S) lipid and fluid (L) lipid. These QCC coexist with L lipid pools in the membrane for temperatures between 28" and 19°C; however, at 19°C physical probes detect the low-temperature onset of the lipid phase separation where the QCC aggregate to form S domains within an L lipid matrix (see Gordon et a / . , 1978, 1980a). For reasons discussed at some length earlier (Gordon e t a / ., 1980a), we believe that penetrant proteins are excluded from both QCC and S domains. Integral enzymes, associated with the external half of the bilayer, will sense the break only at 28"C, as it is the onset of a new phenomenon at this point (i.e., the recruitment of specific lipids from L into QCC) that affects the properties of their lipid microenvironment and hence their activity. The lowtemperature onset at 19°C merely reflects the aggregation of QCC to form larger S pools and will not perturb integral enzymes which are preferentially partitioned into the fluid L domains. It should, however, be pointed out that certain integral enzymes restricted to the cytosol half of the bilayer, such as fluoride-stimulated adenylate cyclase (i.e., the uncoupled activity) and cyclic AMP phosphodiesterase, do not sense this lipid phase separation and exhibit linear Arrhenius plots of activity (Houslay et al., 1976a,b,c). Arrhenius plots of the basal adenylate cyclase activity in rat liver plasma membranes exhibit a discontinuity at around 20-25°C (Pliego and Rubalcava, 1978; Rene et al., 1978; Houslay, 1979), and it is worthwhile to consider whether this break indicates a sensitivity to the low-temperature onset of the lipid phase separation. This does not, however, appear to be the case as the break point is unaltered by the presence of 50 mM benzyl alcohol (Fig. 14), even though the low-temperature onset of the lipid phase separation is depressed by some 6°C (Fig. 4). Thus the use of benzyl alcohol allows us to ascribe the break in the Arrhenius plots of the basal activity at 20-25°C to a protein-mediated effect. Breaks at around 25°C in the Arrhenius plots of basal adenylate cyclase
198
MILES D. HOUSLAY AND LARRY M. GORDON
P C
f
1.0 -
pmsence of
€>
50 mM bensyl alcohol
E
a Y
3 0
>
0 0.5
-
5>
\
absence of /” bensyl alcohol
E0 U
I
D3.1
I
3.2
3.3
3.4
I
4
I
3.5
3.8
3.7
[ 1 / T qx1m
FIG. 14. The break in the Arrhenius plot of basal adenylate cyclase plot is unaffected by changes in lipid fluidity. Experiments were carried out on rat liver plasma membrane adenylate cyclase as before (Dipple and Houslay. 1978). Benzyl alcohol, which depresses lipid phase separations, has no such effect here. This implies that the break is protein- not lipid-mediated (1. Dipple and M . D. Houslay , unpublished).
activity have also been noted in rat brain (Bar, 1974), turkey erythrocytes (Orly and Schramm, 1975),rat enterocyte basolateral membranes (Brasitusand Schachter, 1980), and mouse LM cells (Engelhard et al., 1976). We would like to suggest that this is a characteristic of basal adenylate cyclase and reflects the E’, such presence of an equilibrium between two states of the enzyme, E that E predominates at high temperatures and E’ at low temperatures (below ca. 25°C). These two states of the enzyme would display different activation energies for cyclic AMP production. They would also exhibit very different abilities to be activated by guanine nucleotides (see Orly and Schramm, 1975; Krall et al., 1981). Such a two-state model is not unlike that suggested by Iyengar ef al. (1980) on kinetic grounds. Consistent with such an interpretation are the findings that membrane fluidizing agents such as benzyl alcohol or cis-vaccenic acid have little or no effect on the break temperature (see Fig. 14; Orly and Schramm, 1975; Brasitus and Schachter, 1980) and that conversion to the fully activated state using fluoride, guanine nucleotides, or hormones plus guanine nucleotides abolishes the break or changes it to a lipid-dependent one (Houslay, 1979; 1981a;
-
199
THE LIPID ENVIRONMENT
Bar, 1974; Rene et a / . , 1978; Pliego and Rubalcava, 1978; Engelhard et a / . , 1976; Brasitus and Schachter, 1980). These studies emphasize the importance of distinguishing between protein- and lipid-mediated breaks in Arrhenius plots and also indicate the usefulness of Arrhenius plots of activity in understanding the molecular processes of adenylate cyclase activation. There are many observations (see, e.g., Houslay et al., 1976a,b,c, 1981a; Dipple and Houslay, 1978; Rene et a / . , 1978) that, under conditions in which a mobile receptor mechanism is obeyed, the glucagon-stimulated adenylate cyclase reaction displays a lower activation energy at temperatures above the break (at 28°C) than below. In contrast to this, Keirns et ul. (1973), while reporting a break at a similar temperature, observed an increase in the activation energy at temperatures above the break point. I . Dipple and M . D. Houslay (unpublished) have now managed to resolve this apparent controversy by noting that Keirns et a / . (1973) used weanling rats. It is well known that during the weanling period dramatic changes occur both in the cholesterol content and the degree of unsaturation of phospholipids in membranes. Indeed, marked lipidmediated changes in the activity of P-hydroxybutyrate dehydrogenase have been noted (Houslay et a/., 1978). We can now reproduce the results of Keirns et a / . (1973) by using 4-week-old animals to prepare membranes; however, by the time the animals attain 6 weeks of age, the adult situation, as reported by us previously (Houslay et a/., 1976a,b,c), is attained (Table I). This effect appears to be lipid-mediated rather than reflecting a change in the protein, as similar results were obtained using 5'-nucleotidase (1. Dipple and M. D. Houslay, unpublished results), another integral enzyme that is sensitive to lipid fluidity (Gordon et al., 1980a; Dipple et a/., 1982).
C H A N G I S IN 'IHL.. FORMO b
TABLE I ARRHENIUS PLOT-S O b
LIVER PIASMA Mt:MBKANt
WITH MA.T\JRATION OI. 'IHL
ENZYMES
RATS"
Ligand-stimulated adenylate cyclase activity Glucagon
Break point ("C) Activation energy above break (kJ moleActivation energy below break (kJ mole ~
5'-Nucleotidase activity
Fluoride
4 weeks
8 weeks
4 wceks
28 82
28 13
L -
36
60
70
8 weeks
4 weeks
8 wceks
L
28 57
28 47
84
44
95
~
I)
I)
Arrhenius plotb were conducted on enzyme activities of the liver plasma membrane enzymes from male Sprague-Dawley rats at 6 and 8 weeks old. L, Linear.
200
MILES D. HOUSLAY AND LARRY M. GORDON
Temperature studies on the adenylate cyclase activity of turkey erythrocytes are somewhat confusing as there appears to be disagreement on both the presence of break points and the temperatures at which they occur (Orly and Schramm, 1975; Rimon et a / . , 1980). This is partly due to insufficient data points and a restricted range of observations. However, the low-temperature break point observed by both groups at 24"-26"C in the partially activated state, which is insensitive to changes in bilayer fluidity (Orly and Schramm, 1975) and disappears upon full preactivation (Orly and Schramm, 1975; Rimon et al., 1980), is undoubtedly protein mediated, as discussed above. The upper break at 35°C detected by Rimon et af. (1980) for the enzyme in the fully activated state could well be lipid mediated; nevertheless, there is no independent evidence to provide strong support for this hypothesis at present. For although a number of apparent discontinuities in the temperature dependence of the fluorescence intensity of dansyl phosphatidylethanolamine-labeledmembranes were observed (Rimon et a / . , 19801, previously these workers had claimed the absence of lipid phase separations based upon fluorescence polarization studies with diphenylhexatriene (DPH) (Hanski et al., 1979). Clearly this is a fascinating system to study the interrelationships of both the P-receptor and adenosine receptor with adenylate cyclase in view of the elegant models proposed for their interaction (Tolkovsky and Levitzki, 1978). However, it would seem rather premature at this stage to assign (Rimon et a / ., 1980) asymmetric lipid-mediated effects to this system. Often the abolition of a break point in an Arrhenius plot that occurs upon detergent solubilization is taken to indicate that the break observed with the TABLE II THETHLKMODEPENDENCE OF THE ACTIVITY OF ADENYLATE CYCLASE IS MODULATED B Y THE DETERGENT USED FOR SOLUBILIZATION"." Enzyme activity break point in Arrhenius plot ("C) Solubilizing detergent
Detergent mp ("C)
Fluoride-preactivated adenylate cyclase
Na-deoxycholate Triton X-100 Lubrol G Lubrol 12A9 Lubrol N13 Lubrol 17AIO
174 7 (PP) -10 (PP) 16 20 23
n.d. n.d. Linear 16.2 20.4 22.8
5'-Nucleotidase Linear 6.8 Linear 16
19.7 23
Data adapted from Dipple et a / . (1978) and Dipple and Houslay (1979). mp, Melting point: n.d., not determined due to inactivation of enzyme by these detergents; pp. pouring point. I'
THE LIPID ENVIRONMENT
201
native membranes is due to the presence of a lipid-mediated effect (see Kimelberg, 1977, for review). However, this now appears to be a rather premature conclusion, for it has been demonstrated (Dipple et a / . , 1978; Dipple and Houslay, 1979a) that solubilized forms of both adenylate cyclase and 5'-nucleotidase can exhibit very different Arrhenius plots, dependent upon the nature of the detergent used to effect solubilization. In these instances the enzymes are liberated as detergent-lipid-protein complexes, where temperature-dependent rearrangements of the micellar structures formed can influence the thermodependence of the enzyme activity (see Dipple and Houslay, 1978). Similar effects (Dipple and Houslay, 1978) are seen with both adenylate cyclase and 5'-nucleotidase, and these correlate very well with the melting points or so-called pouring points of the detergents (see Table 11). Indeed, over the temperature range studied (0"-45"C), the linear Arrhenius plots were obtained when melting points were outside of this range, whereas biphasic plots occurred if the melting point was within this range. Clearly, this demonstrates the dramatic sensitivity of adenylate cyclase activity to changes in the physical properties of its environment.
D. Manipulation of Phospholipid Species The phospholipid headgroup composition of membranes can be altered in vivo by either dietary means or by additions to cell culture medium; however, this can lead to adaptive, homeoviscous changes which will also affect the cholesterol content and the nature of the phospholipid acyl chains (see Kimelberg, 1977). Manipulation in vitro can be achieved by using detergent-mediated lipid substitution techniques or by fusing phospholipids with the target membrane (Houslay et at., 1976a,b,c). The disadvantages of such techniques are that, unless properly controlled, membrane proteins may be denatured, membrane asymmetry may be lost, and cholesteroliphospholipid or protein/lipid ratios may be altered. Nevertheless, the use of in vitro lipid substitution and fusion experiments has provided strong supporting evidence that lipid fluidity does indeed modulate the activity of adenylate cyclase in various ligand-stimulating states (see Houslay et al., 1976a,b,c; Dipple and Houslay, 1978; Bakardjieva e r a / . , 1979). The insertion of defined phosphatidylcholines with very different physical properties (phase transitions) shows not only that the lipid phase separation temperature in the membrane can be shifted or abolished, but also that the activation energies for the reaction can be affected. These effects are undoubtedly due to changes in fluidity that are elicited, but one can also expect to see a contribution due to the nature of the headgroup of the phospholipid species inserted (see Section 111,E). In rat liver plasma membranes the incorporation of a phospholipid with a high transition temperature elevates the lipid phase separation in the membrane,
202
MILES D. HOUSLAY AND LARRY M. GORDON
~
Activation energy (kJ mole- I ) Percentage defined lipid in lipid pool
Fused complex Native Dimyristoyl phosphatidylcholine Dioleoyl phosphatidylcholine "
Data adapted from Houslay
>' L, Linear.
-
61 60
Break point ("CI
Above hreak
Below bredk
28 22 L
25
84
61
I60
71
el ul. ( 1976a.h).
whereas defined phospholipids, with low phase transitions, depress the lipid phase separation temperature (Table 111). Essentially similar results were obtained using cultured Chang liver cells (Bakardjieva et nl., 1979). E. Cholesterol
Although cholesterol can be expected to exert a profound effect on membrane fluidity, and hence the activity of integral enzymes, it can also perturb enzyme function in a number of other ways. For example, it can interact preferentially with certain phospholipid species (Demel er d., 1977) to form cholesterol-rich and -poor domains. As proteins tend to segregate in cholesterol-poor domains (Kleeman and McConnell, 1976; Houslay and Palmer, 1978), alterations in membrane cholesterol will modulate both the chemical composition of the lipid pool available to interact with the protein and the concentration of protein within its available lipid pool. Each of these parameters could influence enzyme activity, as perhaps could the direct interaction of cholesterol with the protein itself. All of the studies attempting to describe the actions of cholesterol on adenylate cyclase activity that have been reported so far have involved either treating intact cells with cholesterol-rich and -poor liposomes or by using cell mutants defective in cholesterol biosynthesis (Sinha et al., 1977; Insel et al., 1978; Sinensky et al., 1979). Both of these methods can have severe drawbacks that markedly affect interpretation of results. If viable whole cells are employed, then attempts to manipulate plasma membrane cholesterol levels by adding it to the serum are indeed successful, but it is likely that the cells will attempt to adapt to this change in fluidity by altering their phospholipids (see Kimelberg, 1977) and have indeed been noted recently in such mutant cell lines (Sinensky, 1980). While mam-
THE LIPID ENVIRONMENT
203
malian cells are not exceptionally successful at achieving this, adaptive changes in membranes have always been observed (see Kimelberg, 1977). An additional difficulty in analyzing such experiments is the finding that serum lipoproteins can activate adenylate cyclase activity (Pairault et al., 1977; Ghiselli et al.. 19811. Conceivably, the introduction of cholesterol to cells grown in serum also modifies the expression of adenylate cyclase activity by perturbing the ability of serum lipoproteins to activate the enzyme. In cells incubated with liposomes, it is probable that considerable fusion can occur which would be expected to introduce exogenous phospholipids capable of affecting both the lipid composition of the membrane and the lipid-protein ratio and hence adenylate cyclase activity. Klein et NI. (1978) demonstrated that incubation of fibroblasts with cholesterol-enriched liposomes led to an increase in cell, and presumably plasma membrane, cholesterol content. Such treatment also mediated a loss of basal, fluoride-, and prostaglandin E,-stimulated adenylate cyclase activity. Although these observations would be consistent with elevated cholesterol levels’ decreasing membrane fluidity and inhibiting the enzyme, further experimentation is needed before making such an assignment. Comparable results were obtained using either cholesterol-egg lecithin or cholesterol-dipalniitoyl phosphatidylcholine, even though fusion of the cells with liposomes occurred (Klein et al., 1978), which implies that the inhibitory effect was achieved by gross overloading with cholesterol. Similar studies were performed on human platelets t o assess the relationship between membrane cholesterol and adenylate cyclase activity (Sinha et a / . , 1977; Insel e t a / ., 1978). However, Insel eral. (1978) were unable to confirm the earlier findings of this group working on platelets (Sinha et d.,1977) that elevated cholesterol levels suppressed fluoride-, hormone-, and guanine nucleotide-stimulated activities. They now maintain that platelets incubated with cholesterol-rich liposomes exhibit an enhanced basal activity but similar hormone-stimulated activities, i.e., there is a net decrease in the fold stimulation achieved by hormone. Since lipid analyses were not carried out on the native and cholesterol-manipulated platelet plasma membranes, it is not possible to evaluate accurately the action of membrane cholesterol on adenylate cyclase activity in these studies. Sinensky el al. ( 1979) have modulated the plasma membrane cholesterol content of a mutant Chinese hamster ovary (CHO) cell line, defective in cholesterol biosynthesis, by supplementing the medium with cholesterol. These workers observed an enhanced basal activity with increasing cholesterol levels, although the relative stimulation achieved by fluoride or prostaglandin E, fell dramatically. The increased cholesterol content of the membrane was associated with a decrease in the bilayer fluidity detected with a fatty acid spin label, and it was suggested that the increased membrane acyl chain ordering was responsible for the activation of basal adenylate cyclase (Sinensky et al., 1979). Their view
MILES D. HOUSLAY AND LARRY M. GORDON
204
that decreases in membrane fluidity play a controlling factor in activating basal adenylate cyclase is somewhat at odds with their finding that decreases in fluidity achieved by temperature reductions are associated with decreases in basal adenylate cyclase activity (Sinensky et a / ., 1979). The interpretation of Sinensky et al. (1979) also appears somewhat anomalous, inasmuch as there is considerable evidence that for adenylate cyclase and other enzymes (see Gordon et af.,1980a) increased membrane fluidity leads to enhanced activity. As the basal activity appears to be relatively insensitive to the lipid fluidity (see Section III,C), and considering the myriad interactions of cholesterol in biological membranes, it would seem more likely that in platelets and CHO cells the increased cholesterol content is complexing an inhibitory phospholipid species. This would enhance the basal activity, but the decreased fluidity would be expected to inhibit the hormone responses. Such factors would explain the reduced net stimulation over basal observed activity in these systems. Thermodependence studies of the adenylate cyclase activity of membranes from native and cholesterol-supplemented CHO cells were also carried out (Sinensky et al., 1979). Unfortunately, the number of data points (4-5) is insufficient to make any meaningful evaluation, save that the activation energies for adenylate cyclase in cholesterol-rich membranes are greater than for the native membranes. This would be consistent with our interpretation stated above that the more rigid environment would render the reaction less favorable. In an attempt to define the effect of cholesterol upon adenylate cyclase A. D. Whetton, L. M. Gordon, and M. D. Houslay (unpublished results) have developed a technique, using liposomes, to both increase and decrease the cholesterol content of rat liver plasma membranes. The manipulations are carried out at 0°-4"C, so that adenylate cyclase is not denatured, and conditions are such that fusion of liposomes with the plasma membrane is negligible. From the results of such experiments it would appear that cholesterol optimizes the functioning of adenylate cyclase in these membranes (Fig. 15). Any increase or decrease in the cholesterol content from that exhibited by native membranes [0.65-0.72, cholesteroUphospholipid ( U P ) molar ratio] leads to a reduced activity of the enzyme. These effects are reversible upon further manipulation of cholesterol levels. We should note that if suboptimal cholesterol concentrations exist in a membrane, and the responses are similar to those described here, then over an appropriate range an increase in cholesterol concentration may actually enhance enzyme activity. Furthermore, basal activity seems remarkably insensitive to increased cholesterol concentrations compared to the hormone-stimulated activity. Thus the degree of activation of adenylate cyclase, over basal activity, by glucagon will drop precipitously upon increased cholesterol levels up until they become very high ( U P 1 .O). A decreased cholesterol content will also diminish the net stimulation by hormone. Such results imply that the cholesterol content of membranes has an important influence on adenylate cyclase function,
-
THE LIPID ENVIRONMENT
205
FIG, 15. Cholesterol optimizes the functioning of adenylate cyclase. The cholesterol content of native rat liver plasma membranes (CIP = 0.72) was manipulated using liposomes either loaded with or free from cholesterol under conditions where all the cholesterol was transferred by exchange and no liposomal fusion occurred with the membranes. The steady-state activity of the enzyme at 30°C was followed as before (see Houslay et d . . 1980a) with various activating ligands (A. D. Whetton and M. D. Houslay. unpublished).
206
MILES D. HOUSLAY AND LARRY M. GORDON
TABLE IV ALTERATIONS IN THE CHOLESTEROL CONTENT O t RAT LIVER PLASMAMEMBRANESMODULATE THE BILAYEK FLUIDITV WITH THE 5-NlTROXIUE STEARATE FATTY ACID DETECTED SPIN PROBE CholesteroUphospholipid molar ratio
S(Tl1)”
0.36
0.43
0.72“
0.82
0.94
0.715c (0.005)d
0.686 (0.003)
0.665 (0.004)
0.720 (0.004)
0.715 (0.005)
Native membrane preparation. The polarity-uncorrected order parameter S(T11) was determined from native and cholesterol-modulated rat liver plasma membranes at 30°C. Mean determined from duplicate measurements. Values in parentheses indicate I SD calculated from duplicate determinations.
as changes can lead to as much as a 50% decline in the degree of hormone stimulation. How does cholesterol exert its effects on adenylate cyclase? Certainly an increased membrane cholesterol content is usually accompanied by decreased membrane fluidity (see Kimelberg, 1977), and this is just what we observed from spin-label studies (Table IV). Increases in cholesterol levels may inhibit adenylate cyclase activity, at least in part, by making the membrane more rigid. Another possibility is that incorporation of cholesterol into the bilayer decreases the size of those cholesterol-poor domains sampled by the protein, thereby inhibiting enzyme activity either by promoting protein-protein interactions or by restricting the availability to the enzyme of phospholipids that are critical for maintaining maximal activity. This may be somewhat analogous to lowering the temperature below T , (see Fig. 2). Additional experiments show that the enzyme in a high-cholesterol environment is more potently activated by benzyl alcohol than is the native enzyme. Indeed the maximal specific activity achieved by optimal benzyl alcohol concentrations is identical for the enzyme in both native and high-cholesterol membranes. Accordingly, inhibition at high cholesterol concentrations may be due entirely to the increased rigidity of the membrane and to the restricted size of lipid domains sampled by the enzyme, as it can be fully reversed by benzyl alcohol, an agent capable of ‘fluidizing liver plasma membranes (Gordon et al., 1980a) and disrupting cholesterol-rich domains in model membranes (Colley and Metcalfe, 1972). Surprisingly, decreasing the cholesterol content over the range examined also appears to lower the fluidity of the membrane (Table IV). This at first rather
THE LIPID ENVIRONMENT
207
unexpected result may well reflect the inherent domain structure of the membrane. Here, cholesterol in the native membrane may be preferentially clustering specific, rather rigid lipids such as sphingomyelin or acidic phospholipids to form domains excluding spin probe and adenylate cyclase. However, the release of such lipids, upon decreasing the cholesterol content, would lead to their mixing in the lipid pool, thereby increasing the rigidity of the environment of the enzyme and the probe. Certainly, adenylate cyclase appears to be somewhat more activated by benzyl alcohol, but the effect is nowhere near as dramatic as with high-cholesterol membranes. On this basis we would like to suggest that the inhibition observed at low cholesterol/phosphoIipid levels is due to the release of inhibitory phospholipid species from complexes in cholesterol-rich domains. Indeed, these observations are entirely in accord with our earlier experiments using the polyene amphoptericin B (Dipple and Houslay, 1979b) and with those of others using filipin (Puchwein et a / . , 1974; Lad et al.. 1979). These drugs form high-affinity, specific complexes with cholesterol in the membranes and so would be expected to mimic the effects of depleting cholesterol. This is in fact what happens as both fluoride- and glucagon-stimulated activities are inhibited by increasing concentrations of these drugs. These effects can be shown to be fully reversible in the case of amphoptericin B (Dipple and Houslay, 1979b). In line with our arguments, identical changes in lipid phase separation temperatures and the production of a new lipid phase separation are seen either by decreasing the cholesterol content (A. D. Whetton, L. M. Gordon, and M. D. Houslay, unpublished experiments) or by using amphoptericin B (Dipple and Houslay, 1979b). Presumably this is due both to the depletion of cholesterol and the release of specific phospholipids into the lipid domains interacting with adenylate cyclase and sampled by the spin probe. We cannot, however, rule out entirely the possibility that cholesterol itself is required to interact with the protein in order to optimize its function.
F. Hormone-Mediated Alterations in Lipid Fluidity There have been numerous reports employing extrinsic fluorescent probes that in vitro additions of hormones (e.g., insulin and growth hormone) could elicit changes in plasma membrane fluidity, thereby altering cellular functions (see Sauerheber et al., 1980, for review). All of the above studies must, however, be viewed as preliminary inasmuch as none have been confirmed by independent investigators using similar or complementary techniques. Indeed, recent experiments employing spin labels tend to disprove such contentions (Amatruda andFinch, 1979; Sauerheber et a/., 1980). The view that changes in the membrane fluidity will mediate the action of hormones appears untenable, since perturbations in the bilayer fluidity will influence the activity of a wide range of enzymes (see Kimelberg, 1977; Gordon et ul., 1980a) and thus lack the specificity that is
208
MILES D. HOUSLAY AND LARRY M. GORDON
the hallmark of a classic hormone effector. Moreover, if such a hypothesis were correct, then agents that modulate the membrane fluidity (e.g., local anesthetics and cholesterol) should act as hormones; this clearly is not the case. Recently, it has been suggested (Hirata and Axelrod, 1980) that agonist occupancy of P-adrenergic receptors, benzodiazepine receptors, and mast cell IgE receptors stimulates the plasma membrane to synthesize phosphatidylcholine from phosphatidylethanolamine.This is apparently achieved by the liganded receptor activating two asymmetrically oriented S-adenosylmethionine (SAM)required methyltransferases in the plasma membrane. The first enzyme exposed at the cytosol surface of the membrane monomethylates phosphatidylethanolamine and is presumed to trigger the transfer of this lipid to the external half of the bilayer, where it cad be further methylated. In in vitro experiments with DPH-labeled erythrocytes (Hirata and Axelrod, 1978), it has been claimed that monomethylation induces an increase in bilayer fluidity, leading to the activation of hormone-responsive adenylate cyclase. As the fluoride-stimulated activity was unaffected, it is possible that any bilayer perturbation is restricted to the outer monolayer. The interpretation of these results in terms of a perturbation of the membrane fluidity (Hirata and Axelrod, 1978, 1980) has been severely criticized (Vance and de Kruijff, 1980) on the basis that the minute amounts of lipid transformed in the process would be insufficient to fluidize the bilayer. As yet, the report by Hirata and Axelrod (1 978) that monomethylation triggers a membrane fluidization has not received independent corroboration. Thus the effects may not be due to a change in fluidity detectable by extrinsic reporter groups but instead may be exerted by a direct action of the modified lipid on the protein. However, it is unlikely that this mechanism will be of general importance in hormone action, although it may prove to have a key role in the transfer of lipid molecules across the membrane of the endoplasmic reticulum (Higgins, 1981) during the biosynthesis of lipids and the generation of membrane lipid asymmetry.
IV. SELECTIVE MODULATION OF ADENYLATE CYCLASE BY ASYMMETRIC PERTURBATIONS OF THE MEMBRANE BILAYER The previous section established that alterations in the bilayer fluidity may act as effectors of the activity of adenylate cyclase. Since the adenylate cyclase complex is asymmetrically oriented with respect to the bilayer, it is reasonable to suppose that agents which perturb the fluidity of one or the other half of the lipid bilayer will selectively modify the expression of this enzyme activity. Here, we consider the results of earlier structural and functional studies to determine whether such a relationship indeed exists for such perturbants as charged local anesthetics, Ca2+, and mitogenic agents.
THE LIPID ENVIRONMENT
209
A. Positively and Negatively Charged Local Anesthetics Sheetz and Singer (1974) have suggested that, owing to the now well-documented asymmetry of the lipid composition of plasma membranes (see Rothman and Lenard, 1977), in which negatively charged phospholipids predominate at the cytosol-facing surface, drugs of opposite charge may act preferentially at one or the other side of the bilayer. One might well expect such drugs to act selectively on functioning proteins that are themselves asymmetrically orientated in the bilayer. In recent studies (Houslay et al., 1980b, 1981a) we have sought to test the above hypothesis by examining the actions of positively and negatively charged local anesthetics on the activity of hepatic adenylate cyclase. Such an investigation seemed warranted, since Higgins and Evans (1978) have demonstrated that, similar to the surface membranes of other eukaryotic cells, the acidic phospholipids are restricted to the cytosol surface of the bilayer of rat liver plasma membranes. Previous work has also defined the topology of glucagon-stimulated adenylate cyclase relative to the bilayer, where the receptor is exposed at the external surface of the plasma membrane and the catalytic unit and guanine nucleotide regulatory component are at the internal surface (Houslay et af., 1980a). In the absence of glucagon, the receptor and catalytic units are able to undergo free lateral diffusion as independent entities (Houslay et al., 1977; Houslay, 1981a) and the activity of the free catalytic unit (i.e., the uncoupled activity) is sensitive to the lipid environment of the cytosol side of the bilayer only (Houslay and Palmer, 1978; Houslay, 1979). However, in the presence of glucagon (coupled activity), the occupied receptor and catalytic unit interact to form a transmembrane complex under conditions in which a mobile receptor model is obeyed, spanning the lipid bilayer. This complex allows adenylate cyclase to respond to the lipid environment of both halves of the bilayer (see Section 111,B). Thus, the measurement of adenylate cyclase in the coupled and uncoupled states provides us with a useful system to probe the lipid properties of both halves of the bilayer. The experimental protocol that we employed to study the effects of charged drugs on rat liver plasma membranes is as follows. Dose-response curves were performed at 30°C (i.e., above the high-temperature onset of the lipid phase separation) to assess the actions of the agents on the glucagon-stimulated (coupled) activity and the fluoride-stimulated (uncoupled) activity of the membranebound enzyme. Parallel ESR studies were conducted on 5-nitroxide stearatelabeled membranes to determine the role that changes in the bilayer fluidity might play. To discriminate between direct effects on the catalytic unit and those transmitted through the native lipid bilayer, the actions of the agent on the activity of detergent-solubilized adenylate cyclase were also examined. Finally, thermodependence studies on the activity in the membrane-bound and solubilized states and on the membrane fluidity were conducted with selected con-
210
MILES D. HOUSLAY AND LARRY M. GORDON
6ol
40 20
1, 1
I
-0.5
0
I
I
1.0 1.5 log [Rilocaine] mM
0.5
2.0
Fic. 16. Effect of prilocaine on the adenylate cyclase activity of rat liver plasma membranes.
(A) Glucagon-stimulated activity (mobile receptor model); ( 0 ) fluoride-stimulated adenylate lubrol-solubilized, fluoride-preactivated enzyme at 30°C. (Data from Houslay cyclase; (0) 1980b.)
rt
a / .,
centrations of the agent. These experiments allow us to evaluate whether a given agent is perturbing the thermotropic lipid phase separation occurring in the outer half of the bilayer, or, alternatively, inducing a lipid phase separation in the inner half of the bilayer. The effects of the positively charged amine local anesthetics prilocaine, nupercaine, and carbocaine on the structural and functional properties of rat liver plasma membranes were investigated (Houslay er a / . , 1980a; Gordon er a / . , 1980b). As shown in Fig. 16, concentrations of prilocaine above 4 mM led to an augmentation of fluoride-stimulated adenylate cyclase activity, up to a maximum of 150% of its original activity at 10 mM prilocaine; further increases in prilocaine concentration led to a progressive inhibition of the activity. When these experiments were repeated on an enzyme preparation that had been solubilized with the nonionic detergent Lubrol 12A9 the fluoride-stimulated activity exhibited no response to these concentrations of prilocaine. In contrast to its effect on the fluoride-stimulated activity, the glucagon-stimulated activity began to be inhibited by prilocaine at concentrations where the fluoride-stimulated activity began to increase. Further addition of this compound led to a progressive inhibition of the glucagon-stimulated activity (Fig. 16). All these functional effects were fully reversible upon washing to remove the anesthetic. Spin-label studies of liver plasma membranes demonstrated that, at concentrations of prilocaine greater than 4 mM, the bilayer fluidity was progressively augmented up to the highest drug concentration tested (33 mM) (Fig. 17). Carbocaine and nupercaine
21 1
THE LIPID ENVIRONMENT
*r
-I
-+= - 4 v)
a
-6
d.5 l o ;1. log [Prilocaine] mM
&
FIG. 17. Prilocaine incrcases the fluidity of rat liver plasma membranes labeled with 5-nitroxide stearate. Dependence of AS(T11 ) on prilocaine concentration at 30°C. (Data from Houslay ef ul., I980b. )
(dibucaine) exerted similar effects on the activities of the coupled and uncoupled adenylate cyclase and the fluidity of liver plasma membranes (Gordon et al., 1980b). Although carbocaine achieved these perturbations over a drug concentration range identical to that of prilocaine, nupercaine manifested its actions on both membrane fluidity and adenylate cyclase activity at a 10-fold lower concentration. The activation of the fluoride-stimulated activity mediated by prilocaine, carbocaine, and nupercaine is most likely due to the drug-induced fluidization of the rat liver plasma membrane bilayer. Such an interpretation agrees with our previous observations that low benzyl alcohol concentrations (up to 30-40 nM) progressively activated the fluoride-stimulated activity and also increased the lipid fluidity to the same degree seen with the cationic drugs (Figs. 3,5,and 16). Further support for this hypothesis comes from the finding the nupercaine initiates increases in both the bilayer fluidity and the fluoride-stimulated activity at a concentration that is an order of magnitude less than that noted for either prilocaine or carbocaine. Clearly, these effects are not due simply to the direct action of the drug on those portions of the enzyme exposed to the aqueous buffer, since incubation of the cationic drugs did not perturb the activity of the detergentsolubilized enzyme (Fig. 16). Since anionic lipids predominate at the cytosolfacing surface of the bilayer, and cationic local anesthetics efficaciously act to increase the fluidity of such lipids in model systems, it is reasonable to suppose that the increase in fluidity achieved by the positively charged drugs (Fig. 17) is due to the interaction of the drug with the inner half of the bilayer (see Houslay et
21 2
MILES D. HOUSLAY AND LARRY M. GORDON Anionic drugs act here
TSrn 'Native
+phewdtai
+pttbcairA
28
16
20
-
11
outer
Acidic phospholipids Fluoride., guanine nuclaotldestirnulated
\
Catlonlc drugs act here Giucagon.stlrnulated
Frc. 18. Sensitivity of ligand-stimulated adenylate cyclase in liver plasma membranes to asymmetric perturbations. Ts,Lipid phase separation temperature; R. receptor; C. catalytic unit; g, G/F coupling protein. This demonstrates the states of the enzyme when stimulated by fluoride or guanine nucleotides and when stimulated by glucagon and suboptimal concentrations (0.08 F M ) of GTP where a mobile receptor model is obeyed. ( f ) indicates inserted fatty acid spin label; h, glucagon. (Adapted from Houslay ef (11.. 1981 .)
1980b, 1981). This selective fluidization then leads to the activation of the fluoride-stimulated adenylate cyclase, an uncoupled activity that responds only to the lipid environment of the cytosol-facing half of the bilayer (Fig. 18). Of particular interest is our finding that these cationic drugs did not cause an enhancement of the glucagon-stimulated activity but instead began to inhibit the enzyme at concentrations where activation of the fluoride-stimulated activity commenced (Fig. 16). One might expect that, since the uncoupled catalytic unit was activated due to an increase in the fluidity of the surrounding lipid, the coupled catalytic unit would be similarly activated; this clearly was not the case. That the inhibition occurred at a concentration similar to the activation of the uncoupled catalytic unit suggests that they have a common origin. Presumably, this is the interaction of the cationic drug with the bilayer leading to an increase in fluidity, an event that we have suggested is preferentially localized to the inner half of the bilayer. However, this may well affect the activity of the coupled enzyme differently than that of the uncoupled enzyme. It was earlier proposed that the vertical positioning of the catalytic unit in the bilayer alters upon its coupling to the glucagon receptor (Dipple and Houslay, 1978), and such a change might lead it to react unfavorably with the surrounding lipid or drug. An alternative explanation focuses on the demonstration that negatively charged phospholipids might be involved in the coupling process between the receptor and catalytic unit (see Section 111,E). Because there is good evidence for a strong interaction between cationic anesthetics and negatively charged phospholipids, the involvement of such lipids in the coupling process may well yield an inhibitory response that would mask any activation caused by an increase in bilayer fluidity. Additional evidence that the cationic anesthetic prilocaine perturbs rat liver a/.,
THE LIPID ENVIRONMENT
1.0
213
-
0.9-
f
0.8 -
d
-
/
# 0 0.7 0.6 -
0.5 3.1
I
3.2
I
3.3
1
3.4
I
35
1
3.6
1
3.7
103
T O FIG. 19. Effect of 10 mM prilocaine on the temperature dependence of the order parameter for a S(T11) (0).and S ( T , ) fatty acid spin probe incorporated into rat liver plasma membrane. S (A), (W). (Adapted from Houslay ef al., 1980b.)
plasma membranes asymmetrically has been provided by a study of the thermodependence of both the activity of adenylate cyclase and of the membrane fluidity detected with a fatty acid spin label (Houslay er ul., 1980b). Prilocaine (10 mM) had no significant effect on the thermotropic lipid phase separation occurring at 28°C that could be detected with either the spin probe (Fig. 19) or Arrhenius plots of glucagon-stimulated adenylate cyclase (Fig. 20). These data suggest that 10 mM prilocaine does not perturb the lipids of the external half of the bilayer, but instead selectively fluidizes the lipids of the inner half of the bilayer. Consistent with this hypothesis are our observations that the Arrhenius plots of the fluoride-stimulated adenylate cyclase activity, which were normally linear over the temperature range studied, exhibited a well-defined break at around 11°C when 10 mM prilocaine was present in the assays (Fig. 20). The
214
MILES D. HOUSLAY AND LARRY M. GORDON
3.c
2.5
- 2.0 T
0,
E
ln
c .-
C
->3 1.5 0 7
0 -
1.0
0.5 3.1
I
3.2
1
3.3
1
I
3.4 3.5
I
I
3.6
3.7
FIG. 20. Effect of 10 mM prilocaine on Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity in liver plasma membranes. For fluoride- (01and glucagon- (A)stimulated activies. (Adapted from Houslay el a/.. 1980b.)
occurrence of this prilocaine-induced break was also apparent in Arrhenius plots of glucagon-stimulated adenylate cyclase activity (Fig. 20), which, unlike the fluoride-stimulated activity, is sensitive to the lipid environment of both halves of the bilayer. As Arrhenius plots for the solubilized enzyme were unaffected by 10 mM prilocaine, the break in the Arrhenius plots occurring at around I 1"C does not appear to be due to an effect of prilocaine on the protein, but is instead a consequence of a lipid phase separation that is reported by the spin probe (Fig. 19). The simplest explanation of these data is that the prilocaine- (10 mM) induced lipid phase separation is localized to the inner (cytosol-facing) half of the bilayer (Fig. 18). The inhibitory effects of fluoride-stimulated adenylate cyclase activity, observed at the higher cationic anesthetic concentrations (Fig. 16), have been attributed to the drug preventing annular/boundary lipid from interacting with
THE LIPID ENVIRONMENT
21 5
sites of the protein (i.e., the annular lipid displacement model; Gordon et al., 1980a,b; Houslay et a l . , 1980b). Clearly, this inhibition is not simply due to a direct action of cationic anesthetics on the protein, as the detergent-solubilized enzyme was unaffected for each agent tested. By contrasting the effects on adenylate cyclase activity induced by increases in fluidity achieved by temperature elevations or anesthetic additions, we have also ruled out the cationic drugmediated inhibition as being a consequence of a “too-fluid” bilayer (Gordon et al., 1980a; Houslay et al., 1980b). Instead, we suggest that occupancy of annular sites by the anesthetic leads to reduced activity, because either the anesthetic itself was inhibitory or the displaced lipid was essential for activity. Since the lipid asymmetry of cell plasma membranes, in which anionic lipids predominate at the cytosol-facing surface, appears to be a widespread phenomonon (see Rothman and Lenard, 1977), the adenylate cyclase activity of a number of other tissues might well be expected to respond to cationic anesthetics in a manner similar to that noted in rat liver plasma membranes. Indeed, the fluoride- and hormone-stimulated activities in such diverse systems as beef thyroid membranes (Wolff and Jones, 1970), rat adipocyte plasma membranes (Hepp et al., 1978), pigeon erythrocyte membranes (Salesse and Gamier, 1979), and rat liver plasma membranes (Houslay et ul., 1980b, 1981a) are all modulated similarly by positively charged drugs. Although the model presented by us to account for the actions of cationic drugs on rat liver plasma membranes (see above) may also be invoked to explain the functional effects of cationic anesthetics on these other membranes, additional experimentation will be needed before making any definitive assignments. The anionic drugs phenobarbital, pentobarbital, and salicylic acid were also examined for their actions on rat liver plasma membranes (Gordon et al., 1980b; Houslay et al., I98 1). Phenobarbital initially inhibited the glucagon-stimulated (coupled) activity at concentrations of about 1 mM, above which concentrations a dramatic activation ensued, that reached a maximum between 4 and 6 mM. Any further increases in phenobarbital concentration led to a progressive loss in the coupled activity. On the other hand, no effect of phenobarbital was observed on the fluoride-stimulated (uncoupled) activity of either native membranes or a Lubrol-solubilized enzyme (Fig. 21). All of the changes in activity were readily reversed upon washing to remove phenobarbital. Qualitatively similar results were obtained with the anionic drugs pentobarbital and salicylic acid. Thermodependence studies were also conducted to assess the effects of phenobarbital on the activity of hepatic adenylate cyclase and the membrane fluidity detected with a fatty acid spin probe. Addition of optimum (4 mM) phenobarbital concentrations to the assays had a dramatic effect on the Arrhenius plot for the glucagon-stimulated activity, such that the break was depressed from 28” to 16°C and the activation energies detected above and below the break were considerably less than those exhibited in the absence of phenobarbital (Fig. 2 2 ) . Howev-
MILES D. HOUSLAY AND LARRY M. GORDON
216
" 2oo/
h
Glucagon
[phenobarbital 1 rnM
FIG. 21. Sensitivity of liver plasma membrane adenylate cyclase to phenobarbital. (0) Glucagon + suboptimal GTP; (0) fluoride and (B) lubrol-solubilized. fluoride-preactivated enzyme at 30°C. (From Houslay el d., 1981.)
3.5
3.0
-
i"
\ C .-
E 2.5
-
\ u
-5 0
>
m
2 2.0
1.5
& ;
1.0 3.1
Fluorlds Native
3.2
3.4
3.3 (OIT '
3.5
3.6
3.7
(K)
FIG.22. Arrhenius plots,of liver plasma membrane adenylate cyclase activity in the presence of 4 mM phenobarbital. Fluoride; (0) glucagon + suboptimal CTP concentration. (Data from Houslay et al., 1981.)
(m)
21 7
THE LIPID ENVIRONMENT 1.o
0.90
-
OB0
-
A m -
+Phenobarbital
Sprll) 1ooc
-
0.60 f I 1 3.2
I 3.3
3.4
3.5
3.6
FIG. 23. Phenobarbital's effect on the temperature dependence of the order parameter ST for a fatty acid spin probe inserted into liver plasma membranes. ( 0 )Treated and (A)native membranes. (Data from Houslay er al., 198 1 , )
er, the Arrhenius plot of the fluoride-stimulated (uncoupled) activity was apparently unaffected by the presence of phenobarbital. Arrhenius-type plots of the order parameter of 5-nitroxide stearate-labeled rat liver plasma membranes indicated that phenobarbital not only lowered the high-temperature onset of the lipid phase separation from 28" to 16°C but also increased the membrane fluidity for temperatures between 10" and 30°C (Fig. 23). The activation of the coupled adenylate cyclase activity that we observe appears to be due to a selective fluidization of the external half of the bilayer by phenobarbital (Fig. 18), since the drug-induced depression of the break temperature in the Arrhenius plot of the coupled activity (Fig. 22) closely parallels the reduction of the high-temperature onset of the lipid phase separation identified with our spin probe (Fig. 23). Further evidence of a selective fluidization of the external half of the bilayer by phenobarbital is gained from our observation that the activation energies for the coupled activity were reduced, whereas for the uncoupled activity not only does the Arrhenius plot stay linear but the activation energy for the reaction remained identical (Fig. 22). The fluidization of rat liver plasma membranes for temperatures between 10" and 30°C achieved by phenobarbital is consistent with the disordering that this drug exerts on spin-labeled phospho1ipid:cholesterol (2: 1) bilayers (Pang and Miller, 1978), and these effects may be a consequence of phenobarbital disrupting intrinsic phospho1ipid:cholesterol associations. It is significant that phenobarbital is much more effective in activating glucagon-stimulated adenylate cyclase at temperatures below 28°C than above (Fig. 22). The coupled enzyme is probably restricted to relatively cholesterol-
218
MILES D. HOUSLAY AND LARRY M. GORDON
free lipid domains; otherwise, Arrhenius plots of the glucagon-stimulated activity of native membranes would be unable to exhibit a well-defined break at 28°C with a sharply increased activation energy below the break temperature (Fig. 22; see Houslay and Palmer, 1978; Dipple and Houslay, 1979b, for discussion). Indeed, increases in the cholesterol content of rat liver plasma membranes (from cho1esterol:phospholipid ratios of 0.65 to 0.94) using the liposome methodology described in Section III,E. abolished the break at 28°C normally observed in Arrhenius plots of the coupled activity and the order parameter of 5-nitroxide stearate-labeled membranes (A. D. Whetton, L. M. Gordon, and M. D. Houslay, unpublished results). The elevated activation energy noted for the coupled enzyme of native membranes below 28°C may be due directly or indirectly to the formation of cholesterol-rich QCC in the L matrix. For example, the increase in QCC as the temperature is lowered may segregate the coupled enzyme into a relatively small L phase, perhaps inhibiting enzyme activity by promoting protein-protein interactions (see Fig. 3). Support for such a hypothesis has been provided by earlier reports indicating that integral proteins are nonrandomly distributed in liver plasma membranes (Montesano et al., 1979) and that the presence of either QCC or S in other membranes inhomogeneously segregates proteins in the plane of the bilayer (Melchior and Steim, 1979). Alternatively, the formation of QCC may restrict the availability to the coupled enzyme of phospholipids that are critical for maintaining maximal activity. Consequently, phenobarbital may be such a potent activator of glucagon-stimulated adenylate cyclase at low temperatures partly because the bulk lipid fluidity is increased, but perhaps also because this drug facilitates disruption of clusters of QCC domains, or proteins, or both. A similar explanation has been proposed to account for the marked activation of glucagon-stimulated adenylate cyclase induced by 40 mM benzyl alcohol at low temperatures (Gordon et al., 1980a). The inhibition of the glucagon-stimulated activity that is observed at low concentrations of phenobarbital (Fig. 21) and other anionic drugs (Gordon et al., 1980b) may be due to these drugs having a direct effect on the receptor or acting at the coupling interface (see Houslay and Palmer, 1979; Whetton and Houslay, 1980), although it is possible that they could disrupt the interaction with specific lipids that may be required for activity (see Rubalcava and Rodbell, 1973). The inhibition of activity seen at high phenobarbital concentrations (Fig. 21) is similar to that observed with a number of neutral and charged local anesthetics (Figs. 5 and 16), and is probably due to the displacement of annular lipid from around the enzyme (see above). Thus, our studies of the effects of positively and negatively charged anesthetics on rat liver plasma membrane adenylate cyclase activity and fluidity present us with striking confirmation of the "bilayer-couple" theory of Sheetz and Singer (1974). However, the above results suggest only that charged drugs exhibit a greater tendency to interact with one or the other half of the bilayer of
THE LIPID ENVIRONMENT
219
liver plasma membranes and do not prove that any of these agents exclusively reside in only one half of the bilayer. It is likely, for example, that the anionic fatty acid spin probe used in this study, 5-nitroxide stearate, distributes between both halves of the bilayer. Clearly, some fraction of the label samples the outer half of the bilayer, since 5-nitroxide stearate detects the lipid phase separation in the outer leaflet (Houslay et al. 1979b,c; Gordon et al., 1980a) and is acutely sensitive to phenobarbital, which depresses the phase separation (Fig. 23). Inasmuch as the incorporated 5-nitroxide stearate probably retains a partial negative charge, it would not be unexpected for this probe to reside in the outer half of the bilayer, since that is where positive and neutral lipids are concentrated. Nevertheless, there is evidence suggesting that 5-nitroxide stearate also partitions into the inner half of the bilayer (Houslay et al., 1980b). Since the cationic local anesthetics nupercaine, carbocaine, and prilocaine each begins to stimulate fluoride-stimulated adenylate cyclase (i .e., an enzyme localized in the cytosol-facing leaflet) at the same concentrations that decrease S(T II ), a certain fraction of the 5-nitroxide stearate probe must be restricted to the inner half of the bilayer to report on this fluidization (Houslay et af., 1980a; Gordon et al., 1980b). That phenobarbital induces such dramatic effects on the lipid phase separation monitored by 5-nitroxide stearate (Fig. 23) indicates that the proportion of probe may be somewhat greater in the outer than in the inner half of the bilayer, perhaps owing to selective charge interactions (Fig. 18). The use of cationic and anionic drugs provides us with powerful tools capable of modulating the fluidity of each half of the bilayer of rat liver plasma membrane independently, Since a wide variety of liver plasma membrane enzymes are influenced by changes in the bilayer fluidity (Gordon et al., 1980a), one might predict that charged drugs may be used to define the vertical positioning of these integral proteins with respect to the bilayer. Indeed, recent studies on the effects of positively and negatively charged anesthetics on hepatic 5'-nucleotidase [i.e., an enzyme activity sensitive to the 28°C thermotropic lipid phase separation (Dippleand Houslay, 1978)and responsive to increases in lipid fluidity achieved by the neutral anesthetic benzyl alcohol (Gordon et af., 1980a)l demonstrate that this ectoenzyme is regulated by only the external half of the bilayer (Dipple et al., 1982). We propose that, once appropriate concentrations of anionic and cationic drugs have been determined for a given plasma membrane system from structural and functional studies, these agents may be of general use in defining the topology of integral enzymes relative to the bilayer.
B. Calcium Ca2+ exerts multiple effects on biological membrane processes. It is well known that Ca2 participates in a number of membrane-associated functions, +
220
MILES D. HOUSLAY AND LARRY M. GORDON
including regulation of enzyme activities, transduction of hormonal information, stimulus secretion coupling, functioning of transport systems, neuronal conduction, and muscular contraction (see for reviews Rasmussen and Goodman, 1977; Triggle, 1972; Gordon and Sauerheber, 1982). In view of the facts that millimolar concentrations of Ca2+ reduce the fluidities of plasma membranes from a wide variety of tissues (see Gordon and Sauerheber, 1982) and that changes in bilayer fluidity modulate the activities of a number of membrane enzymes (Gordon et al., 1980a), it would not be untoward to hypothesize that Ca2+ perturbs certain of the aforementioned membrane functions by altering the lipid fluidity. L. M. Gordon, A . D. Whetton, S. Rawal, J. A. Esgate, and M. D. Houslay (unpublished results) have recently explored in some detail the relationships that may exist between the effects of Ca2 on the fluidity and lipid organization of rat liver plasma membranes and adenylate cyclase activity. As demonstrated by us before (Gordon et al., 1978), ESR studies on rat liver plasma membranes probed with 5-nitroxide stearate demonstrated that Ca2 decreased the lipid fluidity, as indicated by marked increases in the order parameter S. These effects are a concentration-dependent binding process reaching saturation at about 2.8 mM CaCI, and could be reversed with the use of a divalent cation-chelating agent. Over a range of cation concentrations similar to that which lowered the bilayer fluidity, the fluoride-stimulated (uncoupled) activity of the liver plasma membrane-bound enzyme was inhibited by Ca2 with an ID,, (concentration yielding 50% inhibition) of I mM. To discriminate between direct effects on the catalytic unit and those transmitted through the native lipid bilayer, the actions of Ca2+ on the activity of adenylate cyclase in the solubilized state were also examined. The fact that the fluoride-stimulated activities of the membrane-bound enzyme of a Lubrol-solubilized preparation were identically inhibited for Ca2 concentrations less than 2 mM suggests that Ca2+ achieves this effect not by reducing the bilayer fluidity but instead either by liganding to the protein and substrate (ATP) components or by binding to associated annular lipid. Only at Ca2 concentrations greater than 2 mM, where the membrane-bound enzyme was somewhat more inhibited than the solubilized preparation, is it probable that Ca2 +-mediated increases in lipid ordering influence the uncoupled activity. The glucagon-stimulated (coupled) activity is, on the other hand, more sensitive to Ca2+ inhibition with an ID,, of 0.2 mM. Such effects are clearly not due to either changes in lipid fluidity or alterations in the binding of ’2s1-labeled glucagon to its receptor, which was unaffected by CaZ+ concentrations up to 10 +
+
+
+
+
mM.
Although the inhibitory effects on the coupled and uncoupled hepatic adenylate cyclase activities are undoubtedly primarily due to the direct interaction of Ca2+ with the enzyme complex, it is, nevertheless, possible that the actions of Ca2+ on the lipid structures of the outer and inner halves of the bilayer might exert “second-order” perturbations on the temperature dependence of these
22 1
THE LIPID ENVIRONMENT
activities. Arrhenius-type plots of the order parameters indicated that the thermotropic lipid phase separation, which occurs in the outer half of the bilayer of native membranes from 28" to 19"C, is perturbed by millimolar concentrations of Ca2 due to the binding of cation to the outer leaflet such that the high-temperature onset is elevated to 32"-34"C, but the low-temperature onset is apparently undisturbed. Ca*+ was also found to bind to the cytosol-facing half of the bilayer, inasmuch as pretreatment of liver membranes with 10 mM prilocaine, an agent that selectively fluidizes the inner leaflet by interacting with acidic lipids residing there, inhibited the ability of Ca2+ to decrease the fluidity detected by the spin probe. It is of particular interest then that, with 1 mM CaCI,, Arrhenius plots of the glucagon-stimulated activity indicated breaks at 32" and 16"C, while those of the fluoride-stimulated activity showed a single break at 17°C. We achieves these effects by asymmetrically perturbing the suggest that Ca' bilayer, such that the high-temperature onset in the 'outer leaflet is elevated to 32"C, and a second lipid phase separation at 16"-17"C is induced by Ca2+ binding to acidic phospholipids residing in the inner leaflet. The glucagonstimulated activity responds to Ca2 -induced perturbations occurring in both halves of the bilayer because it is a transmembrane complex, while the fluoridestimulated activity senses only the lipid phase separation at 16-17°C because the catalytic unit, when dissociated from the glucagon receptor, is restricted to the inner half of the bilayer. The relative insensitivity of the activity of adenylate cyclase to Ca2 -dependent changes in the fluidity of 5-nitroxide stearate-labeled liver plasma membranes is somewhat anomalous, since increases in fluidity achieved by temperature elevations or low concentrations of anesthetics markedly activate this enzyme (see Sections III,C and D). The stimulation that ensued in these earlier studies was attributed to an increase in the conformational flexibility of the enzyme achieved by a relief of a physical constraint imposed by the bilayer upon the protein (Dipple and Houslay, 1978). One explanation of these disparate findings is that the decrease in fluidity mediated by Ca2 occurs in lipid domains of the membrane from which adenylate cyclase is excluded. On the other hand, the direct binding of Ca2+ that takes place under our assay conditions may "desensitize" adenylate cyclase to alterations in lipid fluidity. The latter hypothesis was tested by examining whether 1 mM CaCI, modified the ability of either temperature alterations or benzyl alcohol additions to perturb liver plasma membrane fluidity and adenylate cyclase activity. Although pretreatment of liver membranes with CaCI, did not alter the percentage change in S(T 11 ), S(T,) induced by raising the temperature from 30" to 37"C, the increases in the fluoride- and glucagon-stimulated activities achieved by this temperature elevation were each significantly reduced for the Ca2 -treated membranes when compared to that observed for native membranes (Fig. 24). Incubation of liver membranes with CaCI, also did not influence the response of either the bilayer +
+
+
+
+
+
222
MILES D. HOUSLAY AND LARRY M. GORDON
I
Initial Condition: Native at 30%
I
I
T
Perturbant: TDC ralsed
t037
1 mM CaCi, at 30%
Native at 30°C
TC raised to 37
50 mM Benzyl Alcohol
mi CaCl,
31 50 mM Benzyi Alcohol
Native at 3oDc
Native at 3(pc
1 rnMCaC1,
50rnM Benzyi Alcohol + 1 mM CaCI,
FIG. 24. Does Ca2+ desensitize adenylate cyclase to changes in lipid fluidity? The presence of Ca2+ apparently reduces the sensitivity of adenylate cyclase to changes in fluidity caused by temperature and benzyl alcohol, and to Ca2+ itself. G SAC, Glucagon-stimulated activity; F SAC, fluoride-stimulated activity.
fluidity or the fluoride-stimulated activity to 50 mM benzyl alcohol at 30°C, but did inhibit the ability of this neutral anesthetic to activate the glucagon-stimulated activity (Fig. 24). Finally, Fig. 24 shows the respective actions of 1 mM CaCl,, or the joint addition of 50 mM benzyl alcohol and I mM CaCI,, on the fluidity and adenylate cyclase activity of liver membranes. Treatment with CaCl, decreased the fluidity of the liver membranes and was a potent inhibitor of the fluoride- and glucagon-stimulated activities. Simultaneous addition of 50 mM benzyl alcohol and 1 mM CaCl, served to override the Ca2+-dependent lipid ordering such that the bilayer was more fluid than untreated membranes, but was unable to return the fluoride- and glucagon-stimulated activities to the baseline values observed with native membranes. The results in Fig. 24 suggest that the binding of Ca2+ “desensitizes” the activity of the coupled enzymes and, to a lesser extent, that of the uncoupled enzyme to increases in bilayer fluidity. Once bound to adenylate cyclase, Ca2+ may alter the protein conformation such that the enzyme exists in a relatively stable state that resists changes mediated by the lipid fluidity. However, Ca2+ does not affect the ability of the enzyme to detect lipid phase separations occurring in the liver plasma membrane.
THE LIPID ENVIRONMENT
223
C. Mitogenic Agents The stimulation of lymphoid cells to mitosis achieved by a variety of agents may be viewed as a model of gene activation in higher organisms. Mitogenic agents appear to ligand to specific receptors on the surface membranes of these cells, and trigger a number of alterations in membrane-linked processes, inciuding influx of K + , Ca2+, amino acids, glucose, and uridine and changes in activity of nucleotide cyclases. Mitogenic agents trigger the patching and capping of plasma membrane proteins components and, in doing so, mediate the redistribution of plasma membrane lipid to form relatively rigid, glycosphingolipid-rich domains and more fluid, glycosphingolipid-poor domains in the outer half of the bilayer (Curtain et al., 1980). The stimulated lymphocytes have increased cellular cyclic AMP levels, which, using fluorescent antibody techniques, exhibit an identical localization to the patched mitogen and glycosphingolipid (Curtain, 1979; Curtain ef al., 1980). It may be that the glycosphingolipids are hydrogen bonded to receptor-bearing membrane components and migrate with them into the ligand-induced patches and caps. Although other explanations may be invoked to account for the above observations, the activation of lymphocyte adenylate cyclase may simply be a consequence of changes in the enzyme's lipid environment induced by the clustering of the glycosphingolipids. The formation of glycosphingolipid-rich and -poor domains, initiated by mitogenic agents, might well be expected to modulate the fluid properties of the outer half of the lipid bilayer. However, this lipid redistribution could also influence the fluidity of the cytosol-facing half of the bilayer, since in lymphocytes more than 50% of the glycosphingolipid fatty acids contain 22 or more carbon atoms. If the longer acyl chains of the glycolipid were to interdigitate into the inner leaflet, then alterations in the topographical distribution of this lipid could modulate the activity of adenylate cyclase (Curtain, 1979) either through direct interaction with the catalytic unit or by perturbing the fluid properties of the cytosol-facing lipid leaflet. Support for such a view has been obtained from experiments in which addition of gangliosides to rat cerebral cortex membranes substantially increases the activity of basal adenylate cyclase (Partington and Daly, 1979).
V.
PHOSPHOLIPID HEADGROUP COMPOSITION AND ADENYLATE CYCLASE ACTIVITY
To date there is only one clear example of an enzyme that requires a specific type of phospholipid in order to function. This enzyme is P-hydroxybutyrate dehydrogenase, which requires the choline headgroup of phosphatidylcholine in
224
MILES D. HOUSLAY AND LARRY M. GORDON
order to bind its coenzyme, NAD (see Houslay et al.. 1975). Many attempts have been made to try to assess whether a specific type of phospholipid is required for the functioning of adenylate cyclase (see Fain, 1978; Ross and Gilman, 1980). However, the inability, at present, to reconstitute homogeneous preparations of receptors, guanine nucleotide coupling protein, and catalytic unit in their appropriate orientations in defined lipid bilayers has meant that, of necessity, indirect approaches or impure preparations have been used. Indeed studies with nonionic-detergent-solubilized preparations which purported to show that acidic phospholipids could reconstitute hormone-stimulated activity have not been able to be reproduced by others (see Ross and Gilman, 1980). Moreover, the reconstitution of such a system would prove to be extremely difficult as, in order to function, all three componets need to be present in their correct, asymmetric orientations within the same liposome. If this is to be achieved by random insertion during reconstitution, we can expect that the number of fruitful reconstitutions is likely to be very low indeed. The effect of specific lipid headgroups on the functioning of an enzyme can be ascertained by a number of different means. These methodologies include detergent-mediated lipid substitution, the insertion of defined lipids into membranes by lipid fusion, phospholipase treatment, supplementing the cell culture medium either with precursors or inhibitors, and also dietary manipulation (see Kimelberg, 1977). However, all of these methods require careful consideration as, in many cases, a number of parameters other than merely headgroup composition can be affected. For example, although certain phospholipases can preferentially attack specific species of phospholipids, lysophospholipids, which are a product of the action of phospholipase A,, can both enhance and inhibit the activity of adenylate cyclase depending upon their concentration (Houslay and Palmer 1979; Lad et al., 1979). Furthermore, alterations of the phospholipid headgroup composition of membranes by dietarylculture media manipulation can lead to adaptive responses which affect the fluidity of the membranes, their fatty acyl chain, and cholesterol content which would lead to alterations in domain structure. Lipid fusion studies involving defined synthetic lipids can be expected to have effects on the cholestero1:phospholipid ratio, domain structure, and 1ipid:protein ratio, all of which have been shown to affect the activity of adenylate cyclase. Thus it is fair to state that at present there is no unambiguous evidence for the involvement of specific headgroup phospholipids in the action of adenylate cyclase. Nevertheless, there are a number of intriguing reports which do suggest that alterations in the levels of certain phospholipid species may affect the functioning of adenylate cyclase. Treatment of liver plasma membranes with a pure preparation of phospholipase C from Bacillus ureus, which hydrolyzes preferentially acidic phospholipids by cleaving off their headgroup to leave the innocuous diglyceride, causes a marked loss of the glucagon-stimulated response. This is in contrast to the action of the
225
THE LIPID ENVIRONMENT
enzyme from Clostridium perfringens which hydrolyzes neutral phospholipids preferentially and, even after a substantial hydrolysis, has little effect on the hormone receptors (Rubaclava and Rodbell, 1973). These effects imply that certain acidic phospholipids are necessary for the hormone response. However, both the structure and properties of the membrane are being perturbed, and so the altered activities could be due to second-order effects. Indeed such a loss of activity would need to be shown to be reversed by the fusion of defined acidic phospholipids with the membrane. In contrast to these results rather large changes in the lipid pool content achieved by fusion with defined synthetic phosphatidylcholines or phosphatidylethanolamines caused no dramatic loss of hormone-stimulated activities in liver membranes, although some reduction was observed (Houslay et al., 1976b; Bakardjieva et al., 1979). However, as acidic phospholipids are only a minor component of these membranes, it could well be argued that the enzyme would have a high affinity for any such specific phospholipid. Engelhard et al. (1978) have investigated the effect of supplementing the medium of mouse LM cells with polar headgroup analogs. These led to rather large changes in the ratio of phosphatidylethanolarnine (PE) to phosphatidylcholine (PC) in the plasma membranes of these cells as well as to the incorporation of the analogs into phospholipids. However, the amounts of acidic phospholipids present remained relatively constant. In this case there were marked changes in both basal and prostaglandin E ,-stimulated adenylate cyclase activity, whose increases seemed to correlate well with the PE/PC ratio. However, it remains to be seen whether these changes in activity are due to specific headgroup effects or changes in the physical properties (fluidity) of the lipid domains occupied by the enzyme. In contrast to these studies the incorporation of a solubilized enzyme from brain into liposomes consisting of mixtures of phosphatidylcholine and another lipid has demonstrated that increasing amounts of phosphatidylethanolamine, phosphatidylserine, and cholesterol progressively inhibit enzyme activity (Hebdon et af., 1979). However, such studies are no doubt complicated by the presence of a nonionic detergent, impure lipids and proteins, and the fact that pure phosphatidylethanolamine and cholesterol do not form bilayers into which the enzyme can insert. It would thus seem that the existing evidence for the involvement of phospholipid headgroup composition on the functioning of adenylate cyclase is at best equivocal.
VI.
DISEASE STATES
There have been reports indicating. that the lipid structures/fluidities of the surface membranes from a number of tissues are altered in such disease states as
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transformation, atherosclerosis, spur-cell anemia, abetalipoproteinemia, muscular dystrophy, cystic fibrosis, diabetes mellitus, and multiple sclerosis (see Kimelberg, 1977, and Houslay and Stanley, 1982, for reviews). In view of the sensitivity of adenylate cyclase to its lipid environment (see above), it might well be profitable to explore whether any abnormalities in the functioning of the adenylate cyclase complex observed in the above disease states are due to alterations in the plasma membrane lipid structure/fluidity. In this regard, Gidwitz et al. ( 1980) have demonstrated that the kinetic properties, thermodependence, and thermostability of adenylate cyclase in chicken embryo fibroblasts are markedly different from the properties of this enzyme obtained from cells transformed with Rous Sarcoma virus. However, upon detergent solubilization the enzyme activity from the two sources behaved identically. This implies that transformation caused a change in the membrane environment of the enzyme which led to marked effects on the enzyme's properties. As it is possible to alter membrane lipid composition and properties by dietary means (see Kimelberg, 1977) and also to influence membrane properties using drugs, it may be fruitful to explore their uses in alleviating certain problems associated with such cellular malfunctions. Clearly, however, rigorous investigations employing the experimental protocols outlined in this article must be performed in these various disease states before assignments as to the significance of membrane perturbation can be made. ACKNOWLEDGMENTS Work in the authors' laboratories was supported by a Medical Research Council (U.K.) project grant (M.D.H); NATO Research Grant RG/218.80 (M.D.H); a Wellcome Trust Travel Fellowship (M.D.H); grants-in-aid from the American Diabetes Association, Southern California Affilitate, Inc.; and NIH Grants AM-21290. AM-28431. and HLIAM-27120 (L.M.G). We would like to thank Elizabeth M. Wright for typing the manuscript. REFERENCES Ahkong, Q. F., Botham, G. M., Woodward. A. W., and Lucy, J. A. (1980). Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol. Biochem. J . 192, 829-836. Amatruda, J. M., and Finch, E. D. (1979). Modulation of hexose uptake and insulin action by cell membrane fluidity. J. B i d . Chem. 254, 26 19-2625. Amir, S. M., Mubrow, N. I . , and Ingbar, S. H. (1981). Phenol, a potent stimulator of adenylate cyclase in human thyroid membranes. Endocrine Res. Commun. 8, 83-95. Bakardjieva, A., Galla, H. J., and Helmreich, E. I . M. (1979). Modulation of the P-receptor adenylate cyclase interactions in cultured Chang liver cells by phospholipid enrichment. Biochemisrry 18, 3016-3023. Bar, H.-P. (1974). On the kinetics and temperature dependence of adrenaline-adenylate cyclase interactions. Mol. Pharmacol. 10, 597-604. Brasitus, T. A., and Schachter, D. (1980). Lipid dynamics and lipid-protein interactions in rdt enterocyte basolateral and microvillus membranes. Biochemistry 18, 2763-2769. Colley, C. M., and Metcalfe, J. C. (1972). The localisation of small molecules in lipid bilayers. FEES Lett. 24, 241-246.
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Curtain. C. C. ( 1979). Lymphocyte surface modulation and glycosphingolipids. Immunology 36, 805-8 10. Curtain. C. C.. Looney. F. D.. and Smelstorius. J. A. (1980). Lipid domain formation and ligandinduced lymphocyte membrane changes. Biuchim. Biophys. Aria 596, 43-56. Demel, R . A., Jansen. J. W. C. M., van Dijck, P. W. M.. and van Deenan, L. L. M. (1977). The preferential interaction of cholesterol with different classes of phospholipids. Biochim. Bio p h w . Actu 465, 1-10, Dipple. I . , and Houslay, M. D. (1978). The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment. Biochem. J . 174, 179-190. Dipple. I . . and Houslay, M. D. (1979a). The temperature dependence of adenylate cyclase activity solubilized using various Lubrol detergents. Biochem. B i o p h y . Res. Commun. 90, 663-666. Dipple. I . , and Houslay. M. D. (l979b). Aniphoptericin B has very different effects on the glucagonand fluoride-stimulated adenylate cyclase activities of rat liver plasma membranes. FEBS Lett. 106, 21-24. Dipple, I.. Elliot, K . R. F.. and Houslay, M. D. (1978). Detergents modify the form of Arrhenius plots of 5’-nucleotidase activity. FEBS Lcrt. 89, 153- 156. Dipple. I . , Gordon. L. M., and Houslay, M. D. (1982). The activity of 5’-nucleotidase in liver plasma membranes is affected by the increase in bilayer fluidity achieved by anionic drugs but not by cationic drugs. J . B i d . Chem. 257, I81 I-1815. Engelhard, V. H.. Esko, J . D., Storm. D. R . , and Glaser, M. (1976). Modification of adenylate cyclase activity in LM cells by manipulation of the membrane phospholipid composition in vivo. Proc. Null. Acud. Sci. U.S.A. 73, 4482-4486. Engelhard. V. H., Glaser, M.. and Storm. D. R . (1978). Effect of membrane compositional changes on adenylate cyclase in LM cells. Biochemistry 17, 3 I9 1-3200. Fain. J . N. (1978). Hormones. membranes and cyclic nucleotides. I n “Receptors and Recognition” ( M . F. Greaves and P. Cuatrecasas. eds.). Ser. A.. Vol. 6. pp. 3-62. Chapman & Hall, London. Ghiselli, G . , Sitori. C. R.. and Nicosia, S . (1981). Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes. B m h r m . J . 196, 899-902. Gidwitz, S., Toscano, W. A.. Toscano, D. G . . Weber, M. J., and Storm, D. (1980). A comparison between adenylate cyclase solubilized from normal and Rous Sarcoma virus-transformed chicken embryo fibroblasts. Biochim. Biophys. Acrm 627, I - 16. Cordon. L. M . , and Sauerheber, R. D. (1982). Calcium and membrane stability. In “Calcium in Normal and Pathological Biological Systems” (L. J . Anghileri. ed.). CRC. Boca Raton, Florida, in press. Gordon. L. M . , Sauerheber, R. D., and Esgate, J. A . (1978). Spin label studies on rat liver and heart plasma membranes: Effects of temperature, calcium and lanthanum on membrane fluidity, J . Siiprumoi. Srruct. 9. 299-326. Gordon, L. M., Sauerheber, R. D., Esgate. J. A,. Dipple, I.. Marchmont. R. J.. and Houslay, M. D. (1980a). The increase in bilayer fluidity of rat liver plasma membranes achieved by the local anesthetic benzyl alcohol affects the activity of intrinsic membrane enzymes. J. Biol. Chrm. 255, 4519-4527. Gordon. L. M., Dipple, I . . Sauerheber, R. D., Esgate, J . A,. and Houslay, M. D. (1980b). The selective effects of charged local anaesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma membranes. J . Suprumol. Struct. 14, 21-32. Hanski, E . , Rimon. G.. and Levitzki. A. (1979). Adenylate cyclase activiation by the P-adrenergic receptors as a diffiision-controlled process. Biochemisrry 18, 846-853. Hebdon, G . M.. LeVine, H., Minard. R . B.. Sahyoun. N. E.. Schmitges, C. J.. and Cuatrecasas, P. ( 1979). Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles. J . Biol. Chem. 254, 10459-10465.
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Hepp, K. D., Rinninger, J . , Langley, 1.. and Renner, R. (1978). Inhibition of catecholaminestimulated adenylate cyclase in fat cells by local anaesthetics. FEES Lett. 91, 325-328. Higgins, J . A. ( 1981). Biogenesis of endoplasmic reticulum phosphatidylcholine. Translocation of intermediates across the membrane bilayer during methylation of phosphatidylethanolamine. Eiochim. Biophys. Acta 640, 1-15. Higgins, J . A,, and Evans, W. H. (1978). Transverse organisation of phospholipids across the bilayer of plasma-membrane subfractions of rat hepatocytes. Biochem. J. 174, 563-567. Hirata, F.. and Axelrod. J. ( 1978). Enzymatic methylation of phosphatidylethanolamine increases erythrocyte membrane fluidity. Narure (London) 275, 2 19-220. Hirata, F., and Axelrod, J. (1980). Phospholipid methylation and biological signal transmission. Science 209, 1082-1090. Houslay. M. D. (1979). Coupling of the glucagon receptor to adenylate cyclase. Eiochem. Soc. Trans. 7 , 843-846. Houslay, M. D. (1981a). Mobile receptor and collision coupling mechanisms for the activation of adenylate cyclase by glucagon. Adv. Cyclic Nucleoride Res. 14, I 1 I - 119. Houslay, M. D. (1981b). Membrane phosphorylation: A crucial role in the action of insulin, EGF and pp60src. Eiosci. Rep. 1, 19-34. Houslay, M. D., and Palmer, R. W. (1978). Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation. Biochem. 1. 174, 909-919. Houslay, M. D., and Palmer, R. W. (1979). Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase in rat liver plasma membranes. Eiochern. J. 178, 2 17-221, Houslay, M. D., and Stanley, K. K. (1982). “Dynamics of Biological Membranes: Influence on Synthesis, Structure and Function.” Wiley, New York. Houslay, M. D., Warren. G. B., Birdsall, N. J . M., and Metcalfe, J. C. (1975). Lipid phase transitions control P-hydroxybutyrate dehydrogenase activity in defined-lipid protein complexes. FEESLert. 51, 146-151. Houslay, M. D.. Metcalfe, J . C., Warren, G. B., Hesketh, T. R., and Smith, G. A. (1976a). The glucagon receptor of rat liver plasma membranes can couple to adenylate cyclase without activating it. Eiochim. Biophys. Acra 436, 489-494. Houslay. M. D., Hesketh, T. R., Smith, G. A., Warren. G . B., and Metcalfe, J. C. (1976b). The lipid environment of the glucagon receptor regulates adenylate cyclase activity. Eiochim. B i o p h p . Acru 436, 495-504. Houslay. M. D., Johannsson, A,, Smith, G. A,. Hesketh, T. R., Warren, G. B., and Metcalfe. J. C. (1976~).On the coupling of the glucagon receptor to adenylate cyclase. Nobel Found. S.ymp. 34,331-344. Houslay, M. D., Ellory, J . C., Smith, G . A . , Hesketh, T. R . , Stein, I. M . , Warren, G. B., and Metcalfe, I. C. (1977). Exchange of partners in glucagon receptor-adenylate cyclase complexes: Physical evidence for the independent mobile receptor model. Biochim. Eiophys. Acra 467, 208-2 19. Houslay. M. D., Palmer, R. W., and Duncan. R. J . S . (1978). The action of the local anaesthetic, benzyl alcohol and the monoamine oxidase inhibitor, clorgyline on the P-hydroxybutyrate dehydrogenase activity of adult and weanling rat brain mitochondria. J. Pharm. Pharmucol. 30, 711-714. Houslay, M. D., Dipple, 1.. and Elliot, K . R. F. (1980a). Guanosine 5’-triphosphate and guanosine 5’-[pa-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and a catalytic unit of adenylate cyclase. Eiochem. J. 186, 649-658. Houslay, M. D., Dipple, I . , Rawal. S., Sauerbeber, R. D., Esgate, J. A , , and Gordon, L. M. ( 1980b). Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner
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half of the liver plasma membrane bilayer achieved by the local anaesthetic prilocaine. Biochem. J . 190, 131-137.
Houslay, M. D., Dipple, I . , and Gordon, L. M. (1981). Phenobarbital selectively modulates the glucagon-stimulated activity of adenylate cyclase by depressing the lipid phase separation occurring in the outer half of the bilayer of liver plasma membranes. Biochem. J . 197, 675-68 I . Insel, P. A., Nirenberg. P., Turnbull, J . , and Shattil, S. J . (1978). Relationships between membrane cholesterol, a-adrenergic receptors and platelet function. Biochemistry 17, 5269-5274. Iyengar, R., Birnbaumer, L., Schulster, D.. Houslay. M. D.,and Michell, R. W. (1980). Mode of membrane receptor-signal coupling. In “Cellular Receptors for Hormones and Nuerotransmitters” (D. Schulster and A. Levitzki, eds.), pp. 55-81. Wiley, New York. Katz, A . M., and Messineo, F. C. (1981). Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ. Res. 48, 1-16. Keirns, J. J., Kreiner, P. W . , and Bitensky, M. W. (1973). An abrupt temperature-dependent change in the energy of activation of hormone-stimulated hepatic adenylyl cyclase. J . Supramol. Struct. I, 368-378. Kimelberg, H. K . (1977). The influence of membrane fluidity on the activity of membrane-bound enzymes. In “Dynamic Aspects of Cell Surface Organisation” ( G . Poste and G. L. Nicolson, eds.), pp. 205-293. Elsevier, Amsterdam. Kimura, N., and Nagata, N. (1977). The requirement of guanine nucleotides for glucagon stimulation of adenylate cyclase in rat liver plasma membranes. J . Biol. Chem. 252, 3829-3835. Kleeman, W., and McConnell, H. M. (1976). Interactions of proteins and cholesterol with lipids in bilayer memhranes. Biochim. Biophys. Acta 419, 206-222. Klein, I . , Moore, L., and Pastan, I . (1978). Effect of liposomes containing cholesterol on adenylate cyclase activity of cultured mammalian fibroblasts. Biorhim. Biophys. Acta 506, 4253. Krdll, J . F., Leshw, S . C., Frolich, M., and Korenman, S. G. (1981). Activation of uterine smooth muscle adenvlste cyclase by guanyl nucleotide. J . Biol. Chem. 256, 5436-5442. Lad, P. M., Preston, M. S . , Welton, F., Nielsen, T. B . , and Rodbell, M. (1979). Effects of phospholipase A2 and filipin on the activation of adenylate cyclase. Biochim. Biophys. Acta 551, 368-381. Martin, B. R., Stein, J. M., Kennedy, E. L., Doberska, C. A,, and Metcalfe, J. C. (1979). Transient complexes. Biochem. J . 184, 253-260. Marinetti, G . V . , and Crain, R. C. (1978). Topology of amino phospholipids in the red cell membrane. J . Supramol. Strucr. 8, 191-213. Melchior, D. C., and Steim, J. M. (1979). Lipid-associated thermal events in biomembranes. In “Progress in Surface and Membrane Science” (D. A. Cadenhead and J . F. Danielli, eds.), Vol. 13, pp. 21 1-289. Academic Press, New York. Montesano, R., Perrelet, A , , Vassali, P., and Orci, L. (1979). Absence of filipin-sterol complexes from large coated pits on the surface of cultured cells. Proc. Nutl. Acad. Sci. U.S.A. 76, 6391 -6395. Neer, E. J . (1976). The size of adenylate cyclase and guanylate cyclase from the rat renal medulla. J . Supramol. Strurt. 4, 5 1-6 I . Orly, J . , and Schramm, M. (1975). Fatty acids as modulators of membrane functions: Catecholamine-activated adenylate cyclase of the turkey erythrocyte. Proc. Nuti. Acad. Sci. U.S.A. 72, 3433-3437. Pairault, J . , Levilliers, J . , and Chapman, M. J . (1977). Human serum lipoproteins activate adipocyte plasma membrane adenylate cyclase. Nature (London) 269, 607-609. Pang, K.-Y. Y., and Miller, K. W. (1978). Cholesterol modulates the effect of membrane perturbers in phospholipid vesicles and biomembranes. Biochim. Biophys. Acra 511, 1-9.
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Partington, C. R., and Daly, J. W. (1979). Effect of yangliosides on adenylate cyclase activity in rat cerebral cortical membranes. Mol. Pharmucol. 15, 484-491. Pliego, J . A , , and Rubalcava, B. (1978). The effect of temperature on the activity of the adenylate cyclase of liver plasma membranes. Eiochem. Eiophys. Res. Commun. 80, 609-615. Puchwein, G., Pfeuffer, T.. and Helmreich, E. J . M. (1974). Uncoupling of catecholamine activation of pigeon erythrocyte membrane adenylate cyclase by filipin. J . Biol. Chem. 249, 3232-3240. Rasrnussen, H., and Goodman, D. B. P. (1977). Relationship between Ca2+ and cyclic nucleotides in cell activation. Physiol. Rev. 57, 421-509. Rene, E., Pecker, F., Stengel, D., and Hanoune, I. (1978). Thermodependence of basal and stimulated rat liver adenylate cyclase. J . Eiol. Chem. 253, 838-84 I . Rimon, G., Hanski, E., Braun, S . , and Levitzki, A. (1978). Mode of coupling between hormone receptors and adenylate cyclase elucidated by modulation of membrane fluidity. Narure (London) 276, 394-396. Rimon, G., Hanski, E., and Levitzki, A. (1980). Temperature dependence of P receptors, adenosine receptor, and sodium fluoride-stimulated adenylate cyclase from turkey erythrocytes. Eiochemistry 19, 445 1-4460. Ross, E. M., and Gilman A. G . (1980). Biochemical properties of hormone-sensitive adenylate cyclase. Annu. Rev. Biochem. 49, 533-564. Roihman, J. E., and Lenard, J. (1977). Membrane asymmetry. Science 195, 743-753. Rubaclava, B., and Rodbell, M. (1973). The role of acidic phospholipids in glucagon action on rat liver adenylate cyclase. J . Eiol. Chem. 248, 3831-3837. Salesse, R., and Gamier, J. (1979). Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer. Eiochim. Eiophjs. Acta 554, 102-1 13. Sauerheber, R. D., Lewis, U. J . , Esgate, I. A,, and Gordon, L. M. (1980). Effect of calcium, insulin, and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts. Eiochim. Eiophys. Acfu 597, 292-304. Schrdmm, M., Orly, J . , Eimerl, S . , and Komer, M . (1977). Coupling of hormone receptors to adenylate cyclase of different cells by cell fusion. Nature (London) 268, 3 10-3 13. Sheetz, M. P., and Singer, S. J . (1974). Biological membranes as bilayer couples. A molecular mechanism of drug-erythrocyte interactions. Proc. Narl. Acad. Sri. U.S.A. 71, 4457-4461. Sinensky, M. (1980). Adaptive alteration in phospholipid composition of plasma membranes from a somatic cell mutant defective in cholesterol biosynthesis. J . Cell Eiol. 85, 166-169. Sinensky, M., Minneman, K. P., and Molinoff, P. B. (1979). Increased membrane acyl chain ordering activates adenylate cyclase. J . B i d . Chem. 254, 9135-9141. Sinha, A. K., Shattil, S. J . , and Colman, R. W .(1977). Cyclic AMP metabolism in cholesterol-rich platelets. J . Eiol. Chem. 252, 3310-3314. Tolkovsky, A. M., and Levitzki, A. (1978). Mode of coupling between the P-adrenergic receptor and adenylate cyclase in turkey erythrocytes. Biochemistry 17, 3795-38 10. Triggle, D. J . (1972). Effects of calcium on excitable membranes and neurotransmitter action. In “Progress in Surface and Membrane Science” (J. Danielli, P. M. Rosenberg, and D. Cadenhead, eds.), Vol. 5, pp. 267-331. Academic Press, New York. Vance. D. E., and de Kruijff, B. (1980). The possible functional significance of phosphatidylethanolamine methylation. Nature (London) 288, 277-278. Warren, G. B., and Houslay, M. D. (1980). Membrane structure and receptor organisation. In “Cellular Receptors for Hormones and Neurotransmitters” ( G . Schulster and A. Levitzki, eds.), pp. 29-54, Wiley, New York. Warren, G. B., Houslay, M. D., Metcalfe. J . C., and Birdsall, N. J . M. (1975). Cholesterol is excluded from the phospholipid annulus surrounding an active calcium transport protein. Nafure (London) 255, 684-687.
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Whetton. A. D., and Houslay, M. D. (1980). The effect of vinblastine on the glucagon, basal and GTP-stimulated states of the adenylale cyclase from rat liver plasma membranes. FEES Leu. 111, 290-294. Wolff. J . . and Jones. A . B. (1970). Inhibition of hormone-sensitive adenylate cyclase by phenothiazines. Proc. Nail. Acad. Sci. U.S.A. 65, 454-459.
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I 8
The Analysis of Interactions between Hormone Receptors and Adenylate Cyclase by Target Size Determinations Using Irradiation Inactivation B . RICHARD MARTIN Department of Biochemistry Universit.v of Cambridge Cambridge. England
I. If. 111. IV .
Irradiation Inactivation: General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Practical Considerations in Irradiation Inactivation Studies on Membranes ......... ................. ............
Membrane Adenylate Cyclase . . . . . . . . . . . . . . ....................... Model of Hormone Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Effects of Fluoride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Evaluation of the Model in Relation to the Results of Other Approaches . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V.
1.
233 236 231 239 241 246 248 253
IRRADIATION INACTIVATION: GENERAL CONSIDERATIONS
The method of irradiation inactivation as an approach to the determination of the molecular weight of proteins had been used as long ago as the 1930s. The first systematic study was conducted by Kepner and Macey in 1968. They examined a number of proteins whose molecular weights had already been determined by conventional methods. In most cases the activity of the enzyme decayed as a monoexponential with increasing doses of electron irradiation, and they were able to determine empirically the expression Molecular weight = (6.5 233
X
1OS)/D37 Copyright Q 1983 by Academic Press, Inc. All rishi? of re~roduclionin any form rcwrved.
ISBN 0-12-153318-2
234
B. RICHARD MARTIN
where D37 is the radiation dose in Mrad at which 37% of the original activity remains. A theory to account for this behavior was formulated-the single hit hypothesis. This states that a single hit by one electron is sufficient to destroy the activity of the protein completely. Thus for a given dose of electron radiation the likelihood of a protein molecule being hit will depend upon its size and, accordingly, upon the molecular weight. This method of protein size determination was particularly attractive as a means of examining association and dissociation of proteins in membranes. All that is required is a measure of the activity of the protein under study. There is no requirement for the protein to be pure or for the protein to be in solution. Thus, the method offers the opportunity to examine the size of proteins in intact biological membranes. Before applying the method to adenylate cyclase in plasma membranes, however, a number of potential problems need to be considered. At the time we started these experiments there were very few determinations of molecular weights of adenylate cyclase or indeed of other membrane integral enzymes available for comparison with the results of the target size analysis. Many of the values which had been determined for adenylate cyclase largely depended on gel filtration experiments using preparations solubilized in nonionic detergents (Welton et af., 1977, 1978). Under these conditions there is a tendency for large polydisperse aggregates to form, giving overestimates of the molecular weight. The possibility must therefore be considered that the target size determined for a membrane integral protein may reflect the molecular weight of not only the protein but also some attached lipid. In fact, subsequent molecular weight determinations for adenylate cyclase by a variety of methods have yielded values which are in quite good agreement with the values obtained by irradiation inactivation (Neer, 1974; Newby et af., 1978). In any case, we are more interested in alterations in the molecular weight of the components in response to specific effectors than in the absolute values of the molecular weight. A second, more serious consideration relates to the validity of the method in determining changes in states of associations. Where multisubunit enzymes have been examined by irradiation inactivation the target size sometimes corresponds to the whole enzyme, which is obviously the state of affairs which is desirable in this case, and sometimes corresponds to the monomer molecular weight (for review see Kempner and Schlegel, 1979). If this is the case with adenylate cyclase, the method will not yield any useful results. The only way around this problem is to perform the experiments on an empirical basis and to see if the results conform to a rational model which can be supported by other types of data. There are two types of approach to the study of protein-protein interaction by target size analysis. In the first case the preparation, in our case rat liver plasma membranes, is irradiated in the absence of any effectors or in the basal state. The
TARGET SIZE ANALYSIS
235
activity under study, for example, the catalytic activity of adenylate cyclase, is then determined in the usual way, and the target size for the basal state can be calculated. The activity can also be determined in the presence of an activator such as a hormone. If the increased activity does not depend upon the association of a second component, in other words, if in this case the binding site for the hormone is on the same protein unit as the catalytic site of adenylate cyclase then the target size will not change. If, on the other hand, the increased activity in the presence of the effector does require the association of a second distinct component, then the target size will increase. This is because the increased activity depends upon both the catalytic unit and the receptor, and both are affected by the irradiation. It is important to stress, however, that the extent of the increase in target size will depend upon the extent to which the activity is dependent on the second component, that is, the larger the activation the larger the increase. The increase will also depend on the relative size of the two components. If, for example, the hormone receptor is small relative to the catalytic unit the loss of receptor activity will also be less for a given radiation dose and the increase in target size will be small. In theory this approach should also be applicable to twocomponent systems where the regulatory component results in an inhibition, although in this case the addition of the inhibitor in the assay will result in a reduction rather than an increase in the target size. However, I am not aware of any publications in which irradiation inactivation has been used to examine inhibitory processes. It should be stressed that the target size increases determined under these conditions are not a direct measure of a change in molecular weight but simply reflect the fact that more than one separate component is involved in the change in activity. In the second type of general approach the membranes are preincubated with the effector prior to irradiation. In this case the preincubations should, so far as possible, be identical to the conditions which are normally employed in the assay of the activity which is under study, although minor modifications may be necessary. For example, it has been found that the presence of thiol reagents such as dithiothreitol during irradiation leads to extensive nonspecific loss of activity of many enzymes. In experiments in which the system is exposed to the effector before the irradiation, any changes in target size should reflect an actual change in the molecular weight of the component responsible for the activity which is measured. However, in interpreting this type of result care must be taken to ensure that the conditions under which the enzyme is assayed do not lead to any further apparent changes in target size resulting from the involvement of another component. In general the interpretation of the results of the first type of approach is relatively straightforward. The pretreatment approach yields more interesting information but more care is required in the interpretation of the results.
236
8 . RICHARD MARTIN
II. PRACTICAL CONSIDERATIONS IN IRRADIATION INACTIVATION STUDIES ON MEMBRANES A number of approaches have been used in the preparation of plasma membranes for irradiation. Schlegel et al. (1979) irradiated membranes in aqueous suspension. In this case the suspension must be frozen, and they conducted their irradiations at - 170°C using liquid nitrogen as the freezing agent. This approach has the advantage that there is no possibility of a damaging rise in temperature due to the irradiation which may result in a loss in activity due to heating rather than to the impact of the electrons. It also has the advantage that recoveries of activity in nonirradiated samples in frozen suspension are likely to be close to loo%, and since the preparation simply has to be allowed to thaw the preparation of the samples for assay is simple. The main disadvantage with the use of frozen samples is that with a temperature as low as - 170°C the expression derived by Kepner and Macey ( 1968) no longer holds and a conversion factor of 1.7 has to be used. This results in the need for very high radiation doses of the order of 20-30 Mrad to achieve 90% inactivation of quite large proteins of molecular weights of 100,000or more. One then has to be concerned about'the calibration of the radiation dose. This is most commonly done by measuring changes in the optical density of pieces of Perspex of standard thickness and quality. The operative range for this type of calibration is 0-3 Mrad and the advisability of extrapolating the calibration to doses 10 or 20 times higher is doubtful. As discussed above, a general cause for concern in the application of the method is whether or not association of different components is always reflected in an increase in the target size. This seems more likely to be a problem when the irradiation is carried out at very low temperatures. We have preferred to use the alternative approach of freeze-drying the membranes after preincubation when appropriate and before irradiation. At the end of the preincubation the preparation is rapidly frozen by immersing the tube in liquid nitrogen and is then lyophilized. Obviously a critical first step is to ensure that the bulk of the activity of a preparation which has not been irradiated can be recovered after the rehydration of the membranes. In the case of adenylate cyclase we were able to recover more than 90% of the enzyme activity routinely. It is important, however, that the freeze-drying process should be carried out very carefully since in our experience any loss of vacuum during the drying process leads to substantial and variable loss of activity. When freeze-dried membranes are used the irradiation is carried out at about 10"-15°C and the apparatus can be cooled by a flow of chilled air. It should be borne in mind, however, that large irradiation doses of 10 Mrad or more may lead to unacceptable increases in temperature. In either approach oxygen should be excluded from the system. In the case of irradiation in aqueous suspension the medium should be flushed with nitrogen before use. In the case of dry samples the ideal
237
TARGET SIZE ANALYSIS
solution is to irradiate under vacuum, or, if this is not possible, under a nitrogen atmosphere. In the majority of cases the irradiations will be carried out using a linear accelerator belonging to a radiotherapeutics facility. One is therefore dependent upon the goodwill of technical staff whose prime concern is the treatment of cancer patients, so to some degree the simpler the irradiation protocol that can be used the better.
111.
ANALYSIS OF DATA
Typical irradiation plots are shown in Fig. 1. In this case all the plots are linear, which indicates that the protein which is responsible for the activity under study is present as a single homogeneous species. If this is not the case a curved plot will result (Fig. 2). Great care is needed in the analysis of this type of data. It may be tempting to use a computer to fit the data to two straight lines to determine the target sizes of the two species present. However, a number of problems should be borne in mind. First, it is necessary to be satisfied that there are only two species and not three or four, in other words, that the curve is not fitted better by assuming three or four species. In order to do this with any degree of confidence the curve must be very well defined indeed, with a very large number of data points. Care must be taken in the choice of the mathematical expression used to fit the curve and it is probably advisable to take expert advice. If other data are available to give an independent measure of the target size, that is, target sizes obtained from linear plots obtained under other conditions, the situation is more straightforward. In this case one can ask the question, Does the nonlinear plot give a good fit to a combination of two linear plots whose parameters are known'? Of course one should have a biochemical rationale for the assumption that the curve represents these two particular species. It should always be borne in mind, however, that given a particular set of instructions a computer will usually batter a set of data into some sort of submission but the results may not be particularly useful. In dealing with nonlinear plots alternative explanations should be considered as well as the existence of more than one species. One should not lose sight of the fact that a preparation such as a plasma membrane contains many enzymes, all of which will be affected by irradiation. The possibility that this may give rise to spurious effects should be considered. In the case of adenylate cyclase, for example, care must be taken to ensure that the ATP regenerating system in the assay is adequate to maintain ATP levels throughout the assay time course. If this is not the case, the fact that the ATPase activity in the plasma membranes will also be reduced by irradiation may cause an apparent curvature in the irradiation plot. This is particularly likely to be the case if very low concentrations of ATP are used so as to increase the specific activity of the ATP used in the assay in an
238
8.RICHARD MARTIN
30' C
30'C
(315,000)
I , 0
.I
1
2
3
4
5
I,...., 0
1
2
o'c
3
4
5
0%
0
1
2
3
4
5
Mrad
Mrad
FIG. 1. Effect of glucagon and p(NH)ppG on irradiation inactivation of adenylate cyclase. Rat liver plasma membranes were preincubated in the presence of 25 mM Tris-HCI buffer, pH 7.4, 0. I mM ATP, 0. I mM cyclic 3'5'-AMP, 10 mM MgC12, 5 mM phosphocreatine, and 5 units of creatine kinase for 10 minutes at either 30 or 0°C. Further additions were (0) no additions, (0)p(NH)ppG (0.1 mM), (0) glucagon ( I pM), and glucagon (1 pht) + p(NH)ppG (0.1 mM). After irradiation adenylate cyclase assay was determined at 30°C for 20 minutes in a medium containing 25 mM Tris-HCI, pH 7.4,0.5 mM ATP, 1 FCi of [a-32P]ATP, 0.1 mM cyclic 3'5'-AMP, 10 mM MgCI,, I mM dithiothreitol, 5 mM phosphocreatine, and 5 units of creatine kinase. Numbers in parentheses indicate target sizes. From Martin et al. (1979).
(m)
239
TARGET SIZE ANALYSIS
30°C
30°C
No Additions
(331,000)
,
3
I
1
2 3 M rad
4
5
I
1
0
1
1
2 3 Mrad
L
5
FIG. 2. Effect of GTP on irradiation inactivation of adenylate cyclase. Rat liver plasma membranes were preincubated at 30°C in the presence of (0) no additions, ( 0 )GTP (1 mM), and ((?) GTP ( 1 mM) + glucagon ( 1 w). Other conditions were as described in the legend to Fig. I . From Martin et al. (1979).
attempt to make the detection of very low adenylate cyclase activities easier. In general, it is desirable to conduct irradiations under extreme conditions. If an activation is under study it should be ensured that activation is maximal. This increases the chances of generating a single homogeneous species and hence a linear irradiation activation plot which will be much easier to interpret. Conditions which give a partial effect and hence nonlinear plots are also much easier to interpret in the light of additional information derived from extreme states.
IV. THE APPLICATION OF TARGET SIZE ANALYSIS TO RAT LIVER PLASMA MEMBRANE ADENYLATE CYCLASE The approach of irradiation inactivation was first used on rat liver plasma membrane adenylate cyclase by Houslay and his colleagues (Houslay et al., 1977). The aim was to see if the method would yield any evidence in support of the mobile receptor hypothesis. They examined the effect of glucagon on both the target size of adenylate cyclase catalytic activity and on the target size of the specific glucagon binding activity which was taken to reflect the amount of functional glucagon receptor. When liver plasma membranes were irradiated in the absence of any effectors the target size for adenylate cyclase in the presence of either fluoride or guanylyl imidodiphosphate [p(NH)ppG] was 160,000. In the presence of glucagon, however, added in the assay after the irradiation, the target
240
8. RICHARD MARTIN
size increased to 389,000. Under the same conditions the specific glucagon binding activity had a target size of 217,000. The increase in target size on addition of glucagon after the irradiation indicates the involvement of a second component in the glucagon activation other than the component containing the catalytic site. When the plasma membranes were exposed to glucagon at 0°C prior to irradiation the target size for both the catalytic activity and the glucagon binding activity increased to about 380,000. Thus the data were consistent with the binding of the hormone to the receptor causing the association of the receptor with the catalytic unit of adenylate cyclase. A particularly attractive aspect of this study was that the activity of both of the components could be determined independently, the catalytic unit by measuring adenylate cyclase activity and the hormone receptor by measuring glucagon binding. The results of both types of measurements were internally consistent. This study provided convincing support for the mobile receptor hypothesis in which the binding of hormone promotes the association of the hormone receptor with the catalytic unit and the complex formed represents the activated state. In the initial study of Houslay and his colleagues, all the preincubations with glucagon were carried out at 0°C in the absence of any other effectors and in the absence of the substrates necessary for catalytic activity. In particular, no attempt was made to examine the effects of guanine nucleotides which are essential for the full expression of hormone stimulation of adenylate cyclase. We therefore decided to examine the effects of glucagon and also of guanine nucleotides under conditions in which we could be certain that activation of adenylate cyclase had taken place (Martin et al., 1979). To this end all the pretreatments of the plasma membranes were carried out under conditions identical with those routinely used for the determination of adenylate cyclase activity with the single exception that dithiothreitol was omitted from the medium. Under these conditions we were able to essentially repeat Houslay’s original observations of the effects of pretreatment of membranes with glucagon at 0°C. In this case the target size of adenylate cyclase catalytic activity increased from 300,000 to 470,000, again reflecting the association of the hormone receptor with the catalytic unit of the enzyme. At 0°C the addition of either GTP or p(NH)ppG had no effect on the target size in either the presence or absence of glucagon, and hence apparently no effect on the association of the hormone receptor with the enzyme. The effect of glucagon and guanine nucleotides on pretreatment at 30°C presented a very different picture, however. Pretreatment with p(NH)ppG caused a reduction in the target size from 300,000 to 200,000 and this was not affected by the presence of glucagon (Fig. 1). Thus, in the presence of p(NH)ppG which in liver plasma membranes is capable of activating adenylate cyclase to its maximal extent, there was no evidence of any association of the hormone receptor with the catalytic unit of adenylate cyclase. It also appeared that some component of the system dissociates. We then examined the effect of the natural guanine nucleotide GTP
241
TARGET SIZE ANALYSIS
at 30°C. On preincubation in the presence of GTP alone we found a nonlinear inactivation plot indicating that more than one species was responsible for the activity of adenylate cyclase. Preincubation in the presence of GTP together with glucagon gave a linear plot indicating a reduction in target size from 300,000 to 200,000, comparable to that seen with p(NH)ppG either alone or in the presence of glucagon (Fig. 2 ) . Thus it appears that GTP promotes the same dissociation observed with p(NH)ppG but that the effect is incomplete. At this stage we attempted to devise a model which would account for the data and provide a framework for the design of further experiments.
V.
MODEL OF HORMONE ACTION
The model for the effects of glucagon and guanine nucleotides on the target size of adenylate cyclase is outlined in Fig. 3 . We suggest that the effect of p(NH)ppG is to cause the dissociation of a regulatory subunit (G) from the catalytic subunit (C). The catalytic subunit is assumed to be fully activated in the
b
GI ucagon
I
FIG. 3. Model of the mechanism of activation of adenylate cyclase by glucagon and guanine nucleotides. The figure describes the model proposed for the alterations in the aggregation state of adenylate cyclase during activation by (a) guanine nucleotides and (b) guanine nucleotides in the presence of glucagon. The glucagon receptor is represented by R, the catalytic unit by C, and the regulatory unit by C. From Martin el al. (1979).
242
0. RICHARD MARTIN
dissociated state and the regulatory subunit is assumed to be a guanine nucleotide binding protein. The dissociation promoted by p(NH)ppG is unaffected by the independent hormone receptor which, in the absence of glucagon, does not interact with the (GC) dimer (Fig. 3a). The effect of p(NH)ppG is assumed to be essentially irreversible and complete. In contrast, the effects of GTP alone are reversible and incomplete in that an equilibrium is established between the activate dissociated state (C) and the inactive associated state (GC); this is consistent with the nonlinear irradiation inactivation plot and with the lower degree of activation observed in the presence of GTP than that observed with p(NH)ppG (Table 1). All the effects of guanine nucleotides showed a marked dependence on temperature and did not occur at 0°C. The model for the action of glucagon is summarized in Fig. 3b. In the presence of glucagon the receptor (R) associates with the inactive complex (GC) to form a ternary complex (RGC). This effect is not temperature dependant and can be detected at 0°C. In fact, for reasons to be discussed below, it is more readily detected at 0°C when the effects of guanine nucleotides are not present than at 30°C. At 30°C in the presence of either p(NH)ppG or GTP the complex (RGC) dissociates, releasing free (C) and hence activating adenylate cyclase. Glucagon and GTP together are able to promote complete dissociation of the complex (RGC) to release the activated state ( C ) , whereas GTP alone will promote only TABLE I EFFECTSOF GUANINE NuCLEOTlDES A N D GLUCACON ON ADENYLATE CYCLASE I N RAT LIVERPLASMAMEMBRANES".~
Additions to assay No preincubation None GTP ( I mM) p(NH)ppG (0.1 mM) Glucagon ( 1 pkf) Glucagon ( I pkf) + GTP ( I mM) Glucagon ( I pM) + p(NH)ppG (0.1 mM)
A.
Adenylate cyclase (nmole/20 minutes per mg of protein)
0.465 1.40 3.69 1.16 4.64 5.01
k
0.012
f
0.047
5.11 5.15
f 0.136
2 0.126 f 0.014
2 0.008 2 0.057
2 0.104
From Martin et a/. (1979). Adenylate cyclase was determined in rat liver plasma membranes as described in the text. Membranes either without pretreatment (A) or pretreated with p(NH)ppG (0.I M ) (B) were incubated for 10 minutes at 30°C in MgC12 (10 mM). Results are means 5 SEM for three observations. 1'
h
TARGET SIZE ANALYSIS
243
partial dissociation of the complex (GC). Thus, in common with Cassel and Selinger and many other workers, we propose that the effect of the hormone receptor complex is to facilitate the activation of adenylate cyclase by GTP. At this stage a number of features of the model were undefined and at least one important assumption had been made. In this study only the target size of adenylate cyclase catalytic activity was determined, so that we were unable to say whether the ternary complex dissociates completely to give free (C), (G), and (R) or whether component (G) and the receptor (R) remain associated as a complex (GR) and a further dissociation event is required to regenerate free (R) and (G). Under physiological conditions the free regulatory component (G) must eventually become available to regenerate the inactive complex (GC), and in the absence of any evidence one way or the other we made the simpler assumption that the dissociation takes place in one step to release free (C), (G), and (R). The second, more important assumption relates to the nature of the regulatory subunit which dissociates. As described in the first article, there is a large body of evidence to suggest that the adenylate cyclase system contains a protein component which mediates the activation of the enzyme by guanine nucleotides and also has a GTPase activity which is responsible for the decay of the activation. We made the assumption that the regulatory subunit (G) which dissociates under the influence of guanine nucleotides can be identified with this component of the system. It should be pointed out that there is no direct evidence for this assumption. To obtain such evidence we would have to conduct irradiation inactivation analysis of the guanine nucleotide binding activity associated with the activation of adenylate cyclase and of the specific hormone-sensitive GTPase activity. Unfortunately, in rat liver plasma membranes this is not technically possible. Together with a number of other groups (Cassel and Selinger, 1977; Lin et al., 1978), we have found that the background of nonspecific GTPase activity and nonspecific guanine nucleotide binding activity in rat liver plasma membranes is far too high to allow the measurement of the specific activities associated with the regulation of adenylate cyclase. Thus it was not possible to conduct an experiment analogous to those performed by Houslay et al., who were able to measure both adenylate cyclase catalytic activity and the specific glucagon binding activity. This means that we cannot exclude the possibility that the binding site for guanine nucleotides is on the same component (C)as the catalytic site of adenylate cyclase or indeed that both the catalytic unit (C) and the regulatory unit ( G ) contain a binding site for guanine nucleotides. The model gave rise to a number of predictions, some of which were very simple to test. It appears that p(NH)ppC alone can cause complete dissociation of the regulatory subunit from the catalytic subunit, and we propose that the free catalytic subunit (C) represents the fully activated state of the enzymes. It follows from this that glucagon should not cause any further increase in the activity of the enzyme in the presence of p(NH)ppG. At first sight this did not appear to
244
B. RICHARD MARTIN
be the case. If glucagon and p(NH)ppG were added simultaneously at the beginning of the assay, then glucagon apparently caused a small but consistent and significant increase in activity (Table IA). However, if the liver plasma membranes were preincubated in the present of p(NH)ppG for 10 minutes, the period used for preincubations prior to irradiation, the addition of glucagon produced no further activation (Table IB). Rodbell et al. (1975) showed that there was a marked lag in the response of rat liver plasma membrane adenylate cyclase to p(NH)ppG and that this lag was abolished in the presence of glucagon. The final extent of the activation was not, however, affected by the hormone. It seems, therefore, that glucagon is able to increase the rate of activation by p(NH)ppG but not the extent of the activation, and that our preincubation was sufficiently long to allow full activation by the guanine nucleotide. The target size analysis implies that GTP in the presence of glucagon is as effective as p(NH)ppG in promoting the dissociation of the regulatory subunit. These conditions should therefore be equally effective in activating adenylate cyclase. Table I A shows that this was the case. A further prediction was that at 0°C there should be little or no activation by any effector or combination of
FIG. 4. Effect of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides. Adenylate cyclase activity was determined as described in the legend to Fig. 1 at GTP ( I M).([7)p(NH)ppG (0.1 mM). ( 0 )glucagon various temperatures in the presence of (0) ( I pM). glucagon ( I p W ) + GTP (I mM), and (A) glucagon ( I p M ) + p(NH)ppC (0.1 mM). From Martin el a / . (1979).
(m)
245
TARGET SIZE ANALYSIS
effectors, since the target size analysis gave no indication of any dissociation to release the free active catalytic unit at 0°C. The maximum activation at 30°C was 800%, observed with glucagon together with p(NH)ppG. At 0°C the corresponding activation was 50%. This would represent a proportional dissociation to yield free (C) which would be well below the limits of sensitivity of the target size analysis (Fig. 4). The model suggests that at 30°C in the absence of guanine nucleotides glucagon should cause an increase in target size reflecting the formation of the ternary complex (RGC), which in the absence of guanine nucleotides should persist. In fact, under these conditions, we observed a nonlinear plot which suggested the presence of a mixture of several different species (Fig. 5). This is to be expected, since glucagon alone activates the enzyme to a limited extent (Table IA). This is probably due to the presence of small amounts of GTP contaminating either the plasma membrane preparation or the ATP used as substrate in the assay. It is well established that the complete purification of the system from contaminating GTP is very difficult (Kimura et al., 1976). More recently, however, a number of ATP preparations have become available with greatly improved purity with respect to GTP compared to the preparations available at the time of this study. If we were to repeat this experiment now, the highmolecular-weight species corresponding to the ternary complex should predominate to a much greater extent.
Glucagon
GT P
.?
\
bGlucagon
\
ol 0
1.0.
Control
I
0
1
2
3
4
5
Mrad
FIG. 5 . Effect of glucagon on irradiation inactivation of adenylate cyclase at 30°C. Rat liver plasma membranes were preincubated at 30°C as described in the legend to Fig. I in the presence of (0)no additions, (0) glucagon (1 @), and ( 0 )glucagon ( 1 @) + GTP ( 1 mM). Adenylate cyclase activity was determined as described in the legend to Fig. 1 .
246
B. RICHARD MARTIN
VI.
EFFECTS OF FLUORIDE
The mechanism of activation of adenylate cyclase by F- ions i s still not entirely clear. One point which has been established, however, is that the effect appears to be mediated by the same protein component of the system that mediates the action of guanine nucleotides. This conclusion is based on the studies of Gilman and his colleagues (Ross er al., 1978), who showed that cyc- S49 lymphoma cells, where the adenylate cyclase lacks the capacity to respond to guanine nucleotides, have also lost the capacity to respond to fluoride. When we pretreated rat liver plasma membranes with F- in the presence of an adenylate cyclase assay medium at 30"C, the target size was reduced from 300,000 to 200,000 (Fig. 6, Martin et al., 1980). This effect was exactly comparable with the effect of p(NH)ppG or of GTP in the presence of glucagon. It seemed likely, therefore, that the activation of the enzyme by F- ions involved the dissociation of the same regulatory subunit which was released on activation by guanine nucleotides. In contrast to the effects of guanine nucleotides, however, the reduction in target size with F- was observed when the preincubation was carried out at 0°C as well as at 30°C (Fig. 6). Since we proposed that the reduction in target size reflects a necessary step in the activation mechanism,
2.0-
--
>1
> .c
"1.5-
a & =
D
,o 1.0-
No Addltions
No Additions (329.000)
0
0
1
2
3 M rad
4
5
6
b
1
2
3
4
5
6
Mrad
FIG.6 . Effect of F- ions on the irradiation inactivation of rat liver plasma membrane adenylate cyclase. Rat liver plasma membranes were preincubated as described in the legend to Fig. 1 at 0 and or presence (0)of 10 mM NaF. Adenylate cyclase activity was deterat 30°C in the absence (0) mined after incubation as described in the legend to Fig. I . From Martin et a/. (1980).
247
TARGET SIZE ANALYSIS
TABLE I1 EWM-TOF TEMPEKATURt O N T H t ACTIVATION Ob R x r LIVERPLASMA MEMBRANE ADtNYl.ATh C Y C L A SB~Y F- "." Adenylate cyclase activity (nmolei10 minutes per mg of protein) Temperature ("C)
No addition
10 mM NaF
Increase in activity (9%)
0 30
0.01 19 f 0.0004 0.365 t 0.016
0.09.5 !I0.0014 3.587 2 0.067
no0 980
From Martin rr a / . (1980). Rat liver plasma membranes were incubated in 0.1 ml of assay medium as described in the text with further additions as given in the table. Incubations were for 40 minutes with 0. I mg of membrane protein at 0°C and for 10 minutes with 0.04 mg of membrane protein at 30°C. Results are means 2 SEM for three parallel incubations.
then F- ions in contrast to p(NH)ppG should be capable of activating adenylate cyclase at 0°C. Table I1 shows that this was in fact the case. The extent of activation in the presence of F- ions at 0°C was comparable with the activation observed at 30°C. There was a very marked temperature dependence of the adenylate cyclase activity in e.ither the presence or absence of F- ions. In both cases, the increase in activity from 0" to 30°C was more than 30-fold. Similar results have been reported by Houslay et al. (this volume), in a much more extensive study of the effects of temperature on adenylate cyclase activity, and other membrane integral enzymes have been shown to have a similar marked dependence of temperature (Houslay and Palmer, 1976). In common with other workers (for review see Bimbaumer, 1973), we observed that the activation of adenylate cyclase by F- ions was consistently about 20 or 30% less than the activation observed with p(NH)ppG. Since F- ions were as effective as p(NH)ppG in promoting a reduction in target size and since we proposed that the low-molecular-weight species represents the fully activated state of the enzyme, we would expect the two effectors to produce the same activation. When the effects of varying the concentration of F- were examined, it was found that the response of adenylate cyclase activity was biphasic. The activity first increased with increasing F- concentration and then decreased (Fig. 7). If the adenylate cyclase was first fully activated by preincubation in the presence of glucagon and p(NH)ppG before the addition of F - , then all concentrations of F- were inhibitory (Fig. 8). It seems, therefore, that F- has two effects, one to activate and one to inhibit. The lower activation observed in the presence of F- ions in comparison to p(NH)ppG can therefore be explained by the inhibitory effect's becoming significant at F- concentrations at which the
248
B. RICHARD MARTIN 5r
10
20
30
40
[F-] (mM1
FIG.7. Effect of F- ions on rat liver plasma membrane adenylate cyclase activity. Adenylate cyclase activity was determined as described in the legend to Fig. I for 20 minutes at 30°C in the presence of varying concentrations of NaF. From Martin er nl. (1980).
activation effect is not complete. The data are therefore consistent with the view that the activation of adenylate cyclase by F- involves the release of the same regulatory subunit that is released on activation by guanine nucleotides.
VII. EVALUATION OF THE MODEL IN RELATION TO THE RESULTS OF OTHER APPROACHES In conclusion, we should consider how consistent our model, derived from target size analysis, is with data derived by other workers by more conventional approaches. The model makes two major suggestions. The first is that the association of the hormone receptor with the catalytic unit is transitory and that, as the activation process is completed by the involvement of a guanine nucleotide, the receptor dissociates. An implication of this proposal is that the receptor will then be available to promote the activation of further adenylate cyclase complexes. Its effectiveness in doing this will depend upon the relative rates of interaction with further complexes compared to the rate of dissociation of the bound hormone which will, of course, render the receptor ineffective as an activator of the enzyme. The second major proposal was that the activation of
249
TARGET SIZE ANALYSIS
-G E
0
4-
\
c
c
e
a
0'
' 0
10
20
30
40
[F-] ( m M )
FIG.8. Effect of F- ions on rat liver plasma membrane adenylate cyclase after preactivation with glucagon and p(NH)ppG. Rat liver plasma membranes were irradiated for 10 minutes at 30°C in 25 mM Tris, pH 7.4, in the presence of glucagon ( I M),p(NH)ppG (0.1 mM), and MgCI2 (10 mM). Adenylate cyclase activity was determined at the same concentrations of glucagon and p(NH)ppG and varying concentrations of NaF as described in the legend to Fig. I . From Martin efal. (1980).
adenylate cyclase by guanine nucleotide involves the release of a regulatory unit. We also suggested that this regulatory unit corresponds to the guanine nucleotide binding protein, whose existence has been established by a large number of different groups. The suggestion that the association of the hormone receptor is transient helps to explain a number of earlier observations and is also supported by the work of other groups which was conducted at about the same time as our original study. Rodbell and his colleagues examined the relationship between the specific binding of glucagon to rat liver plasma membranes and the activation of adenylate cyclase (Rodbell et al., 1974). They found that in the absence of GTP, the rate of dissociation of glucagon from the plasma membranes was very slow, and that accordingly the apparent binding affinity was very tight. Our model predicts that in the absence of GTP and in the presence of glucagon, the predominant species will be the ternary complex of receptor, regulatory unit, and catalytic unit (RGC). We suggest that the rate of dissociation of glucagon from this complex is slow. In the presence of GTP, the ternary complex will break down to release the free hormone receptor (R). We suggest that the rate of dissociation of glucagon
250
B. RICHARD MARTIN
from the free receptor is relatively fast. This would explain the effects of GTP on the binding of glucagon to the hormone receptor. A more careful consideration of Rodbell’s data suggests that this may be an oversimplification, since the GTP concentration dependence for the effect on glucagon binding and the activation of adenylate cyclase differs. This might suggest that the complex of receptor and regulatory unit (RG) does persist after dissociation of the ternary complex (RGC). The same group also found that at physiological concentrations of glucagon, the addition of GTP resulted in a marked increase of adenylate cyclase activity, while the amount of glucagon bound was reduced to 20% or less (Rodbell et al., 1974). To put this another way, maximal activation by glucagon in the absence of added GTP or more likely in the presence of very low concentrations of GTP, since the problem of GTP contamination had not been recognized when these experiments were done, requires 100% occupancy of the glucagon receptors. In the presence of optimal GTP, maximal activation by glucagon, which was also greatly increased in comparison to the activity in the absence of GTP, required only 20% occupancy of the glucagon receptors. It seems likely, therefore, that in the presence of GTP, one hormone receptor complex is able to promote the activation of several adenylate cyclase catalytic units. When the complex (RGC) is dissociated by the action of GTP at the same time as the activation process is completed, the free hormone receptor complex will be available to promote the activation of further catalytic units. The concentration of free hormone receptor complex which is available to do this will be dependent upon the concentration of free hormone to which the receptors are exposed. This model proposing the transient nature of the association of the hormone receptor with the catalytic unit is very similar to the collision coupling model proposed by Tolkovsky and Levitzki (1978). They came to essentially the same conclusions using a very different experimental approach. Their model was based upon a detailed study of the kinetics of activation of turkey erythrocyte plasma membrane adenylate cyclase by catecholamines. Houslay and his coworkers have made use of the effects of phase transitions in rat liver aqd hamster liver plasma membranes to examine the state of association of the glucagon receptor with the catalytic unit of adenylate cyclase (Houslay et al., 1980). This study gave rise to the same conclusion, that, in the presence of GTP, the association of the receptor with thd’catalytic unit does not persist. Both these studies are described in detail elsewhere in this volume. Lefkowitz and his colleagues have examined the effects of GTP on the binding affinity of P-adrenergic agonists and antagonists (Lefkowitz et al., 1981). In the case of P-adrenergic agonists, they found a situation similar to that observed by Rodbell and his co-workers in their studies of glucagon binding. In the absence of GTP, the agonists bound with an apparently high affinity which was reduced by the addition of GTP. Adrenergic antagonists, however, displayed only the low-affinity binding, and the binding affinity was unaffected by GTP. Antago-
TARGET SIZE ANALYSIS
251
nists are thought to compete for binding to the recognition site for a hormone on a hormone receptor but to be incapable of producing the appropriate conformational change in the receptor to produce the appropriate response. Lefiowitz and his colleagues suggested that agonists are capable of promoting an interaction of the hormone receptor with the GTP binding protein and that, in the absence of GTP, this association is long-lived. The rate of release of hormone from this complex is relatively slow, with the result that the apparent binding affinity is high. On the binding of GTP, they suggest that the free receptor is released and that the rate of release of hormone from the free receptor is relatively fast, resulting in an apparent reduction in binding affinity. Since the antagonists are incapable of promoting the interaction of the hormone receptor with the GTP binding protein in the first place, the receptor remains free in both the presence and absence of GTP and only the low-affinity state is observed. There is a considerable body of evidence arising from several different groups of workers using a wide variety of different experimental techniques to support the concept of the transient association of the hormone receptor with the other components of the system. The essential conclusion is that the role of the hormone receptor in the activation is catalytic and that the coupling of the hormone receptor to the adenylate cyclase catalytic unit is not necessary in order to maintain activation. This type of mechanism has been described by Tolkovsky and Levitzki (1978) as collision coupling. The second major aspect of our model was the proposal that the reduction in target size on activation by p(NH)ppG alone or by GTP in the presence of glucagon or by F- reflected the dissociation of the GTP binding protein. As already discussed, the evidence for this aspect of the model was less satisfactory due to the technical impossibility of determining the target size of the guanine nucleotide binding protein independently of its effects on the adenylate cyclase catalytic activity. A consideration of subsequent work by other groups suggests that this aspect of the model, while consistent with the original data, is likely to be an oversimplification of the true situation. There is now a large body of evidence to support the view that the component of the adenylate cyclase system which binds guanine nucleotides and mediates the activation of the enzyme by GTP and p(NH)ppG remains associated with the catalytic unit in the activated state. At the simplest level, we can consider the properties of the adenylate cyclase of cyc- S49 lymphoma cell membranes. These cells appear to lack a functional guanine nucleotide binding protein but retain a fully competent catalytic unit (Ross et al., 1978). If activation requires the dissociation of the guanine nucleotide binding protein, we might expect that the adenylate cyclase in these cells would be fully active, but in fact it displays very low activity. It is possible to argue that the guanine nucleotide binding protein is in fact still present but has lost its ability to interact with GTP while retaining its ability to suppress the activity of adenylate cyclase. However, this is difficult to reconcile with the
252
6. RICHARD MARTIN
ability of active guanine nucleotide binding protein to restore the response of adenylate cyclase to guanine nucleotides. Pfeuffer ( 1977) has shown that the treatment of detergent-solubilized preparations with GTP immobilized on agarose beads and the removal of the agarose beads lead to a loss in adenylate cyclase activity in the presence of p(NH)ppG. The conclusion is again that the removal of the guanine nucleotide binding protein leads to a loss of activity rather than to increased activation. A problem with this particular study is that treatment with GTP agarose does not affect the response of the enzyme to F- ions to the same extent as would be expected if the same protein subunit is involved in activation by fluoride as is involved in activation by guanine nucleotides. It has also been shown by gel filtration studies of detergent-solubilized preparations that treatment with p(NH)ppG tends to increase the molecular weight of adenylate cyclase rather than to decrease the molecular weight (Neer et af., 1980). These studies can be criticized on the grounds that detergent solubilization causes a major disruption of the system and that the size changes determined, while consistent, are very small in relation to size determined for the enzyme in the absence of p(NH)ppG. While none of these studies is conclusive, they do all point to the same conclusion that the component containing the guanine nucleotide binding site needs to be associated with the catalytic unit for the maintenance of guanine nucleotide activation. In the light of this, we should reconsider our conclusion that the regulatory subunit which dissociates under the influence of guanine nucleotides can be identified with the guanine nucleotide binding protein responsible for activation. In this connection, a recent suggestion by Rodbell (1980) is of considerable interest. He points out that in fat cell plasma membranes, GTP demonstrates an inhibitory effect on adenylate cyclase as well as an activation (Harwood et a/., 1973). Furthermore, it is well established that the effects of inhibitory hormones such as a2-agonists on adenylate cyclase also appear to be mediated through a guanine nucleotide binding protein (Jakobs et af., 1981). Thus, the possibility arises that there are two distinct guanine nucleotide binding proteins, one responsible for mediating activation and the other responsible for mediating inhibition. The strength of the approach of target size analysis by irradiation inactivation is exemplified by the results achieved in studies of the effects of hormones. In this case, the method has yielded direct physical evidence of the nature of the interactions of the hormone receptor with other components of the system and the conclusions are well supported by other types of evidence. The major potential pitfall of the approach is illustrated by our original conclusions regarding the guanine nucleotide binding protein. It is not much of an exaggeration to say that, in this case, it was possible to do only half the experiment, since independent determination of the activity of the guanine nucleotide binding protein was not possible. We started the study with the idea derived from a large body of
253
TARGET SIZE ANALYSIS
literature that there are three components in the system and, indeed, the data provided evidence for the existence of three separate components. It was then very difficult to avoid identifying the three components for which we had experimental evidence with the three components which made up our mental picture of the system. As it turns out in the case of the guanine nucleotide binding protein, the picture is not so simple, and other possibilities should be considered. ACKNOWLEDGMENTS The work described in this article was supported by grants from the MRC and the SRC. I would also like to thank Dr. J. M. Stein, who was closely involved in the studies, and Drs. J. C. Metcalfe and M. D. Houslay, with whom I have had many helpful and stimulating discussions. REFERENCES Birnbaumer, L. (1973). Hormone sensitive adenylate cyclases. Useful models for studying hormone receptor functions in cell free systems. Biochim. Biophys. Acru 300, 129-158. Cassel, D., and Selinger, Z. (1977). Mechanism of activation of adenylate cyclase by cholera toxin. Inhibition of GTP hydrolysis at the regulatory site. Proc. Nut/. Acad. Sci. U.S.A. 74, 3307-33 1 I . Cassel, D., and Selinger, Z. (1978). Mechanism of adenylate cyclase activation through the p adrenergic receptor: Catecholamine induced displacement of bound GDP by GTP. Proc. Nutl. Acad. Sci. U.S.A. 75, 4155-4159. Harwood, J . P., Low, H., and Rodbell, M. (1973). Stimulatory and inhibitory effecs of guanyl nucleotides on fat cell adenylate cyclase. J . Eiol. Chem. 248, 6239-6245. Houslay, M. D., and Palmer, R. W. (1978). Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma membrane enzymes occurring on hibernation. Biochem. J . 174, 909-919. Houslay, M. D., Ellory, J. C., Smith, G . H . , Hesketh, T. R . , Stein, J . M., Warren, G. B . , and Metcalfe, I. R. (1977). Exchange of partners in glucagon receptor adenylate cyclase complexes. Physical evidence for the independent, mobile receptor model. Biochim. Biophys. Acta 467 208-2 19. Houslay, M. D., Dipple, I . , and Elliot, K. R. F. (1980). Guanosine triphosphate and guanosine 5'[py-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase. Biochem. J . 186, 649-658. Jakobs, K. H., Aktories, K., and Schulz, G. (1981). Inhibition of adenylate cyclase by neurotransmitters and hormones. Adv. Cyclic Nucleotide Res. 14, 173- 189. Kempner, E. S . , and Schlegel, W. (1979). Size determinations of enzymes by radiation inactivation. Anal. Biochem. 92, 2- 10. Kepner, G. R., and Macey. R. I. (1968). Molecular size determinations by radiation inactivation. Eiochim. Biophvs. Acfa 163, 188-203. Kimura, N., Nakane, K . , and Nagata, N. (1976). Activation by GTP of liver adenylate cyclase in the presence of high concentrations of ATP. Biochem. Biophys. Res. Commun. 70, 1250-1256. Lefkowitz, R. J . , DeLean. A , , Hoffman, B., Stadel, J. M.. Kent, R., Michel, T., and Limbird, L. (1981). Molecular pharmacology of adenylate cyclase-coupled a and p adrenergic receptors. Adv. Cvclic Nucleoride Res. 14, 145-163. Lin, M. C., Welton, A. F., and Berman, M. F. (1978). Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment. J . Cyclic Nucleotide Res. 4, 159-168. Martin, B. R. Stein, J. M., Kennedy, E. L., Doberska, C. A , , and Metcalfe, J . C. (1979). Transient I
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complexes. A new structural model for the activation of adenylate cyclase by glucagon. Biochem. J . 184, 253-260. Martin, B. R., Stein, J. M., Kennedy, E. L., and Doberska, C. A. (1980). The effect of fluoride on the state of aggregation of adenylate cyclase in rat liver plasma membranes. Biochem. J . 188, 137-140. Neer, E. J. (1974). The size of adenylate cyclase. J. Biol. Chem. 249, 6527-6531. Neer, E. J . , Echeverria, D., and Knox, S. (1980). Increase in the size of soluble brain adenylate cyclase with activation by guanosine 5’-(py-imino)triphosphate.J . B i d . Chem. 255, 9782-9789. Newby, A. C., Rodbell, M., and Chramback, A. (1978). Adenylate cyclase in polyacrylamide gel electrophoresis solubilised but active. Arch. Biochem. Eiophys. 190, 109-1 17. Pfeuffer. T. (1977). GTP-binding proteins in membranes and the control of adenylate cyclase activity. J . Biol. Chem. 252, 7224-7234. Robinson, G. A., Butcher, R. W., and Sutherland, E. W. (1967). Adenyl cyclase as an adrenergic receptor. Ann. N.Y. Acad. Sci. 139, 703-723. Rodbell, M. (1980). The role of hormone receptors and GTP regulatory proteins in membrane transduction. Nature (London) 284, 17-22. Rodbell, M., Lin, M. C., and Salomon, V. (1974). Evidence for interdependent action of glucagon and nucleotides on the hepatic adenylate cyclase system. J . Eiol. Chem. 249, 59-65. Rodbell, M., Lin, M.C.. Salomon, Y.,Londos, C., Harwood, J. P., Martin, B. R., Rendell, M., and Berman, M. (1975). Role of adenine and guanine nucleotides in the activity and response of adenylate cyclase systems to hormones. Evidence for multisite transition states. Adv. Cyclic Nucleotide Res. 5, 3-29. Ross, E. M., Howlett, A. C., Ferguson, K. M., and Gilman, A. G . (1978). Reconstitution of a hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J . Eiol. Chem. 253, 6401-6412. Schlegel, W., Kempner, E. S., and Rodbell, M. (1979). Activation of adenylate cyclase in hepatic membranes involves interactions of the catalytic unit with multimeric complexes of regulatory proteins. J. Biol. Chem. 254, 5168-5176, Tolkovsky, A., and Levitzki, A. (1978). Collision coupling of the P-adrenergic receptor with adenylate cyclase. In “Hormones and Cell Regulation” (J. Dumont and 1. Nunez, eds.), Vol. 2, pp. 89-105. North-Holland, Amsterdam. Welton, A. F., Lad, P. M., Newby, A. C., Yamamura, H.,Nicosia, S., and Rodbell, M. (1977). Solubilisation and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide sensitive states. J. Eiol. Chem. 252, 5947-5950. Welton, A. F., Lad, P. M., Newby, A. C., Yamamura, H., Nicosia, S., and Rodbell, M. (1978). The characteristics of lubrol solubilised adenylate cyclase from rat liver plasma membranes. Eiochim. Biophys. Arlo 522, 625-639.
Part I I
Receptors Not InvoIving Adeny Iate Cy clase
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I8
Vasopressin Iso receptors in Mammals: Relation to Cyclic AMPDependent and Cyclic AMPIndependent Transduction Mechanisms SERGE JARD Centre CNRS-INSERM de Pharmarologie-Endorrinologie Montpellier. France
. . . . . . . . . . . . . 255 1. Introduction ...................... 11. 111. Kinetics of Hormone Binding to Vasopressin Receptors . . . . . . . . . . .
IV.
D. Other Vasopressin-Responsive Cells ............ V. Effects of Nucleotides and Other Putative ceptors. . . . . . A. Kidney Receptors ............................................ ........
270 272 272 272
vr. v11. Recognition Patterns of Vasopressin lsoreceptors . . . . . . . . . . . . . . . . . . . . . . . . 275 VIII. Summary and Conclusions. . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
1.
INTRODUCTION
Vasopressin was first recognized as a pressoric principle present in postpituitary extracts (Oliver and Shafer, 1895). Shortly thereafter its antidiuretic action 255
Copyright 0 19x3 by Academic Press, Inc. All rights of reproduction i n any form reserved. ISBN 0- 12-153318-2
256
SERGE JARD
was discovered and its role in the overall regulation of body fluid osmolarity was clearly established (Verney, 1947). The physiological relevance of the vasopressor action of vasopressin in mammals has often been questioned (see, for instance, Saameli, 1968). Indeed, in intact animals the doses of vasopressin needed to elicit a vascular response are much higher than those producing a clearcut antidiuretic response. However, there is increasing experimental evidence that endogenous vasopressin might play a role in controlling blood pressure under several physiopathological conditions such as hemorrhage (Rocha da Silva and Rosenberg, 1969) or hypertension induced by deoxycorticosterone and high salt intake (Mohring et al., 1977). In these situations where plasma argininevasopressin rises, vasopressin antagonists (Cowley et a l . , 1980; Crofton et al., 1979) or antivasopressin antibodies (Mohring et al., 1977) produce the hypotensive responses expected from blocking of the effects of endogenous vasopressin on vascular tone. In the past few years a large variety of biological effects of vasopressin have been described (see Table I), the physiological relevance of which in most instances has yet to be established. Most of the biological effects cited in Table I are elicited by vasopressin doses which are in the range (or at the upper limit) of physiological blood levels. Pharmacological data obtained with vasopressin structural analogs revealed in most instances a high degree of specificity, suggesting that all the observed effects are mediated by specific vasopressin receptors. It is also clearly established (Sawyer et al., 1981) that several structural modifications of the natural vasopressin could affect its biological activities in a differential manner depending on the target tissue considered. In addition, it has been recognized (see for instance Orloff and Handler, 1967; de Wulf et al., 1980) that vasopressin exerts its biological activities through cyclic AMP-dependent (antidiuretic) or cyclic AMP-independent (pressoric and glycogenolytic, among others) effects. It is therefore very likely that several types of vasopressin receptors (vasopressin isoreceptors) exist. The main purpose of the present article is to review available pharmacological and biochemical data on vasopressin receptors in mammals and to discuss the validity of the different criteria which have been or could be used to distinguish vasopressin isoreceptors.
II. METHODOLOGICAL BASIS FOR THE CHARACTERIZATION OF VASOPRESSIN SORECEPTORS The only absolute criterion for the identification of vasopressin isoreptors is the determination of their primary structure in terms of amino acid sequence. This determination is not presently possible, and the use of indirect criteria is therefore necessary. Michell et al. (1979) have proposed two types of vasopressin receptors, V1 and V2, distinguished on the basis of their functional
257
VASOPRESSIN ISORECEPTORS
TABLE I Bioi.oC‘icAi. Ef-wcrs of- VASOPRE\
Liver Blood vessels Heart Adenoh ypophysis Platelets Bone niarrow Chondrocytes in culture Thyniocytes Mouse 3T3 fibroblasts Ciliary body Blood-brain barrier Central newous system
Biological response
Reference
Increased water reabsorption by collecting ducts Increased solute transport by the ascending limb of Henle’s loop Contraction of glomerular mesangial cells Increased prostaglandin synthesis by medullary interstitial cells Inhibition of isoproterenolinduced renin release Increased glycogenolysis and neoglucogenesis Contraction Positive chronotropic and negative inotropic effects Increased corticotropin secretion
Crantham and Burg (1966); Wirz (1956) Hall and Varney 1980)
Aggregation Mitogenic effect Mitogenic effect Mitogenic effect Mitogenic effect Increasd fluid secretion Increased fluid secretion Increased firing rate of specific neuronal groups Affects animal behavior, in particular memory retention Localized changes in catecholamine turnover
Ausiello et a / . (1980a) Zusman and Keiser (1977) Konrads er
ci/. (
1978)
Hems and Whitton (1973) Saameli (1968) Saameli (1968) Doepfner (1968); Cillies er a]. ( 1978) Hasiam and Rosson ( 1972) Hunt rt UI. (1977) Miler rr [ I / . (1977) Whittield rt a / . (1970) Rozengurt er ul. ( 1979) Nagasubramian ( 1977) Raichle and Grube (1978) Mbhlethaler and Dreyfuss (1982) De Wied and Bohus (1978); De Wied and Versteeg (1979) Tanaka rt u / . ( 1977)
coupling either to adenylate cyclase (V2 receptors) or to the cellular mechanisms involved in the regulation of calcium entry in the cell and (or) mobilization of cellular calcium (V I receptors). The use of functional criteria to distinguish isoreceptors (with the precise meaning that isoreceptors are the products of different genes) can be questioned. The ability of V 1 and V2 receptors to interact specifically with a given type of effector (adenylate cyclase or “X”) does not necessarily imply that these two classes of receptors are structurally different. Several other mechanisms, such as selective distribution of receptor and effector molecules in specialized membrane domains, could account for coupling selectivity. Conversely, isoreceptors might well be functionally coupled to the same
258
SERGE JARD
effector. Indeed, it is clearly established that in the same cell receptors for different peptidic hormones or neurotransmitters can be coupled to the same adenylate cyclase. The same limitations apply to other functional criteria which could be used to distinguish isoreceptors. As far as the kinetics of hormonal binding is concerned, it is now clear that parameters such as the equilibrium dissociation constant of hormone receptor complexes are dependent on several effectors (e.g., guanylnucleotides, magnesium) (for review see Rodbell, 1980). Data obtained on different systems and under different experimental conditions are therefore not directly comparable. When binding studies are performed using intact cells actual concentration of active effectors can hardly be controlled. It seems obvious that the identification of isoreceptors must imply the determination of both functional and structural characteristics. The only structural characteristics which can be presently determined are gross physicochemical parameters such as Stokes radius, sedimentation coefficient, and apparent molecular weight. Finally, the most valuable information on structural differences between receptors can be derived from the analysis of structure-activity relationships if one accepts the validity of the assumption that an observed difference between the respective recognition patterns of two receptors very likely reflects a difference in the structures of their respective binding sites or in parts of the receptor molecules which contribute to the stabilization of slightly different conformations of the binding site. The classic approach for the definition of the recognition pattern of a given class of receptor consists in measuring apparent affinity constants (Aso) values of a series of structural analogs using standardized bioassay procedures. It must be pointed out that the existence of slight differences between the apparent recognition patterns determined on two target organs or target cells does not necessarily indicate that the specific receptors involved in the two biological responses are in fact different. It does not constitute a sufficient basis for the distinction of isoreceptors. For a large number of hormones and neurotransmitters it is now well documented that several cellular components distinct from the receptor itself are involved in the transduction of the regulatory signal into a biological response. In most instances, the overall transduction process is a complex sequence of events. The magnitude of the response measured at a given step is not a linear function of the response which could be measured at the preceding step. Thus, in cyclic AMP-dependent mechanisms triggered by adenylate cyclasecoupled receptors and involving the participation of cyclic AMP-dependent protein kinases, the rate of cyclic AMP (CAMP)production by adenylate cyclase is a saturable function of the number of occupied receptors, and activation of protein kinases a saturable function of CAMP concentration. Such mechanisms can ensure a marked amplification of the hormonal signal, i.e., a final response of maximal magnitude can be elicited by hormone binding to a small fraction of the population of receptors present on the target cell (spare-receptor phenomenon).
VASOPRESSIN ISORECEPTORS
259
In a situation in which the so-called “spare-receptor phenomenon” is highly operative, a partial agonist which could be recognized as such at the primary step of its action can in fact behave like a full agonist at a more distal step. The A,, value determined at that final step will depend on both its affinity for the receptor and its intrinsic activity. Although much valuable information could be derived from the data obtained by using standardized bioassay procedures, it is clear that ( I ) the correct definition of the recognition pattern of a receptor will imply a direct determination of the dissociation constants for the binding of a series of structurally related ligands to the receptor, and (2) a correct estimation of the intrinsic activity of a given active ligand could be derived only from the determination of the maximal magnitude of the primary effect elicited by full receptor occupancy by that ligand. These conclusions do not hold for pure competitive antagonists. In this case there will be an identity between the dissociation constant for antagonist binding to the receptor and the inhibition constant (concentration of antagonist that reduces the response of 2X units of agonist to equal the response to IX units in the absence of antagonist). For the above reasons, the comparison of vasopressin receptors in terms of recognition patterns will be restricted to those receptors for which binding data are presently available. To our knowledge, vasopressin receptors have been directly characterized on the following materials: membranes derived from the inner portions of porcine (Bockaert et al., 1973; Roy et al., 1975a,b), bovine (Hechter et al., 1978a,b), rat (Rajerison et al., 1974; Butlen et a l . , 1978), and human (Guillon et al., 1982) kidneys; kidney cells from the LLC-PK1 line (Roy and Ausiello, 1981); isolated rat hepatocytes and rat liver membranes (Cantau et al., 1980); and rat aortic smooth muscle cells in primary culture (Penit et al., 1982). The following sections will be devoted to a comparison of vasopressin receptors in mammals with respect to ( I ) the kinetics of hormonal binding, (2) the nature of their primary effectors, (3) the nature of the putative modulators of receptor-effector coupling, (4) the known physiocochemical characteristics of solubilized receptors, and (5) respective recognition patterns. 111.
KINETICS OF HORMONE BINDING TO VASOPRESSIN RECEPTORS
A. Vasopressin Binding to Kidney Membranes and Kidney Cells Specific vasopressin binding sites which could be identified with vasopressin receptors involved in vasopressin-induced adenylate cyclase activation (see below) have been characterized on membranes prepared from the inner portions of
260
SERGE JARD
porcine (Bockaert et a!., 1973; Roy et al., 1975a,b), bovine (Hechter et al., 1978a,b), rat (Rajerison er al., 1974; Butlen et af., 1978), and human (Guillon et af., 1982) kidneys. The medullopapillary portion of the mammalian kidney contains the major parts of vasopressin-responsive segments of the mammalian nephron, i.e., collecting ducts (Imbert et af., 1975a) and ascending limbs of Henle’s loop (Imbert el af., 1975b). Renal vasopressin receptors have been characterized by using tritiated lysine(Pradelles et af., 1972) or arginine-vasopressin (Flouret et af., 1977). Radioiodinated vasopressin can hardly be used for vasopressin receptor labeling, since iodination of the molecule leads to an almost complete loss in biological activity (see Table IV). The main characteristics of the detected binding sites are summarized in Table 11. Data presented deserve several comments. (1) In all cases the population of vasopressin binding sites appeared to be homogeneous. Dose-dependent binding curves generated linear Scatchard plots. This suggests that there is no cooperativity in hormonal binding. This conclusion is confirmed by the similarity of the dissociation constants as evaluated independently from equilibrium saturation and kinetic data. The apparent homogeneity of the population of vasopressin receptors present in renal membrane preparations also indicates that vasopressin receptors from the two vasopressin-responsive segments (see above) have similar affinities for vasopressin. (2) The dissociation constants measured for the natural vasopressins (lysine-vasopressin in the pig and arginine-vasopressin in the other mammalian species studied) all fall in the same concentration range. They are much higher than circulating vasopressin concentrations. This suggests the existence of a large number of spare receptors and the existence of a marked amplification between the primary signal in vasopressin action on the kidney and the final antidiuretic response. Anyway dissociation constants in the range of circulating vasopressin level (5- 10 pM in man) (Robertson et al., 1973) would very likely imply the formation of very slowly reversible hormone receptor complexes and therefore be hardly compatible with one physiologically important characteristic of vasopressin action on the kidney, i.e., a very rapid (time constant in the minute range) adjustment of the antidiuretic response to changes in circulating vasopressin levels. The fairly low affinity of vasopressin receptor associated with a marked amplification of the hormonal signal could be considered as the optimal conditions ensuring a rapid regulation of kidney function with a reasonably low vasopressin secretion rate. Indeed, the ability of mammals to change vasopressin blood levels within a few minutes is made possible by the existence of very efficient elimination and inactivition mechanisms of circulating vasopressin (see, e.g., Ginsburg, 1968). Hence, the maintenance of high vasopressin levels would also imply a very high hormonal secretion rate. Vasopressin binding sites have been recently characterized on a pig kidney cell line (LLC-PKl) (Roy and Ausiello, 1981). This established cell line maintains
TABLE I1 KINETICS OF VASOPRESSIN BINDING TO VASOPRESSIN RECEPTORS
Animal species
ox
Receptor source Renal medullary membranes
Pig
Renal medullary membranes
Rat
Renal medullary membranes
Human
Renal medullary membranes
Equilibrium dissociation constant (M) 1.
H a
5-25 10-20c 28-380 0.4 + 0.1“ 4.2 + 0.7‘
Association rate constant ( M - 1 min-1 x 10-7)
Dissociation rate constant (min-1)
2.3-6.7 1.2-3.4 2.3
0.09-0.12 0.11-0.23 0.035
Binding capacity 1.3-4b
Hechter er al. (1978a)
1.Ob
Bockaert er al. (1973)
0.22
40
Reference
+
0.02b
Rajerison el a / . (1974)
0.4748
Guillon ei al. (1982)
Roy and Ausiello (1981)
20c Pig
Rat
LLC-PKl cells In monolayer EDTA-suspended Isolated hepatocytes Purified liver membranes
Rat
Aortic myocytes in primary culture
3 and 30d 12c 7.90 15c 3.2” 4.9c 12O 30(
0.13-0.34 0. I4
O.le
1.1
1-1.4
6.2
0.96
0.32e
2
0.1
0.83” 0.04p
Cantau
Values corresponding to arginine-vasopressin. Picomoles per milligram protein. Values corresponding to lysine-vasopressin. d Values corresponding, respectively, to the high- and low-affinity state of the receptor (C. Roy, unpublished results). e Picomoles per 106 cells
(1
b
el al.
(1980)
Penit ef al. (1982)
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characteristics of polar epithelial cells in culture and responds to vasopressin by increasing intracellular CAMPcontent (Ausiello et al., 1980b; Mills et al., 1979) and activating CAMP-dependent protein kinase. The basic characteristics of the vasopressin receptors on LLC-PK1 cells are similar to those found in renal membranes with respect to their recognition pattern. The concentration of lysinevasopressin leading to 50% receptor occupancy (10 nM) is in good agreement with the value determined on pig kidney membranes (Bockaert et al., 1973) or membranes derived from LLC-PKl cells. However, binding studies on intact cells revealed a heterogeneity in the population of binding sites. The dosebinding curves generated curvilinear Scatchard plots. A precise analysis of the kinetics of hormonal binding including determination of the time course of formation, and dissociation of hormone receptor complexes led Roy and Ausiello (1981) to conclude that neither negative cooperativity nor binding to two or more independent populations of binding sites could adequately account for the observed apparent heterogeneity in the population of vasopressin receptors. On the other hand, the experimental data could be fitted with a model involving a multimeric (dimeric) receptor and hormone-induced receptor transition. With the binding of hormone to one site of the dimer, the affinity of the other site is lowered to a value about 10 times lower. Roy et al. (198 I ) provided convincing evidence suggesting that receptor transition might be involved in the rapid desensitization of adenylate cyclase activity which was demonstrated on LLC-PK 1 cells. Indeed, the acute decrease in hormone-stimulated activity correlated with increased occupancy of low-affinity binding sites. Receptor transition was not apparent in EDTA suspended cells which did not demonstrate desensitization. These studies by Roy et af. (1981) on LLC-PK 1 cells reveals the important fact that the functional integrity of those cellular mechanisms involved in the regulation of hormonal action can affect the characteristics of hormonal binding to the receptor. Conversely, comparison of the binding characteristics on intact cells and membrane preparation might help in identifying such mechanisms. As will be discussed in the following section, differences in binding characteristics on intact cells and purified plasma membrane fractions could also be demonstrated on other vasopressin-responsive tissues.
8. Vasopressin Binding to Liver Membranes and Isolated Hepatocytes Specific [3Hlvasopressin binding sites have been found on isolated rat hepatocytes and purified liver membranes prepared according to Neville’s procedure (Cantau et al., 1980). These vasopressin binding sites were identified with the physiological receptors involved in phosphorylase activation on the
VASOPRESSIN ISORECEPTORS
263
following grounds: (1) [3H]vasopressin binding was inhibited by vasopressin structural analogs which were shown to inhibit competitively vasopressin-induced phosphorylase activation; (2) the same order of potency was found when measuring the activities of a series of vasopressin analogs by their ability either to promote phosphorylase activation or to inhibit [3H]vasopressin binding. On both isolated hepatocytes and liver membranes vasopressin binding was reversible, time-dependent, and saturable. The binding time course had the characteristics of a pseudo-first-order reaction. Vasopressin binding to isolated hepatocytes was faster than binding to purified liver membranes. At a free [3H]vasopressin concentration close to the apparent dissociation constant, the half-time for hormone binding to membranes was about 1.7 minutes and the corresponding value for the binding to intact cells was 0.3 minute. Vasopressin binding to isolated hepatocytes and purified membranes was adequately described by a Michaelian-type relation. Scatchard plots of binding curves were linear. If any, deviation of Scatchard plots of the dose binding curves from linearity fell within the range of experimental errors in binding measurements. The apparent dissociation constants for ['H]vasopressin binding to isolated cells and membranes were different (15 and 5 nM, respectively). As will be discussed later, this difference very likely reflects the effects of agonist-specific modulators of receptor function operating in intact cells, and evidence will be provided suggesting that one of these modulators might be GTP. Maximal vasopressin binding capacity of isolated hepatocytes was 320 fmole/lO6 cells, a value about three times higher than found for LLC-PKI cells (see Table 11). On the other hand, the apparent dissociation constant for vasopressin binding to rat liver membranes is very similar to that found for rat kidney membranes. It therefore appears that kidney and liver receptors can hardly be distinguished on the basis of clearly different binding properties. The marked difference in the respective sensitivities of liver and kidney to vasopressin cannot be accounted for by a difference in receptor affinity. It very likely reflects different efficacies of the cellular mechanisms, which in the two systems ensures the amplification of the hormonal signal.
C. Vasopressin Receptors in Blood Vessels Direct characterization of vasopressin vascular receptors is rendered difficult by the high degree of cellular heterogeneity of the vascular wall and by the technical problems encountered in preparing pure plasma membrane fractions derived from smooth muscle cells. We recently showed that rat aortic smooth muscle cells in primary culture might constitute a convenient biological material for a direct characterization of vascular vasopressin receptor (Penit er al., 1982).
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Rat aortic smooth muscle cells were isolated using the method described by Chamley et a / . (1977) as modified by Travo et a / . (1980) and maintained in primary culture. After 2-3 days, cells recovered their contractile phenotype and could be induced to contract in response to vasopressin and angiotensin 11. Specific vasopressin sites could be detected using tritiated lysine-vasopressin (Pradelles et ul., 1972). Vasopressin binding was a time-dependent and fairly rapid process. Using a submaximal [3HH]vasopressinconcentration, an equilibrium state for hormone binding was reached within less than 4 minutes incubation at 37°C. Determination of dose-dependent binding revealed the presence of an apparently homogeneous population of binding sites. The maximal binding capacity was about 40 fmole/106 cells, i.e., two and eight times lower than found for LLC-PKl cells and isolated hepatocytes, respectively (see Table 11). The apparent dissociation constant for lysine-vasopressin binding was 40 nM. Determination of the relative affinities for a series of vasopressin structural analogs for rat aortic smooth muscle cells revealed a high degree of specificity (for details see Table IV). The Kd values determined for a series of 20 vasopressin analogs were distributed over a very large concentration range (more than three orders of magnitude). The peptide which was the more efficient in inhibiting [3H]vasopressinbinding was arginine-vasopressin, the natural vasopressin in the rat ( K , = 12 nM). A clear correlation between vasopressor activities and the corresponding affinity constants for binding to aortic smooth muscle cells could be demonstrated for the analogs which were active in eliciting a vasopressor response. For a series of five antivasopressor peptides a close correspondence was found between the corresponding Kd and pA2 values (pA2 is the negative logarithm of the molar concentration of antagonist that reduces the response to 2X units of agonist to equal the response to 1X units in the absence of antagonist). These observations strongly suggested that vasopressin receptors detected on aortic smooth muscle cells in culture are representative of the main class of vasopressin receptors involved in the pressoric response to vasopressin in vivo. However, it must be pointed out that the apparent dissociation constant for the binding of arginine-vasopressin to rat aortic smooth muscle cells (12 nM) is high compared to the doses of exogenous arginine-vasopressin which elicit a hypertensive response in the pithed rat (Sawyer, 1966). In the rat, the half-maximal response is obtained with about 1 mU arginine-vasopressin, i.e., about 2.3 pmole. Division of that amount by the plasma volume (the virtual distribution space of vasopressin in several mammalian species) led to a plasma concentra'The antivasopressor peptides tested were I -deamino-[4-valine,8-o-arginine]-vasopressin, [( I-prnercapto-p,p-cyclopentamethylenepropionicacid),4-valine,8-1~-arginine]-vasopressin, [ 1-dearninopenicillamine,4-~aline,8-~-arginine]-vasopressin, [ 1-deaminopenicillamine,8-arginine]-vasopressin, [ I -deamino,2-O-methyltyrosine,4-valine,8-~-arginine]-vasopressin, and [ I -deaminopenicillamine,4threonine]oxytocin. (For references to synthesis and biological activities, see Table IV.)
VASOPRESSIN ISORECEPTORS
265
tion (K, value) of about 0.25 nM, i.e., a concentration 50 times lower than the dissociation constant ( K d )measured on aortic smooth muscle cells. This observation does not invalidate the conclusion that the detected binding sites are the receptors involved in the pressoric response. Marked differences between apparent K, and K, values deduced from parallel determinations and binding dose dependencies have been observed for several hormones and in several systems such as ACTH-responsive adrenal cells (McIlhinney and Schulster, 1975) or (see above) vasopressin-sensitive isolated hepatocytes (Cantau et al., 1980). As previously discussed, such differences are to be expected in all situations in which the biological response is triggered by a series of sequential events and where saturation kinetics occur between two or more successive steps. In line with the point under discussion, it is interesting to note that for antagonists of the pressoric response a fairly good identity between pK, and pA2 values could be demonstrated. Comparison of the kinetics of vasopressin binding to intact hepatocytes and to aortic smooth muscle cells reveals the existence of striking similarities between the two types of receptors with respect to their affinity for vasopressin and analogs (for a more detailed discussion see Section V). More generally available data on the kinetics of hormone binding to vasopressin receptors from different tissular origin* do not reveal marked differences which could be related either to the type of biological response elicited or to the type of effector coupled to these receptors (see Section IV).
IV. TRANSDUCTION MECHANISMS TRIGGERED BY VASOPRESSIN RECEPTORS A. Vasopressin Receptors in Kidney Numerous physiological and biochemical studies have clearly established that both the effects of vasopressin on the osmotic permeability to water of mammalian collecting ducts (Grantham and Burg, 1966) and solute transport by the ascending limb of Henle’s loop (Hall and Varney, 1980) are mediated by an increase in CAMP production by adenylate cyclase (Imbert er al., 1975a.b). The relation of kidney vasopressin receptors to adenylate cyclase has been extensively reviewed (see, e.g., Orloff and Handler, 1967; Jard et d.,1975; Jard, 1981; Jard and Bockaert, 1975). The main conclusions which can be drawn from presently available data are:
*A preliniinary characterization of vasopressin receptors on rat glomerular mesangial cells indicates the presence of specific binding sites. The dissociation constant for lysine-vasopressin (10 nM) is close to that found in other vasopressin-rcsponsive cells (Ausiello et ul., 1980a).
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I . Vasopressin-specific binding sites detected on kidney membrane fractions can be identified with the receptors involved in adenylate cyclase activation on the basis of the following: similar distributions within the cortical and medullary portions of the kidney of vasopressin binding sites (Rajerison et u/., 1974) and vasopressin-sensitive adenylate cyclase activity (Chase and Aurbach, 1968); identity in the recognition patterns deduced from binding studies and determinations of dose-dependent adenylate cyclase activation by large series of vasopressin structural analogs including full agonists, partial agonists, and antagonists (Guillon et af., 1982; Hechter et a / . , 1978b; Roy e t a / . , 1975a,b; Butlen et al., 1978); demonstration of parallel modifications in the number of vasopressin receptors and the magnitude of maximal vasopressin-induced adenylate cyclase activation under a variety of experimental and physiopathological situations [hereditary or chronically induced diabetes insipidus in the rat (Rajerison et a/., 1977), adrenalectomy and glucocorticoid administration (Rajerison e t a / ., 1974), vasopressin-induced desensitization (Rajerison et ul., 1977), parallel ontogenic development of vasopressin binding sites and vasopressin-sensitive adenylate cyclase activity (Rajerison et al.. 1976; Butlen rt ul.. 1980)l. 2. Adenylate cyclase activation appears to be a saturable function of receptor occupancy (nonlinear coupling). Half-maximal adenylate cyclase activation is obtained for a fractional receptor occupancy less than 0.5. The nonlinearity in the coupling of hormone binding to response, as estimated by the ratio of vasopressin concentrations leading to half-maximal binding and half-maxirnal adenylate cyclase activation, varies somewhat depending on the mammalian species considered: 40, 5 , 5 , and 1.2 for porcine (Jard et al., I975), bovine (Hechter et a/., 1978a), rat (Butlen et a/., 1978), and human (Guillon er al., 1982) renal adenylate cyclases, re~pectively.~ The nonlinear receptor-enzyme coupling ensures a significant amplification of the hormonal signal. However, it accounts for only a small part of the total amplification observed between hormone binding to receptors and the final antidiuretic response (for detailed discussion see Jard, 1981), suggesting that a marked amplification also occurs at steps beyond the cAMP production step, In accord with this conclusion is the fact that vasopressin structural analogs recognized as partial agonists of low intrinsic activity at the adenylate cyclase activation step are able to induce a full antidiuretic response (Butlen et af. 1978).
B. Vasopressin Receptors in Liver There is much evidence that the glycogenolytic response to vasopressin is triggered by CAMP-independent mechanisms: ( I ) Vasopressin does not increase the concentration of cAMP in liver (Kirk and Hems, 1974); ( 2 ) in experimental %'slues are those corresponding
to the natural vasopressin in the species considered.
VASOPRESSIN ISORECEPTORS
267
conditions in which both activation (by glucagon) and inhibition (by angiotensin and qadrenergic agonists) of rat liver membrane adenylate cyclase could be demonstrated, vasopressin did not produce any detectable change in enzyme activity (Jard et al., 1981); (3) vasopressin-induced activation of phosphorylase a of isolated hepatocytes (Keppens and de Wulf, 1975) is not accompanied by any change in the activity of phosphorylase b kinase or CAMP-dependent protein kinase (Keppens er al., 1977; Garrison et al., 1979). On the other hand, the primary involvement of changes in cell calcium fluxes leading to a rise in cytosolic calcium) in the glycogenolytic response to vasopressin stimulation of isolated hepatocytes was convincingly established (for a recent review see de Wulf et al., 1980): (1) the absence of calcium from the incubation medium prevents the activation of glycogen phosphorylase by vasopressin, but is compatible with the CAMP-dependent activation of the enzyme by glucagon (Keppens et a/., 1977); (2) the ionophore A 23187 produces the same degree of activation of phosphorylase as vasopressin, provided that calcium is present in the incubation medium (Keppens et a / ., 1977; Assimacopoulos-Jeannet el al., 1977); (3) vasopressin in the concentration range needed to observe the glycogenolytic response increases 4sCa uptake from the incubation medium (Keppens er a / ., 1977); (4) phosphorylase b kinase, the enzyme which converts phosphorylase b to a. is allosterically stimulated by concentrations of calcium within the range (10 nM- 10 J&) probably occurring in liver cytosol (Khoo and Steinberg, 1975). The mechanisms by which vasopressin (or a-adrenergic agonists and angiotensin) raises cytosolic calcium in liver is not well understood. Two main schemes have been proposed: stimulation of calcium influx from the extracellular medium and mobilization from intracellular stores. The main evidence for a stimulation of calcium influx is the transient increase in 4sCa uptake l i m d under vasopressin stimulation (Keppens et al., 1977). However, as discussed by Exton (1981), 45Ca fluxes may not reflect net calcium movements because of the presence of several calcium pools of different specific radioactivity in liver cells. Studies of calcium movements by atomic absorption, metallochrome dye, chlortetracycline fluorescence, and calcium electrode techniques failed to reveal detectable net calcium uptake (for review see Exton, 1981). In fact, adrenergic stimulation causes a large release of calcium. These results favor the view that hormonal stimulation mobilizes calcium from intracellular sources. Although several studies suggest that mitochondria are the major source of mobilized calcium, the possibility that other cellular organelles, such as endoplasmic reticulum and plasma membrane, might contribute to the hormoneinduced rise in cytosolic calcium can hardly be excluded (for extensive review see Exton, 1981). Incubation of rat hepatocytes with vasopressin increases the incorporation of 32Pinto phosphatidylinositol (Kirk et a l . , 1977). Stimulated phosphatidylinositol breakdown followed by compensatory resynthesis is a response of a wide variety
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of cells to many hormones and neurotransmitters acting through CAMP-independent mechanisms. Michell and collaborators (Michell et al., 1979; Billah and Michell, 1979; Jones et al., 1979; Michell, 1975) have developed the view that phosphatidylinositol breakdown might be a reaction intrinsic to the same unitary mechanism whereby these hormones bring about calcium mobilization in their target cells. There is general agreement (Billah and Michell, 1979; Jones et al., 1979) that the primary event in the hormone-induced increase in phosphatidylinositol turnover is a stimulated breakdown of phosphatidylinositol by a phosphatidylinositol-specificphospholipase C leading to the production of 1,2diacylglycerol. The diglyceride formed during phosphatidylinositol cleavage is converted to labeled phosphatidic acid in the presence of ATP. Phosphatidic acid reacts with GTP to give phosphate and CDP-diacylglycerol. This reaction is virtually irreversible. The final reaction sequence is a reversible step in which free inositol reacts with CDP-diacylglycerol to form phosphatidylinositol and CMP. As pointed out by Tolbert et al. (1980) the use of labeled inositol to measure phosphatidylinositol synthesis in unreliable since it appears to reflect the activity of the freely reversible reaction of CDP-diacylglycerol with inositol. In addition, there is evidence that the inositol pool involved in the formation of phosphatidylinositol is in a compartment which exchanges very slowly with inositol in the medium. The following arguments favor the proposal by Michell and collaborators that a causal relationship exists between hormone-induced increase in phosphatidylinositol breakdown and calcium mobilization. ( I ) The phosphatidylinositol response appears to occur independently of hormone-induced changes in cytosolic calcium concentration (Michell, 1975; Billah and Michell, 1979). Thus, phosphatidylinositol breakdown and labeling are resistant at least partially to calcium elimination from the incubation medium, a situation in which phosphorylase activation by vasopressin is abolished. Furthermore, admission of calcium into hepatocytes with the ionophore A 23 187 does not elicit the phosphatidylinositol response (Billah and Michell, 1979). (2) Stimulation of phosphatidylinositol breakdown by vasopressin is rapid. The effect is clearly detectable within 1-2 minutes after addition of vasopressin (Kirk et al., 1977; Tolbert et al., 1980). Although precise information on the time course of the phosphatidylinositol effect is lacking, the available data are compatible with the observed time course of vasopressin binding to intact hepatocytes (Cantau et al., 1980) and that of vasopressin-induced phosphorylase activation (see, e.g., Keppens et al., 1977). (3) Studies by Kirk et al. (1981a,b) using vasopressin structural analogs clearly indicated that dose-response curves for phosphorylase activation and for enhanced phosphatidylinositol metabolism are parallel, suggesting that the same receptor population is responsible for triggering both responses. (4)Comparison of the dose dependencies for the four different detectable effects of vasopressin
VASOPRESSIN ISORECEPTORS
269
on isolated hepatocytes, namely, binding of the hormone to the receptor (Cantau et al., 1980), increase in phosphatidylinositol turnover (Kirk et ul., 1981a,b), increase in calcium fluxes, and the final glycogenolytic response, indicate that the A,, value for the phosphatidylinositol effect is almost identical with the dissociation constant for vasopressin binding to hepatocytes. On the other hand, the vasopressin-induced increase in 4sCa uptake can be detected upon exposure of hepatocytes to 0.1 nM vasopressin; phosphorylase activation is detectable at about 10 times less concentration. These results are compatible with the supposed sequence of events: hormone binding to the receptor, increase in phosphatidylinositol turnover, and rise in cytosolic calcium concentration with subsequent triggering of the calcium-dependent mechanisms of phosphorylase activation. They also suggest that a marked amplification exists between the primary signal in hormone action and the final biological response and that the larger part of this amplification occurs between the phosphatidylinositol breakdown step and calcium mobilization, i.e., very small changes in phosphatidylinositol turnover could result in maximal calcium mobilization. The molecular mechanisms by which phosphatidylinositol breakdown leads to an increase in cytosolic calcium are not known. The fact that the phosphatidylinositol effect can be clearly detected only on intact cells contributes to the great difficulties encountered in the elucidation of these mechanisms. Among the proposed mechanisms are ( 1 ) a release of calcium bound to the plasma membrane, (2) opening of a calcium gate leading to an influx of extracellular calcium, and (3) generation of an intracellular messenger which causes the release of calcium from intracellular organelles, It was hypothesized that inositol 1,2-cyclic phosphate or inositol I -phosphate released during phosphatidylinositol breakdown might be this messenger. This seems unlikely since, as observed by Hughes and Blackmore (in Exton, 1981), these compounds do not increase calcium efflux from mitochondria. Phosphatidic acid has also been proposed as a mediator of muscarinic and adrenergic effects in smooth muscle and parotid gland (Barritt et al., 1981), but it was recently shown that in hepatocytes phosphatidate at low concentrations produces changes in calcium fluxes which are opposite to those induced by adrenergic stimulation. Kirk et ul. ( 1981 a,b) have demonstrated that vasopressin, angiotensin, and epinephrine stimulate the breakdown of phosphatidylinositol 4,5-bisphosphate in hepatocytes. Since di- and triphosphoinositides are known to bind calcium, it is possible that stimulation of their breakdown in the membrane could release bound calcium. Takai et ul. (1979a,b) have recently proposed an interesting hypothesis relating phosphatidylinositol turnover with a calcium-activated phospholipiddependent protein kinase. They showed that small quantities of unsaturated diacylglycerol sharply decreased the calcium and phospholipid concentrations needed for full activation of the enzyme. It is therefore possible that unsaturated
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diacylglycerol, which may be derived from hormone-induced phosphatidylinositol breakdown, could lead to an activation of the so-called protein kinase C (Takai et a/., 1979a,b).
C. Vascular Smooth Muscle Cells Vasopressin is a potent stimulant for the contraction of vascular smooth muscle. This response, like hepatic glycogenolysis, is abolished if the muscle is deprived of extracellular calcium (Altura and Altura, 1977; Altura, 1975), suggesting that a rise in cytosolic calcium concentration is also the initiator of vasopressin-induced increase in vascular tone. There is no indication of any positive or negative coupling of vasopressin receptors to membrane adenylate cyclase in vascular smooth muscle cells. Vasopressin does not produce any detectable changes in intracellular cAMP of rat aortic smooth muscle cells in primary culture which exhibited a contractile response to vasopressin and showed the expected increase in intracellular cAMP following stimulation by P-adrenergic agonists (Penit et al., 1982). Conversely, most of the available evidence indicates that inhibition of adenylate cyclase and/or activation of cAMP phosphodiesterase is not the mechanisms by which smooth muscle contractants exert their biological action. Although the scarcity of available experimental data cannot permit a definite conclusion about the relation of vasopressin receptors with adenylate cyclase in smooth muscle cells, it seems reasonable to conclude that the contractile response of vascular smooth muscle cells to vasopressin stimulation is primarily due to a hormone-induced calcium mobilization. Takhar and Kirk (198 I ) recently showed that in rat aorta, as in hepatocytes, vasopressin stimulates the incorporation of 32P into phosphatidylinositol but not into other phospholipids. Furthermore, the concentration of arginine-vasopressin that provoked half-maximal phosphatidylinositol labeling in rat aorta (5.5 nM) is very similar to the concentration that produces half-maximal phosphatidylinositol labeling in hepatocytes (Kirk er d . , 1981a,b).
D. Other Vasopressin-Responsive Cells As already indicated, vasopressin is able to contract cultured rat glomerular cells of apparent mesangial origin. The exact function of mesangial cells is not clear, but it has been proposed that they contribute to the regulation of glomerular size and blood flow and could be the target cells involved in the hormonal regulation by vasopressin, angiotensin, parathyroid hormone, and prostaglandins of glomerular function. No detectable changes in cAMP and cGMP content of mesangial cells could be detected upon exposure of the cells to vasopressin, or angiotensin at doses which induce cell contraction (Ausiello et al., 1980a).
VASOPRESSIN ISORECEPTORS
27 1
Despite the fact that Ausiello et a/. (1980a) showed that large doses of argininevasopressin (AVP) (200 nM) were able, in the presence of phosphodiesterase inhibitor, to increase twofold the cAMP content of mesangial cells, they concluded that this effect of AVP on cAMP content bears no relationship to the contractile response observed with low doses AVP (in the range of 0.1 nM). The same conclusions apply to the vasopressin-induced platelet aggregation which can be demonstrated in several but not all mammalian species (Haslam and Rosson, 1972). There is compelling evidence that the two responses of platelets to stimulation by aggregating agents, namely, aggregation per se and the secretion of granule constituents, depend on the release of calcium ions in the cytosol from intracellular binding sites including the dense tubular system which may be analogous to the sarcoplasmic reticulum of muscle. Most of the aggregating agents including vasopressin do not affect platelet adenylate cyclase where inhibitory agents, PGE, and adenosine, increase intracellular cAMP content through an activation of adenylate cyclase (for review see Haslam el al., 1978). It is therefore very likely that vasopressin receptors in platelets are not functionally coupled to adenylate cyclase and that they trigger platelet aggregation by mobilizing intracellular calcium stores. It would be of interest to investigate whether, as can be expected according to Michell’s hypothesis, vasopressin increases phosphatidylinositol breakdown in platelets. Gillies et at. (1978) reported that removal of calcium and magnesium from the perfusate of rat anterior pituitary cells causes a decrease in arginine-vasopressinstimulated corticotropin release, suggesting that calcium ions might be involved in the action of vasopressin on corticotropin secretion. Studies by Rozengurt et al. (1979) on the mitogenic action of vasopressin on quiescent cultures of Swiss 3T3 cells deserve a special comment. The addition of vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates ion movements across the membrane (Mendoza er d . , 1980). A stimulation of active transport of Rb by the Na-K pump is detectable within 2 minutes after addition of the hormone. Vasopressin increases significantly K cell content but has no significant effect on cell Na. When the Na-K pump is inhibited by ouabain, vasopressin markedly increases cell Na, while cell K is not changed. Finally, vasopressin accelerates the rate of Na entry in Swiss 3T3 cells. Altogether these results indicate that the primary effect of vasopressin is an increase in Na entry resulting in stimulation of the Na-K pump, which maintains cell Na essentially unchanged while increasing K uptake. The vasopressin action on Swiss 3T3 cells thus resembles the vasopressin action on amphibian skin and urinary bladder epithelial cells. There is general agreement that vasopressin stimulates transepithelial active sodium transport by these structures by increasing Na entry through amiloride-sensitive Na channels at the apical plasma membrane (for review see Jard and Bockaert, 1975). Since there is compelling evidence that the so-called natriferic action of vasopressin on amphibian epithelial cells is medi-
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ated by cAMP (for review see Jard and Bockaert, 1975) it would be of interest to investigate a possible relation of vasopressin receptors to adenylate cyclase in Swiss 3T3 cells and other cells exhibiting a mitogenic response to vasopressin stimulation: bone marrow cells (Hunt et al., 1977), thymocytes (Whitfield et af., 1970), and chondrocytes (Miller et al., 1977). Although studies by Whitfield et al. (1970) provide indirect arguments for a possible mediation by cAMP of the stimulation of thymocyte proliferation by vasopressin, it is worth mentioning that cAMP is generally considered an inhibitor of cell growth (Kram et al., 1973). To our knowledge there are at present no published results concerning the possible involvement of calcium and/or cyclic nucleotides in the action of vasopressin on the central nervous system.
V.
EFFECTS OF NUCLEOTIDES AND OTHER PUTATIVE EFFECTORS ON VASOPRESSIN RECEPTORS
A. Kidney Receptors As could be expected from the well-established functional coupling of vasopressin receptors in kidney to adenylate cyclase (see above), guanylnucleotides affect both hormone binding and adenylate cyclase activation. Although vasopressin-induced stimulation of adenylate cyclase activity in crude or purified kidney membranes is demonstrable without addition of GTP to the incubation medium, it can be reasonably concluded that the so-called guanylnucleotide regulatory protein is involved in renal adenylate cyclase activation by vasopressin. Thus, in rat (Rajerison, 1979) and human (Guillon et al., 1982) kidney membranes GTP and the nonhydrolyzable GTP analog 5’-guanylyl imidodiphosphate [Gpp(NH)p] markedly reduce the vasopressin concentration eliciting halfmaximal adenylate cyclase activation. Under the same experimental conditions, Gpp(NH)p significantly reduces apparent receptor affinity for agonists but not for antagonists (Guillon et al., unpublished). A protein involved in renal adenylate cyclase activation by NaF and Gpp(NH)p could be identified in the soluble fraction of detergent-treated kidney membranes (Guillon et al., 1981). The lack of a GTP effect on vasopressin-sensitive adenylate cyclase as reported in several studies can very likely be attributed to the presence of GTP in the membrane preparations used or synthesis of the nucleotide during the course of membrane incubation in the presence of ATP.
B. Liver Receptors In a previous report (Cantau et al., 1980) we showed that GTP also affected the characteristics of vasopressin binding to rat liver vasopressin receptors. GTP
VASOPRESSIN ISORECEPTORS
273
(0.1 mM) reduced receptor affinity for vasopressin by a factor of three to four. The GTP-induced reduction in receptor affinity could be accounted for by an increase in the dissociation rate of vasopressin-receptor complexes. GTP affected the binding of vasopressin and several active vasopressin analogs but did not modify the binding of analogs acting as antagonists of rhe glycogenolytic response. It was also shown that there was a marked difference between the dissociation constants for the binding of vasopressin and active analogs to isolated hepatocytes and the corresponding values determined on purified membranes incubated in the absence of GTP. In contrast, good correspondence between the dissociation constants for binding to intact hepatocytes and to membrane incubated in the presence of GTP could be demonstrated. From these observations we concluded that vasopressin binding to hepatocytes is very likely under the control of endogenous modulators and that GTP might be one of these modulators. This also raised the interesting possibility that adenylate cyclasecoupled vasopressin receptors and receptors coupled to the effector(s) involved in calcium mobilization in hepatocytes could be influenced by the same modulators. More recent studies (Bono et u / . , unpublished observations) clearly showed that the GTP effects on liver vasopressin receptors can be distinguished from the effects on adenylate cyclase-coupled renal receptors. We take advantage of the possibility to compare on the same preparations of purified liver membranes and under strictly identical experimental conditions the effects of nucleotides on vasopressin receptors and the adenylate cyclase-coupled glucagon receptors. These studies revealed marked differences in the dose dependencies and specificities of the nucleotide effects on these two types of receptors. On vasopressin receptors, GTP was active in a millimolar range, ATP was as active as GTP, and the nonhydrolyzable analogs Gpp(NH)p and App(NH)p (5’-adenylyl imidodiphosphate) were completely inactive. In line with the results previously obtained by Rodbell and colleagues (for review see Rodbell, 1980), glucagon was affected by GTP in a micromolar concentration range. Gpp(NH)p was at least as active as GTP. The nucleotide effect was highly specific; ATP and App(NH)p were very poorly active. The observed characteristics of the nucleotide effect on liver vasopressin receptors is compatible with the possible involvement of a phosphorylation reaction in the nucleotide-induced change in receptor affinity. The “agonist-specific” character of the nucleotide effect might indicate that ATP or GTP plays a role in the functional coupling of liver vasopressin receptors to their still unknown primary effectors. The characteristics of the nucleotide effect on vasopressin liver receptors are somehow reminiscent of the ATP-, Mg-dependent, hormone-induced desensitization of LH-sensitive adenylate cyclase from corpus luteum (Bockaert et al., 1976) and that of vasopressin-sensitive adenylate cyclase from pig kidney membranes (Roy et a / . , 1976). Much additional work is obviously needed to evaluate the possible physiological significance of the effect of ATP and GTP on vasopressin binding to liver receptor.
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It has long been recognized that extracellular magnesium ions influence the response of neurohypophyseal hormone-sensitive target cells [rat myometrium (Krejci et a / ., 1967; Walter et a[., 1968), mammary gland (Somlyo et al., 1966; Walter et af., 1968), and blood vessels (Munsick et al., 1960; Somlyo ef a / . , 1966)]. There is evidence (Pearlniutter and Soloff, 1979) that the presence of magnesium in the incubation medium of rat mammary gland homogenates is necessary for optimal binding of oxytocin to the specific binding sites detectable in this preparation. We recently investigated (Bono et al., unpublished observations) the effect of magnesium on vasopressin binding to rat liver receptors. Chelation of magnesium in the incubation medium of rat liver membranes completely abolished specific vasopressin binding to these membranes. Normal binding could be restored by addition of magnesium (apparent K , value for magnesium, 1 d). The effect of reducing magnesium concentration to inframaximal value is to introduce an apparent heterogeneity in the population of vasopressin binding sites. Upon progressive reduction of the magnesium concentration, an increasing number of vasopressin receptors were found in a low-affinity state (K,, about 100 M ) .Studies with agonists and antagonists of the glycogenolytic response indicated that the magnitude of the magnesium effect was dependent on the nature and/or properties of the peptides tested. Magnesium did not modify the binding of the three antagonists tested and affected to different degrees that of the three agonists. It is therefore possible that the magnesium effect on vasopressin liver receptors is agonist specific and might, for that reason, reflect a role of magnesium in the receptor-effector coupling. The magnesium effect does not appear to be specific for liver receptors, since it was shown that binding of vasopressin to adenylate cyclase-coupled receptors in LLC-PK 1 cells exhibits an absolute magnesium requirement (Roy and Ausiello, 198 1).
VI.
PHYSICOCHEMICAL CHARACTERISTICS OF SOLUBILIZED VASOPRESSIN RECEPTORS
Vasopressin receptors from pig and rat kidney membranes and rat liver membranes can be solubilized by treatment with nonionic detergents (Roy et al., 1975; Guillon et a / . , 1980). Solubilization of nonoccupied receptors results in a marked impairment in their ability to bind vasopressin specifically. However, experimental conditions can be found which prevent dissociation of vasopressin-receptor complexes formed on the membrane prior to solubilization. It was therefore possible to determine the main physicochemical characteristics of these complexes. The results shown in Table I11 indicate the existence of striking similarities between liver and kidney receptors. Both receptors appear to be hydrophobic proteins which bind significant amounts of detergent. They cannot be clearly distinguished on the basis of any of the determined physicochemical
275
VASOPRESSIN ISORECEPTORS
Liver receptor Stokes radius (nm) Apparent sedimentation coefficient measured in H2O gradient, Sap, ( S ) D2O gradient, Sap,, (S) Standard sedimentation coefficient, szo,w ( S ) Partial specific volume (mlig) Frictional ratio, FIFO Apparent molecular weight, M , Detergent bound (mg/mg protein) Molecular weight of protein Moiety, M ,
5.4 3.7 3.4 3.7 0.75
* (5)
Kidney receptor
5.6
-t
(7)
92,000 0.09
3.5 -t 0.2 (6) 3.0 2 0.1 (6) 3.7 0.78 t 0.02 (14) I .77 101,000 0.22
83,000
80,000
? -C
0.1 (6) 0.1 (6)
* 0.01 (10)
1.81
"Modified from Guillon et a / . (1981).
characteristics (Stokes radius, sedimentation coefficient, frictional ratio, specific partial volume, and apparent molecular weight).
VII. RECOGNITION PATTERNS OF VASOPRESSIN ISORECEPTORS For reasons indicated in Section 11 the following discussion will be restricted to those vasopressin receptors from the same mammalian species for which direct measurements of receptor affinity for natural vasopressins and analogs have been performed, namely, kidney, liver, and smooth muscle receptors. Presently available data are summarized in Table IV. As discussed above (for more details see Cantau et al., 1980), the affinity of vasopressin receptors for the natural vasopressins and anlogs depends on the receptor environment. For example, it is clear from the data in Table I11 that there are marked differences between corresponding pK, values determined on intact hepatocytes and purified rat liver membranes for a series of agonists of the glycogenolytic response. There is evidence that these differences may reflect the influence of endogenous modulators on vasopressin binding to hepatocytes. In order to evaluate an eventual difference in the respective recognition patterns of vasopressin receptors from different target cells, it appears necessary, therefore, to compare affinity constants determined under identical or homologous experimental situations. Comparison of data obtained on rat aortic smooth muscle cells in culture and isolated rat hepatocytes reveals striking similarities in the recognition patterns of the vasopressin receptors present on these two cell types. The largest difference
SERGE JARD
276 TABLE IV
RLCOGNITION PATTERNSOF VASOPRESSIN ISORECEFTORS
INTHE
RAT^
pKd values for binding to ~
Rat liver membranes (2)
Rat kidney membranes (3)
8.5 8.1
9.4 9.3
Rat aortic
Rat
myocytes
hepatocytes
(1)
(2)
7.9 7.5 6.0
7.4 6.0
7.0
5.9 7.4 5.5
6.0 7.I 5.6
7.0 8.0 6.3
9.6 9.5 9.6
6.I
6.5h
6.5
9.6
6.3
6.4h
7.0
9.6
7.5
8.4
9.2
9.I
6.0
-
6.4
9.1
7.2
7.7h
7.6
8.9
7.9
7.7"
7.3
7.9c
8.2
9.2~
Peptide ~~
Arginine-vasopressin Lysine-vasopressin I -Dearnino-arginine-vasopressin(4) 1 -Deamino-[8-~-arginine]-vasopressin
(4) [4-Valine]arginine-vasopressin( 5 ) [4-Valine,8-o-arginine]-vasopressin( 5 ) 1-Dearnino-[4-valine,8-~-arginine]vasopressin (5) 1-Deamino-[4-threonine,8-~-arginine]vasopressin (6) [ I -( L-a-Hydroxy-P-mercaptopropionic acid)]arginine-vasopressin (7) [ I -( L-a-Hydroxy-P-rnercaptopropionic acid),4-~a~ine,8-~-arginine]-vasopressin (7) [ 1-Deaminopenici~lamine,4-va~ine,8-oargininel-.vasopressin(8) [ I -(P-Mercapto-P,P-cyclopentarnethylenepropionic acid).4-valine, 8-~-arginine]-vasopressin(9) [ I -(P-Mercapto-P,P-cyclopentarnethylenepropionic acid),2-O-ethyltyrosine,4-valine]arginine-vasopressin
8.1
(10)
u References: ( I ) Penit et a/. (1982); (2)Cantau et al. (1980);(3)Butlen ef a / . (1978);(4)Manning et a/. (1976);( 5 ) Sawyer et a/. (1974);(6)Manning et al. (1976);(7)Lowbridge et a/. (1977);(8) Manning et al. (1977);(9)Lowbridge er al. (1978);(10) Sawyer et a / . (1981). Antagonist of the glycogenolytic response.
Antagonist of vasopressin-inducedadenylate cyclase activation.
observed between paired relative pK, values is 0.32; it corresponds to a twofold difference in relative affinities (a value which falls within the range of experimental errors in the determination of dissociation constants of unlabeled vasopressin analogs). The mean difference in relative pK, values is 0.10 0.24 (SD) corresponding to a 25% difference in paired Kd values. It therefore appears reasonable to conclude that vasopressin receptors from hepatocytes and rat aortic smooth muscle cells have identical recognition patterns. This constitutes direct
*
VASOPRESSIN ISORECEPTORS
277
confirmation, at the receptor level, of pharmacological data indicating the existence of a close correspondence between the relative glycogenolytic and vasopressor activities of a large series of vasopressin structural analogs including agonists and antagonists (Keppens and de Wulf, 1979; Kirk et al., 1981a,b). On the other hand, comparison of pK, values determined on rat liver membranes and rat kidney membranes reveals marked differences in the recognition patterns of vasopressin receptors in liver and kidney. For 7 of the 14 analogs tested there was a more than 10-fold difference between the corresponding relative affinities for liver and kidney receptors. The main structural modifications in the vasopressin molecule leading to enhanced selectivity are ( I ) the substitution of D-arginine for L-arginine in the eighth position of deamino vasopressin. The affinity of l-deamino-[8-~-arginine]-vasopressin for kidney receptor is 1.4 times higher than that of vasopressin, while its affinity for liver receptors is reduced by a factor of 31 as compared to arginine-vasopressin; (2) introduction of a penicillamine residue; (3) substitution of HO for the free a-amino group [ l - ( ~ - a hydroxy-P-mercaptopropionic acid)]arginine-vasopressin; and (4) substitution of valine in the 4-position together with one of the above-mentioned modifications. [4-Valine,8-~-arginine]-vasopressin has a 160-times-reduced affinity for liver receptors and a twofold increased affinity for kidney receptors. The above conclusions derived from direct binding studies are in agreement with the main conclusions which can be drawn from measurement of biological activities using the in vivo rat blood pressure and antidiuretic assays (for review see Lowbridge et at., 1977; Manning and Sawyer, 1977). However, the precise comparison of pharmacological data and more direct biochemical data clearly illustrates the point raised in Section I about the difficulty in determining the recognition pattern of receptors on the basis of the sole determinations of the magnitude of the final responses in vivo. Let us consider the analog [l-(p-mercapto-p,pcyclopentomethylenepropionic acid),4-valine, 8-~-arginine]-vasopressin(cyclo dVDAVP). It has been described (Lowbridge et a l . , 1978) as a specific antivasopressor peptide on the basis of the observed inhibition of the vasopressininduced pressor response (pA2 of 7.68) and of its low but significant antidiuretic activity of 0.10 U/mg, i.e., about 4000 times less than that of arginine-vasopressin. Direct measurement of the affinity of cyclo dVDAVP for rat renal membranes gave a value which is only 30 times less than that of vasopressin. In fact, at the level of the vasopressin-sensitive adenylate cyclase cyclo dVDAVP behaves like a partial agonist of very low intrinsic activity (Butlen et a ] . , 1978). A likely explanation for this apparent discrepancy between in vivo and in vitro data is the existence of a large number of spare vasopressin receptors in kidney. Due to the marked amplification of the hormonal signal which very likely operates in vasopressin-sensitive tubular cells (see above), cyclo dVDAVP is able to induce a full antidiuretic response. However, as a consequence of its decreased intrinsic activity at the primary step of the antidiuretic response, the antidiuretic
278
SERGE JARD
activity of cyclo dVDAVP expressed in terms of units per milligram appears much lower than what might have been expected from the actual affinity of cyclo dVDAVP for vasopressin kidney receptors. Finally, the apparent selectivity in the antagonistic properties of cyclo dVDAVP might reflect a difference in the respective efficacies of the cellular mechanisms ensuring the amplification of the hormonal signal in kidney and in the vascular smooth muscle cells. In connection with the above discussion, it must be pointed out that all the vasopressin analogs which behave as antagonists of the pressoric and glycogenolytic responses to vasopressin exhibited a reduced intrinsic activity on kidney receptors when tested at the level of vasopressin-sensitive kidney adenylate cyclase (Butlen el al., 1978; Guillon er al., 1982). Thus, the design of specific blockers which might be very useful for the characterization of vasopressin isoreceptors remains an open problem. The pharmacological data obtained with vasopressin structural analogs on other vasopressin target cells are too scarce to allow any valid conclusion on the characteristics of the recognition patterns of the receptors present on these cells as compared to those of vascular and hepatic receptors on the one hand and those of kidney receptors on the other hand. In their study of vasopressin-induced human blood platelet aggregation, Haslam and Rosson (1972) reported that equal pressor activities of argininevasopressin and [ 8-ornithine]-vasopressin had almost identical effects on human blood platelets, although the ratio of pressor to antidiuretic activities of [8ornithinel-vasopressin is four times that of arginine-vasopressin. Furthermore, [2-phenylalanine]lysine-vasopressin,which has a high ratio of pressor to antidiuretic activity, was slightly more effective in inducing platelet aggregation than equal pressor activities of arginine-vasopressin. It therefore appears that vasopressin receptors in platelets resemble those involved in the pressor action of vasopressin. Similar conclusions can be drawn from the available data on the structure-activity relationships for the C.R.F.-like effect of vasopressin on the adenohypophysis (Gillies ef al., 1978). Studies by Walter et al. (1978) on the effects of structural modifications of the vasopressin molecule on its ability to induce modifications of conditioned behavior of rats (increased resistance to extinction of a pole-jumping avoidance response) indicated that behavioral activities of vasopressin are more resistant to modification of the hormone in positions 8 and 9 than activities of vasopressin on peripheral organs. This raises the possibility that vasopressin receptors in the central nervous system might have recognition patterns different from those of vasopressin receptors at the periphery. However, it seems obvious that a more direct analysis of structure-activity relationships derived from binding studies of vasopressin and analogs in brain or from simplest bioassay procedures such as vasopressin-induced changes in the electrical activity of identified groups of neurons (Miihlethalerand Dreyfuss, 1982) is needed to allow more definite conclusions on the nature of
VASOPRESSIN ISORECEPTORS
279
vasopressin receptors in the central nervous system. Muhlethaler and Dreyfuss ( 1982) showed that 1-deamin0-[8-~-arginine]-vasopressin, which is virtually devoid of pressor activity, had a much weaker action on hippocampal neurons than vasopressin. This suggests that vasopressin receptors on hippocampal neurons could resemble vascular receptors.
VIII.
SUMMARY AND CONCLUSIONS
Specific vasopressin binding sites have been characterized in isolated cells or membrane fractions derived from three vasopressin-responsive tissues: kidney, fiver, and blood vessels. In all cases it has been convincingly established that these binding sites are the receptors involved in the biological effects of vasopressin on these tissues: vasopressin receptors in kidney, liver, and blood vessels have similar properties with respect to the kinetics of hormone binding. The equilibrium dissociation constants for vasopressin binding (1 - 10 nM) (depending on the biological material used) are much lower than circulating vasopressin levels in mammals. There is much experimental evidence indicating that this apparent discrepancy reflects the existence of very efficient mechanisms ensuring a marked amplification of the hormonal signal. Unlike vasopressin receptors triggering the tubular effects (increase in water and solute transport) of vasopressin on the kidney, the receptors present on hepatocytes and vascular smooth muscle cells are not functionally coupled to adenylate cyclase. The primary involvement of a rise in cytosolic cell calcium in the glycogenolytic response of hepatocytes and in the contractile response of vascular smooth muscle cells to vasopressin has been established. It is likely that several other biological effects of vasopressin such as a platelet aggregation and corticotropin release by the adenohypophysis are also elicited by calcium-dependent, CAMP-independent mechanisms. In liver and blood vessels vasopressin increases phosphatidylinositol breakdown in an at least partially calcium-independent manner. Presently available data are compatible with the proposal by Michell that the increase in phosphatidylinositol breakdown is part of the transduction mechanism responsible for the vasopressin-induced rise in cytosolic calcium. Pharmacological studies using large series of vasopressin structural analogs including full and partial agonists and antagonists showed identical recognition patterns for vasopressin receptors in liver and vascular (aortic) smooth muscle cells, suggesting that these receptors might be structurally identical, i.e., might be products of the same gene. There are marked differences in the respective recognition patterns of vasopressin receptors in liver and blood vessels on the one hand and receptors present in the vasopressin-sensitive segments of the nephron on the other hand. It can thus be concluded that at least two types of vasopressin
280
SERGE JARD
receptors exist in mammals. Pharmacological data obtained with other vasopressin-responsive cells or tissues (platelets, chondrocytes, thymocytes, central nervous system, etc.) are too scarce to determine whether vasopressin receptors present in these structures are of one of the two types of vasopressin isoreceptors presently well characterized or are of another type. REFERENCES Altura, B. M. ( 1975). Dose-response relationships for arginine-vasopressin and synthetic analogs on three types of rat blood vessels: Possible evidence for regional differences in vasopressin receptor sites within a mammal. J. Pharmacol. Exp. Ther. 193, 413-423. Altura, B. M.. and Altura, B. T. (1977). Vascular smooth muscle and neurohypophyseal hormones. Fed. Proc. Fed. Am. Soc. Exp. B i d . 36, 1853-1860. Assimacopoulos-Jeannet, F. D., Blackmore, P. F., and Exton, J . H. (1977). Studies on a-adrenergic activation of hepatic glucose output. J. Biol. Chem. 252, 2662-2669. Ausiello, D. A,, Kreisberg, J. I., Roy, C., and Kamovsky, M. J . (1980a). Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin 11 and arginine-vasopressin. J. Clin. Invest. 65, 754-760. Ausiello, D. A,, Hall, D. H., and Dayer, J. M. (1980b). Modulation of cyclic AMP-dependent protein kinase by vasopressin and calcitonin in cultured porcine renal LLC-PK I cells. Biochem J. 186, 773-780. Barritt, G . J., Dalton, K. A , , and Whiting, J. A. (1981). Evidence that phosphatidic acid stimulates the uptake of calcium by liver cells but not calcium release from mitochondria. FEBS Leff. 125, 137-140. Billah, M. M., and Michell, R. H. (1979). Phosphatidylinositol metabolism in rat hepatocytes stimulated by glycogenolytic hormones. Effects of angiotensin, vasopressin, aderenaline, ionophore A23187 and calcium-ion deprivation. Biochem. J . 182, 661-668. Bockaert, J.. Roy, C.. Rajerison, R., and Jard, S. (1973). Specific binding of ('H)-lysine-vasopressin to pig kidney plasma membranes. Relationship of receptor occupancy to adenylate cyclase activation. J. B i d . Chem. 248, 5922-593 I . Bockaert, J., Hunzicker-Dunn, M., and Birnbdumer, L. ( I 976). Hormone-stimulated desensitization of hormone-dependent adenylate cyclase: Dual action of luteinizing hormone on pig graafian follicle membranes. J. B i d . Chem. 251, 2653-2663. Butlen, D., Guillon, G.,Rajerison, R. M., Jard, S . , Sawyer, W. H., and Manning, M. (1978). Structural requirements for activation of vasopressin-sensitive adenylate cyclase. hormone binding and antidiuretic action: Effects of highly potent analogues and competitive inhibitors. Mol. Pharmacol. 14, 1006-1017. Butlen, D., Guillon, G..Cantau, B., and Jard. S. (1980). Comparison of the developmental patterns of vasopressin, glucagon and alpha-adrenergic receptors from rat liver membranes. Mo/. Cell. Endocrinol. 19, 275-289. Cantau, B.. Keppens, S., De Wulf, H., and Jard, S. (1980). Vasopressin binding to isolated rat hepatocytes and liver membranes: Regulation by GTP and relation to glycogen phosphorylase activation. J. Receptor Res. 1, 137-168. Chamley, J. H., Campbell, G.R., McDonnell, J. D., and Groschel-Stewart, V. (1977). Comparison of vascular smooth muscle cells from adult human monkey and rabbit in primary culture and subculture. Cell. Tiss. Res. 177, 503-522. Chase, L. R., and Aurbach, G. D. (1968). Renal adenylate cyclase: Anatomically separate sites for parathyroid hormone and vasopressin. Science 159, 545-547. Cowley. A. W . , Jr., Switzer, S. J., and Guinn, M. M. (1980). Evidence and quantification of the vasopressin arterial pressure control system in the dog. Circ. Res. 46, 58-67.
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Crofton. J . T., Shade, L.. Shade, R. E.. Lee-Kwon, W. J., Manning, M.. and Sawyer. W. H. ( 1979). The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the rat. Hypertension I, 31-38. De Wied, D., and Bohus, B., (1978). The modulation of memory processes by vasotocin, the evolutionarily oldest neurosecretory principle. ProK. Bruin Res. 48, 327-336. De Wied. D., and Versteeg, D. H. G. (1979). Neurohypophyseal principles and memory. Fed. Proc. Fed. Am. SOC. E.rp. Biol. 38, 2348-2354. De Wulf, H.. Keppens, S . , Vandenheede, J. R.. Haustraete. F., Proost, C., and Carton, H. (1980). Cyclic AMP-independent regulation of liver glycogenolysis. I n “Hormone and Cell Regulation” (J. Nunez and J . Dumont, eds.). North-Holland Publ. Amsterdam. Diamond. J. (1978). Role of cyclic nucleotides in control of smooth muscle contraction. Adv. Cyclic Nucleotide Res. 9, 327-340. Doepfner, N. (1968). The influence of neurohypophysial polypeptides on adenohypophysial function. I n “Neurohypophysial Hormones and Similar Polypeptides” (B. Berde, ed.), Handbook of Experimental Pharmacology, Vol. 23. Springer-Verlag. Berlin and New York. Exton, J. H. (1981). Molecular mechanisms involved in alpha-adrenergic responses. Mol. Cell. Endocrinol. 23, 233-264. Flouret, G . , Terada, S. H., Nakahara, T.. and Hechter, 0. (1977). lodinated neurohypophyseal hormones as potential ligands for receptor binding and intermediates in synthesis of tritiated hormones. Biochemistry 16, 2 I 19-2 124. Garrison. 1. C., Borland, M. K.,Florio, V. A , , and Twible, D. A. (1979). The role of calcium ion as a mediator of the effects of angiotensin 11, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes. J. Biol. Chem. 254, 7147-7156. Gillies, G., Van WimersmaGrei Danus, T. B., and Lowry, P. J. (1978). Characterization of rat stalk eminence vasopressin and its involvement in adrenoconicotropin release. Endocrinology 103, 528-534. Ginsburg, M. ( 1968). Production, release, transportation and elimination of the neurohypophyseal hormones and similar polypeptides. In “Handbook of Experimental Pharmacology” (B. Berde, ed.), Vol. 23, pp. 286-371. Springer-Verlag. Berlin and New York. Grantham, J. J.. and Burg, M. B. (1966). Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules. Am. J. Ph.vsio1. 211, 255-259. Guillon, G . , Couraud. P. 0.. Butlen, D., Cantau, B.,and Jard, S. (1980). Size of vasopressin receptors from rat liver and kidney. Eur. J. Biochem. 111, 287-294. Guillon, G., Cantau. B., and Jard, S. (1981). Effects of thiol-protecting reagents on the size of solubilized adenylate cyclase and on its ability to be stimulated by guanylnucleotides and fluoride. Eur. J . Biochem. 117, 401-406. Guillon, G., Butlen, D., Cantau, B.. Barth, T., and Jard, S. (1982). Kinetic and pharmacological characterization of vasopressin membrane receptors from human kidney medulla: Relation to adenylate cyclase activation. Eur. J . Phurmucol. (in press). Hall, D. A., and Varney, D. M. (1980). Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle’s loop. J. Clin. Invest. 66, 792-802. Haslam, R. J., and Rosson, G. M. (1972). Aggregation of human blood platelets by vasopressin. Am. J. Physiol. 233, 958-967. Haslam, R. J., Davidson, M. M. L., Davies, T . , Lynham, J. A., and McClenaghan, M. D. ( 1978). Regulation of blood platelet function by cyclic nucleotides. Adv. Cyclic Nucleotide Res. 9, 533-552. Hechter, 0 . .Terada S . , Nakahara T., and Flouret, G., (1978a). Neurohypophyseal hormone-responsive renal adenylate cyclase. 11. Relationship between hormonal occupancy of neurohypophyseal hormone receptor sites and adenylate cyclase activation. J. Biol. Chem. 253, 3219-3229.
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Hechter, 0.. Terada, S.. Spitsberg, V., Nakahara, T., Nakagawaga. S. H., and Flouret, G. (1978b). Neurohypophyseal hormone-responsive renal adenylate cyclase. 111. Relationship between affinity and intrinsic activity in neurohypophyseal hormones and structural analogs. J. Biol. Chem. 253, 3320-3327. Hems, D. A.. and Whitton, P. D. (1973). Stimulation by vasopressin of glycogen breakdown and glucogeneogenesis in the perfused rat liver. Biochem. J. 136, 705-709. Hunt. N . H.. Perris, A. D., and Sandford, P. A. (1977). Role of vasopressin in the mitotic response of rat bone marrow cells to hemorrhage. J. Endocrinol. 72, 5-16. Imbert, M., Chabardes, D., Montegut, M., Clique A,. and Morel F. (l975a). Vasopressin dependent adenylate cyclase in single segments of rabbit kidney tubule. P’ugers Arch. 357, 173-186. Imbert. M., Chabardes. D., Montegut. M., Clique A,, and Morel F. (1975b). Prisence d’une adinyl cyclase stirnulie par la vasopressine dans la branche ascendante des anses du niphron de rein de lapin. C.R. Acad. Sci. Paris 280, 2129-2132. Jard. S. (1981). Les isorecepteurs de la vasopressine dans le foie et dans le rein: Relation entre fixation d’hormone et riponse biologique. J. Phvsio/. Paris 77, 62 1-628. Jard. S . , and Bockdert, J. (1975). Stimulus-response coupling in neurohypophysial peptide target cells. Physiol. Rev. 55, 489-536. Jard. S., Roy C., Barth T . , Rajerison R.. and Bockaert J. (1975). Antidiuretic hormone-sensitive kidney adenylate cyclase. Adv. Cvclic Nucleoride Res. 5, 31-52. Jard, S., Cantau, B., and K. H. Jakobs (1981). Angiotensin I1 and or-adrenergic agonists inhibit rat liver adenylate cyclase. J. Bio/. Chem. 256, 2603-2606. Jones, L. M.. Cockcrofts, S.. and Michell, R. H. (1979). Stimulation of phosphatidylinositol turnover in various tissues by cholinergic and adrenergic agonists by histamine and by caerulein. Bbchem. J. 182, 669-676. Keppens, S.. and De Wulf, H. (1975). The activation of liver glycogen phosphorylase by vasopressin. FEBS Left. 51, 29-32. Keppens, S . , and De Wulf, H. (1979). The nature of the hepatic receptors involved in vasopressininduced glycogenolysis. Biochim. Biophy. Acta 588, 63-69. Keppens, S . , Vandenheede, J. R.. and De Wulf. H. (1977). On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase. Biochim. Biophys. Act0 496, 448-457. Khoo, J. C . , and Steinberg, D. (1975). Stimulation of rat liver phosphorylase kinase by micromolar concentrations of Ca. FEBS Leu. 57, 68-72. Kirk, C. J . , and Hems, D. A. (1974). Hepatic action of vasopressin: Lack of role for adenosine 3’-5’ monophosphate. FEBS Lerr. 47, 128-1 31. Kirk, C. J . , Verrinder, T. R., and Hems, D. A . (1977). Rapid stimulation by vasopressin and adrenaline of inorganic phosphate incorporation into phosphatidylinositol in isolated hepatocytes. FEES Len. 83, 267-27 1. Kirk, C. J.. Rodrigues, L. M., and Hems, D. A. (1979). The influence of vasopressin and related peptides on glycogen phosphorylase activity and phosphatidylinositol metabolism in hepatocytes. Biochem. J. 178, 493-496. Kirk, C. J.. Michell, R. H.,and D. A. Hems (1981a). Phosphatdylinositol metabolism in rat hepatocytes stimulated by vasopressin. Biochem. J. 194, 155- 165. Kirk, C. J., Creba, I. A., Downes, C. P., and Michell, R. H. (1981b). Ca-dependent polyphosphoinositide breakdown in stimulated rat hepatocytes. Biochem. Soe. Trans. 9, 135. Konrads, A., Hofbaver, K . G., Werner, U., and Gross, F. (1978). Effects of vasopressin and its deamino-marginine analogue on renin release in the isolated perfused rat kidney. Pflugrrs Arch. 377, 81-85.
Kram, R., Mamont, P., and Tomkins. G. M. (1973). Pleiotypic control by adenosine 3’:5’-cyclic monophosphate: A model for growth control in animal cells. Proc. Natl. Acad. Sci. U.S.A. 70, 1432- 1436.
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Krejci. I . , Polacek, I . , and Rudinger, J. (196?). The action of 2-0-methyltyrosine-oxytocin on the rat and rabbit uterus: Effect of some experimental conditions on change from agonism to antagonism. E r . J . Pharmucol. Chemother. 30, 506-5 17. Lowbridge, J . , Manning, M . , Haldar, J . , and Sawyer, W. (1977). (1-(~-2-Hydroxy-3-mercaptopropionic acid) analogues of arginine vasopressin. (8.1)-arginine) vasopressin, and (4-valine, 8-Darginine) vasopressin. J . Med. Chem. 20, 1173-1 176. Lowbridge, J.. Manning, M., Haldar. J . , and Sawyer, W. H. (1978).(I-(P-Mercapto-P,P-cyclopentamethylenepropionic acid)4 valine, 8-o-arginine) vasopressin, a potent and selective inhibitor of the vasopressor response to arginine-vasopressin. J . Med. Chem. 21, 313-315. McIlhinney, R. A. J . . and Schulster, D. (1975). Studies on the binding of '15 I-labelled corticotropin to isolated rat adrenocortical cells. J . Endocrinol. 64, 175- 184. Manning, M., and Sawyer, W. H. (1977). Structure-activities studies on oxytocin and vasopressin 1954- 1976: From empiricism to design. Neurohyophvs. Int. Corzf. Key Eiscuyne. pp. 9-2 I. Manning, M.,Balaspiri, L.. Moehring, S., Haldar. J . . and Sawyer, W. H. (1976). Synthesis and some pharmacological properties of deamino (4-threonine. 8-o-arginine) vasopressin and deamino (8-o-arginine) vasopressin, highly potent and specific antidiuretic peptides, and ( 8 . ~ arginine) vasopressin, and deamino arginine vasopressin. J . Med. Chem. 19, 842-845. Manning, M., Lowbridge, J . , Stier, C. R., Jr., Haldar, J . , and Sawyer, W. H. (1977). (1-Dea highly potent inhibitor of the vasoaminopenicillamine, 4-valine)-8-~-arginine-vasopressin, pressor response to arginine-vasopressin. J . Med. Chem. 20, 1228- 1230. Mendoza, S. A.. Wigglesworth, N. M., and Rozengurt, E. (1980). Vasopressin rapidly stimulates Na entry and Na-K pump activity in quiescent cultures of mouse 3T3 cells. J . Cell. Physiol. 105, 153-162. Michell, R . H. ( 1975). Inositol phospholipid and cell surface receptor function. Biochim. Eiophys. Acta 415, 81-147. Michell, R. H., Kirk, C. J . , and Billah, M. M. (1979). Hormonal stimulationofphosphatidylinositol breakdown, with particular reference to the hepatic effects of vasopressin. Biochem. Soc. Trans. 7 , 861-865. Miler. R. P., Husain. F., Svensson, M., and Lohin, S. (1977). Enhancement of "methyl thyinidine incorporation and replication of rat chondrocytes grown in tissue culture by plasma tissue extracts and vasopressin. Endocrinology 100, 1365- 1375. Mills, J. W., Macknight, A. D. C., Dayer, J. M., and Ausiello, D. A. (1979). Localization of 'H ouabain-sensitive Na pump sites in cultured pig kidney cells. Am. J . Physiol. 236, C 157-Cl62. Mohring, J . , Mohring, B., Petri, M., and Haack, D. (1977). Vasopressor role of ADH in the pathogenesis of malignant DOC hypertension. Am. J . Physiol. 232, F260-F269. Muhlethaler, M.. Dreyfuss, J . J . , and Gawiler, B. H. (1982). Vasopressin causes excitation of hippocampal neurons. Nutitre (London) 296, 749-75 I. Munsick, R. A , , Sawyer, W . H., and Van Dyke, H. B. (1960).Avian neurohypophysial hormones, pharmacological properties and tentative identification. Endocrinology 66, 860-87 I . Nagasubramanian, S.( 1977). Role of pituitary vasopressin in the formation and dynamics of aqueous humour. Trans. Ophtulmol. Soc. U . K . 97, 686-701. Oliver. G . , and Shafer. E. A,, (1895). On the physiological action of extracts of pituitary body and certain other glandular organs. J . PhvsioL. London 18, 277-279. Orloff. J., and Handler, J. S. (1967). The role of adenosine 3',5'-phosphate in the action of antidiuretic hormone. Am. J . Med. 42, 757-768. Pearlmutter. A. F., and Soloff, M. S.. (1979). Characterization of the metal ion requirements for oxytocin-receptor interaction in rat mammary gland membranes. J . B i d . Chern. 254, 3899- 3906. Penit. J . , Faure, M., and Jard, S. (1982). Vasopressin and angiotensin I1 receptors in rat aorta smooth muscle cells in culture. Am. J . Phvsiol. (in press).
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Pradelles, P., Morgat, J-L., Fromageot, P., Camier, M., Bonne, D., Cohen, P., Bockaert, J., and lard, S. (1972). Tritium labeling of (8-lysine)-vasopressin and its purification by affinity chromatography on sepharose-bound neurophysins. FEBS Lett. 26, 189- 192. Raichle, M. E., and Grubb Jr. R. L. (1978). Regulation of brain water permeability by centrally released vasopressin. Brain Res. 143, 191- 194. Rajerison, R. M. (1979). Aspects mol6culaires de la regulation de la sensibilite A I’hormone antidiuretique de l’adenyl cyclase de reins de mammiferes. These de Doctorat d’Etat. Universitt Pierre et Marie Curie, Paris. Rajerison, R., Marchetti, J., Roy, C.. Bockaert, J., and Jard, S., (1974). The vasopressin-sensitive adenylate cyclase of the rat kidney: Effect of adrenalectomy and corticosteroids on hormonal receptor-enzyme coupling. J . Biol. Chem. 249, 6390-6400. Rajerison, R., Butlen, D., and Jard, S . (1976). Ontogenic development of antidiuretic hormone receptors in rat kidney: Comparison of hormonal binding and adenykdte cyclase activation. Mol. Cell. Endocrinol 4, 271-285. Rajerison, R. M., Butlen, D., and Jard, S. (1977). Effects of in vivo treatment with vasopressin and analogues on renal adenylate cyclase responsiveness to vasopressin in virro. Endoctinofom 101, 1-12. Robertson, G. L., Ermelinda, A,, Mahr, Sahahid Athar, and Tushar Sinha. (1973). Development and clinical application of a new method for the radioimmunoassay of arginine vasopressin in human phsma. J . Clin. Invest. 52, 2340-2352. Rocha da Silva, M., Jr.. and Rosenberg, M. (1969). The release of vasopressin in response to haemorrage and its role in the mechanism of blood pressure regulation. J . Physiol. (London) 202, 535-557. Rodbell, M. (1980). The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature (London) 284, 17-22. Roy, C., and D. A. Ausiello (1981). Characterization of (8-lysine)-vasopressin binding sites on a pig kidney cell line (LLC-PKI). J . Biol. Chem. 256, 3415-3422. Roy, C., Barth, T., and Jard, S. (1975a). Vasopressin-sensitive kidney adenylate cyclase. Structural requirements for attachment to the receptor and enzyme activation. Studies with oxytocin analogues. J . Biol. Chem. 250, 3157-3168. Roy, C., Barth, T., and Jard, S. (1975b). Vasopressin-sensitive kidney adenylate cyclase. Structural requirements for attachment to the receptor and enzyme activation. Studies with vasopressin structural analogues. J . Biol. Chem. 250, 3 149-3 156. Roy, C., Rajerison, R., Bockaert, J . , and Jard, S. (1975~).Solubilization of the 8-lysine vasopressin receptor and adenylate cyclase from pig kidney plasma membranes. J. Biol. Chem. 250, 7885-7893. Roy, C., Guillon, G., and Jard, S. (1976). Hormone-dependent desensitization of vasopressinsensitive adenylate cyclase. Biochem. Biophys. Res. Commun. 72, 1265- 1270. Roy, C., Hall, D., Karish, M., and Ausiello, D. A. (1981). Relationship of (8-lysine)-vasopressin receptor transition to receptor functional properties in a pig kidney cell line (LLC-PKI). J. Biol. Chem. 256, 3423-3427. Rozengurt, E., Legg, A,, and Curd Pettican, P. (1979). Vasopressin stimulation of mouse 3T3 cell growth. Proc. Natl. Acad. Sri. U.S.A. 76, 1284-1287. Saameli, K. (1968). The circulatory actions of the neurohypophysial hormones and similar polypeptides. in “Neurohypophysial Hormones and Similar Polypeptides” (B. Berde, ed.), Handbook of Experimental Pharmacology, Val. 23, pp. 545-612. Springer-Verlag, Berlin and New York. Sawyer, W. H. (1966). Biological assays for neurohypophysial principles in tissues and in blood. In “The pituitary gland” (G. W. Harris and B. T. Donova, eds.). Vol. 3. Butterworths, London. Sawyer, W. H., Acosta, M., Balaspiri, L., Judd, J., and Manning, M. (1974). Structural changes in
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the arginine vasopressin molecule that enhance antidiuretic activity and specificity. Errdocrinology 94, 1106- I 115. Sawyer, W. H., Crzonka, Z., and Manning, M. (1981) Neurohypophysial peptides: Design of tissue specific agonists and antagonists. Mol. Cell. Endocrinol. 22, 1 17- 134. Somlyo, A. V., Woo. C., and Somlyo, A . P. (1966). Effect of magnesium on posterior pituitary hormone action on vascular smooth muscle. Am. J. Ph.vsiol. 210, 705-714. Takai, Y.,Kishimoto, A., Kikkawa, V., Mori, T., and Nishizuka, Y. (1979a). Unsaturated diacylglycerol as a possible messenger for the activation of calcium-activated, phospholipiddependent protein kinase system. Biochem. Biophys. Res. Commun. 91, 12 18-1224. Takai, Y.,Kishoimoto, A., Iwasa, Y., Kawahara, Y.. Mori, T., and Nishizuka, Y. (1979b). Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids. J. Biol. Chem. 254, 3692-3695. Takhar, A. P. S., and Kirk, C. J. (1981). Stimulation of inorganic phosphate incorporation into phosphatidylinositol in rat thoracic aorta mediated through VI-vasopressin receptors. Biochem. J. 194, 167-172. Tanaka, M., de Kloet, E. R., DeWied, D., and Versteeg, D. H. G. (1977). Arginine-vasopressin affects catecholamine metabolism in specific brain nuclii. Life Sci. 20, 1799- 1803. Tolbert, M. E. M., White, A. C.. Aspry, K., Cutts, J . , and Fain, I. N . (1980). Stimulation by vasopressin and alpha-catecholamines of phosphatidylinositol formation in isolated rat liver parenchymal cells. J. Biol. Chem. 255, 1938-1944. Travo, P . , Barrett, G., and Burnstock. (1980). Differences in proliferation of primary cultures of vascular smooth muscle cells taken from male and female rats. Blood Vessels 17, 110-1 16. Verney, E. B. (1947). The antidiuretic hormone and the factors which determine its release. Proc. R . Soc. Sci. 6 135, 27-106. Walter, R.. Dubois, B. M., and Schwartz, I. L. (1968). Biological significance of the aminoacid residue in position 3 of neurohypophyseal hormones and the effect of magnesium on their uterotonic action. Endocrinology 83, 979-983. Walter, R.. Van Ree, J. M., and DeWied, D. (1978). Modification of conditioned behavior of rats by neurohypophyseal hormones and analogues. Proc. Nutl. Acad. Sci. U S A . 75, 2493-2496. Whitfield, J . P., MacManus, J. P., and Gillan. D. J. (1970). The possible mediation by cyclic AMP of the stimulation of thymocyte proliferation by vasopressin and the inhibition of this mitogenic action by thyrocalcitonin. J. Cell. Physiol. 76, 65-76. Zusman, R. M., and Keiser, H. R. (1977). Prostaglandin biosynthesis by rabbit renomedullary interstitial cells in tissue culture. J . Clin. Invest. 60, 2 15-223.
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I8
Induction of Hormone Receptors and Responsiveness during Cel Iular Different iatio n MlCHAEL C. LIN AND SUZANNE K . BECKNER Laboratory of Cellulor and Developmental Biology National Institute c$ Arthritis, Diabetes, Digestive and Kidney Diseases National Institutes oj' Health Bethesda. Maryland
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Properties of the Hormone-Sensitive Adenylate Cyclase System ....... B. Hormone Responsiveness as a Differentiated Function . . . . . . . . . . . . . . . . . . . . . C. Use of Cell Cultures for the Study of the Differentiation Process. . . . . . . . . . . . . D. Chemical Induction of Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Model Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. 3T3-LI Adipocytes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Liver Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Granulosa Cells .................... ................ D. Madin-Darby C (MDCK) Cells.. .................... III. Conclusion ............................................................. A. General Requirement for Induction of Hormone Receptors .................. B. Biochemical Mechanisms for Induction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Biological Significance ..... ................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
1.
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307 307 307 309 310
INTRODUCTION
Many hormones initiate their effects on the target cells by binding to specific cell surface receptors. In numerous systems, this interaction leads to the generation of the second messenger, cyclic AMP, which initiates a complex cascade of events, responsible for the cellular responsiveness to that hormone. Normal development and malignant transformation are accompanied by numerous bio287 Copyright 0 IYR3 by Audemic Press. Inc All righ6 of rcproduction In any form reserved ISBN 0-12-153318.2
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chemical and morphological changes, including changes in hormone sensitivity. This article will focus primarily on the hormone-sensitive adenylate cyclase system as one indication of the state of cellular differentiation.
A. Properties of the Hormone-Sensitive Adenylate Cyclase System Knowledge of the molecular organization of the hormone-sensitive adenylate cyclase system, which produces cyclic AMP, provides a basis for understanding the alteration in hormone responsiveness. As this enzyme system has been covered by several articles in this series (see this volume for reference), only its general characteristics and fundamental principles will be discussed to provide a sufficient background for understanding the regulation of this system. The adenylate cyclase system consists of at least three major components, the catalytic component, the hormone receptor, and coupling factors, the guanine nucleotide regulatory component being the best characterized. The catalytic component is the enzyme which catalyzes the formation of cyclic AMP from ATP, while receptors serve to recognize the hormone and initiate the- sequence of events leading to the eventual hormone action. The nucleotide regulatory component governs the expression of the activity of the catalytic component by its interaction with guanine nucleotides (Rodbell, 1980). The ability of guanine nucleotides to modulate catalytic activity is attenuated by the coupling of the nucleotide component to hormone receptors, while hormones act to release this restrictive effect through interaction with their receptors (Lin er af., 1979). Thus, hormone receptors provide the discrimination which governs the ultimate specificity intrinsic to the responsive system.
B. Hormone Responsiveness as a Differentiated Function During the process of differentiation cells acquire various specialized functions, and the responsiveness of cells to hormones is often associated with a more differentiated state. For example, during fetal development, rat liver cells become increasingly more responsive to glucagon and achieve maximal sensitivity after birth (Blazquez et af., 1976). The acquisition of responsiveness to catecholamines and adenosine in rat cerebral cortex during neonatal development (Perkins and Moore, 1973) is another example of the appearance of hormone sensitivity as a function of differentiation. Since differentiation characteristics appear in an orderly fashion during development, they must be precisely programmed or regulated. Thus the evolution of hormone responsiveness can be
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expected to be highly regulated; however, factors governing this development remain poorly understood. Several recent studies suggest that the appearance of hormone sensitivity may be regulated by other hormones. These observations indicate that certain types of hormone sensitivity appear early during differentiation and are crucial for subsequent elaboration of other differentiated functions. The hormones involved in this type of regulation can be considered as developmental hormones. On the other hand, some hormone responses do not appear until later development, and their functions mainly concern the regulation of cellular metabolism during the matured state. This second class of hormones can be termed metabolic hormones. The nature of both types of hormones varies depending on the cell type. The same hormone may serve either a developmental or metabolic role in different cells.
C. Use of Cell Cultures for the Study of the Differentiation Process Differentiation is a complex process. Even when studying a single function, such as hormone sensitivity, numerous other factors must be considered. Therefore, by using cultured cells, the cellular environment can be brought under better control, thus reducing the number of variables within the experimentation. Both primary and established cell cultures have been used in studies of cellular development. The primary culture often reflects more closely the in vivo characteristics; however, this type of culture tends to be less homogeneous and less able to survive subculturing. On the other hand, established cell lines provide a continuous supply of an essentially homogeneous population of cells. Due to adaptation to artificial growth conditions, established cell cultures often become partially dedifferentiated and, in many cases, no longer retain the initial cellular characteristics.
D. Chemical Induction of Differentiation Certain chemical compounds have been found to induce differentiated functions in several established cell lines. Friend erythroleukemic cells (a line of erythroblasts transformed with Friend leukemia virus) and HeLa cells (Gey et al., 1952), derived from a human cervical carcinoma, have been well characterized in this regard. Following exposure to dimethyl sulfoxide (Friend et a / . , 1971). other polar compounds (Reuben er u / . , 1976; Tanaka et a/., 1975), or butyrate (Leder and Leder, 1 9 7 3 , Friend cells differentiate with a program characteristic of normal
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erythropoiesis (Friend et a / . , 1971) and thus represent a good model system of hematopoietic stem cell differentiation. During differentiation, the cells exhibit altered morphology, accumulate hemoglobin mRNA (Ross et al., 1972), and express different sets of cell surface glycoproteins (Fukada et a l . , 198I ) . Siniilarly, butyrate induces morphological (Ginsburg et at., 1973) and biochemical changes (Simmons et a / . , 19751, including increases in the secretion of human chorionic gonadotropin (hCG) (Ghosh and Cox, 1976) and follicle-stimulating hormone (FSH) (Ghosh and Cox, 1977) in HeLa cells. In both cell lines, these differentiation inducers also increase the number of P-adrenergic receptors and responsiveness (Tallman et al., 1977). However, the mechanism of the induction of differentiation by these chemical inducers remains unknown. Because both Friend and HeLa cells are oncogenic they represent good model systems to study the relationship between the expression of differentiated characteristics and the neoplastic state. Recently, several cell culture systems have been utilized to examine the regulation of differentiation from a more developmental aspect. The regulation of hormone responsiveness, as an indicator of the differentiated state, in several of these systems is discussed below.
II. MODEL SYSTEMS A. 3T3-Ll Adipocytes 1 . CHARACTERISTICS OF THE CELLS
Resting cultures of 3T3 murine fibroblasts accumulate small droplets of lipid when confluent. Subsequently, several clones of this line were isolated which accumulated triglyceride to a large degree (Green and Meuth, 1974), 3T3-LI preadipocytes being the best in this regard. When these cells achieve confluency , they spontaneously differentiate and acquire morphological and biochemical characteristics of mammalian adipose tissue (Green and Kehinde, 1975), including responsiveness to lipolytic and lipogenic hormones. Differentiating 3T3-L 1 preadipocytes also accumulate various enzymes associated with triglyceride synthesis. Levels of acetyl-CoA carboxylase, ATP citrate-lyase (Mackall et a/., 1976), pyruvate carboxylase (Freytag and Utter, 19801, glycerophosphate dehydrogenase (Pairault and Green, I979), fatty acid synthetase (Weiss et a / ., 1980), glycerophosphate acyltransferase, and malic enzyme (Kuri-Harcuch and Green, 1977) have all been found to rise dramatically during the differentiation process. In most cases, this increased activity is a result of greatly increased de now synthesis of enzyme, and probably involves complex coordination of gene expression (Pekala et al., 1981).
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2. RESPONSIVENESSTO HORMONES The mature mammalian fat cell is characterized by an adenylate cyclase system that is reponsive to a number of hormones (Birnbaumer, 1973) which control lipolysis as well as receptors for insulin which control lipogenesis (Winegrad and Renold, 1958). The nondifferentiated 3T3-L 1 preadipocyte does have insulin receptors (Rubin et al., 1977), but these receptors are not coupled to hexose transport or hexose conversion. Once the differentiation process begins, there is a large increase in high-affinity insulin receptors, from 7000 to 250,000 siteskell (Rubin et a / . , 1978). These newly synthesized insulin receptors (Reed et af., 1981) are coupled (Fig. 1) to the hexose transport system and glucose metaboliz-
/
10'0
CONFLUENT
10-8
10-6
INSULIN ( M )
FIG. I . Relationship between insulin binding and stimulation of glucose oxidation in 3T3-LI cells from different stages of growth and differentiation. The increment in glucose oxidation produced at a given insulin concentration is plotted (A). Basal glucose oxidation was approximately 10 nmoles/106 cells/2 hours. The amount of insulin bound as a function of insulin concentration is given (B). (Reproduced from Karlsson e t a / . , 1979, with permission. 0 1979, The Endocrine Society.)
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MICHAEL C. LIN AND SUZANNE K. BECKNER
ing enzymes (Karlsson er a[., 1979), although the time course of the development of insulin responsiveness does not parallel that of insulin receptor synthesis (Rosen et al., 1978). When 3T3-Ll preadipocytes were induced to differentiate (see Section II,A,3), insulin-stimulated glucose uptake and conversion to CO, was increased three- to fivefold, but there was no increase in insulin binding. During the next 72 hours, insulin binding increased six- to tenfold with a concomitant increase in insulin-stimulated hexose transport and conversion. As with mature adipocytes, insulin stimulated the phosphorylation of a ribosomal protein (Smith et al., 1979). Numerous other hormone-responsive systems are altered upon the differentiation of 3T3-Ll preadipocytes to adipocytes (Table I). The adenylate cyclase of adipocytes exhibits an increased responsiveness to P-adrenergic agonists and also responds to adrenocorticotropic hormone. However, there is only a slight increase in the responsiveness to prostaglandin E, , and neither preadipocytes nor adipocytes respond to glucagon (Rubin et al., 1977), while both are responsive to fluoride, indicating a functional cyclase system.
3. FACTORSAFFECTING DIFFERENTIATION The spontaneous differentiation of 3T3-Ll cells occurs over a period of 2-4 weeks after the cells achieve confluence, and can be accelerated by high concentrations of serum (Green and Meuth, 1974).
EFFECTS
OF
TABLE I HORMONES ON ADENYLATE CYCLASE ACTIVITYI N HOMOGENATES OF ADIPOCYTES AND PREADIFOCYTES~".~
-
Adenylate cyclase activity (pmole CAMP formediminutelmg protein) Addition
Preadipocytes
Adipocytes
None (basal activity) Isoproterenol (2.5 pkf) ACTH (2 pbf) Glucagon (2 )LM) Prostaglandin E l (3 )LM) NaF (20 mM)
3.5 8.9 3.5 3.1 7.3 21.2
4.5 65.2 17.6 3.9 16.6 47.5
Adapted from Rubin el a / . (1977), with permission. Homogenates were prepared from confluent 3T3-LI cells cultured in the presence or absence of inducers and assayed for adenylate cyclase activity at 37°C with or without hormones as indicated. The assay medium consists of 0.5 mM [a-'ZP]ATP (150 cpdpmole). 5 mM MgCI2, 1 mM CAMP, 20 mM creatine phosphate, 2.5 pg creatine phosphokinase (0.4 unit), and 40 pg cell homogenate protein in 25 mM Tris-CI buffer, pH 7.4. Cyclic AMP formed was measured as described by Salomon (1979). a
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The differentiation process can be compressed into a period of 7 days by the addition of dexamethasone and isobutylmethylxanthine, and inhibitor of phosphodiesterase activity (Rubin et a / . , 1978). Although the mechanism of this induction is unknown, Murray and Russell (1980) have found that this process can be inhibited, reversibly, if retinoic acid is present along with these inducers. Whether the effect of retinoic acid is related to its recently discovered role in development and differentiation (Rao er al., 1979) remains to be established. Several lipolytic and lipogenic hormones also affect the adipocyte conversion process. Insulin markedly stimulated the synthesis and accumulation of triglycerides during adipocyte conversion (Green and Kehinde, 1975)as well as enhanced the conversion process. Furthermore, insulin was required for the induction of differentiation by dexamethasone and isobutylmethylxanthine (Miller and Carrino, 1980) or by serum (Miller and Carrino, 1981). However, insulin alone was not sufficient to trigger differentiation (Serrero ef ul., 1979), under defined media conditions. In addition, insulin facilitated adipocyte conversion in combination with indomethacin, a lipogenic agent (Williams and Polakis, 1977), hydrocortisone (Miller and Carrino, 19801, isobutylmethylxanthine, and prostaglandin F,, (Russell and Ho, 1976). The finding that adipocyte conversion could be triggered by prostaglandin F,, and isobutylmethylxanthine (Russell and Ho, 1976) suggested that cyclic nucleotides may have a role in the conversion process. Both compounds were found to have a rapid and irreversible effect on the conversion process. However, this effect could not be mimicked by dibutyryl cyclic AMP or cyclic GMP. Subsequently, cyclic AMP was shown to retard the differentiation process (Williams and Polakis, 1977). An inhibitory role for cyclic AMP in the adipocyte conversion process is supported by the recent findings of Hopkins and Gorman ( 198 1). They found that the predominant prostaglandin produced by 3T3-L 1 adipocytes (prostacyclin) activated adenylate cyclase to a greater extent than other prostaglandins. If prostacyclin synthesis was selectively inhibited, the insulin-stimulated conversion process was accelerated, an effect which was reversed by the addition of exogenous prostacyclin. In addition, as 3T3-Ll cells differentiated, the ability of prostacyclin to stimulate adenylate cyclase was diminished, suggesting that the rate of differentiation may be controlled by prostacyclin synthesis or the sensitivity of the adenylate cyclase system to this agent. These data may explain why indomethacin accelerated the differentiation process (Williams and Polakis, 1977), as this agent inhibits the synthesis of all prostaglandins. Similarly, steroidal antiinflammatory agents (dexamethasone and hydrocortisone) which accelerated differentation have also been found to decrease prostaglandin synthesis in other systems (Flower and Blackwell, 1979). Obviously, a role for cyclic AMP in mediating the differentiation process is complicated by the fact that cyclic AMP mediates lipolysis in the fat cell. It has been found that agents that induce
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lipolysis also reduce the synthesis of lipogenic enzymes during adipose differentiation (Spiegelman and Green, 198I ) , suggesting independent control of morphological change and enzyme synthesis during adipose differentiation.
REMARKS 4. CONCLUDING The 3T3-LI cell line represents an excellent model system for exploring factors which regulate the differentiation of hormone-responsive systems. Unfortunately, factors which trigger the differentiation of these cells are not yet completely identified. Clearly, serum contains all the factors required for the conversion process. The conversion can be reversibly and specifically inhibited by bromodeoxyuridine (Green and Meuth, 1974), known to inhibit differentiation in other systems (Rutter et 01.. 1973). This inhibitory effect could be reversed in another preadipocyte cell line, ob 17, by the addition of indomethacin (an inhibitor of prostaglandin synthesis and a lipogenic agent) or by clofenapate (a hypolipidemic drug), presumably by reversing the inhibition of endogenous fatty acid synthesis (Verrando et af., 1981). It does seem clear that cyclic AMP prevents adipocyte conversion. Whether endogenously produced prostaglandins inhibit differentiation by elevating cyclic AMP levels is unknown, but the acceleration of differentiation by agents (steroids) known to decrease prostaglandin synthesis would support this idea. It is unclear how inhibitors of phosphodiesterase activity accelerate conversion. However, cyclic AMP can increase the concentration of insulin receptors (Thomapoulos et al., 19781, so it is possible that the effect of cyclic AMP on differentiation is twofold. While an increase in the number of insulin receptors would accelerate differentiation, the inhibitory effect of cyclic AMP could be exerted at the level of synthesis of lipogenic enzymes. Certainly insulin occupies a central role in the differentiation process. If insulin is withdrawn, the adipocyte conversion is reversed. It is likely that insulin is required for all factors that induce conversion, although this has not been rigorously demonstrated. The fact that insulin receptors of 3T3-LI cells are not down-regulated by insulin (Chang and Polakis, 1978) supports the idea that functionally coupled insulin receptors are somehow integral to the differentiation process. Whether lipogenesis (stimulated by insulin or indomethacin) triggers differentiation or merely supports the process remains to be established.
B. Liver Cells 1. CHARACTERISTICS OF THE CELLS The liver has long been the subject of intense study due to the central role of this organ in carbohydrate metabolism, detoxification, and numerous other bio-
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chemical processes. Additionally, the liver represents a good system for examining differentiation, as fetal liver cells produce a markedly different array of gene products as compared to adult liver. During adulthood, fetal gene products are not produced in any substantial quantity, except perhaps during oncogenesis or diseased states. Many studies of liver function have utilized freshly isolated hepatocytes and primary cultures of hepatocytes. Conditions have been developed to maintain viability and differentiated functions in such cells (Dickson and Pogson, 1977; Leffert et al., 1978). In general such cell preparations are less than optimal due to heterogeneous cell populations, cell damage during isolation, and loss of viability. Although some differentiated functions can be retained in primary liver cultures, such cells eventually deteriorate and lose most of their differentiated functions. Several continuous liver cell lines have been developed. Bausher and Schaeffer ( 1 974) have developed a cloned line of stably diploid hepatocytes from rat, designated RL-PR-C, which retains many differentiated characteristics of liver. Following approximately 300 population doublings, these cells spontaneously transform, at which time they acquire many characteristics of transformed cells, including serum-independent growth, chromosomal changes, and oncogenicity (Schaeffer and Polifka, 1975). K16 cells, a normal rat liver epithelial cell line (Makarski and Niles, 1978), also retain some differentiated properties in culture. Several stable cell lines (RLA clones) have been developed by transformation of fetal liver cells with simian virus 40 (Schlegel-Haueter et al., 1980) and shown to produce a-fetoprotein, albumin, and transferrin (Chou and Schlegel-Haueter, 198 1 ) . Numerous continuous cell lines of hepatoma cells have also been developed and have been used extensively in carcinogenesis studies. However, as these cells are oncogenic, they retain few differentiated functions, although several of these lines do exhibit some differentiated characteristics. 2 . RESPONSIVENESS TO HORMONES
Carbohydrate metabolism in the liver is regulated by the hormone-responsive adenylate cyclase system and insulin. The adenylate cyclase of RL-PR-C hepatocytes is regulated by glucagon and epinephrine (Beckner et al., 1980), and that of K 16 cells by epinephrine, isoproterenol, norepinephrine, and prostaglandin E, (Makarski and Niles, 1978). RLA cells also have glucagon receptors coupled to adenylate cyclase. RL-PR-C cells also respond to insulin (Petersen et al., 1978). Different hepatomas and cell lines derived from them have varying degrees of hormone responsiveness, including that to glucagon, insulin, and isoproterenol (see Section II,B,3).
296
MICHAEL C. LIN AND SUZANNE K. BECKNER
3. FACTORS AFFECTING DIFFERENTIATION The most common change associated with altered differentiation of liver cells is seen in the hormone-responsive adenylate cyclase system. Numerous changes in hormone responsiveness have been observed following transformation by a variety of means. When K16 cells were chemically transformed, they no longer responded to P-adrenergic hormones or prostaglandin E, (Makarski and Niles, 1978), although the adenylate cyclase system was still responsive to fluoride, suggesting that transformation resulted in a loss of hormone receptors. Following spontaneous transformation, insulin binding and responsiveness were decreased in RL-PR-C hepatocytes (Petersen and Blecher, 1979). There were also numerous changes in the adenylate cyclase system of these cells (Fig. 2). Cyclic AMP levels of normal RL-PR-C cells increased as the cells achieved confluence, while those of transformed cells declined. Although the adenylate cyclase system was more sensitive to epinephrine following spontaneous transformation, the cells were no longer responsive to glucagon (Beckner et a [ . , 1980). It was later demonstrated that transformation of RL-PR-C cells is accompanied by a loss of high-affinity glucagon binding sites, although low-affinity sites increased from 10,000 to 1 12,000 siteskell after transformation (Reilly and Blecher, 1981). Whether the loss of glucagon responsiveness can be prevented or restored by conditions favoring differentiation remains to be demonstrated. In RLA cells, the response to glucagon (Fig. 3) was evident only at the nonpermissive temperature and in the presence of cortisol (Schlegel-Haueter et al., 1980); therefore, this transformed liver cell line can potentially be used for studying the induction of glucagon sensitivity by glucocorticoids when viral expression is suppressed. Pezzino et al. (1979) found that glucagon binding capacity and insulin binding affinity were decreased in various hepatomas as compared to normal liver. The decrease in insulin binding seemed to correlate well with the increased rate of tumor growth. Mire1 et af. (1978) found that poorly differentiated hepatomas lacked glucagon receptors and responsiveness whereas well-differentiated hepatomas still retained good responsiveness to the hormone; the responsiveness to epinephrine and fluoride showed no such variation. Normally, in liver, carcinogenesis is accompanied by enhanced responsiveness to catecholamines, and Lacombe et al. (1976) have suggested that normal liver has P2-type receptors, while hepatoma cells have p, -type receptors. Hormone sensitivity of liver tissue also changes during development. Blazquez et al. (1976) found that both insulin and glucagon binding and responsiveness increased during normal fetal development (Fig. 4). The insulin response seemed to appear more quickly during the developmental process, presumably so that anabolic processes required for normal fetal development were favored. Similarly, Sicard and Aprille ( 1977) found that glucagon sensitivity increased
297
INDUCTION OF HORMONE RESPONSIVENESS
0NORMAL TRANSFORMED
T I
40 20
t BASAL
CHOLERATOXIN (Ipg/ml)
EPINEPHRINE (0.5pM)
GLUCAGON ( 1 pM)
FIG.2. Total cAMP production by normal and transformed RL-PR-C hepatocytes in response to various ligands. Cells in monolayer were incubated for 1.5 hours, 37"C, with cholera toxin; 15 minutes, 37°C. with epinephrine; or 2 minutes, 22°C. with glucagon and 0.3 mM papaverine. all in phosphate-buffered saline with 15 mg/ml bovine serum albumin, pH 7.4. Values represent the sum of cAMP of the cells plus that of the media. Although normal RL-PR-C excrete much more cAMP than transformed cells, there was no effect of hormones on this excretion. Each value represents the average of triplicate determinations 2 SD. Basal cAMP values were identical under the conditions used for maximal hormonal stimulation. (Reproduced from Beckner et a/.. 1980, with permission.)
4r
298
MICHAEL C. LIN AND SUZANNE K. BECKNER
C
I
3
-1
~0 D
- 4
-3
:I,,)#$ 3
-2
,
-1
1 2 3 4 5 6 7 8 9
' 0 1 2 3 4 5 6 7 8 9
0
DAYS
3. Effect of glucagon on intracellular accumulation ofcAMP in RLA255-4 cells. RLA255-4 cells were grown in 24-well plates in a-modified minimal essential medium containing 10% fetal bovine serum. Medium was changed daily. The arrow indicates the temperature shift to 40°C and the addition of cortisol (10 pM). Glucagon (0. I p~!4)was added to the cultures after preincubation with papaverine (0.5 IM),an inhibitor for phosphodiesterase, for 5 minutes. The reaction was stopped 3 minutes after the addition of glucagon and CAMP was determined by a radioimmunoassay. Culture conditions were ( A ) control. 33°C; (B)control, 40°C; (C) cortisol, 33°C; (D)cortisol, 40°C. 0 , Papaverine; glucagon plus papaverine. (Adopted from Schlegel-Haueter et a / ., 1980. with permission.) FIG.
A,
during normal liver development, while adenylate cyclase sensitivity to fluoride and guanine nucleotides was unchanged, suggesting that the adenylate cyclase system appears early during development, and that glucagon receptors are limiting. The hormone responsiveness of liver cells can also be manipulated by chemical means. Glucagon responsiveness, usually undetectable in fetal hepatocytes, could be induced by butyrate, isobutylmethylxanthine, hydrocortisone, and insulin (Lin, 1980). Glucagon receptors so induced persisted for up to 7 weeks in culture and were functionally coupled to the adenylate cyclase system.
299
INDUCTION OF HORMONE RESPONSIVENESS BI5TH
I
PRENATAL
I
POSTNATAL I
I
I
T
ADULT
I
100 -
W
3 80-
3
5
3 0
2 600 W
; 0
40-
u
[L
W
a 20
-
'a 15
-17
k
21
RESPONSE TO GLUCAGON (10-9 M )
i ~
I
5
10
"
20
"
i I
30"
DAYS
FIG.4. Comparison of binding of [12sI]iodoglucagon (2 nM) and the response to glucagon ( 1 nM) of adenylate cyclase activity in partially purified plasma membranes prepared from rat liver obtained at various times during prenatal and postnatal life, expressed as percentage of the adult level. (Adopted from Blazquez cr al.. 1976, with permission. 0 1976, The Endocrine Society.)
4. CONCLUDING REMARKS
Mechanisms of regulation of liver function obviously are of interest from a developmental point of view, as well as providing a model of factors involved in the development of the neoplastic state. The appearance of differentiated functions during normal liver development suggests that a highly regulated program of specific gene expression exists, although not much is known of the regulatory processes involved. Glucocorticoids seem to have a modulatory role in liver development. Both dexamethasone and hydrocortisone were found to increase the production of a-fetoprotein (de Nechaud et al., 1976) and lactate and malate dehydrogenase (Yo0 et al., 1979) in various clones of Morris hepatoma. In another hepatoma cell line, dexamethasone also stimulated a-fetoprotein synthesis, an effect attenuated by insulin (Tsukada et al., 1979). The induction of glucagon receptors by butyrate is, as in other systems, not
300
MICHAEL C. LIN AND SUZANNE K. BECKNER
understood, but it has been found that butyrate induces the formation of prostacyclin in a liver cell line (Koshihara e? af.,1981). Hopefully, manipulation of differentiation in well-characterized liver culture systems (RLA, RL-PR-C) will provide insight into factors which regulate hepatic differentiation during development.
C. Granulosa Cells 1. CHARACTERISTICS OF THE CELLS Granulosa cells derived from the ovarian follicles differentiate in culture as they do in vivo. Such cells provide an ideal system for examining the mechanisms of control of hormone-dependent cell differentiation. During Graafian follicle development there are numerous changes in hormone responsiveness (see Section II,C,2) in addition to morphological and functional changes. During follicular maturation, granulosa cells form antrum and microvilli (Richards and Midgley, 1976), as well as intercellular junctions (Albertini and Anderson, 1974). It is the granulosa cells which are responsible for the nutrient supply to the oocyte and the secretion of progesterone which occurs during differentiation (Richards and Midgley, 1976). Recently, it has been demonstrated that granulosa cells can grow and differentiate in a defined medium (Erickson e? al., 1979), permitting study of the development process under carefully controlled conditions. TO HORMONES 2. RESPONSIVENESS
During Graafian follicle development, granulosa cells sequentially develop receptors for FSH (Nimrod et al., 1976) and luteinizing hormone (LH) (Nimrod et al., 1977), both of which are coupled to adenylate cyclase. The increase in LH receptors is thought to prepare the follicle for the preovulatory surge of LH, which initiates luteinization of granulosa cells. Both types of receptors via cyclic AMP (Kolena and Channing, 1972) are responsible for the enhanced estrogen and progesterone production that occurs (Erickson et af., 1979). Differentiated, but not undifferentiated, granulosa cells also have prolactin receptors, which stimulate progesterone production. Granulosa cells also have receptors for gonadotropin-releasing hormone, normally produced in the hypothalamus and responsible for regulating the release of both FSH and LH from the pituitary (Clayton et al., 1979). Gonadotropinreleasing hormone inhibits follicular development and ovulation (Ying and Guillemin, 1979), and prevents steroidogenesis induced by FSH (Hsueh and Erickson, 1979) and LH (Clayton et al., 1979) in granulosa cells.
301
INDUCTION OF HORMONE RESPONSIVENESS
3. FACTORS AFFECTING DIFFERENTIATION FSH induces LH receptors in vivo (Zeleznik et al., 1974) as well as in granulosa cells cultured in defined media (Erickson et al., 1979). Insulin also induces LH receptors (Fig. 5 ) and enhances the effect of FSH on this process (May et al., 1980). The presence of FSH seems to be required for normal aggregation and communication of granulosa cells, as in the absence of the hormone the cells retain a flattened smooth shape and do not aggregate into multilayered clusters (Amsterdam et al.. 1981). The new LH hormone receptors are located mainly on the
" 160 1 0 z 140 0
Y
9
120
A
W
m
a
+ 100
I
N n
00 k
-z
3z w
60 40
W
a 20
2
0
4 DAY OF CULTURE
6
FIG. 5 . The specific binding of ['2~1]iodo-hCGby porcine granulosa cells during monolayer culture. Aliquots of freshly harvested cells were assayed for the initial [ W]iodo-hCG-binding capacity (0.024 2 0.002 to 0.053 0.001 pmoles hCG/mg cell protein; mean k SE). The remaining cells were cultured as monolayers in the continuous presence of insulin (IN: 25 mIU/ml), hFSH (300 ngiml), or insulin and FSH. Control cultures contained no exogenous hormones. At 2 . 4 , and 6 days, the [ 'ZSI]iodo-hCG-binding capacity of granulosa cell monolayers was expressed as a percentage of the initial level. The figure is formulated from several independent experiments (mean 2 SE). The 2day value for insulin-treated cultures represents the mean 2 SE for all treatment groups. The [iZsl]iodohCGconcentration used was 150 ng/ml. (Adapted from May eral.. 1980, with permission. 0 1980, The Endocrine Society.)
*
302
MICHAEL C. LIN AND SUZANNE K. BECKNER
microvilli of granulosa cells. Both the induction of LH receptors and follicular organization can also be achieved by cholera toxin and cyclic AMP (Nimrod, 1981; Amsterdam et al., 1981). FSH also induces prolactin receptors of granulosa cells (Wang et al., 1979) (Table 11). The induction of LH receptors by FSH (Table 11) can be inhibited by gonadotropin-releasing hormone (Amsterdam et ul., I98 1). It seems likely that gonadotropin-releasing hormone exerts its antigonadal effects by increasing phosphodiesterase activity (Knecht et al., 1981), as the hormone inhibits FSHmediated increases in cyclic AMP, as well as steroidogenesis stimulated by agents which increase cyclic AMP levels (Hsueh et al., 1980). Hormone receptors of granulosa cells can also be modulated by various growth factors. The induction of LH receptors by FSH was prevented by epidermal and fibroblastic growth factors (EGF and FGF) and stimulated by platelet-derived growth factor (Mondschein and Schomberg, 1981). In cloned Leydig cells (Ascoli, 1981), both LH and cyclic AMP enhanced steroidogenesis. While EGF decreased the number of LH receptors and responsiveness, there was no impairment in the ability of cyclic AMP to increase steroidogenesis, suggesting that the effect of EGF is also mediated at the receptor level. 4. CONCLUDING REMARKS
The granulosa cell system represents an ideal model system in which to examine the sequential development of differentiated functions under defined conditions, as the maturation of ovarian function seems to be a highly regulated event with both positive and negative hormonal regulation. The precise mechanism of
INDUCTION
OF
TABLE 11 RECEP~ORSFOR hCG A N D PROLACTINI N GRANULOSA CELLSO Receptor concentration (fmole/106 cells)
Addition to culture None FSH FSH + GnRH
Bound hCG 2.0 22.4" 2.6b
Bound prolactin I .O 3.5c
11 Granulosa cells from hypophysectomized rats were isolated and cultured in McCoy's 5A medium with or without hormones, as indicated, for 2 days. Binding of '2SI-labeled hCC and 12s1-labeled prolactin to the suspended cells was measured. FSH (200 ngiml) and GnRH (0.1 phf) were used. Adapted from Amsterdam er uf. (1981). with permission. FSH (100 ng/ml) was used. Adapted from Wang et ul. (1979). with permission.
'
INDUCTION OF HORMONE RESPONSIVENESS
303
FSH induction of LH receptors is not clear. The finding that the induced LH receptors appeared mainly on aggregated cells suggests that cell aggregation and communication are important in this process. All of these effects of FSH seem to be mediated by cyclic AMP, as is the effect of FSH on steroidogenesis. Also unclear are the mechanisms responsible for the inhibitory effects of gonadotropin-releasing hormone, EGF, and FGF on FSH induction of LH receptors. Granulosa cells have specific receptors for these factors, and their effects do not seem to be mediated by direct inhibition of FSH binding.
D. Madin-Darby Canine Kidney (MDCK) Cells 1. CHARACTERISTICS OF THE CELLS
MDCK cells were established in 1958 by Madin and Darby (Gaush et al., 1966) from the kidney of a normal cocker spaniel. In monolayer culture, these cells form sheets with “blisters” or “ulcers” which result from the accumulation of water between the sheet and the culture dish (Leighton et al., 1969). The presence of microvillus projections and tight junctions on the surface of these blisters suggests that these kidney cells retain polarity in culture (Leighton et al., 1969). Salt and water are transported from the mucosal side, facing the media, to the serosal surface, facing the culture dish (Misfeldt et al., 1976). In addition, MDCK cells are tumorigenic (Leighton et al., 1969), but retain kidney tubulelike structures in vivo, when injected into nude mice (Rindler er al., 1979). MDCK cells can be grown in a serum-free medium supplemented with insulin, transferrin, prostaglandin E, (PGE,), triiodothyronine, and selenium (Taub er af.,1979). MDCK cells have been transformed by Harvey murine sarcoma virus (Shih et al., 1980) and shown to produce a specific viral protein. Such cells no longer form “blisters” and have been found to selectively lose responsiveness to glucagon (Lin et al., 1981). In addition, the production of prostaglandins E, (PGE,) and F,, (PGF,,) is decreased by 90% following transformation. Alrhough these cells take up arachidonic acid as readily as do untransformed MDCK cells, the release of arachidonic acid and its conversion to prostaglandins are depressed by 80% (Lin et al., 1981, 1982). 2. HORMONERESPONSIVENESS Many hormones regulate solute transport by kidney tubule epithelium (Katz and Lindheimer, 1977). In addition, the kidney itself is an endocrine organ, secreting both prostaglandins and renin, which also serve to regulate kidney function. The adenylate cyclase of MDCK (Table 111) is regulated by a number of hormones including PGE, , 6-adrenergic agonists, glucagon, vasopressin, and
304
MICHAEL C. LIN AND SUZANNE K. BECKNER
TABLE 111
EFPEC~ OF HORMONES ON cAMP PRODUCTION B Y MDCK CELL SO.^
Addition
Production of cAMP (pmole/hour/mg protein)
None Prostaglandin E l (IpM) Arginine-vasopressin (I pM) Isoproterenol (I0 pM) Glucagon (70 pM) PTH ( I 0 nM)
325 76 28 55 26
18
Adapted from Rindler et al. (1979). with permission. MDCK monolayer cultures were washed with phosphate-buffered saline. and 2 ml of Dulbecco's modified Eagle's medium containing 0. I mM isobutylmethylxanthine, an inhibitor of phosphodiesterase, was added with hormones as indicated. After 2 4 hours incubation at 37°C. 5% COz, medium was collected and boiled. Cyclic AMP in the medium was extracted, purified, and measured by the protein-binding assay of Gilman (1970).
oxytocin (Rindler er ul., 1979). Cyclic AMP enhances hemicyst formation in MDCK cells (Rindler et ul., 1979), which suggests that these hormone receptors are functionally coupled and that the cell line retains the differentiated characteristics of the kidney epithelial cell of origin. 3. FACTORSAFFECTING DIFFERENTIATION The selective loss of glucagon receptors following viral transformation of MDCK cells provides a model system for studying factors regulating differentiated functions. The loss of glucagon responsiveness (Table IV) is accompanied by a loss of glucagon receptors (Lin et af., I98 1, 1982). Various agents have been shown to restore glucagon receptors and responsiveness to transformed MDCK cells (Table V), including butyrate, PGE, and Ro20-1724 [4-(3butoxy-4-methoxybenzyl)-2-imidazolidinone], a potent phosphodiesterase inhibitor (Lin et al., 1981, 1982). This induction of glucagon receptors can be completely prevented by tunicamycin (an inhibitor of glycosylation) and cycloheximide, suggesting that the induction process involves the de n o w synthesis of glucagon receptors. Although the restored glucagon receptors are coupled to adenylate cyclase, the restored system seems defective in the coupling process. The concentration of glucagon required for the half-maximal cyclic AMP response is higher in the induced cells, although the isoproterenol dose-response curve is not shifted (Lin et al., 1981, 1982). The induction of glucagon receptors in transformed MDCK cells by PGE, and a phosphodiesterase inhibitor suggests cyclic AMP involvement in the induction
305
INDUCTION OF HORMONE RESPONSIVENESS TABLE IV HORMONE RESPONSIVENESS OF MDCK CELLS" Intracellular cyclic AMP (pmoleil06 cellsi3 minutes)
+ Glucagon + Vasopressin + Cell type
No addition
Parental Transformed Transformed and butyrate-treated
21.2 9.0 8.6
(2 p M ) 127
(6)
9.2 (I) 34.5 (4)
(2
W)
170 (8) 26 (3) 33 (4)
Isoproterenol
+
PGE
W)
(1
M)
(1
318 (15) 144 (16) 87 (10)
616 (29) 331 (37) 85 (10)
" Parental, transformed, and butyrate-treated (4 mM butyrate for 3 days in culture) MDCK cells were washed once with PBS. Dulbecco's modified Eagle's medium (2 ml) containing 20 phf Ro20-1724, an inhibitor of phosphodiesterase, and 20 mM Hepes buffer, pH 7.4. was added for 30 minutes at 25°C. Aliquots (20 PI) of hormone stocks were added to achieve a final concentration as indicated for 3 minutes, medium was removed, and boiling water was added to terminate the reaction and to extract intracellular CAMP. After scraping and boiling for 5 minutes, the concentration of cAMP in the supernatant was measured by a radioimmunoassay. Each value represents the average of triplicate determinations. Values in parentheses represent fold activation by hormone, which is the ratio of activity obtained with hormone to that without hormone. Onefold activation represents the basal activity.
I N D U C T I O N OF
TABLE V GLUCAGON RESPONSIVENESS I N TRANSFORMED MDCK CELLS UNDER DEFINED CONDITIONS" Intracellular cAMP (pmolei 106 cells/3 minutes)
Addition to culture medium
Basal
None Prostaglandin E l (0.I @) Prostaglandin Fza (0. I pM) Ro20-1724 (20 W ) Fetal bovine serum (10%)
8.4 12.3 8.4
~
~~~
7.4 11.3
+
Glucagon 11.1 64.5 16.0 39.6 13.6
Glucagonibasal 1.3
5.2 I.9 5.4
I .2 ~
~~
~
('Transformed MDCK cells were plated in Dulbecco's modified Eagle's medium containing insulin (5 pgiml), transferrin (5 pgiml), triiodothyronine ( 5 pM), hydrocortisone (50 nM). selenium oxide (10 nM). and Hepes buffer (10 mM), pH 7.4. On the second day cells were fed media with additions as indicated. After one more change of media on the fourth day, hormone sensitivity was tested on the seventh day. Production of intracellular cAMP was determined as described in Table IV. Each value represents the average of triplicate determinations.
306
MICHAEL C. LIN AND SUZANNE K. BECKNER
process. However, glucagon responsiveness could not be induced by cholera toxin and, in fact, induction seems to be inhibited by high concentrations of cyclic AMP (Beckner and Lin, 1982). Lever (1979) has shown that blister formation, clearly a differentiated function, can be accelerated in normal MDCK cells by a variety of agents, including polar compounds (dimethyl sulfoxide), butyrate, adenosine, PGE, , and agents which elevate intracellular cyclic AMP. All of these effects were blocked by inhibitors of protein and RNA but not DNA synthesis. Roy et al. (1980) found that a clone derived from a cell line of normal pig kidney epithelial cells had only 5% of the vasopressin receptors as the parental line. Treatment of these cells with insulin or fetal bovine serum increased the number of vasopressin receptors, which were coupled to adenylate cyclase. Although the effects of insulin and serum were additive, the mechanism of induction is unclear, as there was no induction of vasopressin receptors with butyrate or cyclic AMP. REMARKS 4. CONCLUDING MDCK cells represent an excellent system to study differentiated functions of the kidney, such as fluid transport and its regulation by hormones. In addition, the finding that glucagon receptors and responsiveness are selectively lost following viral transformation permits this system to serve as a model for the study of factors affecting differentiation. The interesting finding that transformed MDCK cells have a defect in arachidonic acid metabolism suggests that prostaglandins may serve as local developmental hormones in the regulation of this differentiated function (glucagon receptors). The fact that PGE, induces glucagon responsiveness supports this idea. But the situation is obviously more complicated. Preliminary experiments indicate that butyrate, the most potent inducer of glucagon receptors, has no effect on prostaglandin production. In addition, the effect of cyclic AMP on the induction remains to be defined, as does the mechanism of action of Ro 20-1724, an inhibitor of phosphodiesterase. Glucagon receptors cannot be induced with other phosphodiesterase inhibitors, at concentrations which inhibit phosphodiesterase. Similarly, adenosine was ineffective in inducing glucagon responsiveness. Although the induction of glucagon receptors seems to involve de novo protein synthesis, the factors regulating this induction are poorly defined and undoubtedly complex. The data suggest that prostaglandins may play some role in regulating differentiated functions, but it is not clear if prostaglandins are responsible for maintaining the differentiated state or actually induce it. Similarly undefined is the nature of the prostaglandins involved and whether endogenous and exogenous prostaglandins have equivalent effects.
INDUCTION OF HORMONE RESPONSIVENESS
111.
307
CONCLUSION
A. General Requirement for Induction of Hormone Receptors In general, the cellular environment favorable for differentiation promotes the appearance of hormone receptors and responsiveness. The factors which facilitate cellular development include various chemical inducers of differentiation, such as butyrate and dimethyl sulfoxide; metabolic inhibitors, such as inhibitors for phosphodiesterase and protein synthesis; and naturally occurring hormones, such as insulin, hydrocortisone, other peptide hormones, and prostaglandins. While growth arrest has been thought to precede differentiation, in many cases hormone sensitivity evolves in the presence of continuous growth of cultured cells. Therefore, growth and differentiation do not seem to be mutually exclusive. In all the studies reviewed, the changes in hormone responsiveness appear to reflect changes at the level of hormone receptors, as no consistent alterations in the catalytic and nucleotide regulatory components of the adenylate cyclase system have been observed during the induction of hormone sensitivity. Genetic and developmental analyses suggest that expression of each component of the adenylate cyclase system is governed by an independent regulatory process (Ross and Gilman, 1980). Deletion of the guanine nucleotide regulatory component has been observed in a variant of lymphoma cells (Ross and Gilman, 1980), and human erythrocytes are known to be deficient in the catalytic component (Nielsen et al., 1980). However, changes at the receptor level are more commonly seen either as a result of environmental changes or as a function of cellular development. It has been shown that he appearance of p-adrenergic receptors in rat cerebral cortex (Harden et af., 1977) is responsible for the acquisition of padrenergic responsiveness. Neonatal development leads to evolution of glucagon receptors and responsiveness in rat liver (Blazquez et al., 1976). Changes in the density of hormone receptors after chemical or viral transformation have also been shown in many studies. Therefore, the hormone receptor is the predominant component of the hormone-sensitive adenylate cyclase system subject to regulation. Judging from the model systems described above, variation in the density of hormone receptors is also the major event which occurs during differentiation of cultured cells.
B. Biochemical Mechanisms for Induction The hormone sensitivity of cells depends on numerous factors. The lipid environment, particularly the chemical composition of phospholipid, affects the
308
MICHAEL C.LIN AND SUZANNE K. BECKNER
hormone responsiveness of the adenylate cyclase system (Engelhard et al., 1978). Hormones are known to induce acute desensitization of the system, a process which often involves loss of hormone receptors and which can be recovered rapidly upon withdrawal of the hormone (Su et al., 1980). Hormone responsiveness also depends on the cell cycle (Penit et al., 1977). In addition, there are physiological conditions known to regulate hormone sensitivity, such as the change of P-adrenergic receptors and response resulting from increased levels of thyroid hormone (Williams er al., 1977) or a decreased concentration of adrenocorticoids (Wolfe et al., 1976). However, these conditions do not appear to change the state of differentiation of cells, since some of these effects can be seen in isolated plasma membrane preparations. The major type of regulation of hormone sensitivity, as reviewed here, relates to the developmental process of cells. During cellular differentiation, the appearance of hormone receptors does not merely involve unmasking of cryptic receptors but seems to require de novo synthesis of receptors. The induction of glucagon receptors in MDCK cells at least involves glycosylation and protein biosynthesis, since the appearance of functional receptors is completely blocked by inhibitors for both types of activities. The requirement for protein and RNA synthesis is also implicated in the induction of P-adrenergic receptors in HeLa cells and Friend erythroleukemic cells. Therefore, the effect of butyrate most certainly is expressed at the transcriptional level. However, the detailed biochemical mechanisms responsible are unknown. There is also a lack of knowledge of the mechanism of action for other inducers. In many cases, whether the inducer acts alone or in concert with other factors remains unestablished. The number of other proteins induced in conjunction with hormone receptors is undetermined in most cases; it is unlikely that the inducers described are specific for the induction of hormone receptors. Therefore, a possible general mechanism might involve the ability of inducers to allow cells to commit to differentiation, and the appearance of hormone receptors represents just one of the events occurring during this process. Several possible mechanisms at the molecular level can be considered. Whether the site of action is at the transcriptional or translational level, the inducers most likely act indirectly, i.e., the induction is mediated by a primary effect of physical or chemical nature. The inducers could interact with genetic material directly or via a carrier as do steroid hormones. Alternatively, a chemical signal, such as cyclic AMP, could be generated in response to the inducer, which modulates subsequent activity by exerting its effect on a specific component in the biosynthetic apparatus. Cyclic nucleotide-dependent protein kinase is a potentially good candidate, although direct involvement of cyclic nucleotidedependent phosphorylation has not been shown to mediate the induction of hormone receptors in any system. Mobilization of cellular Ca2 represents yet another possible mechanism of mediating the effect of inducers. Recent studies +
INDUCTION OF HORMONE RESPONSIVENESS
309
indicate that calmodulin, which modulates numerous cellular activities, may be responsible for many Ca2 -dependent phenomena (Means and Dedman, 1980). The ability of calmodulin to activate phosphodiesterase could potentially regulate the cellular concentration of cyclic nucleotides, for example. Our major difficulty encountered in the elucidation of detailed biochemical mechanisms relates to the minute quantity of the induced hormone receptors. A true understanding of the mechanism of action for various inducers would be forthcoming only when highly sensitive tools become available. +
C. Biological Significance The orderly appearance of differentiated characteristics requires precise programming or regulation. The elaboration of hormone receptors and responsiveness represents one of numerous functions that are scheduled to evolve during development. Understanding factors that regulate the appearance of hormone receptors undoubtedly would provide insight into cellular development. In erythroleukemic cells, the observation that the appearance of P-receptors precedes the induction of other functions suggests possible sequential relationships between various differentiated characteristics (Lin and Lin, 1979); whether Preceptors and response regulate subsequent differentiation of stem cells is an intriguing question. The synergistic effect of (3-agonists and erythropoietin on the maturation of bone marrow cells (Brown and Adamson, 1977) supports the significance of the early appearance of P-receptors in erythroleukemic cells. In MDCK cells, prostaglandins seems to play a unique role in the development of hormone sensitivity. The observation that normal MDCK cells produce prostaglandins and that prostaglandins induce glucagon sensitivity in a transformed cell line which cannot produce prostaglandins suggests a causal relationship between the local production of prostaglandins and the subsequent development of glucagon responsiveness. This example of a locally produced developmental hormone which promotes cellular differentiation may turn out to be more common than has been documented in the literature. The decreased sensitivity of 3T3-L 1 adipocytes to prostacyclin during differentiation supports this idea. Most cells produce prostaglandins of various types. The role of prostaglandins in the modulation of both differentiation and transformation is becoming increasingly appreciated. Since cellular functions operate in a coordinated fashion, the appearance of differentiated characteristics needs to be sequentially organized. Needless to say, the evolution of a cellular function has to precede or coincide with the needs of the cell during development. For example, in rat liver, receptors for P-adrenergic hormones are well developed during the fetal stage (Lin, unpublished observation); by contrast, the same receptors in cerebral cortex are not fully elaborated
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until after birth (Perkins and Moore, 1973). These observations may be related to the temporal requirement of P-adrenergic regulation in both tissues during development. In the systems reviewed above, the evolution of hormone receptors is often governed by other hormones. This type of regulation obviously requires that the appearance of developmental hormone receptors precedes the second type of receptor. For example, follicle-stimulating hormone is needed for appearance of lutenizing hormone receptors in granulosa cells, insulin governs the evolution of receptors for adrenocorticotropin in 3T3-L I cells, and prostaglandins are essential for subsequent elaboration of glucagon receptors in MDCK cells. Therefore, by studying the evolution of hormone receptors in cultured cells during their development, further insight into the process of differentiation can be gained. New tools-for example, antireceptor antibody for detection of hormone receptors-are urgently needed to allow further understanding of the regulation of hormone receptors during development. REFERENCES Albertini, D. F., and Anderson, E. (1974). The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap functions. J. Cell Biol. 63, 234-250. Amsterdam, A., Knecht, M., and Catt, K. G. (1981).Hormonal regulation of cytodifferentiation and intercellular communication in cultured granulosa cells. Proc. Nufl. Acud. Sci. U.S.A. 78, 3000-3004. Ascoli, M. (1981). Regulation of gonadotropin receptors and gonadotropin responses in a clonal strain of Leydig tumor cells by epidermal growth factor. J. Biol. Chem. 256, 179-183. Bauscher, J . , and Schaeffer, W. I. (1974). A diploid rat liver cell culture: Characterization and sensitivity to Aflatoxin B,. In Vitro 9, 286-293. Beckner, S . K.. and Lin, M. C. (1982). Role of cyclic AMP in the induction of glucagon responsiveness in canine kidney cells. J. Cell. Biochem. Suppl. 6 , 184. Beckner. S. K.. Reilly, T., Martinez, A., and Blecher, M. (1980). Alterations of CAMPmetabolism and hormone responsiveness of cloned differentiated rat liver cells (RL-PR-C) upon spontaneous transformation. Exp. Cell Res. 128, 151-158. Birnbaumer, L. ( 1973). Hormone-sensitive adenylyl cyclases. Useful models for studying hormone receptor functions in cell-free systems. Biochim. Biophys. Actu 300, 129- 158. Blazquez, E., Rubalcava, B., Montesano, R.. Orci, L., and Unger, R. (1976). Development of insulin and glucagon binding and the adenylate cyclase response in liver membranes of the prenatal, postnatal and adult rat: Evidence of glucagon resistance. Endocrinology 98, 10 14- 1023. Brown, J. E., and Adamson, J. W. (1977). Modulation of in vitro erythropoiesis. J . Clin. Invest. 60, 70-77. Chang, T.-H.,and Polakis, S. E. (1978). Differentiation of 3T3-Ll fibroblasts to adipocytes. J. Biol. Chem. 253, 4693-4696. Chou, J . Y . , and Schlegel-Haueter, S. E. (1981). Study of liver differentiation in vitro. J. CellEiol. 89, 216-222. Clayton, R. N . , Hanvood, J. P.. and Catt, K. J. (1979). Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production. Nature (London) 282, 90-92. de Nechaud, B., Becker, J . E., and Potter, V. A. (1976). Effect of glucocorticoids on fetoprotein
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production by an established cell line from Morris hepatoma 8994. Biochem. Biophvs. Res. Commun. 68, 8-15. Dickson. A. J . , and Pogson, C. I. (1977). The metabolic integrity of hepatocytes in sustained incubations. FEES Leu. 83, 27-32. Engelhard, V. H., Glaser, M., and Storm, D. R. (1978). Effect of membrane phospholipid compositional changes on adenylate cyclase in LM cells. Biochemistry 17, 3191-3200. Erickson, G. F., Wang, C., and Hseuh, A. J. (1979). FSH induction of functional LH receptors in granulosa cells cultured in a chemically defined medium. Nature (London) 279, 336-338. Flower, R. J., and Blackwell, G . J. (1979). Anti-inflammatory steroids induce biosynthesis of a phospholipase AZ inhibitor which prevents prostaglandin generation. Nature (London) 278, 456-459. Freytag, S. O., and Utter, M. F. (1980). Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-Ll cells during differentiation. Proc. Narl. Acad. Sci. U.S.A. 77, 1321-1325. Friend, C., Scher, W., Holland, J. G., and Sato. T. (1971). Hemoglobin synthesis in murine virusinduced leukemic cells in vitrot Stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natl. Acad. Sci. U.S.A. 68, 378-382. Fukuda, M., Koeffler, H. P., and Minowada, J. (1981). Membrane differentiation of human myeloid cells: Expression of unique profiles of cell surface glycoproteins in myeloid leukemic cell lines blocked at different stages of differentiation and maturation. Proc. Narl. Acad. Sci. U.S.A. 78, 6299-6303. Gaush, C. R., Hard, W. L., and Smith, T. F. (1966). Characterization of an established line of canine kidney cells (MDCK). Proc. Soc. Exp. Biol. Med. 122, 931-935. Gey, G. 0..Coffman, W. D.,and Kubicek, M. T. (1952). Tissue culture studies on the proliferative capacity of cervical carcinoma and normal epithelium. Cancer Res. 12, 264-265. Ghosh, N. K., and Cox, R. P. (1976). Production of human chorionic gonadotropin in HeLa cell cultures. Nature (London) 259, 416-417. Ghosh, N. K., and Cox, R. P. (1977). Induction of human follicle-stimulating hormone in HeLa cells by sodium butyrate. Nature (London) 267, 435-437. Gilman, A. G. ( 1970). A protein binding assay for adenosine 3':5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67, 305-312. Ginsburg. E., Salomon, D., Sreevalsan, T., and Freese, E. (1973). Growth inhibition and morphological changes caused by lipophilic acids in mammalian cells. Proc. Narl. Acad. Sci. U.S.A. 70, 2457-2461. Green, H., and Kehinde, 0. (1975). An established preadipose cell line and its differentiation in culture 11. Factors affecting the adipose conversion. Cell 5, 19-27. Green, H.,and Meuth, M. (1974). An established pre-adipose cell line and its differentiation in culture. Cell 3, 127-133. Harden, T. K., Wolfe, B. B., Sporn, J. R., Perkins, J. P., and Molinoff, P. B. (1977). Ontogeny of P-adrenergic receptors in rat cerebral cortex. Brain Res. 125, 99-108. Hopkins, N. K., and Gorman, R. R. (1981). Regulation of 3T3-LI fibroblasts differentiation by prostacyclin (prostaglandin 12). Biochim. Biophys. Acta 663, 457-466. Hsueh, A. J. W., and Erickson, G . F. (1979). Extrapituitary action of gonadotropin-releasing hormone: Direct inhibition of ovarian steroidogenesis. Science 204, 854-855. Hsueh, A. 1. W., Wang, C., and Erickson, G . F. (1980). Direct inhibitory effect of gonadotropinreleasing hormone upon follicle-stimulating hormone induction of lutenizing hormone receptor and aromatase activity in rat granulosa cells. Endocrinology 106, 1697- 1704. Karlsson, F. A., Grunfeld, C., Kahn. C. R., and Roth, J. (1979). Regulation of insulin receptors and insulin responsiveness in 3T3-L I fatty fibroblasts. Endocrino1o;ey 104, 1383- 1392. Katz, A. I., and Lindheimer, M. D. (1977). Actions of hormones on the kidney. Annu. Rev. Physiof. 39, 97-134.
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Rodbell, M. (1980). The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature (London) 284, 17-22. Rosen, 0. M., Smith, C. J.. Fung, C . , and Rubin, C. S . (1978). Development of hormone receptors and hormone responsiveness in virro. J . Biol. Chem. 253, 7579-7583. Ross, E. M., and Gilman, A. 0. (1980). Biochemical properties of hormone-sensitive adenylate cyclase. Annu. Rev. Biochem. 49, 533-564. Ross. J . . Ikawa, Y.. and Leder, P. (1972). Globin messenger-RNA induction during erythroid differentiation of cultured leukemic cells. Proc. Natl. Acud. Sei. U.S.A. 69, 3620-3623. Roy, C.. Preston, A. S . , and Handler, J. S. (1980). Insulin and serum increase the number of receptors for vasopressin in a kidney-derived line of cells grown in a defined medium. Proc. Nut/. Acad. Sci. U . S . A . 77, 5979-5983. Rubin, C. S., Lai, E., and Rosen, 0. M. (1977).Acquisition of increased hormone sensitivity during in virro adipocyte development. J . Biol. Chem. 252, 3554-3557. Rubin, C. S., Hirsch. A., Fung, C., and Rosen, 0. M. (1978). Development of hormone receptors and hormonal responsiveness in vitro. J. Biol. Chem. 253, 7570-7578. Russell, T. R., and Ho. R.-J. (1976). Conversion of 3T3 fibroblasts into adipose cells: Triggering of differentiation by prostaglandin Fzu and I-methyl-3-isobutyl xanthine. Proc. Narl. Acad. Sci. U.S.A. 73, 4516-4520. Rutter, W. J., Pictet, R. L., and Morris. P. W. (1973). Toward molecular mechanisms of developmental processes. Annu. Rev. Biochem. 42, 601-646. Salomon, Y. (1979). Adenylate cyclase assay. Adv. Cvcic Nucleotide Res. 10, 35-55. Schaeffer, W. I.. and Polifka. M. D. (1975). A diploid rat liver cell culture. Characterization of the heteroploid morphological variants which develop with time in culture. Exp. Cell Res. 95, 167- 175. Schlegel-Haueter, S. E., Schlegel. W.. and Chou, 1. Y. (1980). Establishment of a fetal rat liver cell line that retains differentiated liver functions. Proc. Natl. Acad. Sci. U.S.A. 77, 273 1-2734. Serrero, G. R., McClure, D. B., and Sato, G. H. (1979). Growth of mouse 3T3 fibroblasts in serumfree. hormone-supplemented media. In "Hormones and Cell Culture" (G. H. Sato and R. Ross, eds.), pp. 523-530. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Shih. T. Y., Papageorge, A. G.. Stokes, P. E., Weeks. M. 0..and Scolnick, E. M. (1980). Guanine nucleotide-binding and autophosphorylating activities associated with the p2Isrc protein of Harvey murine sarcoma virus. Narure (London) 287, 686-691. Sicard, R. W., and Aprille. J. R . (1977). Adenylate cyclase and cyclic nucleotide phosphodiesterases in the developing rat liver. Biochim. Biophys. Acta 500, 235-245. Simmons. J . L., Fishman, P. H., Freese, E., and Brady, R. 0. (1975). Morphological alterations and gdnglioside sialyltransferase activity induced by small fatty acids in HeLa cells. J . Cell Biol. 66, 414-424. Smith. C. J . . Wejksnora, P. J.. Warner, J. R.. Rubin, C. S., and Rosen, 0. M. (1979). Insulinstimulated protein phosphorylation in 3T3-L I preadipocytes. Proc. Narl. Acad. Sci. U . S . A . 76, 2725-2729. Spiegelman, B. M., and Green, H. (1981). Cyclic AMP-mediated control of lipogenic enzyme synthesis during adipose differentiation of 3T3 cells. Cell 24, 503-510. Su. Y. F.. Harden. T. K . , and Perkins, J. P. (1980). Catecholamine-specific desensitization of adenylate cyclase. J . Biol. Chem. 255, 7410-7419. Tallman, J. F., Smith, C. C., and Henneberry, R. C. (1977). Induction of functional P-adrenergic receptors in HeLa cells. Proc. Narl. Acud. Sci. U.S.A. 74, 873-877. Tanaka, M., Levy. J., Terada, M., Breslow. R., Rifkind, R. A., and Marks, P. A. (1975). Induction of erythroid differentiation in murine virus infected erythroleukemic cells by highly polar compounds. Proc. Null. Acad. Sci. U.S.A. 72, 1003-1006. Taub. M.. Chuman, L.. Saier, M. H., Jr.. and Sato. G. (1979). Growth of Madin-Darby canine
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I X
Receptors for Lysosomal Enzymes and GIyco proteins VIRGINIA SHEPHERD, PAUL SCHLESINGER, AND PHILIP STAHL Deportment of Phvsiologv und Biophysics Wushington University School o j Medicine St. Louis. Missouri
Introduction, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... The Phosphomannosyl Recognition Pathway . . . . . . . ...................... A . Biosynthesis of the Mannose 6-Phosphate Recognition Marker. . . . . . . . . . . . . . . B. Transfer of Newly Synthesized Hydrolases to Lysosom C. Lysosomal Enzyme Packaging through an Alternate Pa 111. Role of Oligosaccharide Moiety in Recognition of Extracehlar Lysosomal Enzymes and Glycoproteins . ................................. IV. Lysosomal Enzymes and the Mannosyl Recognition System. .................... A. Mannosyl Recognition and Macrophages ..................... 8 . Mannose-Binding Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Receptor-Mediated Endocytosis of Glycoconjugates ........................... A. Receptor Recycling and Weak Bases.. . . . . . . . . . . . . . . . . . . . . . . . B. Cycling and Recycling Receptors . .................................. VI. Conclusion. . . . . ........................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.
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INTRODUCTION
Since the discovery of lysosomes almost three decades ago, considerable progress has been made toward elucidating their biochemistry and physiology (Bainton, 1981). It is now widely accepted that lysosomes are found in virtually all eukaryotic cells, where they function principally in the disposal of macromolecules derived from uptake or from the turnover of cellular structures. The enzymes found in lysosomes are held sequestered within a semipermeable membrane presumably designed in such a way as to be immune to the hydrolytic events taking place within, while at the same time allowing the low-molecular317
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weight hydrolytic products generated by the action of the hydrolases to diffuse through the membrane. The products, which make their way into the cytoplasmic compartment, serve as substrates for energy metabolism or for biosynthesis. Lysosomal enzymes which catalyze these hydrolytic reactions operate almost invariably at acid pH. Moreover, they are glycosylated. The low pH found within lysosomes appears to be driven by an ATP-dependent proton pump (Schneider, 1981). The glycosylation may play a dual role: it may serve a protective role (glycosylated macromolecules may be more resistant to denaturation), or second, and more relevant to this article, it may provide recognition signals required for the transport of both newly synthesized and preexisting lysosomal enzymes within and between cells. The intracellular transport of lysosomal enzymes and the biogenesis of lysosomes are subjects of considerable contemporary interest. Some years ago, Novikoff and colleagues (see Novikoff et al., 1981) identified a region within the cell, adjacent to the endoplasmic reticulum, which stained for acid phosphatase and certain Golgi apparatus markers. They referred to this area as Golgi-endoplasmic reticulum-lysosomes (GERL) and reasoned that primary lysosomes were probably formed in this region. Attention slowly drifted away from this concept, but recent studies on the processing of newly synthesized lysosomal enzymes have prompted renewed interest in the GERL. In fact, it is now generally accepted that membranes in the Golgi region are intimately involved in the transfer of lysosomal enzymes to the lysosomal system. A second line of experimentation which initially offered considerable promise as a model for lysosome biogenesis derived from studies with the enzyme P-glucuronidase. This lysosoma1 enzyme has a dual distribution in rodent liver, with about half of the enzyme activity associated with endoplasmic reticulum (Owens et al., 1975). Initially, it was thought that the microsomal enzyme might be the direct precursor of lysosoma1 P-glucuronidase. The enzymes these two locations are the same gene product and have similar chemical and physical properties. Moreover, a protein called egasyn was discovered which bound mouse P-glucuronidase to microsoma1 membranes (Lugis and Paigen, 1977). In the rat, it would appear that the microsomal P-glucuronidase is an enzyme destined for secretion into the extracellular space and is not directly transferred to lysosomes. In fact, the secretion of the microsomal enzyme can be accelerated by injection of organophosphate compounds into rats or mice (Mandell and Stahl, 1977). Just what physiological function the rnicrosomal enzyme may play or why its secretion is prompted by poisoning with organophosphate compounds is unclear. The current concepts of lysosomal biogenesis have been strongly influenced by studies on lysosomal storage diseases and I-cell disease. The latter, in particular, is a mutation initially noted by Leroy et af. (1972) which affects the transport and packaging of a large number of lysosomal enzymes. Studies on storagedisease cells and I-cell disease have provided support for the concept of receptormediated transfer of newly synthesized lysosomal hydrolases to lysosomes
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via recognition signals attached to the enzymes. These recognition signals are carbohydrate in nature. There are at least two recognition signals (and, correspondingly, two cellular receptors) found on lysosomal enzymes which mediate transfer to lysosomes: ( 1) mannose 6-phosphate-terminated oligosaccharides and (2) mannose-terminated oligosaccharides (Sly and Stahl, 1978).
II. THE PHOSPHOMANNOSYL RECOGNITION PATHWAY The mechanism of lysosomal enzyme biosynthesis and transport has been extensively studied in fibroblasts by several laboratories. Based on early work by Neufeld and co-workers (Hickman and Neufeld, 1972) and later by Kaplan et al. (1977) and Sando and Neufeld (1977), the packaging within lysosomes of newly synthesized hydrolases is now known to involve specific receptor-mediated recognition of mannose 6-phosphate residues on the enzyme. The development of this concept was due, in part, to the initial observation that cultured I-cell fibroblasts secrete large quantities of enzymes into the medium, and that intracellular levels of hydrolases in these cells are low. This led to the proposal that a specific recognition marker was normally present on newly synthesized enzyme and that it was missing in I-cell disease. This notion was supported by the finding that various enzymes secreted by normal cells were taken up by 1 cells, suggesting that the defect was in the enzyme structure and not in the packaging mechanism. Hickman and Neufeld (1972) went on to propose that the route of normal enzyme synthesis and packaging involved secretion of the newly synthesized hydrolases into the extracellular fluid, followed by uptake into the cell via specific cell surface receptors and subsequent transport to the lysosome-the ‘ ‘secretion-recapture hypothesis” (Neufeld et al., 1977). The structure of the recognition marker was shown to involve carbohydrate residues, specifically mannose. Kaplan et al. (1977) determined that mannose 6phosphate was the best inhibitor of uptake, and that phosphatase treatment reduced uptake of spleen p-glucuronidase by fibroblasts. These studies were extended to other lysosomal enzymes, and mannose 6-phosphate has subsequently been demonstrated chemically to be a component of the oligosaccharide structure of purified spleen P-glucuronidase (Natowicz et al., 1979) and bovine P-galactosidase (Distler et a l . , 1979).
A. Biosynthesis of the Mannose 6-Phosphate Recognition Marker Since these early kinetic studies showing that mannose 6-phosphate is a competitive inhibitor of enzyme uptake, considerable interest has been focused on deducing the pathway of biosynthesis of the oligosaccharide chains. Lysosomal enzymes contain carbohydrate units with several mannose residues (high man-
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nose oligosaccharides). The mechanism of transfer to the protein chain and processing of these units has been extensively studied in a number of laboratories and is diagrammed in Fig. 1. The core structure of the complex chain is synthesized on a lipid intermediate and transferred as a precursor unit to the nascent glycoprotein in the endoplasmic reticulum. As the glycoprotein progresses through the endoplasmic reticulum and Golgi apparatus, external glucose residues are removed. Tabas and Kornfeld (1980) have recently proposed a scheme for addition of phosphate to the mannose chain, as shown in Fig. 1. Following removal of the two outermost glucose residues, GlcNAc-phosphate is enzymatically transferred from UDPGlcNAc to one or more mannose units resulting in a phosphodiester-linked GlcNAc. At some point in the transport of these glycoproteins (presumably in the Golgi) the blocking group (i.e., GlcNAc) is removed, resulting in an exposed mannose 6-phosphate residue. Later, perhaps before the enzyme reaches the lysosomal compartment, the phosphate groups are removed. The enzymes which add the phosphate group and remove the blocking residue have been recently isolated. Varki and Kornfeld (1981) reported the characterization of a-N-acetylglucosaminylphosphodiesterasefrom rat liver. Based on its activity toward phosphorylated high-mannose oligosaccharides and its subcellular localization in the GolgiIGERL region, they concluded that this was the enzyme responsible for the exposure of the phosphomannosyl recognition marker. Reitman and Kornfeld (1981a) have purified an enzyme which transfers N-acetylglucosamine 1-phosphate from UDPGlcNAc to the 6-hydroxyl s
6
P
Glc N Ac 0 Manrose
.Glucose Galactose *Sialic Acid
-
I . Intracellular processing of high-mannose oligosaccharides and the targeting of enzymes to lysosomes by the transfer of phosphate. (After Tabas and Kornfeld, 1980.) FIG.
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of mannose residues in high-mannose units, supporting the hypothesis that the phosphate group is acquired via this transfer mechanism. Concurrently, Waheed et al. (1981) studied the subcellular localization of both enzymes in rat liver. Both activities were found in the Golgi fraction, suggesting as above that synthesis of the mannose 6-phosphate recognition marker occurs prior to lysosomal packaging. One question which remains to be answered is how the cell determines which glycoproteins should receive the phosphate groups for subsequent routing to the lysosome. The only glycoproteins which have been found to contain mannose 6phosphate are lysosomal hydrolases. Since other glycoproteins contain identical high-mannose oligosaccharide chains, a specific determinant must exist on the newly synthesized lysosomal enzyme to identify it for transfer of N-acetylglucosamine I -phosphate. 8. Transfer of Newly Synthesized Hydrolases to Lysosomes
Once the blocking group (i.e., GlcNAc) on the attached oligosaccharide has been removed in the Golgi, the mannose 6-phosphate residue can be recognized by a specific receptor for transport to the lysosome. Very little is known about this intracellular movement, but intracellular mannose 6-phosphate-specific receptors have been found. In fact, most of the receptors have been found within cells, with only a small fraction on the cell surface (Fischer et al., 1980). This raises the question of the exact role of this receptor at the cell surface. Neufeld et al. ( 1 977) originally proposed that newly synthesized hydrolases were routed to the plasma membrane, where they were secreted (containing the mannose 6phosphate recognition marker) and taken back into the cell via a specific receptor-mediated process. The finding that mannose 6-phosphate in the growth medium failed to reduce intracellular enzyme levels led von Figura and Weber (1978) to suggest that enzymes are not secreted and recaptured, but instead are cycled to the plasma membrane bound to receptor and then internalized without dissociation. This hypothesis was based on specific labeling of four different enzymes at the cell surface by irnmunofluorescence and the release of enzyme activity by treatment with trypsin. Fischer et al. (1980) have recently presented evidence for an alternative to the appearance of enzymes at the cell surface. Based on binding of purified hexosaminidase B to subcellular fractions of rat liver, they reported that most (90%) of the phosphomannosyl-enyzme receptors are in the endoplasmic reticulum (ER), Golgi, and lysosomes, with only 9.5% in the plasma membrane fraction. Furthermore, they found that growth of fibroblasts in the presence of mannose 6-phosphate did not reduce intracellular enzyme levels and did not affect extracellular levels. The above observations would support the
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view that these receptors function internally to route acid hydrolases from the ER to lysosomes. It has been postulated that receptor-bound hydrolases collect in vesicles which bud off from the GERL or the Golgi apparatus and deliver hydrolases to lysosomes. Once in the lysosomes, the receptor-enzyme complex dissociates, leaving the enzyme packaged in the lysosome and the receptor free to recycle back to the ER. Equally possible is that the receptor never reaches the lysosome but rather recycles through acid-filled vesicles. The point at which the enzymes lose their mannose 6-phosphate recognition site is still not certain. Fischer ez al. (1980) reported that most of the endogenous hexosaminidase in isolated lysosomes from rat liver was released by detergent alone (suggesting it was vesicle enclosed, but not receptor bound), while most of the enzyme in the ER, plasma membrane, and Golgi fractions could be displaced only after addition of mannose 6-phosphate. Furthermore, the lysosomal enzyme appeared to lack the mannose 6-phosphate group necessary for high uptake, while the enzyme isolated from the ER and Golgi was phosphorylated. However, Miller er al. (198 1) have recently reported that hexosaminidase isolated from lysosomes of normal human fibroblasts is approximately 8% high-uptake form. They suggested that the phosphomannosyl recognition marker is not totally removed once the enzyme has been segregated into lysosomes. The mannose 6phosphate receptor may then also function in the lysosome to prevent release of enzymes during lysosome-plasma membrane fusion and reduce self-degradation within the lysosome.
C. Lysosomal Enzyme Packaging through an Alternate Pathway I-cell disease has been used as a model for investigating the intracellular packaging of hydrolases which do not contain a mannose 6-phosphate recognition site. Several workers have shown that acid hydrolases from I-cell fibroblasts are not phosphorylated, and consequently are secreted by the cells instead of being targeted to lysosomes (Bach et al., 1979; Hasilik and Neufeld, 1980; Tabas and Komfeld, 1980; Hasilik et al., 1981). Subsequently, it has been shown that I cells are deficient in N-acetylglucosaminylphosphotransferase(Reitman et al., 1981). However, I-cell fibroblasts are known to contain residual activities of a number of acid hydrolases. Miller et al. (1981) have recently reported that these enzymes are located predominantly in the lysosomes of 1 cells, and the oligosaccharide chains on normal and I-cell lysosomal enzymes are high-mannose units. Furthermore, at least two enzymes in I-cell fibroblastsP-D-glucosidase and acid phosphatase-are at normal levels. It is possible then that certain enzymes lacking the phosphomannosyl recognition site are packaged
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via an alternate mechanism such as the mannose receptor (discussed below) or specific sites on the peptide chain (Reitman and Kornfeld, 1981b). The possibility that some site on the peptide chain serves as a recognition marker for uptake is not out of the question, especially considering that lysosomal enzymes are specifically recognized by a phosphotransferase. Moreover, Rome and Miller (1980) have reported that modification of arginyl groups of a-iduronidase reduces the receptor-mediated uptake of this enzyme. One might expect to find a defect in receptor levels in patients Gith lysosomal storage disorders (such as I-cell disease), but this has not been found. Robbins and Myerowitz ( 1981) have isolated receptor-deficient mutants which have altered localization of lysosomal enzymes. As in I-cell disease, intracellular levels of enzymes were low, but in this case the enzymes synthesized by the mutants contained the mannose 6-phosphate recognition site and the cells lacked the proper receptor. Again, as in I cells, some residual activities were found in lysosomes. Although it cannot be ruled out that the presence of small amounts of mannose 6-phosphate receptors was responsible for the intralysosomal packaging, it is interesting to postulate that a second mechanism directs delivery of the enzymes.
111. ROLE OF OLIGOSACCHARIDE MOIETY IN RECOGNITION OF EXTRACELLULAR LYSOSOMAL ENZYMES AND GLYCOPROTEINS Most plasma glycoproteins have complex oligosaccharides terminated in sialic acid. The presence of sialic acid as the terminal sugar on the oligosaccharide chain protects plasma glycoproteins from being recognized in vivo. This conclusion derives from the classic work of Ashwell and Morel1 (1974) who demonstrated that following intravenous infusion, asialoglycoproteins are rapidly cleared from the plasma compartment via a receptor associated with liver hepatocytes. The receptor which mediates uptake into liver hepatocytes has been isolated by affinity chromatography on asialoorosomucoid-Sepharose.The receptor is a glycoprotein and requires Ca2+ for optimal binding. Subcellular fractionation experiments indicate that the bulk of the binding activity occurs on intracellular membranes. Some of this intracellular binding activity may be receptors en route to the plasma membrane, although it is not clear whether most of these intracellular receptors are involved in receptor-mediated pinocytosis. It is possible that they play some other intracellular transport function in hepatocytes. As will be discussed later, the galactose-specific pinocytosis receptor appears to be internalized with its ligand bound. However, whereas the ligand is transported to lysosomes, the receptor is recycled. The physiologic role of the hepatocyte
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galactose-specific receptor is still unresolved. It appears unlikely to play a significant role in lysosome biogenesis since it is liver specific.
IV. LYSOSOMAL ENZYMES AND THE MANNOSYL RECOGNITION SYSTEM Intravenous infusion of purified lysosomal glycosidases into the anesthetized animal results in rapid clearance of the infused activity (Stahl et al., 1976). Clearance is rapid and dependent on the presence of an intact oligosaccharide chain, and can be blocked by simultaneous administration of competing doses of mannose-, N-acetylglucosamine-, or fucose-terminated glycoproteins (Shepherd et al., 1981). Thus, this clearance pathway has been referred to as both the mannose/N-acetylglucosamine-specificpathway or as the mannose/fucose-specific pathway. Unlike galactose-terminated glycoproteins, mannoselN-acetylglucosamineterminated glycoproteins and most lysosomal hydrolases are cleared from the plasma via the spleen and nonparenchymal cells of liver, particularly Kupffer cells and hepatic endothelial cells (Schlesinger et al., 1978; Hubbard and Stukenbrok, 1979). In the absence of liver and spleen, the bone marrow-mediated clearance of mannose-terminated glycoproteins becomes exaggerated, suggesting that the mannose receptor is a feature of the reticuloendothelial system (Schlesinger et al., 1980). This notion has been confirmed in studies on isolated macrophages (Stahl and Gordon, 1982).
A. Mannosyl Recognition and Macrophages The presence of mannose receptor activity in macrophages was first unequivocally demonstrated in pulmonary lavage macrophages (Stahl et al., 1978). Alveolar macrophages rapidly take up 1251-labeledP-glucuronidase (rat preputial). Uptake is saturable (Kuptake= lo-' M ) and linear with time over an extended period and fully inhibited by yeast mannan. The sugar specificity of the macrophage receptor system has been disclosed by two types of studies. First, ligands terminating in mannose (e.g., ovalbumin, P-glucuronidase, horseradish peroxidase) (Stahl et al., 1978; Kaplan and Nielsen, 1978), N-acetylglucosamine (agalactoorosomucoid) (Stahl et al., 1978), and L-fucose (L-fucose-BSA) (Shepherd er al., 1981) are taken up by macrophages via a receptor-mediated process. Second, inhibition of I2Tlabeled P-glucuronidase uptake by synthetic glycoconjugates (Shepherd et al., 1981) (Fig. 2) shows the sugar specificity of the recognition mechanism. The nearly equal potency of L-fucose and o-mannose can be explained on the basis of the similarity of the orientation around C-2,3,4 in D-mannose as compared with C-4,3,2 in L-fucose (when L-fucose is flipped 180"
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lOOr 90 -
80
-
70-
5
60-
c .-
n
2 50c -
g 4030 20-
10-
p g added
FIG. 2 . Inhibition of IZSI-labeled P-glucuronidase uptake into macrophages by various neoglycoproteins. Cells ( 5 X 106/ml) in buffered culture media were incubated with 2.5 pg of 1251labeled P-glucuronidase and increasing concentrations of neoglycoproteins for 10 minutes at 37°C. The uptake of ligdnd was terminated by centrifugation through oil. The extent of substitution of the neoglycoprotein is indicated by the subscript. (From Shepherd ef al., 1981.)
C-4,3,2 is very similar to C-2,3,4 of D-mannose). Glucose and N-acetylglucosamine, 2-epimers of D-mannose, are only 10% as active. D-Galactose, a 4-epimer of D-mannose, is inactive. Recent studies by Maynard and Baenziger (1981) and Holt and Stahl (unpublished) indicate that all the information necessary for recognition and uptake of mannose-terminated glycoproteins resides in the oligosaccharide moiety. The mannose receptor has been found in a variety of macrophages. Freshly harvested peripheral blood monocytes are negative. Following several days in culture the macrophage phenotype becomes evident as the monocyte differentiates. At this point, they become highly positive for mannose receptor activity (Shepherd et al., 1982). Similarly, bone marrow cells are negative for mannose receptor activity but their incubation with colony-stimulating factor (CSF- 1) stimulates expression of mannose receptor activity (Konish et a l . , 1982). Most macrophage-like cell lines have been found to be mannose receptor negative. Recently, it has been possible to prepare macrophage hybrids which both divide in culture and express the mannose receptor on their surfaces. In these experiments, a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mutant of the macrophage-like tumor line 5774 (which is negative for mannose-specific recognition and uptake) was fused with a receptor-positive macrophage (alveolar
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macrophages). Subsequent growth and cloning of these macrophage/macrophage hybrids produced stable receptor - and receptor + clones. Fusion of a nonmacrophage cell line (e.g., fibroblast) with a primary macrophage caused extinction of the expression of the mannose receptor. Apparently the tumor macrophage remembered its heritage and permitted expression of this apparent macrophage-specific trait (Stahl and Gordon, 1982). Thus, the mannose receptor expression on the surface of macrophages is selective and appears to be modulated.
6. Mannose-Binding Protein Kawasaki et al. (1978) purified a mannose-binding protein from rabbit liver acetone powder and from rat lymph node (Kawasaki et al., 1980) by affinity chromatography on mannan-Sepharose. Townsend and Stahl ( I 98 1) isolated a similar protein from rat liver. Initially, it was thought that the mannose-binding protein and the mannose-specific pinocytosis receptor were probably the same protein. The sugar specificity of the receptor and the binding protein were quite similar and both required calcium for maximal activity. Antibodies against the binding protein have been found to block uptake of mannose-terminated glycoproteins into macrophages. Moreover, the antibody directed against the binding protein binds to fixed macrophages as detected by a fluorescein-labeled second antibody. It would appear that the binding protein and the receptor share some antigenic determinants (unpublished observations). However, to complicate matters further, mannose-binding protein has been found in plasmdserum (Kozutsumi et al., 1980), in hepatocytes (Maynard and Baenziger, 1982), in fibroblasts (Shepherd et al., in preparation; Strauss, 1981), in lymph node (Kawaski er al., 1980), and in lung (J. Powell, personal communication). These findings indicate that the relationship between the binding protein and the receptor is not a sirnple one. A possible intracellular role for the mannose-binding protein/mannose receptor in nonmacrophages emerges when one considers lysosomal biogenesis in I-cell disease. The I-cell mutation appears to produce a defect or deficiency in the enzyme which transfers N-acetylglucosamine phosphate to the high-mannose chain of a newly synthesized hydrolase. The I-cell mutation affects the transport and packaging of many hydrolases. However, the defect is selective because normal levels of certain enzymes (acid phosphatase) and nearly normal levels of many other hydrolases are found in I-cell disease. Moreover, the I-cell mutation is tissue specific+ertain tissues are normal. These data suggest alternate routes for intracellular lysosome transport; there are two reasonable possibilities. The mannose receptor is a good candidate for the intracellular receptor, especially since all lysosomal enzymes are glycoproteins and have high-mannose chains. Alternatively, some aspect of the protein structure of the lysosomal enzyme
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could be recognized by a pinocytosis receptor. Certainly, the GlcNAc phosphate transferase must recognize some aspect of the lysosomal enzyme other than the oligosaccharide chain, since only lysosomal hydrolases are phosphorylated and nonlysosomal hydrolases bearing high-mannose chains are not phosphorylated (Reitman and Kornfeld, 1981b). Perhaps the transferase, or a related protein capable of recognizing some aspect of the protein structure of a lysosomal enzyme, could serve as receptor in some cells andtor for some enzymes.
V.
RECEPTOR-MEDIATED ENDOCYTOSIS OF GLYCOCONJUGATES
Since the first description of receptor-mediated endocytosis of glycoproteins the most attractive model for a mechanism of action indicates a catalytic role for the receptor. In the systems thus far described, the following mechanism can explain the experimental observations (Fig. 3). The receptor binds the appropriate ligand at the cell surface with high affinity. The ligand is then transferred to an intracellular vesicle (i.e., the internalization step). Subsequently, the ligand is found to have moved to the lysosome and the receptor is presumed to have returned to the cell surface to participate in subsequent endocytic events. Obviously this does not represent a definitive mechanism and the details may vary with different receptors. This model is supported by considerable circumstantial evidence and has been very useful for designing further experiments. In the galactose- (hepatocyte), mannose- (macrophage), and mannose 6-phosphate- (fibroblast) specific uptake systems, cell surface binding without internalization can be demonstrated at reduced temperatures. This indicates a limited number of specific high-affinity receptor sites on the cell surface that can interact with the ligand. In the case of galactose- and mannose-specific systems, this supports the earlier observations that in vivo clearance is both saturable and Lysasomal Degradation
L
Minimal model for recycling receptor mechanism. This figure shows the minimal number of steps and intermediates necessary for a recycling receptor mechanism. The portions of the pathway labeled with roman numerals indicate segments of a pathway and may include many individual steps. L, Ligand; R, receptor. (From Tietze et a/..1982.) FIG.
3.
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competitively inhibitable by alternate ligands. In several specific cases the ligand has been localized to the cell surface after binding at low temperatures. This has been particularly carefully studied in the perfused liver with ligands specific for the galactose receptor. Using ligand conjugated with horseradish peroxidase, Hubbard and co-workers (Wall e t a / . , 1980) have been able to show that below 1O"C, ligand is restricted to the plasma membrane. The ligand is found primarily on regions of the plasma membrane that have cytoplasmic coats. At temperatures greater than 16"C, the ligand is found in two types of cytoplasmic vesicles. One is a very small coated vesicle and the other a larger smooth vesicle that is located close to the plasma membrane. When the perfusion was done at higher temperatures (>25"C), the ligand was found to have moved in a time-dependent fashion to cytoplasmic vesicles that display the morphologic and histochemical characteristics of lysosomes (Wall et a / . , 1980). This work corroborates the kinetic studies of many workers demonstrating that at low temperatures, ligand is bound with high affinity to plasma membrane receptors, but that higher temperatures permit internalization to occur also. In alveolar macrophages, it has been demonstrated that surface-bound ligand can be removed by treating the cells with trypsin or calcium chelators (Stahl et al., 1980). When the cells were allowed to bind isotopically labeled ligand at 4°C and the cells were washed to remove unbound ligand and warmed to 37"C, the bound ligand rapidly ( t l l Z 1 minute) became resistant to removal by these agents (Stahl et al., 1980). This has been interpreted as indicating that the bound ligand is internalized by the cell upon warming to 37°C. When warming was continued for a few minutes, radioactivity began to appear in the media. This radioactivity was shown to be fragments of degraded ligand. The obvious interpretation is that within 5- 10 minutes, ligand has reached the lysosome and proteolysis has begun. Although the interpretation of these results gives a clear-cut model of binding followed by rapid internalization of the ligand, there are many questions concerning the mechanism of this process. Which part of the process is modulated by receptor-ligand interaction is a major issue which has not been answered in even the most general sense. This issue can be broken down into three specific questions: ( I ) Are pinocytosis receptors found only in coated pits, or do they move there from a more diffuse distribution after binding ligand? (2) Do multiple receptor-ligand interactions play any role in receptor localization or movement? (3) Are receptors continually internalized, or is the internalization process triggered by interactions with receptor? As interesting as these questions may be, there are no definitive answers to them, and therefore they only provide ideas for future experiments. A second major issue that is directly related to the validity of the model shown in Fig. 3 concerns the fate of the internalized receptor. Are receptors internalized with the ligand, separated from the ligand intracellularly, and then returned to the cell surface to participate in subsequent endocytic events? There is considerable
-
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experimental evidence related to this question of receptor reuse, and it is the question we will focus on in the remainder of this article. Initially, the carbohydrate-specific endocytosis systems were studied by in vivo clearance of infused glycoconjugates. It was demonstrated that very large quantities of ligand could be cleared (Stahl et a / . , 1976; Ashwell and Morell, 1974). When sufficient ligand was infused to saturate the clearance system, a constant amount of the ligand was removed from circulation per unit time. When a large quantity of unlabeled inhibitor was injected with the labeled ligand, the labeled ligand remained in the circulation until the inhibitor had been cleared. These results are so similar to enzyme-catalyzed reactions at concentrations of substrate (and alternate substrate) where the catalysis is obeying steady-state kinetics that it seemed natural to propose that the receptor was playing a catalytic role in the clearance of ligands. Put more simply, it seemed impossible to deplete the supply of receptor available for clearance. Using isolated cells, the question of receptor reuse could be approached more directly. With the galactose- and mannose 6-phosphate-specific systems, it has been possible to estimate the total cellular complement of ligand-binding sites and to show that the endocytosis of ligand quickly exceeds this stoichiometry (Steer and Ashwell, 1980; Sly et al., 1981). Under conditions in which new receptor synthesis is limited (i.e., presence of cycloheximide) endocytosis proceeds linearly with time, a result which would seem to demonstrate that receptors must be reused and that they behave as catalysts. This does not exclude the possibility that a large cellular pool of inactive receptors exists which can be mobilized to maintain endocytosis. However, cycloheximide has been shown to reduce the turnover of some cellular proteins; it is therefore possible that the use of this drug induces an artifactual reuse of receptors because their turnover has been decreased. Another approach to this problem has been to inactivate the cell surface receptors and then to study the resulting uptake by cells. In alveolar macrophages, the mannose receptor is very sensitive to the action of trypsin (Stahl er al., 1980). When the cells were incubated with trypsin at O'C, the cell surface binding activity was found to be substantially reduced. When these cells were then warmed to 37°C and the binding activity measured, there was complete recovery within 2-5 minutes. When uptake was studied after trypsinization at 4"C, the Kuptakewas unchanged but the maximum rate of uptake was reduced. These results indicate that some fraction of the total cellular receptor pool is inactivated by trypsinization and that the remaining receptors function normally. The rapid return of cell surface binding activity upon warming is most likely due to the movement of these receptors from intracellular sites where they are protected from the action of trypsin. Studies on the turnover of the galactose receptor in hepatocytes have not demonstrated any acceleration of receptor turnover during extended periods of clearance (Tanabe et a l . , 1979; Steer and Ashwell, 1980). This does not preclude
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the existence of a large intracellular pool of uniformly labeled but inactive receptors. However, the size of this pool would have to be very large. Since only 5- 10% of the galactose receptor is found on the cell surface and assuming that this fraction accounts for clearance in vivo, it is possible that increased turnover of this small fraction of the total receptor pool might not be detectable. To explore the possibility that the cell surface receptors in the galactose system are an independent pool of receptors responsible for endocytosis, isolated hepatocytes were treated with neuraminidase, exposing many galactose residues on the cell surface (Stockert et a l . , 1980). This treatment inhibited endocytosis of all ligands except for the very high affinity ligand, asialosubmaxillary mucin. These experiments were interpreted as follows: If an intracellular pool of native receptors existed, one would expect to observe a rapid recovery of cell surface binding activity for low-affinity ligands. However, no recovery was found. It was concluded that the entire pool of active receptors is on the cell surface. However, any new receptors recruited from intracellular pools would be competed for by the exposed galactose residues on the cell surface as well as the added ligand. Therefore, uptake will depend upon the ability of the added ligand to compete with cell surface galactose residues which might be at very high local concentrations. Because of this reservation, the existence of separate pools of receptor would still seem to be unresolved. These data are consistent with a model for endocytosis which is much like the formal kinetic mechanism for enzyme catalysis. The receptor passes through a number of intermediates in the endocytosis of ligand which include several intracellular sites. The major distinction between this mechanism (as shown in Fig. 3) and enzyme catalysis is the physical translocation of the receptor from the cell surface. Although the evidence is not absolutely conclusive (e.g., because of the lack of specificity of the inactivation procedures used), until compelling evidence is presented to the contrary it would seem reasonable to assume that the receptor does leave the cell surface and return to it during endocytosis. A. Receptor Recycling and Weak Bases
Further evidence for the internalization of receptor has come from the use of weak bases, especially chloroquine and ammonium chloride, as inhibitors of endocytosis (Sando et a l . , 1979; Livesey et al., 1980). These drugs are potent inhibitors of intracellular ligand accumulation. Studies at 4°C have demonstrated no direct effect by these drugs on ligand binding. In the case of the mannose 6phosphate receptor, amines have been shown to be noncompetitive inhibitors of endocytosis (Gonzalez-Noriega er al., 1980). In the mannose-specific system, the time dependence of ammonium chloride inhibition has been studied (Tietze et al., 1980). The cells accumulate ligand for a time and then stop. The cell-
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associated ligand in this case is not removable by treatments that readily dissociated receptor-ligand complexes, and therefore this ligand must have been internalized. It can be concluded from these studies that weak bases cause an inhibition of one or more steps in the intracellular portion of the endocytic mechanism and that the receptor accumulates intracellularly, leading to the observed timedependent inhibition of endocytosis. Interestingly, for the mannose and galactose receptors, amines cause a reduction in cell surface receptor number after preincubation at 37°C even if ligand is not present (Tietze et al., 1980; Tolleshaug and Berg, 1979). This indicates that either the drugs promote internalization of the receptors or the receptors are internalized constitutively whether they are occupied or not. The reduction in cell surface binding is rapidly reversed upon incubation in the absence of the drug at 37°C (Tietze et al., 1980) or by incubation at pH 6.0 (Tietze et al., 1982). The site of weak base inhibition appears to be beyond the internalization step. In studies on the alveolar macrophage, mannose-BSA which had been bound to cells at 4°C was found to be rapidly internalized upon warming in the presence of weak bases, indicating that the internalization step was not directly affected. The presence of weak bases does inhibit the appearance of degraded ligand in the media. It is not clear whether this is because ligands are not transported to the lysosome or whether it is due to a reduction in lysosomal proteolysis. The pronounced inhibition of lysosomal proteolysis by weak bases, especially chloroquine, has been demonstrated in many other reports. The interpretation of these studies relies upon one very important assumption, that cell surface binding at 4°C is an accurate representation of the active receptor present on the cell surface. If this assumption is correct then these studies indicate that during ligand uptake and perhaps in the absence of ligand the glycoprotein receptors cycle between intracellular membranes and the plasma membrane. This would certainly be consistent with fractionation studies that have demonstrated the galactose receptor and the mannose 6-phosphate receptor on intracellular membranes (Fisher ef al., 1980; Tanabe et al., 1979). In both cases the intracellular pools of receptor are much larger than those found on the cell suilacc. These intracellular pools do not appear to be merely newly synthesixed receptor on its way to the plasma membrane. It has been proposed that the mannose 6-phosphate receptor is the vehicle by which newly synthesized lysosoma1 enzymes are segregated from secretory proteins at the level of the Golgi apparatus and packaged in lysosomes (Sly et al., 1981). If this were its major function then it would be reasonable to find most of the receptor on intracellular membranes. Another possibility is that mannose 6-phosphate receptors can randomly distribute themselves throughout the membranes involved in the biosynthesis, secretion, and degradation of glycoproteins. Then their relative distribution would parallel the relative proportions of plasma membrane to intracellular membranes found in the cells. In any case, no definitive physiologic function has been proposed for the other receptors, and so there is no need to
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attempt to rationalize their cellular distribution at this point. The evidence indicates that weak bases inhibit endocytosis and reduce the number of binding sites that can be found on the cell surface. The observations are similar with all three of the receptors, and therefore it seems that weak bases inhibit a step that is generally seen in receptor-mediated endocytosis. The inhibition by weak bases is consistent with the mechanism shown in Fig. 3. The site of inhibition is in segments I1 or 111 of this mechanism. These segments most probably involve steps and intermediates that are common to many other receptors which mediate endocytosis. The study of a number of systems has shown that for hormone receptors, low-density lipoprotein receptors, a,-macroglobulin receptors, and others internalization is by way of coated pits on the cell surface (Goldstein et al., 1979). Presumably, the next stage is an intracellular coated vesicle which may have a very transitory existence. Following internalization, the ligand is delivered to the endosome, which can be distinguished from the lysosome by morphological and histochemical techniques. Elegant work using the perfused rat liver and ligands specific for the galactose receptor of hepatocytes has demonstrated a similar sequence of events (Wall et al., 1980). The ultimate destination for galactose-terminated ligands in the hepatocyte is the lysosome, as was clearly demonstrated in this study and many others. It would be premature to assume that the plasma membrane + endosome + lysosome sequence is obligatory for all of the receptors that internalize by way of coated pits. However, a common theme is obvious, and the details of the intracellular pathway of endocytosis receptors are relevant to all of the receptors that involve coated pits. Even more complex is the intracellular pathway of the mannose 6-phosphate receptor which has been proposed to transport lysosomal enzymes both from the endoplasmic reticulum to the lysosome and from the plasma membrane to lysosomes. If the ubiquitous coated vesicle is the common carrier for transit between all these vesicular compartments, then how is selectivity achieved in the delivery of the ligand? Are coated vesicles addressable so that they can deliver their contents only to certain other vesicles, or is this some sort of stochastic process involving the proximity of vesicles or trapping mechanisms in the appropriate destinations? There should be a clear distinction between these two general mechanisms if experimental techniques can be devised to study the movement of receptors from one vesicle to another. If the movement between vesicles is in fact concerted, then the movement from the cell surface to the lysosomes in the case of endocytosis should be orderly with a well-defined progression of intermediates. On the other hand, if this is a stochastic process then there should be branch points with the intracellular pools of receptor and ligand moving in many directions simultaneously. Many factors could influence this latter type of process to give unequal flow of ligand and receptor in the various directions. These include the proximity of the vesicles, the relative
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surface area of various vesicle pools, the efficiency of any trapping mechanisms, and relative rates of formation of coated vesicles from the various membranes involved, to mention only a few. Experimentally, it may be very hard to distinguish these two possibilities given our current techniques. However, these are two conceptually distinct ways of looking at cellular organization which warrant consideration in the interpretation of experimental results. In the case of the glycoprotein endocytosis receptors, which all appear to deliver their ligands to the lysosome, an interesting hypothesis has evolved. The lysosome is distinct from other intracellular vesicles in that it is the only one known to have a very acidic pH. Most estimates give it a pH of 4.5 to 5.5 in vivo. The galactose, mannose 6-phosphate, and mannose receptors all exhibit a large increase in the dissociation of receptor-ligand complexes when the pH is lowered from neutrality to 6. The acid pH of the lysosome fulfills the requirements for a way to trap ligands in this compartment. When the receptor-ligand complex reaches an acid vesicle the ligand is discharged and remains in the lumen of that vesicle. The receptor is free to return to the cell surface and to participate in further endocytosis. This hypothesis does not favor either a concerted movement of receptor from one vesicle to another or a random process, but it does provide a mechanism for dissociating the very stable receptor-ligand complexes that these receptors form. The above view offers an explanation for the type of inhibition of endocytosis produced by weak bases. These compounds are known to raise the pH of acid intracellular compartments by mechanisms that have been carefully described elsewhere (Ohkuma and Poole, 1978). If, when this occurs, receptor-ligand complexes were not dissociated, then the total cellular complement of active receptors would be quickly converted to receptor-ligand complexes which are unable to participate in further transport of ligand. This mechanism has been used to explain the time-dependent inhibition of endocytosis by amines of the mannose- (Tietze el al., 1982), mannose 6phosphate- (Gonzalez-Noriega et al., 1980), and galactose- (Tolleshaug and Berg, 1979) specific receptors. Furthermore, it can reconcile the reduction of binding activity on the cell surface. If binding of ligand triggers the internalization of the receptor, or even enhances it, the effect of filling all receptors with ligand will be to shift the distribution of receptors away from the plasma membrane. Considering the role of the mannose 6-phosphate receptor in the packaging of lysosomal enzymes, a similar hypothesis has been used to explain the observation that ammonium chloride causes the enhanced secretion of P-hexosaminidase from cultured fibroblasts (Gonzalez-Noriega et al., 1980). If raising lysosomal pH results in filling of all the cellular mannose 6-phosphate receptors, then newly synthesized enzymes will not be packaged into lysosomes but will be secreted by the cells. The observation that weak bases reduce cell surface binding in the absence of added ligand is more difficult to explain in this way. It might be
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that weak bases directly affect other portions of the endocytosis process leading to a shift of receptor to intracellular membranes, but there is no evidence on this issue at the present time.
B. Cycling and Recycling Receptors The proposal that weak bases prevent the dissociation of receptor-ligand complexes provided an opportunity to use the ligand to follow, in part, the intracellular pathway of receptor in the presence of the weak base. In alveolar macrophages, it can be shown that approximately 20% of the receptor-ligand complexes return to the cell surface intact when ligand degradation and uptake have been inhibited by ammonium chloride (Tietze et al., 1982). Using a ligand resistant to degradation, receptor-ligand complexes return to the cell surface where they can be observed even in the absence of added weak base. This cycling between intracellular and cell surface membranes seems to be on a rapid time scale with the entire cycle taking perhaps 2-5 minutes. These observations raise several important issues. First, this may represent a branch point or perhaps the end result of several branch points in the endocytic pathway. It would appear from these data that receptor-ligand complexes can return to the cell surface from intracellular compartments. Therefore, the endocytosis pathway is not fully concerted and the targeting of receptor-ligand complexes to specific intracellular compartments is not absolute. Second, if the coated vesicle + endosome + lysosome pathway is not unidirectional for the glycoprotein receptors then it may also be bidirectional for other receptors. This would then add a new parameter to be explored in studying the biochemistry of other receptors. The balance between Acid Corn part ment
[nonreieasoble pool]
la-
Lysosomal Degradation
Fic. 4. Expanded mechanism for the mannose-specific endocytosis receptor. This figure includes a free receptor pool and a cycling pool of receptors (subscript c). L, Ligand; R , receptor. (From Tietze et a / . , 1982.)
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internalization and return could be important in regulating receptor number at the cell surface as well as regulating any cellular response. Thus far this rapid cycling between the cell surface and the interior membrane has been studied only in a few instances including recent studies with the hepatocyte galactose receptor (Debanne and Regoeczi, 1981), but it would seem to be a possibility worth exploring further. The presence of this cycling pool of receptor-ligand complexes is shown in Fig. 4. The existence of the cycling pool relies upon the ability to distinguish cell surface from internal receptor-ligand complexes by their sensitivity to media conditions that promote dissociation of the complex. This presumes that ligand internalization involves receptor internalization as well. In these studies, three independent methods of detecting cell surface complexes were used, but it is not inconceivable that another mechanism of ligand internalization without the receptor leaving the plasma membrane could have yielded these results.
VI.
CONCLUSION
During the past decade considerable new information has been revealed concerning the specific recognition and transport of lysosomal enzymes by cells. A new language, coded in the carbohydrate moieties of transported glycoproteins, is emanating from the current research. At least sugar ligands and their corresponding receptors appear to play prominent roles in the intra- and extracellular traffic of lysosomal hydrolases. Others undoubtedly will be uncovered in time. As newer techniques of recombinant DNA, monoclonal antibodies, and methods for the isolation of new mutants and variants are applied in this area, it is not unlikely that equally large strides in our understanding of intracellular transport mechanisms will be forthcoming. REFERENCES Ashwell, G., and Morell, A. (1974). The role of cell surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. 41, 99- 128. Bach, G . , Bargal, R., and Cantz, M. (1979). I-cell disease: Deficiency of extracellular hydrolase phosphorylation. Biuchem. Biophys. Res. Commun. 91, 976-98 1. Bainton, D. (1981). The discovery of lysosomes. J . Cell Biol. 91, 66-76. Debanne, M. T.. and Regoeczi, E. (1981). Subcellular distribution of human asialotransfemn type 3 in the rat liver. J . Biol. Chem. 256, 11266-1 1272. Distler, J., Hieber, V., Sahagian, G., Scjmickel, R., and Jourdian, G. W. (1979). Identification of mannose 6-phosphate in glycoproteins that inhibit the assimilation of P-galactosidase by fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 76, 4235-4239, Fischer, H. D., Gonzalez-Noriega, A., Sly, W. S., and Morre, D. J. (1980). Phosphomannosylenzyme receptors in rat liver. J . Biol. Chem. 255, 9608-9615. Goldstein, J., Anderson, R. G. W., and Brown, M. S. (1979). Coated pits, coated vesicles and receptor-mediated endocytosis. Nature (London) 279, 682-684.
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Gonzalez-Noriega, A,, Grubb, J. H., Talkad, V., and Sly, W. S. (1980). Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling. J . Cell B i d . 85, 839-852. Hasilik, A,. and Neufeld, E. F. (1980). Biosynthesis of lysosomal enzymes in fibroblasts. J . Biul. Chem. 255, 4946-4950. Hasilik, A,, Waheed, A., and von Figura, K. (1981). Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetyl-glucosamine. Absence of the activity in I-cell fibroblasts. Biuchem. Biophys. Res. Commun. 98, 761-767. Hickman, S., and Neufeld. E. F. (1972). A hypothesis for I-cell disease: Defective hydrolases that do not enter lysosomes. Biochem. Biophys. Res. Commun. 49, 992-999. Hubbard, A. L.. and Stukenbrok, H. (1979). An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. 11. Intracellular fates of the lzsI-ligands. J . Cell Biul. 83, 65-8 I . Kaplan, A,, Achord. D. T., and Sly, W. S. (1977). Phosphohexosyl components of a lysosomal enzyme are recognized by pinocytosis receptors on human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 69, 2026-2030. Kaplan, J . , and Nielsen, M. (1978). Pinocytic activity of rabbit alveolar macrophages in vitru. J . Reticuloenduthel. SUC. 24, 673-685. Kawasaki, T., Etoh, R . , and Yamashina, I. (1978). Isolation and characterization of a mannanbinding protein from rabbit liver. Biuchem. Biuphys. Res. Cumrnun. 81, 1018- 1024. Kawasaki, T., Mizuno, Y., Masuda, T., and Yamashina, I . (1980). Mannan-binding protein in lymphoid tissues of rats. Biochem. J. 88, 1891-1894. Konish, M. G., Thomasson, D., and Stahl, P. D. (1982). Expression of mannose receptor by rat bone marrow derived macrophages: Modulation of receptor activity by lymphokines and glucocorticoids. Fed. Pruc. Fed. Am. Sue. Exp. Biol.. 41, 3764. Kozutsumi, Y., Kawasaki, T., and Yamashina, 1. (1980). Isolation and characterization of a mannan-binding protein from rabbit serum. Biuchem. Biuphys. Res. Commun. 95, 658-664. Leroy, J. G . , Ho, N. M., MacBrinn, M. C., Zielke, K., Jacob, J., and O’Brien. 1. S. (1972). I-cell disease: Biochemical studies. Pediat. Res. 6, 752-759. Livesey, G., Williams, K. E., Knowles, S. E., and Ballard. F. J. (1980). Effectsof weak bases on the degradation of endogenous and exogenous proteins by rat yolk sacs. Biochem. J . 188, 895-903. Lugis, A. J., and Paigen, K. (1977). Mechanisms involved in the intracellular localization of mouse glucuronidase in isozymes. Curr. Top. Biul. Med. Res. 2, 63-106. Mandell, B., and Stahl. P. (1977). Effects of di-isopropyl phosphorofluoridate on rat liver microsoma1 and lysosomal P-glucuronidase. Biochem. J . 164, 549-556. Maynard, Y., and Baenziger, J. U. (I981 ). Oligosaccharide specific endocytosis by isolated rat hepatic reticuloendothelial cells. J . B i d . Chem. 256, 8063-8068. Maynard, Y., and Baenziger, J. U . (1982). Characterization of a mannose- and N-acetylglucosamine-specific lectin present in rat hepatocytes. J. Biol. Chem. 257, 3788-3794. Miller, A. L.. Kress, R. C., Stein, R., Kinnon, C., Kern, H., Schneider, J. A., and Harms, E. (1981). Properties of N-acetyl-P-o-hexosaminidase from isolated normal and I-cell lysosomes. J . Biol. Chem. 256, 9352-9362. Natowicz, M. R., Chi, M. M.-Y., Lowry, 0. H., and Sly. W. S. (1979). Enzymatic identificationof mannose-Qphosphate on the recognition marker for receptor-mediated pinocytosis of P-glucuronidase by human fibroblasts. Pruc. Narl. Acad. Sci. U . S . A . 76, 4322-4326. Neufeld, E. F., Sando, G. N., Garvin. A. J., and Rome, L. (1977). The transport of lysosomal enzymes. J . Supramol. Struct. 6, 95-101. Novikoff, P. M., Yam. A., and Novikoff, A. B. (1981). Lysosomal compartment of macrophages: Extending the definition of GERL. Proe. Narl. Acud. Sci. U . S . A . 78, 5699-5703.
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Ohkuma, S.. and Poole, B. (1978). Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Nut/. Acad. Sci. U . S . A . 75, 3327-333 I . Owens, J . W.. Gammon, K. L., and Stahl. P. D. (1975). Multiple forms of P-glucuronidase in rat liver lysosomes and microsomes. Arch. Biochem. Biophy. 166, 258-272. Reitman, M., and Kornfeld. S. (1981a). UDP-N-Acetylglucosamine: Glycoprotein N-acetylglucosamine I -phosphotransferase. J . Biol. Chem. 256, 4275-4281. Reitman. M., and Kornfeld. S. (1981b). Lyaosomal enzyme targeting. J . Biol. Chem. 256, 11977-1 1980. Reitman. M.. Varki. A . . and Kornfeld. S. (19x1). Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5'-diphosphate-N-acetylglucosamine: Glycoprotein N-acetylglucosaminylphosphotransferase activity. J . Clin. Invest. 67, 1574- 1579. Robbins. A . R.. and Myerowitz, R . (1981). The mannose-6-phosphate receptor of Chinese hamster ovary cells. J . Biol. Chem. 256, 10623-10627. Rome, L. H., and Miller. J . (1980). Butanediol treatment reduces receptor binding of a lysosomal enzyme to cells and membranes. Biochem. Bi0phy.s. Res. Commun. 92, 986-993. Sando, G . , and Neufeld, E. (1977). Recognition and receptor-mediated uptake of a lysosomal enzyme a-iduronidase by cultured human fibroblasts. Cell 12, 619-627. Sando, G. N.. Titus-Dillon. P.. Hall, C. W.. and Neufeld, E. F. (1979). Inhibition of receptormediated uptake of a lysosomal enzyme into fibroblasts by chloroquine. procaine and ammonia. Exp. Cell Res. 119, 359-364. Schlesinger. P., Doebber, T . , Mandell, B., White, R . , DeSchryver, C.. Miller, J . , Rodman, J . . and Stahl, P. i1978). Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Biochrrn. J . 176, 103- I1 I. Schlesinger, P., Rodman, J., Doebber, T., Stahl, P., Lee. Y. C., Stowell, C. P., and KuhlenSchmidt, T. (1980). The role of extra-hepatic tissues in the receptor-mediated plasma clearance of glycoproteins terminated by mannose or N-acetylglucosamine. Biochem. J . 192, 597- 606. Schneider, D. L. (1981 ). ATP-dependent acidification of intact and disrupted lysosomes. J . Biol. Chem. 256, 3858-3864. Shepherd, V. L.. Lee, Y. C., Schlesinger, P. H. and Stahl, P. (1981). L-Fucose terminated glycoconjugates are recognized by pinocytosis receptors on macrophages. Proc. Nutl. Acad. Sci. U.S.A. 78, 1019-1022. Shepherd, V . . Campbell, E. J . , Senior. R. M., and Stahl, P. D. (1982). Characterization of the mannose/fucose receptor on human mononuclear phagocytes. J . Reticuloendorhel. Soc.. in press. Sly. W. S . and Stahl, P. D. (1978). Receptor-mediated uptake of lysosomal enzymes. Transport of macromolecules in cellular systems. Duhlern Konf., Berlin pp. 229-244. Sly, W. S . , Natowicz, M., Gonzalez-Noriega, A , , Grubb. J . , and Fischer. H. D. (1981). The role of the mannose 6-phosphate recognition marker and its receptor in the uptake and intracellular transport of lysosomal enzymes. i n "Lysosomes and Lysosomal Storage Diseases" (J. W. Callahan and J . A. Lowden. eds.), pp. 131-140. Raven. New York. Stahl. P. D., and Gordon, S. (1982). Expression of the mannosyl-fucosyl receptor by macrophages and their hybrids. J . Cell. Biol. 93, 49-56. Stahl, P. D.. Schlesinger, P. H . , Rodman, J. S., and Doebber, T. (1976). Recognition oflysosomal glycosidases in vivo is inhibited by modified glycoproteins. Nurure (London) 264, 86-88. Stahl. P. D., Rodman, I. S . , Miller, J. M., and Schlesinger, P. H. (1978). Evidence for receptormediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc. Nut[. Acad. Sci. U . S . A . 75, 1399- 1403. Stdhl, P. D., Schlesinger. P. H.. Sigardson. E., Rodman, J . S., and Lee, Y. C. (1980). Receptor-
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mediated pinocytosis of mannose glucoconjugates by macrophages: Characterization and evidence for receptor recycling. Cell 19, 207-215. Steer. C. J., and Ashwell, G. (1980). Studies on a mammalian hepatic binding protein for asialoglycoproteins: Evidence for receptor recycling in isolated rat hepatocytes. J. B i d . Chem. 255, 3008-3013. Stockert, R. J . , Harvard, D. J . , Morell, A. G . , and Scheinberg, J. H. (1980). Functional segregation of hepatic receptors for asialoglycoproteins during endocytosis. J. Biol. Chem. 255, 90289029. Straus, W. (198 I ). Cytochemical detection of mannose-specific receptors for glycoproteins with horseradish peroxidase as a ligand. J. Cell Biol. 91, 21 la. Tabas, I.. and Kornfeld, S. ( 1980). Biosynthetic intermediates of P-glucuronidase contain high mannose oligosaccharides with blocked phosphate residues. J. B i d . Chem. 255, 6633-6639. Tanabe, T., Pricer, W. E., Jr.. and Ashwell, G . (1979). Subcellular membrane topology and turnover of a rat hepatic binding protein specific for asialoglycoproteins. J. Biol. Chem. 254, 1038- 1043. Tietze, C., Schlesinger, P.. and Stahl, P. (1980). Chloroquine and ammonium ion inhibit receptormediated endocytosis of mannose-glycoconjugates by macrophages: Apparent inhibition of receptor recycling. Biochem. Biophys. Res. Commun. 93, 1-8. Tietze, C., Schlesinger, P., and Stahl, P. (1982). Mannose-specific endocytosis receptor of alveolar macrophages: Demonstration of two functionally distinct intracellular pools and their roles in receptor recycling. J. Cell B i d . 92, 417-424. Tolleshaug, H., and Berg, T. (1979). Chloroquine reduces the number of asialoglycoprotein receptors in the hepatocyte plasma membrane. Biochem. Phurrnucol. 28, 2919-2922. Townsend, R., and Stahl. P. (1981). Isolation and characterization of a mannoseiN-acetylglucosamine/fucose binding protein from rat liver. Biochem. J. 194, 209-2 14. Varki, A,, and Kornfeld, S. (1981). Purification and characterization of rat liver a-N-acetylglucosaminyl phosphodiesterase. J. B i d . Chem. 256, 9937-9943. von Figura, K., and Weber. E. (1978). An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Biochem. J. 176, 943-950. Waheed, A,, Pohlmann, R.. Hasilik, A., and von Figura, K. (1981). Subcellular localization of two enzymes involved in the synthesis of phosphorylated recognition markers in lysosomal enzymes. J. B i d . Chem. 256, 4150-4152. Wall, D. A., Wilson, G., and Hubbard, A. L. (1980). The galactose-specific recognition system of mammalian liver; the route of ligand internalization in rat hepatocytes. Cell 21, 79-93.
CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I X
The Insulin-Sensitive Hexose Transport System in Adipocytes J . GLIEMANN AND W . D . REES Phvsiologv of Aarhus Aarhus. Denmark Institute of University
. . . . . . . . . . . . . . . . 339 Summary of the Present Status . . . . . . . . . . . . . . . . . . . . . Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 . . . . . . . . . . . . . . . . 342 Critical Steps in the Methodology 342 A. The Cell Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Measurement of Fluxes.. . . . . . . . . . . , . , . . . , . , . . . . , . . . . . . . . . . . . . . . . . . . . . 344 348 IV . Kinetic Approaches to the Study of Hexose Transport. , . . , . . A. General Concepts . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 B. Equilibrium Exchange Experiments _ . _ . . _ _ . . . . . . . . . . . 349 ... 35 1 C. Zero trans Experiments. . . . . . . . . . D. Infinite cis Experiments . . . . . . . . . . . , . . . . , . , , . , . , . , . . . . . . . . . . . . . . . . . . . . 353 354 E. Infinite trans Experiments. . . . . . . . . . , , . . , , . , , . . . . . . 355 V . Transport of Nonmetabolizable Sugars and Sugar Analogs in VI. The Requirements for D-GhCOSe Binding to the Adipocyte Hexose Transport System 359 360 VII. Nontransported Competitive Inhibitors of Transport. . . . . . . . . . . . . . VIII. Sugars Which Are Both Transported and Phosphorylated-Rate-Limiting Steps. . . . . 362 IX. Modulation of the Transport System by Glucose Metabolites . .. . . . . . . . . . . . . . . . . . 366 X. Mechanism of Insulin’s Ability to Increase V,,,, , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . 367 . . . . . . . . . . . . . . . . 37 1 XI. Human Adipocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 1 XII. The Transport System in Obesity and Diabetes . . . . . . . . . . . . . . . . . . . . XIII. Reconstitution of the Hexose Transporter . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372 XIV. Concluding Remarks, . . . . . . . . . . . . . . . . . , . . , , , , , . , . . , . , . . . . . . . . . . . . . . . . . . . . 373 373 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I.
11. 111.
1.
SUMMARY OF THE PRESENT STATUS
The plasma membranes of adipocytes are equipped with special structuresoften referred to as carriers-which greatly facilitate the transfer of hexoses 339
Capyright 0 1983 by Academic F’rerr. Inc All rights 01 reproduction i n any form reserved. ISBN 0- 12- I533 18-2
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(e.g., D-glucose) from the extracellular fluid to the cytosol. This mediated transport allows a nonmetabolizable sugar analog with an affinity similar to that of glucose (e.g., methylglucose, 3-0-methyl-~-glucose) to equilibrate across the plasma membrane at a much higher rate (up to lo4 times) than it would do by simple, nonmediated diffusion alone. The transport system shows saturation and can be described kinetically using equations analogous to those applied in enzymology. The K , for glucose is about 8 mM at physiological temperature, i.e., about twice the fasting plasma glucose concentration in mammals. Glucose metabolism is vital for the adipocyte; for example, fatty acids could not be esterified and triglycerides could not be stored in the absence of production of a-glycerophosphate. At low glucose concentrations, the rate of transmembrane glucose transport limits the rate of glucose metabolism and the transport step is therefore a major point of regulation. Several hormones, most notably insulin, influence the permeability of the plasma membrane. The properties of the transport system are also modulated when the cells metabolize glucose at a high rate. Insulin causes an approximately 10-fold increase in V,,,, without influencing K , significantly. Recent work strongly suggests that the insulin-induced increase in V,,, is brought about by a translocation of transporters from a site in which they are nonoperative (presumably intracellular) to the plasma membrane. Some nontransported sugar analogs inhibit transport of sugars only from the extracellularly facing side of the membrane, whereas others inhibit only from the inside. Such studies have helped clarify the orientation of the glucose molecule during transport in both human red blood cells and rat adipocytes. In both systems, the C-1 end of the glucose molecule is initially bound to the extracellularly facing site and the C-4/C-6 end to the intracellularly facing site. In addition, the spatial and hydrogen bonding requirements are similar although not identical. On the other hand, the transport systems of the two cell types are different in the sense that transport of glucose and 3-0-methylglucose exhibits markedly asymmetric parameters in human erythrocytes, whereas these parameters are symmetrical in adipocytes.
II. HISTORICAL BACKGROUND The development leading to the present view as summarized above has taken place over about four decades. Early studies on sugar transport were carried out in human red blood cells using a photometric method developed by 0rskov (1935) to measure volume changes. At that time the mechanism of permeation was thought of as nonmediated diffusion even though clear deviations from Fick’s law were noted (Bang and 0rskov, 1937; Meldahl and Idrskov, 1940). Later, several groups clearly demonstrated saturability, specificity, and a number
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of other features incompatible with nonmediated diffusion (see for instance LeFevre, 1948). Furthermore, the hexose transfer system of human red cells shows an asymmetric behavior as first reported by Wilbrandt (1954). Further detailed studies of this phenomenon have led to the suggestion of various models for the mechanisms involved. The rate of glucose metabolism in human red blood cells is very low as compared to the rate of unidirectional glucose transport at all glucose concentrations, and the very high transport capacity of the red cell serves no obvious physiological function in adult humans. The present knowledge about this system was recently excellently reviewed by one of the founders of the field (Widdas, 1980). The basic concepts and methods of analysis developed through the studies of the red blood cell system have been of great importance for studies of sugar transport in other cell systems, including adipocytes. The concept that the transport of glucose across the plasma membrane in skeletal muscle might limit the rate of metabolism was first put forward by Lundsgaard (1939), who demonstrated that muscle cells contain very little free glucose. Lundsgaard inferred that insulin must act primarily on the transfer of glucose into the cell. Independent of the work of Lundsgaard, the membrane hypothesis was put forward by Levine et a / . (1949) on the basis of the finding that insulin increases the galactose space in the extrahepatic tissue of dogs. Morgan et al. ( 1964) demonstrated glucose-induced countertransport of 3-0methylglucose in striated muscle and concluded that glucose is transported by carrier-facilitated diffusion. It was further shown that insulin increases the activity of the transport system. The interest in adipose tissue was aroused when it became clear in the 1940s and 1950s that it was not only a storage site but actually possessed a very high metabolic turnover. For instance, D-glucose is rapidly metabolized in adipose tissue, such as rat epididymal fat pads, and this process is markedly enhanced by insulin. Crofford and Renold (1965a,b) showed that glucose is mainly transferred across the cell membranes of adipose tissue by carrier-mediated (facilitated) diffusion and that insulin acts on this step in analogy with the results obtained with skeletal muscle. It is difficult or impossible to obtain a quantitative evaluation of the hexose transport system using pieces of tissue. One problem is that diffusion of substrate in the interstitial fluid may be rate limiting for its entry or exit into the cytosol. Another problem is cellular heterogeneity; for example, the adipocytes appear to possess less than half of the intracellular water in adipose tissue (Gliemann e t a / ., 1972). For these reasons, it seems obvious to study the transport system using suspended cells. In 1964, Rodbell prepared isolated fat cells by treating adipose tissue with crude collagenase and showed that the cells were metabolically active and insulin responsive. This was an important milestone and the preparation is now widely used as a model system. A further advantage is that only adipocytes
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have a density of less than 1 and the cell preparation is therefore homogeneous. However, some technical problems had to be solved before the insulin-sensitive hexose transport system could be characterized.
111.
CRITICAL STEPS IN THE METHODOLOGY
A. The Cell Preparation General procedures are given by Rodbell (1964), Gliemann ( 1967), Vega and Kono (1979), and Foiey et al. (1980b,d). The permeability to 3-O-methylD-glucose of isolated epididymal rat adipocytes incubated in the absence of hormones is about 7 X lOW7 cm sec- I (Whitesell and Gliemann, 1979). However, this number, and consequently the “basal” rate of glucose metabolism, is subject to large variations because a number of factors related to the preparation and incubation of the cells may increase the permeability. This phenomenon was first noted in early work on glucose metabolism (Gliemann, 1967) and it has been shown by Vega and Kono ( 1 979) for methylglucose transport. It has been generally observed in our laboratory that cell preparations giving a high “basal” glucose metabolism will always give a high “basal” methylglucose transport. Thus, the first and necessary condition is that the cell preparation is as responsive to hormones, particularly insulin, as the tissue from which it was prepared. As a rule of thumb, epididymal adipocytes from 130- to 200-g rats fed standard chow ad libitum should be at least about 10-fold stimulated by insulin at a high concentration. If this is not the case, the cells probably exhibit an increased permeability to start with. This may be caused by chemical or mechanical stimuli. Crude collagenase, produced by Clostridium histolyticum, which is used to disintegrate the fat tissue, may sometimes contain proteins with “insulin-like” properties. Highly purified collagenase does not disintegrate the tissue and the combined action of collagenase and an acid protease is necessary (Kono, 1969). Even though it might be possible to purify the active components, all workers in the field appear to use the crude commercial preparations. Everybody seems to compare a given batch of collagenase with an old batch which is known to produce “good” cells, i.e., with a low “basal” permeability. If the new batch is suitable, a good supply is bought, since the crude collagenase is stable for years at -20°C. The directions supplied by the manufacturer (for fat cells, for liver cells, etc.) seem of little value based on our experience and it would be a considerable advantage if more exact chemistry was applied to the active components of the collagenase preparations.
HEXOSE TRANSPORT IN ADIPOCYTES
343
The presence of albumin at a high concentration is necessary to maintain cell integrity (Gliemann, 1967). The bovine serum albumin (fraction V) which is usually used may be contaminated with proteins causing insulin-like effects. Small amounts of insulin itself may be present in the albumin preparation. This is easily checked by the addition of antiinsulin antibodies and is usually not a problem. Other contaminating proteins may lose their insulin-like effect by treatment of the albumin preparation with trypsin (Jordan and Kono, 1980). These contaminations may be due to bacterial growth in the albumin preparation since several microorganisms produce proteases with “insulin-like activities. ” For example, this applies to the ubiquitous Bacillus subrilis. Such contaminations may also occur, of course, in the laboratory if the buffer has been standing on the bench too long before use. The mechanical treatment involves shaking or swirling of the tissue with crude collagenase followed by filtration and repeated washings. Such treatments may increase the sugar permeability (Gliemann, 1967; Vega and Kono, 1979). It is difficult to define exactly what strain the cells can tolerate because, for example, one metabolic shaker may not perform mechanically in the same way as another even when set at the same number of cycles per minute. The general recommendation is to cany out all procedures as gently as possible and to make sure that the geometry and surface qualities of the plastic ware are suitable. The “insulinlike” effect of vigorous mechanical treatment, i.e., an increase in the membrane permeability due to carrier-facilitated diffusion, was difficult to understand for many years. However, part of the mechanism has been elucidated recently. Thus, Kono et al. (198 1) have observed that hard centrifugation causes a shift in the distribution of transporters from a nonfunctional (probably intracellular) site to a functional site in the plasma membrane. This mechanism is similar to that observed when the cells are treated with insulin (Cushman and Wardzala, 1980; Suzuki and Kono, 1980; see below). Vega and Kono (1979) have reported that an increased “basal” transport in freshly prepared cells can be reduced by incubation for about 30 minutes with glucose. We have not observed this phenomenon in our laboratory, perhaps because the permeability was not enhanced to start with. It should be noted that an increased permeability, as caused by preparation artifacts, is not necessarily reflected in changes in morphology or an increased release of intracellular enzymes. More vigorous mechanical traumas may, of course, lead to cell rupture. However, this is easy to recognize because the preparation becomes greasy due to triglyceride droplets in the medium. The easiest way to monitor that problem is perhaps to spin a concentrated cell suspension (about 40% v/v) in a hematocrit centrifuge for 30 seconds (Gliemann et d., 1972); there should be no visible or only a thin film of free triglycerides on top of the cell layer.
344
J. GLIEMANN AND W. D. REES
6. Measurement of Fluxes
Measuring the intracellular waterspace is a necessary prerequisite for measuring the fluxes of transportable, nonmetabolizable sugars or sugar analogs which equilibrate across the plasma membrane. In adipocytes, the intracellular waterspace is only about 2% of the cell volume. In early work, the intracellular waterspace was measured using a filtration technique (Crofford et al., 1966), but the trapped extracellular volume was about I0 times larger than the intracellular volume and the latter was therefore determined with low precision. We introduced an oil flotation method (Gliemann et al., 1972) using dinonylphthalate, which has a density between that of buffer and adipocytes, or a silicone oil with a similar density. Upon centrifugation, the cell suspension is separated into three layers with the cells on top followed by an oil layer and then buffer (Fig. 1A). Using a microfuge tube it is easy to cut through the oil layer and thereby obtain the packed cells separate from the buffer. The space between the cells is largely filled with oil, and the trapped extracellular water volume is reduced to about one-third of the intracellular water. The large size of the adipocytes (small surface-to-volume ratio) is actually an advantage since pellets of smaller cells, such as hepatocytes or thymocytes obtained by centrifugation through an oil with a density slightly higher than that of buffer, contain a considerably larger volume of extracellular buffer per volume of cell pellet (Andreasen et al., 1974). In this way the intracellular waterspace could be measured quickly and with sufficient precision using appropriate markers for the total and the extracellular waterspaces. The distribution space for tritiated water was indistinguishable from that of methylglucose. Centrifugation through oil did not influence the ability of the cells to metabolize glucose, and efflux experiments showed that whereas tritiated water was lost almost immediately from the cells, about 30 seconds was required before half of the methylglucose had passed from nonstimulated cells into the medium (Gliemann et af., 1972). However, it soon became clear that the method was not suitable for measuring methylglucose fluxes in insulin-stimulated cells which were expected to show an approximately 10 times lower efflux half time at low methylglucose concentrations. The reason is that it takes 3-4 seconds to obtain separation between cells, oil, and medium, and, more importantly, that the cells first carry a large amount of extracellular water into the oil phase and this is replaced by oil during the centrifugation in the following 30 seconds (Thorsteinsson et af., 1976). Therefore, transport of sugar into or out of the cells cannot be regarded as being stopped when the centrifuge is turned on or even 4 seconds later. Nevertheless, the method may well be used to determine qualitatively whether a given batch of cells responds to insulin (Kono er al., 1977). The method has been used to measure the flux of slowly transported sugars such as D-allose (Loten et al., 1976) and L-arabinose (Foley et af., 1978).
HEXOSE TRANSPORT IN ADIPOCYTES
345
However, these sugars are transported slowly because their Michaelis constants are high (50 mM or more), and it is therefore difficult to measure transport at concentrations higher than K,. The method was consequently modified to measure fluxes of rapidly transported sugars and sugar analogs such as 3-0-methylglucose. Figure IB shows the principles of an efflux method used to measure equilibrium exchange (Vinten ef al., 1976), which implies that the sugar concentration is equal on the two sides of the membrane at any given time. The cells are first incubated with unlabeled methylglucose for a time sufficient to ensure equilibration of the external sugar. The volume is then reduced and the concentrated cell suspension incubated with [ 14C]methylglucosefor a shorter time. At the end of this period, the intracellular sugar concentration should be the same as the extracellular concentration although the specific activity is not necessarily the same. The reason for this twostep procedure is first, that incubation of concentrated suspensions for long times may damage the cells (incubating dilute suspensions with tracer would be too expensive!), and second, that the procedure minimizes the metabolism (and irreversible trapping) of trace contaminations which are sometimes present in the labeled methylglucose preparations. The cells are then centrifuged lightly through silicone oil in a slender tube, ejected into a large bath which, in equilibrium exchange experiments, contains unlabeled methylglucose at the same concentration as that used for the equilibration. Aliquots are transferred to oilfilled microfuge tubes at appropriate time intervals. It is sufficient to centrifuge the suspension for about 4 seconds because the dilution of extracellular buffer is essentially infinite. It should also be noted that backflux of methylglucose is negligible since the intracellular water volume of the adipocytes is very small as compared to the volume of the bath. Using this method, efflux of [‘4C]methylglucose can be followed until the intracellular concentration is a few percent of the starting concentration, as shown in Fig. 2. The efflux of a labeled nonmetabolizable sugar from a population of identical cells with well-mixed extracellular and intracellular compartments should be monoexponential under equilibrium exchange conditions irrespective of the nature of the transport system. The curve (Fig. 2) actually deviates slightly from an exponential course and this is probably due to cellular heterogeneity with a range from “fast” to “slow” cells (Vinten et al., 1976; Gliemann and Vinten, 1974). This minor deviation is neglected in the analysis of net transport data as described below. Figure IC shows the principles of a method used to measure uptake of rapidly equilibrating sugars (Whitesell and Gliemann, 1979). A small volume of concentrated adipocyte suspension is squirted onto a droplet of buffer containing the labeled sugar to ensure mixing. This is followed by the addition of a large volume of 0.3 mM phloretin, a potent and rapidly acting competitive inhibitor of sugar transport. This acts as a stopping solution and arrests efflux of the sugar
J. GLIEMANN AND W. D.REES
346
I4-I
A
3Omin
Density (glcm3) : cells : 0.915 di nonyl phthalate : 0.99 buffer :
1.012
2 - 2Omin
600~11
30MG
plus labeled 3.0M G
-
Equilibrium Exchange
Unlabeled
calls
Decrease in spec. activity in medium
> LOO
B
40pl
12y1
C
4
Loop1
FIG. I . Oil flotation methods. (A) For slowly equilibrating sugars (or other ligands). (B) Efflux method for rapidly equilibrating sugars. (C) Uptake method for rapidly equilibrating sugars. 3-OMG. 3-0-rnethylglucose; 3-OMG*, 3 - 0 4 14C]methyl-~-glucose;3HTG, [3H]triglyceride, for sample volume correction. For further explanation, see text.
HEXOSE TRANSPORT IN ADIPOCYTES
347
Fic. 2. Efflux of 3 - 0 4 ''C]methyl-o-glucose under equilibrium exchange conditions at 37°C in adipocytes treated with a maximally stimulating insulin concentration (0.7 p N ) . The 3-0-methylglucose concentration was 30 mM. A, denotes the intracellular amount of sugar at time t . (Reproduced with permission from Vinten et al., 1976.)
taken up into the cell. Finally, the cells are centrifuged through an oil layer either in the incubation tube or after transfer to a microfuge tube. These two alternatives give the same experimental results. Trapping of the extracellular mixed incubation buffer is of the order of 0.02%, i.e., insignificant. Timing is aided by a metronome, and uptake can be measured precisely and reproducibly at intervals down to 1 second. Since the shortest half-time reported for uptake of methylglucose at tracer concentration in insulin-stimulated adipocytes is about 2 seconds, and since methylglucose is the fastest transported sugar we know of, initial velocities of sugar uptake can be measured or calculated with a good approximation under all conditions. At equilibrium exchange conditions (and in net uptake experiments when the substrate concentration is very low) the uptake curve is nearly exponential as was previously found for the efflux curve (Fig. 2 ) . This is merely a technical control since the theory demands that equilibrium exchange is identical whether efflux or influx of radiolabeled sugar is followed. (This is what theory demands, independent of the model. Flux into the cell and out of the cell is by definition the same under equilibrium exchange conditions.)
J. GLIEMANN AND W. D. REES
340
IV. KINETIC APPROACHES TO THE STUDY OFHEXOSETRANSPORT
A. General Concepts The combination of the transported hexose with a membrane protein to form a transporter-substrate complex leads to a facilitated diffusion system exhibiting saturation kinetics. This system differs from the familiar model of an enzymic reaction with one free enzyme form E (Scheme a), in that two separate binding sites (E, and E,) are available for the substrate (S) on each face of the membrane. Scheme b shows the most widely used model for facilitated diffusion, the carrier model. The subscripts 1 and 2 refer to the two sides of the membrane, and transport is measured as the movement of substrate from side 1 to side 2 or side 2 to side 1.
SCHEME a
SCHEME b
Kinetic analysis of the carrier model for facilitated diffusion will thus show half saturation constants (K,’s) and maximum velocities (Vmax’s) which have different interpretations depending on the direction of flux (i.e., movement from 1 to 2 or 2 to I) and the substrate concentrations on each side of the membrane (S, and S2). Eilam and Stein (1974) showed that for any of the protocols described below the rate of flux (v) will be given by an equation of the MichaelisMenten form (1)
V,,,) for a facilitated It should be noted that the kinetic constants (K, and V,,,,,) diffusion system are analogous to but not identical with those determined for an enzymic reaction. Other transport models (for review see Naftalin and Holman, 1977) can be described by more complex forms of Eq. (I). During facilitated 1977) diffusion the substrate molecule remains unchanged and, therefore, in the absence of metabolism, the substrate will equilibrate to equal concentrations in both bulk solutions as the substrate runs down its electrochemical gradient. As described above, the main technique for measuring transport rates is to follow the flux of a nonmetabolized radiolabeled substrate from the bulk solution at one face of the membrane [the cis face using the nomenclature of Eilam and (1974)] to the bulk solution at the opposite face (the trans face). This Stein (1974)]
349
HEXOSE TRANSPORT IN ADIPOCYTES
nomenclature shall be used whenever possible. The cis to trans flux of substrate can be measured either into (entry or influx) or out of (exit or efflux) the cell. The intracellular waterspace of the cell is very small (Gliemann ef af., 1972) relative to the external medium so that, in an entry experiment, the substrate concentration in the internal solution will change rapidly. On the other hand, the substrate concentration in the external medium will remain essentially unperturbed. In an entry experiment the radiolabeled substrate will accumulate in the internal (trans) solution. Backflux then occurs as part of this radiolabel, then returns in the trans to cis direction, and this reduces the rate of net flux. In an exit experiment backflux of substrate trans to cis (i.e., from the external solution into the cell) is minimal due to the large dilution of the radiolabeled substrate once it enters the bulk solution on the trans face. The small cis volume in exit experiments leads to a rapid drop in substrate concentration at the cis face as the substrate leaves the cell. Eilam and Stein (1974) in their review of the principles of the measurement of facilitated diffusion processes described integrated rate equations for the characterization of such a system. We shall include the appropriate integrated rate treatment for each protocol as adopted from Eilam and Stein (1974).
B. Equilibrium Exchange Experiments In these experiments the unidirectional flux of radiolabeled substrate is followed when there is no concentration gradient across the membrane, i.e., the substrate concentration in the cis solution is equal to that in the trans solution. Under these conditions there is no net flux of a nonmetabolizable substrate such as 3-O-methylglucose, and the rates of unidirectional influx and efflux are the same. As described above (Fig. 2) the exit curve is nearly exponential, and follows with close approximation the equation A, = A,ePkt
where A, and A, denote the amount of intracellular radioactivity at time zero and time t , respectively. These values are corrected for the small amount of radiolabeled sugar remaining in the cells at infinite time. The rate constant k is the fraction of intracellular radioactive sugar lost per unit time, i.e., the transport velocity v (moles sec- X liters intracellular water- I ) divided by the substrate concentration S (moles/liter). Thus Eq. (2) assumes the form k = viS = In( 1 - f,>/f
(3)
where f , (“fraction remaining”) = A,/A,. The equilibrium exchange influx curve follows (with the same approximation
350
J. GLIEMANN AND W.
D. REES
as the influx curve) the equation A, = A,(l
- eckt)
(4)
where A, denotes the amount of intracellular radioactivity at infinite time [equivalent to A, in Eq. ( l ) ] , A, and A, are corrected for the extracellular radioactivity in the cell pellet at zero time. As with Eq. (2), Eq. (4) can be rewritten as k = v1S = ln[l/(l - f , ) ] / t
(5)
where& (“fraction inside”) = A,/A,. Thus, for equilibrium exchange experiments plots of vlS vs t should be linear at any given substrate concentration. The half time for the efflux of 3-O-methyl-~-glucoseat tracer concentrations is approximately 2 seconds in insulin-stimulated adipocytes at 37°C. Therefore, from Eq. (3) k = vIS = 0.6912 = 0.345 sec-’
In equilibrium exchange experiments the transport of sugar from the extracellular compartment to the intracellular (or vice versa) may be measured as the initial velocity from time zero to a given time t . In experiments in which the labeled sugar is present only on the extracellular side, this implies that efflux of labeled sugar from time zero to time t is neglected. This does not introduce any serious error when only up to about 20% of the intracellular compartment is equilibrated. The limitations of the method may be illustrated using cells with maximal permeability to 3-0-methylglucose (half-time 2 seconds). By 1 second, 34.5% would be equilibrated if no backflux occurred (see above). However, the actual equilibration is 29.2% [cf. Eq. (4)]. Alternatively, once uptake has been shown to be exponential, Eq. (3) or ( 5 ) (for efflux or influx, respectively) can be used to calculate initial uptake rates. Then the values of vlS at different substrate concentrations can be plotted using the transformations of Eq. (1) which are used in enzymology. For example, the Slv vs S plot (Hanes’ plot) transforms Eq. (1) to
It should be noted that some models for sugar transport in erythrocytes predict more than one K,, (Holman, 1980; Holman et al., 1981a). As wide a range of substrate concentrations as possible should therefore be used in determining the saturation kinetic parameters. The equilibrium exchange K,, and V,,, (Kee and Vee using Eilam and Stein’s terminology) reflects the properties of both the internal and external sites of the transport system when substrate is being transported in both directions. The equilibrium exchange experiment is therefore well suited to the determination of inhibition constants for inhibitors of transport. The inhibition of tracer flux can
351
HEXOSE TRANSPORT IN ADIPOCYTES
be measured using a range of inhibitor concentrations and vlS values determined using Eq. (3) or (5). The inhibition constant is then given by the relationship
vdv
= 1
+ (I/Ki)
(7)
where vo is the uninhibited rate, v is the inhibited rate, and 1 is the inhibitor concentration. Ki can be determined from a plot of v,/v vs I (Rees and Holman, 1981). As with the equilibrium exchange experiment more than one K , may be apparent (Holman et al., 1981a).
C. Zero trans Experiments In these experiments the flux of substrate from cis to trans is followed when there is initially no substrate in the trans solution. Thus for a zero frans entry experiment substrate and radiolabeled substrate are added to the external solution and the uptake is measured. Substrate enters the cell rapidly and therefore over practical time courses there will be a finite substrate concentration in the trans solution and backflux into the cis solution. It is therefore necessary to calculate the initial rate of uptake from a net entry experiment in order to estimate the unidirectional zero trans influx. The uptake (or progress) curve is nonexponential except when the substrate concentration is very low as compared with K , for transport. Consider a system with symmetric transport of a given sugar, i.e., K , is the same for transport from the outside and in and from the inside and out. The rate constant for entry (and therefore the permeability) of the external sugar is reduced when its concentration is significant as compared with K, [Eq. (l)]. On the other hand, the permeability will be higher for sugar molecules that have entered the cytosol because the sugar concentration will initially be low in this compartment. Consequently, the sugar leaves the cell more rapidly than it would do under equilibrium exchange conditions and the progress curve of net entry becomes “flatter” than the exponential equilibrium exchange curve (Whitesell and Glieniann, 1979). This means that the error on the apparent initial velocity measured at a given time will be larger and corrections should be applied using an integrated rate equation (see below). Taylor and Holman (1981) reported that a 1 second uptake of 1 mM 3-0-methylglucose by insulin-stimulated adipocytes (net entry experiment) underestimated the initial rate of entry by 24%. Eilam and Stein [Eq. (70) in their 1974 paper] described an integrated rate equation for the net entry experiment. Their equation assumed a carrier model for transport (such as the simple model of Scheme b) but the parameters obtained can readily be reinterpreted in terms of other models for transport. The equation of Eilam and Stein contains a term correcting for volume changes due to the presence of the substrate. However, this may be neglected when the substrate con-
352
J. GLIEMANN AND W.
D. REES
centration does not exceed about 40 mM. Using Eilam and Stein's equation, as described by Ginsburg and Stein (1975), the initial rate of a net entry experiment at a given substrate concentration can be obtained by plotting vs -[ln(l - CIS,) + C/S,]/C (8) where t is the time of uptake, C is the internal substrate concentration, and So is the external substrate concentration. Note that C/S, is the fractional filling of the internal sugar space, i.e., equivalent tofof Eq. (5). This plot therefore shows the initial rate as a function of the internal concentration as described by the carrier model IEq. (17) in Eilam and Stein, 19741. By extrapolating the corrected integrated rate replot to zero internal concentration, the initial rate of influx under zero trans conditions is obtained. This process is analogous to the procedure used for calculating initial rates from the exponential equilibrium exchange progress curve as described above. An example of the use of the integrated rate replot is shown in Fig. 3. The initial rate measured at different substrate concentrations can then be replotted in order to obtain the K, and V,, for zero trans entry. The zero trans entry K, (KT;) measures the K, for the outside site and the zero trans entry V,,, (VT$>is likewise the V,,, for unidirectional influx from 1 to 2. In a zero trans exit experiment, the cells are loaded with substrate and radiolabeled substrate until equilibrium is achieved. The extracellular substrate is then rapidly diluted with a large quantity of buffer (usually at least IW-fold), and the loss of radiolabel from the cells is followed. This method does not give true zero trans conditions but the small amount of substrate in the trans solution relative to the cis sohion Ieads to a minimal error. The practically infinite volume on the trans face of the membrane in a zero trans exit experiment leads to minimal flC
C mM
4.0
'
2.0
u 0.1
Tlma
imesl
0.2
0.3
0.4
In~l-C/s,J+C/&,
C
FIG. 3 . (a) A time course for net entry (zero trans entry) of I mM 3-O-methyl-~-glucosein cells pretreated with 10 nM insulin at 37°C. The apparent initial rate calculated from the I second measurement was 0.16 Mlsecond. (b) An integrated rate equation replot of I mM 3-0-methylD-glucose net entry. The initial rate V , = 0.210 &/second. (From Taylor and Holman, 1981.)
353
HEXOSE TRANSPORT IN ADIPOCYTES
backflux of the substrate trans to cis, and for the simple carrier model the rate of exit will be given by the Michaelis-Menten equation. However, there is a rapid change in the substrate concentration at the cis face as efflux proceeds so that an integrated rate equation treatment is required. The data from zero trans exit experiments can be conveniently analyzed by an integrated Michaelis-Menton equation (Karlish eruf., 1972) which can be rearranged (Baker and Naftalin, 1979) to give
-In(S,/S,)/(So - S,)
=
V,,,,t/[K,(S,
- S,)] - 1/K,
(9)
where S, is the starting substrate concentration inside the cell and S, is the internal substrate concentration at time t . This equation is equivalent to a form of the Lineweaver-Burk transformation of the Michaelis-Menton equation 1/S = (VmaX/K,,,)(l/v0) - 1/K,
(10)
Thus, by plotting -ln(S&)/(S,
-
S,) vs
r/(S, - S,)
a value of - I/Knl is obtained from the intercept on the ordinate and a value of l/Vmxxis obtained from the intercept on the abscissa. By comparing Eqs. (9) and (10) it can be seen that when -ln(SI/So)/(So - S,)
=
I/S,
then t/(S, - S,) is equal to I/V,, i.e., the reciprocal of the initial rate at a given substrate concentration. Therefore, one can alternatively calculate the initial rate of exit at a number of internal substrate concentrations and plot the data using Eq. (6). This procedure has the advantage of giving a more faithful reflection of the experimental error. The K , measured by the zero trans exit experiment (K$ ) measures the K, at the internal face of the membrane. The zero trans exit V,,, (Vf, ) measures the V,, for unidirectional efflux from side 2 to side 1. It should be noted that this analysis is based on a carrier model such as Scheme b and assumes that there is a single operational affinity (single K,) for substrate at the inner face of the membrane. There is now evidence for two operational affinities at the inside face of the human erythrocyte hexose transporter (Ginsberg and Stein, 1975) and this may lead to deviations from the curve predicted by Eq. (9) (see Holnian, 1980).
0. Infinite cis Experiments The zero trans experiments provide a means of measuring the kinetic constants of one side when no substrate is available on the opposite side. The K,,, of one side can also be measured when the opposite side is saturated with substrate. Thus, for
354
J. GLIEMANN AND W. D. REES
the infinite cis experiment the net flux cis to trans is measured when the substrate concentration in the cis solution is at a saturating concentration, that is for practical purposes at least 10 times the zero trans K,. The cis side of the transport system is saturated with substrate, and therefore the rate of unidirectional flux from cis to trans will be maximal. On the other hand, the backflux process is dependent on the substrate concentration in the trans solution, and K , on the trans side can therefore be determined by following the rate of net flux into solutions containing different substrate concentrations. In other words, the net flux (cis to trans minus trans to cis) is reduced as the substrate concentration on the trans side increases and the trans to cis flux depends on the K , on the trans side. The infinite cis entry experiment is most easily performed by measuring the time course for the net uptake of a single high concentration of substrate. As the uptake proceeds, the internal substrate concentration will increase from zero until it is finally at equilibrium with the external solution. Thus, the substrate concentration in the trans solution is changing with time, and this allows the determination of the infinite cis entry parameters. Eilam and Stein (1974) showed that their integrated rate equation for net entry could be applied to the infinite cis experiment (see also Ginsburg and Stein, 1975) so that a plot of
ttc vs
ln(l
+ CIS,) + CIS, C
[cf. Eq. (8)l
yields a straight line giving I/Vmax for infinite cis entry as the intercept on the ordinate. The intercept on the abscissa will be -K,ISi ( I + S o h ) , where 7~ is the effective osmotic concentration of the buffer, and K i can thus be calculated (Ginsburg and Stein, 1975; Holman, 1979). K k will be the K , for the internal side. In order to perform infinite cis exit experiments [also known as the Sen and Widdas experiment (Sen and Widdas, 1962)] the cells are first loaded with a saturating concentration of substrate (e.g., 40 mM) and the net efflux of this substrate into solutions containing different concentrations of substrate is followed. With a saturating concentration of substrate in the cis solution the net exit rate remains linear until the internal substrate concentration ceases to be saturating. In adipocytes this means 10-20 seconds even when the cells are treated with insulin. Thus, the rate of net efflux can easily be measured without the need for integrated rate equations. A plot of I / V vs S gives minus the infinite cis exit K, ( K g ) as the intercept on the abscissa. Kt;' measures the K,, for the external side. The infinite cis experiments are technically simpler to perform than the zero trans experiments since they offer the advantage of longer time courses.
E. Infinite trans Experiments In these experiments (which are equivalent to counterflow experiments) the substrate concentration in the trans solution is at a saturating concentration.
HEXOSE TRANSPORT IN ADIPOCYTES
355
These experiments allow an additional measure of the K , for one side (cis) when the other side is saturated with substrate. The infinite trans entry experiment will therefore measure the K , for the external site while the infinite trans exit experiment will measure the K , for the internal site. It is possible to formulate rejection criteria for different kinetic models by comparing the results of the different experimental protocols with the predictions of the models (Lieb and Stein, 1974a,b). Thus, using these criteria, Hankin et al. (1972) were able to show that the simple carrier model can be rejected as a model for hexose transport in the human erythrocyte.
V.
TRANSPORT OF NONMETABOLIZABLE SUGARS AND SUGAR ANALOGS IN THE ADIPOCYTE
Since D-glucose entering the adipocyte is rapidly metabolized, it is impossible to study its transport directly. When the rate of D-glucose transport is rate limiting for metabolism, a measure of its transport rate can be obtained through the use of indirect measurements such as the rate of D-glucose incorporation into lipids or CO,. The value of this approach is limited and it does not allow a full kinetic characterization of the transport system. Before considering the results obtained with nonmetabolizable sugars, it is important to note that the sugar permeability in the isolated adipocyte due to nonmediated diffusion is negligible under most conditions. This conclusion is in part derived from the finding that L-glucose at a tracer concentration exhibits an equilibration half time of about 60 minutes under conditions where the half time is 2-3 seconds for 3-O-methyl-~-glucose. The half time for equilibration of L-glucose is further increased to several hours in the presence of 40 mM methylglucose (Whitesell and Gliemann, 1979). In addition, Vinten (1978) found that the total methylglucose permeability, which could not be inhibited by a large concentration of cytochalasin B, was only a small fraction of the total permeability. The transport of the nonmetabolizable C-3 epimer of D-glucose, D-allose, was studied by Loten et al. (1976) who showed it to be transported slowly. Foley et al. (1978) reported that L-arabinose (a D-galactose derivative lacking the C-6 hydroxymethyl group) was also transported slowly by the adipocyte and not metabolized. Both analogs are transported by the glucose transport system since their transport is competitively inhibited by glucose and the rate of transport is stimulated by insulin. These sugars are transported slowly due to their high K,’s for the transport system. D-Allose has a K , of about 270 mM (Rees and Holman, 1981), and L-arabinose has a K , > 50 mM (Foley et al., 1978). These high K,’s limit the usefulness of D-allose and L-arabinose in a conventional kinetic characterization of the transport system since saturation of the transporter with these sugars requires very high concentrations, which are outside a practical concentration range. The low affinity, and hence slow transport of these sugars does,
356
J. GLIEMANN AND W. D. REES
however, offer a distinct advantage in inhibition experiments allowing uptakes to be followed over a period of several minutes as opposed to seconds with a high affinity sugar. 3-O-Methyl-~-glucoseis a rapidly transported D-glucose analog which is not metabolized (Czaky and Wilson, 1956), and Gliemann et al. (1972) showed that it has a distribution space not different from that of tritiated water and urea. Equilibrium exchange experiments carried out as demonstrated in Fig. 1B showed only one K , value of about 5 mM, and insulin caused a marked increase in V,,, without changing K , (Vinten et al., 1976). Similar data were obtained by Vinten (1978) using the same method and by Whitesell and Gliemann (1979) and Taylor and Holman (1981) using the influx method shown in Fig. 1C. An experiment of this type is shown in Fig. 4. It should be noted that the 3-0methylglucose concentration range used in these experiments was fairly narrow (up to about 20 mM). However, experiments have been carried out with substrate concentrations up to 60 mM (with the correction for nonmediated diffusion, which is necessary under these conditions) and this gave a K , value of 4.5 ? 0.6 mM at 37°C in cells stimulated maximally with insulin (G. D. Holman and W. D. Rees, unpublished data). It should be mentioned at this point that the reason for the insulin-induced increase in V,,, is probably that more transporters are available in the plasma membrane after treatment of the cells with insulin. This hypothesis is a result of the work of Kono, Cushman, and their co-workers, and will be discussed below.
S Methylglucose (mM) FIG. 4. The concentration dependence of 3-@methyl-~-glucoseequilibrium exchange at 37°C in the absence of insulin and in cells pretreated with 10 nM insulin. K,, was 4.4 mM and not significantly different for “basal” and insulin-treated cells. V,,,,, was 0.08 mM s e c - 1 for “basal” cells and 1.12 mM sec-1 for insulin-pretreated cells.
HEXOSE TRANSPORT IN ADIPOCYTES
357
It implies that one is probably looking at the same species of transporters whether or not the cells are treated with hormone. The properties of the transporter will be discussed in the light of this hypothesis. Adrenalin increases the rate of 3-0-methyl-~-glucosetransport in adipocytes by approximately twofold (Ludvigsen et al., 1980), and this stimulation also occurs through an increase in the V,,,, with no change in the K,. As with insulin, the adrenalin effect is preserved in isolated plasma membranes and it is tempting to speculate that insulin and adrenalin stimulate transport through a common mechanism. The effect of adrenalin appears to be mediated through preceptors (Ludvigsen er al., 1980). Glucocorticoids cause a marked decrease in hexose transport rate. However, no kinetic characterization has been carried out of the transport system in glucocorticoid-treated cells (Foley et al., 1978). It is also possible to measure the transport of hexoses in isolated plasma membranes even though the permeability due to nonmediated diffusion is much higher than in intact adipocytes. Since the membrane preparation does not metabolize D-glucose, the transport of D-glucose can be studied directly without the need for nonmetabolizable analogs. Ludvigsen and Jarett (1979, 1980) reported that the K , for D-glucose uptake was 9-26 mM in plasma membranes isolated from insulin-stimulated adipocytes. The value of the measured K , , was dependent on technical details in the preparation of membranes. The question of whether adipocytes show asymmetric transport parameters was first approached by Whitesell and Gliemann (1979), who measured the net entry of 20 mM 3-0-methylglucose (i.e., “almost” an infinite cis experiment). The progress curve did not deviate significantly from that predicted by symmetrical transport parameters. It was concluded that the system was probably symmetrical and that any asymmetry, if present, was certainly not like that described in human red blood cells. Taylor and Holman (1981) carried out a complete kinetic analysis following the principles of Eilam and Stein (1974) as outlined in the preceding section and found no evidence for kinetic asymmetry of 3-0methylglucose transport. Table 1 shows the transport parameters obtained using different protocols. It should be noted that some authors have reported much lower V,,,, values, particularly in insulin-treated cells. The reason may be that the initial velocities were underestimated. These values are not given in Table I [for discussion, see Whitesell and Gliemann ( 197911. Table 11 shows for comparison the kinetic parameters of the most intensively studied hexose transport system, that of the human erythrocyte. It appears that this transporter shows marked asymmetry as recently reviewed by Widdas ( 1980) in Volume 14 of this series. The data are generally obtained using glucose but the results using 3-0-methylglucose also show marked asymmetry ( G . D. Holman, unpublished observations). The kinetic constants vary depending on the experimental protocol, and the most marked asymmetry is observed in the zero trans experiments with zero trans entry showing a low K , and V,,,, while zero trans
TABLE I KINETICPARAMETERS FOR 3-0-METHYL-D-GLUCOSE TRANSPORT IN
THE
RATADIFQCYTE
K, (mM) Experiment Equilibrium exchange
Zero trans entry (measures outside site)
Zero trans exit (measures inside site) Infinite cis entry (measures inside site) Infinite cis exit (measures outside site)
Reference
Basal
Vinten el a/. ( I 976) Whitsell and Gliemann (1979)" Taylor and Holman ( I98 1) I Whitesell and Gliemann ( 1979)" Taylor and Holman (1981)' Taylor and Holman (1981) r Holman and Rees ( 1982) Taylor and Holman (1981F Taylor and Holman (1981)c
About 5 2.5-5 4.22 2 1.24 2.5-5 5.41 2 0.98 4.09 5 1.05
9.03 5 3.28 4.54 2 1.32
Plus insulin About 5
2.5-5 4.45 5 0.26 2.5-5 6.10 5 1.65 2.66 5 0.26 5.65 5 2.05 6.51 t 0.83 3.60 5 1.33
V,,,
(mM sec - 1)"
Basal
Plus insulin
0.07-0.2 0.058 0.058 ? 0.001
1.6- I .9 0.8 0.84 t 0.002
-
0.034 5 0.034 0.153 2 0.023
1.20 -C 0.19 1.19 t 0.07
-
-
0.066 2 0.013 0.106 2 0.026
0.98 2 0.09 1.76 t 0.63
Equivalent to millimolesiliter intracellular waterkcond. Range of values obtained. 2 SE (from regression analysis). Values from Whitesell and Gliemann (1979) were obtained at 22"C, and the other values at 37°C
359
HEXOSE TRANSPORT IN ADIPOCYTES
TABLE I1 K I N ~ I I CPARAMETERS . FOR
D-GLUCOSEIN T H E HUMANERYTHROCYTE
Experiment
Reference
K , (mM)
Equilibrium exchange Zero trans entry (measures outside site) Zero trans exit (measures inside site) Infinite cis entry (measures inside site) Infinite cis exit (measures outside site)
Naftalin and Holman (1977) Lacko el a!. (1972)
34 1.6
6.0 0.6
Karlish et a / . (1972)
25.0
2.15
Hankin et (I/. (1972)
2.8
-
Lacko et a / . (1972)
1.8
-
V,,,
(mM sec
~
1)
exit shows a high K,,, and V,,,,,. However, when the Kn,’s of the inner and outer sites are measured by the two infinite cis procedures, both show symmetrical low K,’s. Equilibrium exchange experiments have until recently been reported to have a high K,,, and V,,,,,. Holman et af. (1981a) have presented evidence for negative cooperativity in the equilibrium exchange of D-glucose in the human erythrocyte, showing nonlinearity in reciprocal plots which reveal two apparent K,’s of 2 and 26 mM. Other cell types also show different kinetic constants for equilibrium exchange and zero trans entry. Whitesell et al. (1977) reported that sugar in the trans solution increased the rate of uptake in thymocytes. Plagemann et af. (1981) have reported similar results with a range of cultured cell types. On the other hand, hepatocyte preparations are similar to the adipocyte with symmetrical zero trans transport parameters for 3-0-methylglucose (Craik and Elliot, 1979). On the basis of kinetic studies it is thus possible to identify at least two classes of mammalian sodium-independent facilitated diffusion systems for hexoses, those which show symmetric, kinetic parameters, and those which show asymmetric parameters.
VI. THE REQUIREMENTS FOR D-GLUCOSE BINDING TO THE ADIPOCYTE HEXOSE TRANSPORT SYSTEM From the K,,, values for transported substrates it is apparent that the relative affinities decrease in the order 2-deoxy-~-glucose (deoxyglucose) > 3-0methyl-D-glucose > (n-glucose) >> L-arabinose > D-allose. To characterize further the binding of D-glucose to the transporter, Rees and Holman (1981) studied the inhibition of D-allose transport by a range of D-glucose epimers, deoxy sugars, fluoro sugars, and other D-glucose analogs [cf. Eq. ( 7 ) ] . From the
360
J. GLIEMANN AND W.
D.REES
relative Ki’s of these analogs it was possible to determine which atoms of the glucose molecule are important for its binding to the transporter. These experiments revealed that the D-glucose molecule binds to the adipocyte transporter and is transported by it in a pyranose ring form. Hydrogen bonds are directed toward the ring oxygen and the oxygen atoms of the hydroxyls at C-1, C-3, and to a lesser extent C-6. The role of the C-4 hydroxyl is not as clear as the other positions but appears to be more important in the absence of a hydroxyl at C-6. There is no requirement for a gluco-configuration C-2 hydroxyl as is the case for the sodium-dependent active sugar transport systems of the intestine and kidney (Crane, 1960; Silverman, 1976). However, it should be noted that these hydrogen bonds need not form simultaneously, but that all are formed at some stage of the transport process. in solution the hexoses will be hydrogen bonded to water and changes in this hydration shell with different analogs may also influence the binding of the molecule to the transporter. The hydrogen bonding requirements of the adipocyte hexose transporter are very similar to those reported for the human erythrocyte by Kahlenberg and Dolansky (1972) and Barnett et af. (1973a), with only slight differences in the affinities of C-4K-6-modified sugars between the two systems. Similar hydrogen bonding requirements have also been reported for hexose transport across the blood-brain barrier (Betz ef af., 1975) and the sodium-independent system of the basal lateral membranes of the small intestine (Wright ef at., 1980).
VII. NONTRANSPORTED COMPETITIVE INHIBITORS OF TRANSPORT Not all competitive inhibitors of transport are transported by the transport system, apparently since spatial restrictions prevent them from passing through the membrane. The use of alkylated D-glucose analogs and disaccharides (Holman et d . , 198I b) has revealed that D-glucose binds to the external site of the transporter through the reducing part of the molecule (C-I) with the nonreducing part of the molecule (C-4) facing the external solution. There is a close approach of the molecule at C-l and C-2 with little space being available around these hydroxyls. There is rather more space around the C-3 hydroxyl which accounts for the transport of 3-O-methyl-~-glucose.Glucose molecules with bulky hydrophobic substitutions at C- 1 or C-416, for example, 4,6-0-ethylidene-~-glucopyranose (4,6-O-ethylidine-~-glucose), n-propyl, or n-butyl-P-~-glucosides,are not transported by the insulin-sensitive hexose transporter, but these compounds are able to enter the cell through an alternative route, probably by nonmediated diffusion (Holman and Rees, 1982). These analogs show asymmetric side-specific competitive inhibition of 3-U-methylglucose transport; 4,6-U-ethylideneD-glucose is an inhibitor on the outside of the cell but does not inhibit on the
HEXOSE TRANSPORT IN ADIPOCYTES
361
inside, while the alkyl-@-r>-glucosidesare effective inhibitors at the inside of the cell membrane but do not inhibit at the outside. A similar situation exists in the human erythrocyte. Baker and Widdas (1973) reported that 4,6-O-ethylidene-~-glucosewas a much more effective inhibitor of the outside site than the inside site. Barnett et al. (1973b, 1975) showed that 6-0alkyl derivatives of D-glucose were inhibitors only outside the membrane while alkyl-@-~-glucosideswere inhibitors only at the inner face of the membrane. On the basis of the results from both the human erythrocyte and the rat adipocyte, similar models for the mechanism of D-glucose transport have been put forward (Fig. 5). The glucose molecule in the external solution binds to the transporter through the C-1 end of the molecule. The transporter protein is then proposed to undergo a conformational change and the glucose molecule is transferred to the inner site with C-1 facing the internal solution. Inhibitor studies have thus revealed an asymmetry of the inner and outer binding sites of the adipocyte glucose transport system which is not shown by the kinetics of hexose transport. In this context it is interesting to note that the
FIG.5 . The proposed structure of the transporter. (a) In the absence ofthe substrate the system is closed. (b) Binding to the external site destabilizes the interface between subunits. Sufficient spacc is available to accommodate a bulky group at C-4. (c) Binding to the internal site opens the internal subunit interface. Sufficient space is available to accommodate a bulky group at C-I. i, 0, Inner and outer sites, respectively. (From Holman and Rees, 1982.)
362
J. GLIEMANN AND W. D. REES
inhibition constants of both the inside and outside-directed side-specific analogs are very similar in both the erythrocyte and the adipocyte. There is no evidence for the 10-fold asymmetry of affinities between the inner and outer sites of the human erythrocyte, which is evident in the zero trans experiments, when the inhibition constants of these analogs are compared in the two cell types. Models such as the asymmetric carrier (Geck, 1971; Regen and Tarpley, 1974) predict such as asymmetry and this observation may provide a further reason for rejecting such models. The observation that D-glucose may inhibit transport by binding to the transporter on the cytoplasmic facing side through the nonreducing part of the molecule should also be considered in experiments with isolated plasma membranes. The currently favoured membrane preparation (McKeel and Jarett, 1970) uses a sucrose-containing buffer for the isolation procedure. If sucrose can gain access to the inner site of the transporter (the inner face of the membrane) as in a membrane preparation it may cause competitive inhibition of transport through the free C-4/C-6 part of the glucose molecule. If so, this will lead to an increase in the apparent K , for transport and may explain the reported K,, of 26 mM for D-glucose (Ludvigsen and Jarett, 1980). It has been suggested that cytochalasin B inhibits hexose transport through binding to the inside facing site in the human erythrocyte (Basketter and Widdas, 1978), and it might therefore by analogy also bind to the inside site of the adipocyte transporter. Therefore, sucrose may also compete for the cytochalasin B binding site reducing the apparent binding in a competitive manner.
VIII.
SUGARS WHICH ARE BOTH TRANSPORTED AND PHOSPHORYLATED-RATE-LIMITING STEPS
2-Deoxy-~-glucoseis phosphorylated by the hexokinase and is not believed to be metabolized any further to any major extent (Wick et a l . , 1951). 2-Deoxyglucose phosphate is trapped in the cells and the total rate of 2-deoxyglucose uptake might therefore be taken as a measure of the rate of 2-deoxyglucose transport when the transport step is rate determining. Olefsky (1 978) used 2-deoxyglucose (3-minute uptakes) in an attempt to characterize the transport system and found a K,, for deoxyglucose of about 1.2 mM (Fig. 2 of the reference) as well as an inhibition constant (Ki) for glucose of about 2 mM. However, it turns out that the hexokinase becomes partially rate limiting for the uptake of 2-deoxyglucose at deoxyglucose or glucose concentrations as low as about 50 pM (Foley er al., 1980b). This shift in the rate-limiting step from transport at a trace sugar concentration to hexokinase at higher sugar concentrations is particularly evident in insulin-stimulated cells due to the high sugar permeability of the plasma membrane. Therefore, the measured inhibition
HEXOSE TRANSPORT IN ADIPOCYTES
363
constants of phosphorylated sugars will depend on the time of incubation: at short times (a few seconds) the measured inhibition constants will reflect mainly K , on the transport system and at infinite time mainly Ki of the hexokinase. At infinite time the apparent Ki for deoxyglucose and glucose is of the order of 100 @ in insulin-stimulated cells; on the other hand, the inhibition constant of deoxyglucose on the initial velocity of methylglucose uptake ( 1 second measure1980b). ments) is about 5 mM (Foley et d.. Recent results have revealed some surprising characteristics of the 2-deoxyglucose transport (Foley and Gliemann, 1981a). In the presence of 2-deoxyglucose at a very low concentration (7 pM) the hexokinase should act as a sink and the uptake should continue at a linear rate in the absence of efflux of 2deoxyglucose phosphate (or 2-deoxyphosphogluconate which is a minor metabolic product of 2-deoxyglucose phosphate). In fact, the uptake curve was linear for only about 10 minutes and this was caused by a slow efflux of free deoxyglucose. Furthermore, a high fraction of the intracellular sugar was present in the free form. Time course studies showed that the intracellular concentration of free deoxyglucose remained essentially zero for about 1 minute. The ratio of intracellular deoxyglucose concentration to extracellular concentration (accumulation ratio) exceeded unity by 3-5 minutes and then continued to increase. By 60 minutes, the intracellular deoxyglucose concentration had exceeded the extracellular concentration by 50-fold. In other words, free deoxyglucose was markedly accumulated in the cell against its concentration gradient. This accumulation was absent in cells depleted of ATP by treatment with dinitrophenol. The mechanism of the accumulation is in part explained by the phosphorylation of newly transported 2-deoxyglucose followed by dephosphorylation. However, it remains to be explained why the 2-deoxyglucose generated by dephosphorylation does not equilibrate with the extracellular medium within seconds. One possihility is that the transport rate of free 2-deoxyglucose out of the cell is much slower than the inward transport. However, this seems highly unlikely, first because the transport system is symmetric with respect to methylglucose and second because the internal deoxyglucose concentration is so low when the accumulation starts that it is difficult to understand how it could exert any inhibition of the transport system. Therefore, it seems necessary to postulate a diffusion barrier between the site of dephosphorylation and the transporter (Fig. 6 ) . In this connection it is worth noting that deoxyglucose phosphate, generated by phosphorylation of deoxyglucose in a tumor cell line, appears to be located in a compartment of a much lower pH (6.4) than that of inorganic phosphate (cytosol, pH 7.1) (Griffith et al., 1981). Other time course experiments (Foley and Gliemann, 1981a) showed that at higher deoxyglucose concentrations, the accumulation of intracellular free deoxyglucose started earlier whereas the steady-state accumulation ratio de-
364
J. GLIEMANN AND W.
D.REES
plasma membrane
5
Hexokinase
/
[14C] DO
-;
l7pMI
(I
rephosphorylation
a
Slow efflux of accumulated [14d DG from postulated compartment
FIG.6 . A model proposed to explain the accumulation of free 2-deoxyglucose against its concentration gradient in adipocytes. DG, Deoxyglucose; DGP, deoxyglucose phosphate. For further explanation, see text.
creased progressively. Thus, a maximum accumulation ratio of 3.5 was reached by 7 minutes using I mM and a ratio of about 1.6 was reached by 3 minutes using 10 mM extracellular 2-deoxyglucose. This phenomenon is probably related to the limited capacity of the hexokinase. It is difficult to predict the effect of intracellular deoxyglucose on the measured transport parameters of other sugars since the accumulation ratios are not necessarily indicative of the internal concentration at the transport site. Recent experiments have shown that high concentrations of phloretin cause a rapid drop in the ATP level of adipocytes and that this is associated with a dephosphorylation of 2-deoxyglucose phosphate (Wieringa et al., 1981). Phloretin was not used in the experiments cited above showing intracellular accumulation of free deoxyglucose. However, the results of Wieringa et al. (1981) demonstrate that the ATP level may be important in regulating the adipocyte phosphatase activity. Marked accumulations of 2-deoxyglucose have previously been reported in mammalian cells, for example, hamster kidney cortex slices (Elsas and McDonell, 1972). However, in this system sugar transport is sodium dependent and active (uphill), and transport clearly precedes phosphorylation. On the other hand, Kleinzeller and McAvoy (1973) have found evidence for a slight accumulation of 0.5 mM deoxyglucose against its concentration gradient following sodium-independent transport across the basolateral membrane of flounder renal cells. Since dephosphorylation of deoxyglucose phosphate also occurs in this system, the mechanism of accumulation might be similar to that postulated for adipocytes. From a physiological point of view, D-glucose is of course the most interesting
HEXOSE TRANSPORT IN ADIPOCYTES
365
sugar. Its inhibition constant on the initial velocity of methylglucose transport is about 8 m M , and this is in agreement with experiments designed to measure uptake of glucose itself (Whitesell and Gliemann, 1979). Similar values have been reported for the inhibition constants of glucose on allose uptake ( 1 3 mM, Loten et al., 1976; and 9 mM, Rees and Holman, 1981) and arabinose uptake (8 mM, Foley et al., 1978). Using the “slow” sugars, the measured inhibition constant would be one of equilibrium exchange ifthe rapidly transported glucose was not metabolized. However, glucose is actually metabolized and transport is rate limiting at low concentrations (Foley et al., 1980d) but not at concentrations above 0.5 mM in insulin-stimulated cells (Gliemann, 1967, 1968). In fact, it has been shown that 2 mM glucose equilibrates across the membrane in insulinstimulated cells (Foley er al., 1980a). Therefore it seems unlikely that K , for net entry is different from that of equilbrium exchange. In other words, the system is probably symmetric with respect not only to 3-O-methyl-~-glucosetransport (as described above) but also to D-glucose transport. In view of the unexpected results with accumulation of free intracellular deoxyglucose, similar experiments were carried out with 7 pV glucose. However, we were unable to detect any accumulation of glucose, which is perhaps not surprising in view of the rapid further conversion of glucose 6-phosphate to metabolites (Foley and Gliemann, unpublished observations). However, this does not rule out that a phosphorylation-dephosphorylation cycle might occur in analogy to the findings with 2-deoxyglucose. The glucose concentration giving half-maximal glucose metabolism is about 1 mM in insulin-stimulated cells (Gliemann, 1968). This is the reason why insulin assays based on glucose metabolism are carried out at a low glucose concentration (Gliemann, 1967; Moody er al., 1974). However, from a physiological point of view this seems paradoxical considering that the plasma glucose concentration varies roughly between 4 and 8 mM. The low “metabolism Km” agrees neither with experiments on epididymal fat pads (Gliemann, 1968) nor with insulin action in vivo. It seems likely that interstitial diffusion gradients exist not only in incubated pieces of adipose tissue, as shown by Crofford and Renold (1965a,b) but also in vivo. Several other metabolizable sugars are transported via the insulin-sensitive glucose transport system but rather little information is available. Mannose at tracer concentration is transported at a slightly slower rate than glucose and is rapidly phosphorylated and metabolized (Foley et al., 1980~). Galactose is transported at about half of the rate of glucose but is phosphorylated at a much slower rate (Vega and Kono, 1978). Fructose is transported slowly by the glucose transporter discussed in this article. However, it should be stressed that the adipocytes possess a specific transport system for fructose which is not influenced by insulin (Schoenle et al., 1979). In “basal” cells, the fructose uptake is almost entirely accounted for by transport via its specific system, whereas the
366
J. GLIEMANN AND W.
D.REES
insulin-sensitive system accounts for about half of the total fructose uptake in insulin-stimulated cells.
IX. MODULATION OF THE TRANSPORT SYSTEM BY GLUCOSE METABOLITES Using 2-deoxyglucose as a probe, it has been found that a high rate of glucose metabolism modulates the transport system. The ability of 2-deoxyglucose to inhibit the initial velocity of 3-0-[ 14C]methylglucoseis slightly greater than that of methylglucose (Foley et af., 1980~).Therefore, it would be expected that 2deoxyglucose was transported at least as rapidly as methylglucose. It is, however, transported at only about one-third of this rate indicating some resistance to the transfer of 2-deoxyglucose across the membrane after its initial binding. Incubation of the insulin-stimulated adipocytes with 10 mM glucose for 30 minutes at 37°C increases the permeability of deoxyglucose to the same level as that of methylglucose (Foley et a/., 1980~).Mannose, which is transported rapidly, phosphorylated, and further metabolized to glucose intermediates, has the same effect. Sugars which are either transported or metabolized slowly have no effect. Thus, a high rate of glucose metabolism modulates the transport system and removes the resistance to transfer of 2-deoxyglucose at a tracer concentration. It is possible that the effect is caused by a feedback of an intermediate of glucose metabolism but no direct evidence is available. It also remains to be clarified whether a high rate of glucose metabolism affects v,, or K,, for transport of 2-deoxyglucose. A model for the hexose transport system would have to account for this phenomenon and we have proposed that shown in Fig. 7. The initial sugar binding occurs at step 1 and here deoxyglucose and methylglucose have equal affinities. The putative glucose metabolite is presumed to act at the cytoplasmic side of the membrane, i.e., at step 3; in the absence of glucose the resistance of step 3 is higher for 2-deoxyglucose than for 3-0-methylglucose, whereas a high rate of glucose metabolism causes a modification of step 3 to give the same resistance for the two sugars. The transmembrane distance between the two points of solution contact of an intrinsic protein or assembly of proteins is rather large as compared with the size of a hexose molecule, and for this reason a diffusive step (step 2) is proposed between the two “discriminators” or “microcarriers” (steps 1 and 3 ) . The properties of the “microcarriers” would be as described by Holman and Rees (1982): the C-1 region of glucose binds to the extracellularly facing (at step 3 pore facing) side; this induces a conformational change and the sugar is let loose on the other side (cf. Fig. 5). Conversely, the C-4 region binds to the cytosolic facing (at step 1 the pore facing) side followed by a conformational change and transfer of the sugar. This model of two re-
HEXOSE TRANSPORT IN ADIPOCYTES
367
Step i 0
FIG. 7. A model of the glucose transporter in adipocytes. The model was proposed to explain the acceleriltion of glucose metabolism on the initial velocity of 2-deoxyglucose uptake. Each of the microcamers is proposed to function as illustrated in Fig. 5. (From Foley and Gliemann, 1981b.)
sistances in series separated by a pore (Fig. 7) eliminates the necessity of a very large protein carrier moving the sugar molecule across the membrane. The kinetic transport parameters of a model of this type will be described by complex equations. The resistances at step 1 and step 3 and the pore volume will determine the flux through the model and detailed predictions depend on the value of these parameters. The model is not incompatible with the available data for transport of 3-0-methylglucose in the adipocyte but more refined experiments are necessary to assign values for the basic model parameters.
X.
MECHANISM OF INSULIN’S ABILITY TO INCREASE V,,,,,
Two groups have recently reported important observations clarifying the cause of the insulin-induced increase in V,,, for 3-0-methylglucose transport. Cushman and co-workers (Wardzala et al., 1978) characterized a specific class of D-glucose inhibitable cytochalasin B binding sites in adipocyte plasma membranes and found that insulin treatment of the cells prior to preparation of the membranes caused a marked increase in the number of sites. The cytochalasin B binding site was taken to be a marker for the transporter and the insulin-induced increase in specific plasma membrane cytochalasin B binding sites should therefore be analogous to the insulin-induced increase in V,,, as shown in Fig. 4. Cushman and Wardzala ( 1980) also found glucose-displaceable cytochalasin B binding sites in a low-density microsomal fraction, and, moreover, treatment of the cells with insulin before the subcellular fractionation caused a marked shift in the distribution of binding sites so that the insulin-induced increase in plasma
368
J. GLIEMANN AND W. D. REES
membrane sites was accompanied by a comparable decrease in the microsomal sites. This shift does not occur in cells depleted of ATP, and neither does the insulin effect on transport (Kono er al., 1977; Siege1 and Olefsky, 1980) or the reversal of the transport activity to the basal state in insulin-pretreated cells (Vega et al., 1980; Laursen et al., 1981). The increment in the plasma membrane cytochalasin B binding sites after treatment with insulin at a high concentration corresponds well to the insulin-induced increase in transport of 3-0methylglucose. Moreover, there is good quantitative agreement between the steady-state insulin dose-response relationships of the transport increase on the one hand and the appearance of cytochalasin B binding sites on the other, as well as between the time course of the two phenomena (Karnieli et af., 1981a). Suzuki and Kono (1980) used a modification of the method described by Shanahan and Czech ( 1977) to solubilize from isolated membranes components which catalyze stereospecific glucose transport. These authors found that insulin caused an increase in the transport activity derived from the plasma membrane and a decrease in the activity derived from a light microsomal fraction. Also the experiments of Kono and co-workers show adequate quantitative correlations between the insulin-induced increase in transport activity of the whole cell and the transport activity that can be extracted from the plasma membrane fraction (Kono et al., 1981). Taken together, these independent experiments provide convincing evidence that hexose transporters are in two pools, one (functional) in the plasma membrane and another (nonaccessible or nonfunctional) at some other location. Moreover, insulin causes a translocation of the transporters from the nonfunctional to the functional pool. The model proposed by Cushmann and co-workers is shown in Fig. 8. There is little doubt that an increase in the number of functional transporters in the plasma membrane is a major effect of insulin in adipocytes and the same mechanism has been proposed in striated muscle (Wardzala and Jeanrenaud, 1981). However, criticism has been raised (Carter-Su and Czech, 1980) and an alternative mechanism has been proposed in the adipocyte (Pilch er al., 1980). The translocation hypothesis explains an observation made by several authors and first by Martin and Carter (1970), namely, that insulin has no effect when added directly to plasma membrane vesicles retaining the stereospecific glucose transport system. This hypothesis is also in agreement with the identical K , values for transport of 3-0-methylglucose in basal and insulinstimulated cells using different experimental protocols (Taylor and Holman, 1981) and with identical K , values for a range of different sugars (Holman et al., 1981b). The question is whether the hypothesis explains the entire insulin effect. Some observations favor at first glance the proposal that insulin also increases the sugar transport across each individual transport unit. Thus, an increase in temperature from 20 to 37°C increases transport of 3-0-methylglucose in insulinstimulated but not in “basal” cells (Czech, 1976a; Whitesell and Gliemann,
369
HEXOSE TRANSPORT IN ADIPOCYTES Dissociation
(9 Translocation
“V
Glucose
-
0 + -. Transport
Glucose
Glucose
\b
\\
\@Fusion
lntracellular Pool
I
d a n s l o c a tion
\-@Binding
7
Plasma
0 Association FIG.8. Schematic representation of a hypothetical mechanism of insulin’s stirnulatory action on 1981.) glucose transport in adipocytes. (From Karnieli et d,,
1979). However, Kono et al. (1981) have observed that decreasing temperature causes a shift in the steady-state distribution of transporters toward the plasma membrane pool, and this may explain the apparent difference in the behavior of transporters in the “basal” and insulin-stimulated state. Sonne et al. (1981) observed that the “basal” but not the insulin-stimulated transport rate increased with increasing pH. There is n o information as to whether this difference might also be due to a shift in the distribution between the pools of transporters. It also remains to be established whether sections of the plasma membrane are pinched off and transferred to an intracellular pool as depicted in the model of Cushrnan and co-workers (Fig. 8). It should be noted that insulin influences various transport systems to different degrees. Thus, transport of the nonmetabolizable amino acid a-aminoisobutyric acid is not stimulated by insulin in the adipocyte (Minemura ef al., 1970) and neither is transport of adenosine (W. D. Rees, unpublished observations). Also, as noted above, transport of fructose through the fructose transporter is not modulated by insulin. Therefore, a model of the type proposed by Kono, Cushman, and their co-workers seems to
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demand that some areas of the plasma membrane are specialized to contain the glucose transporters or that the transporters are able to cluster in such areas. In any case, the transfer, at least from the putative intracellular pool and to the plasma membrane, is surprisingly rapid since the maximal effect of insulin at a high concentration on 3-0-methylglucose transport is manifest by about 1 minute (Whitesell and Gliemann, 1979). At lower (physiological) insulin concentrations the rate-limiting step for activation or deactivation of the transport system appears to be the association or dissociation of insulin from the receptors (Karnieli et af., 1981a). This is in agreement with previous studies on the time course of insulin binding and insulin-induced activation of lipogenesis from glucose (Gliemann et al., 1975). The next question is whether insulin after binding to its receptor causes the formation of a chemical signal which in turn mediates the transfer of transport units from the inactive to the active pool. Larner et al. ( 1979) have extracted a heat- and acid-stable factor from muscle which inhibits cyclic AMP-dependent protein kinase and activates glycogen synthase phosphoprotein phosphatase. Jarett and Seals (1979) have shown that insulin activates pyruvate dehydrogenase in mitochondria provided that plasma membranes are present in the mixture. Later studies showed that insulin can induce the release of a material from adipocyte membranes which appears to mediate its action on pyruvate dehydrogenase and this material was indistinguishable from the “Larner material” (Kiechle et al., 1981). The factor seems to be a peptide with a molecular weight of 1000-4000 (Seals and Czech, 1981; Kiechle et al., 1981) and is perhaps produced from an endogenous substrate by a protease which becomes activated after binding of insulin to its receptor (Seals and Czech, 1980). The putative mediator is probably not a part of the insulin molecule since its release from adipocyte plasma membranes can be initiated by proteases such as trypsin in the absence of insulin (Seals and Czech, 1980). It should also be noted in this connection that irreversible binding of photoaffinity-labeled insulin to adipocytes appears to cause an irreversible activation of the transport system (Ushkoreit et al., 1981). The question remains open as to whether the “Larner material” has a function as a signal for the stimulation of hexose transport. Another possibility is that the intracellular concentration of calcium ions plays a critical role, even though several authors have noted that the effect of insulin on hexose transport is not influenced by extracellular calcium. Clausen and co-workers (Sorensen et al., 1980) have shown that insulin increases the efflux of radiolabeled calcium ion from preloaded adipose or muscle tissue with a similar time course as the increase in transmembrane sugar transport. Thus, insulin may increase the release of calcium ions from intracellular stores and thereby cause a shift in the distribution of transporters. Several other mechanisms have been proposed [see Gliemann et al. (1981) for review].
HEXOSE TRANSPORT IN ADIPOCYTES
XI.
371
HUMAN ADIPOCYTES
Transport of 3-0-methylglucose has been studied using the technique shown in Fig. IC (Ciaraldi et al., 1979; Pedersen and Gliemann, 1981). The transport system appears very similar to that of the rat adipocyte in that K , for net entry as well as for equilibrium exchange of 3-0-methylglucose is about 4 mM. In other words, the system seems to behave symmetrically with respect to 3-0-methylglucose transport. The inhibition constant of glucose on the initial velocity of 3-0-methylglucose uptake is about 8 mM as in the rat adipocyte. Transfer of glucose across the plasma membrane by nonmediated diffusion is also insignificant in the human adipocyte. The main difference between epididymal adipocytes from 200-g rats and abdominal subcutaneous adipocytes from normal weight adult humans is the relatively small response to insulin (two- to threefold) in the human cells. In the “basal” state, the permeability of the human cells is about half of that of the rat cells. Assuming that the turnover of sugar molecules is the same on each transporter from the two species, this indicates that the density of transporters in the human cell is about half of that in the rat cell. In the presence of insulin the permeability of the human adipocyte is about one-tenth of that of the rat adipocyte. It is likely, therefore, that the adipocyte of adult humans is able to recruit only a limited number of additional transport units when treated with insulin. The questions of the orientation of the glucose molecule in the transporter and the spatial and hydrogen binding requirements have not been studied in the human adipocyte but there is no a priori reason to expect any important differences between the species. As in the rat, the rate of metabolism of glucose appears to be limited by the hexokinase and not by transport when insulin is present and the glucose concentration exceeds a few millimoles per liter (Pedersen and Gliemann, 1981).
XII. THE TRANSPORT SYSTEM IN OBESITY AND DIABETES Results from our laboratory using 3-0-methylglucose have shown that the permeability (cdsecond) in the absence of insulin is about the same in small cells from small lean rats and large cells from large obese rats (Foley el al., 1980d). The number of transporters per unit surface area is thus probably quite independent of the cell size. On the other hand, after treatment with insulin the permeability is much smaller in cells from large rats than in cells from small rats. Therefore, it is likely that the cells from obese rats are able to recruit less transporters from the inactive pool after treatment with insulin. Recently, Cushman et al. (198 I ) showed that the number of cytochalasin B binding sites per unit
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area of the plasma membrane was independent of the cell size whereas the insulin-induced increase in the number of binding sites in the plasma membrane was markedly reduced in cells from obese rats. This agrees with the transport studies of Foley et af. (1980d). Earlier studies have shown that the decreased insulin responsiveness with respect to glucose metabolism was related to the degree of obesity of the animals rather than to the adipocyte size per se (Gliemann and Vinten, 1974; Hansen et uf., 1974). These studies were carried out using a low glucose concentration (0.5 mM) and the same conclusion probably applies, therefore, to the glucose transport step. Other authors studying transport in small and large cells (Livingston and Lockwood, 1974; Czech, 1976b; Olefsky, 1976) have obtained different results, probably because 2-deoxyglucose uptake was taken as a measure of transport or because the initial velocity was missed in transport studies using 3-O-methylglucose (for discussion, see Foley et af., 1980d). Streptozotocin-induced diabetes in the rat is associated with a marked reduction in the ability to stimulate glucose transport and metabolism in the adipose cell (Kasuga et af., 1978; Kobayashi and Olefsky, 1979). Recent studies by Cushman and co-workers (Karnieli et af., 1981b) have shown that this, in fact, is associated with a depletion of the pool of cytochalasin B binding sites (and therefore probably hexose transporters) that can be recruited by insulin treatment.
XIII. RECONSTITUTION OF THE HEXOSE TRANSPORTER The reconstitution of intrinsic membrane proteins into an artificial phospholipid bilayer offers a powerful technique for the study of hexose transport. These techniques have been pioneered with the human erythrocyte hexose transporter (Kasahara and Hinkle, 1976) and have now been refined, giving a high efficiency of reconstitution [for recent reviews see Baldwin and Lienhard (1981) and Jones and Nickson (1981)l. Briefly, the results indicate that the purified transporter is a protein of 46,000 molecular weight (Gorga et af., 1979) which binds cytochalasin B in what Baldwin and Lienhard (1981) have suggested to be a 1:l ratio. Studies on the native membrane using radiation inactivation (Jung et al., 1980) suggest, however, that the transporter is larger with a molecular weight of 2 X lo5. Wheeler and Hinkle (1981) have shown that the reconstituted transporter-like the transporter of the intact erythrocyte-shows accelerated sugar transport when sugar is added to the trans side (accelerated exchange). The transporter is incorporated at random in the liposome and the reconstituted system is therefore not asymmetric. However, asymmetry becomes manifest when the liposomes are treated with trypsin which cancels transport in the transporters incorporated “upside down” (Wheeler and Hinkle, 1981).
373
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Reconstitution of the adipocyte hexose transporter has also been reported (Shanahan and Czech, 1977), but since there are few copies of the transporter in the adipocyte membrane, this system has presented greater difficulties. Carter-Su et al. (1980, 1981) have partially purified a protein from rat adipocyte membranes and report that an integral membrane protein can be reconstituted into liposomes which then show stereospecific glucose transport. The transport protein is-in contrast to the glycoprotein insulin receptor-not retained by column chromatography using immobilized concanavalin A (Carter-Su et al., 1981). This does not rule out the possibility that the two proteins are noncovalently associated within the native membrane. The molecular weight of the adipocyte transport protein has not been determined, but the molecule has been reported to have a Stokes radius of 60-80 A (Carter-Su er af., 1981). These molecular dimensions would be sufficient to allow the protein to span the membrane consistent with a model in which substrate binding sites are in contact with each solution. The molecule could therefore provide a channel through which the sugar can move (cf. Fig. 7).
XIV.
CONCLUDING REMARKS
Studies over the last decade have elucidated the kinetics of the binding of insulin to its receptor and the relation between insulin binding and biological effects such as the enhancement of glucose transport. Furthermore, the subunit structure of the insulin receptor has been clarified (for reviews see, for example, Czech, 1980; and Gliemann ef al., 1982). In addition, the characteristics of the insulin-sensitive glucose transporter have been elucidated using adipocytes as a model system. This transporter is similar to that of human erythrocytes with respect to substrate specificity but is different with respect to the kinetic properties of glucose transport. The insulin-induced increase in hexose transport seems to be brought about by a transfer of transporters to the plasma membrane from a storage pool. Future experiments may clarify the important questions of the precise nature of the transfer process and the properties of a possible chemical mediator. ACKNOWLEDGMENTS W. D. Rees is a recipient of a NATO-SERC overseas postdoctoral fellowship. The authors wish to thank Drs. S . W. Cushman, G . D. Holman, and J . Vinten for allowing us to reproduce their published figures. REFERENCES Andreasen, P . , Schaumburg, B . , Plsterlind, K., Vinten, .I.Gammeltoft, , S . , and Gliemann, J . ( 1974). A rapid technique for isolation of thymocytes from suspension by centrifugation through silicone oil. Anal. Biochcm. 59, 110-1 16.
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0rskov. S . L. ( I 935). Eine Methode zur fortlaufenden photographischen Aufzeichung von Volumanderungen der roten Blutkorperchen. Biochem. Z. 279, 241-249. Pedersen, O., and Gliemann, I. (1981j. Hexose transport in human adipocytes; factors influencing the response to insulin and kinetics of methylglucose and glucose transport. Diahetologia 20, 630-635. Pilch. P. F., Thompson, P. A.. and Czech, M. P. (1980). Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulintreated adipocytes. Proc. Natl. Acad. S r i . U.S.A. 77, 915-918. Plageman, P. G . W., Wohlheuter, R. M., Graff. J.. Erbe. J., Wilkie, P. (1981). Broad specificity hexose transport system with differential mobility of loadcd and empty carrier but directional symmetry is a common property of mammalian cell lines. J . B i d . Chem. 256, 2835-2842. Rees. W. D., and Holman, G. D. (1981). Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes. Biochim. Eiophys. Acta 646, 251-260. Regen. D. M., and Tarpley, H. L. (1974). Anomalous transport kinetics and the glucose carrier hypothesis. Biochim. Biophvs. Acta 339, 2 18-233. Rodbell, M. (1964). Mctabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. B i d . Chem. 239, 375-380. Schoenle, E., Zapf, J., and Froesch, E. R. (1979). Transport and metabolism of fructose in fat cells of normal and hypophysectomized rats. Am. J. Physiol. 237, E325-330. Seals, J. R., and Czech, M. P. (1980). Evidence that insulin activates an intrinsic plasma membrane protease in generating a secondary chemical mediator. J. Eiol. Chem. 255, 6529-6531, Seals. J. R., and Czech, M. P. (1981). Characterization of a pyruvate dehydrogenase activator released by adipocyte plasma membranes in response to insulin. J . B i d . Chem. 256, 2894-2899. Sen. A. K., and Widdas, W. F. (1962) Determination of the temperature and pH dependence of glucose transfer across the human erythrocyte membrane measured by glucose exit. J . Physiol. (London) 160, 392-403. Shanahan, M. F., and Czech, M. P. (1977). Purification and reconstitution of the adipocyte plasma membrane o-glucose transport system. J . Biol. Chem. 252, 8341-8343. Siegel, J . , and Olefsky, J. M. (1980). Role of intracellular energy in insulin’s ability to activate 3-0methylglucose transport by rat adipocytes. Biochemistr?, 19, 2183-2 190. Silverman, M. (1976). Glucose transport in the kidney. Biochim. Biophvs. Acta 457, 303-351. Sonne. O., Gliemann, J., and Linde, S . (1981). Effect of pH on binding kinetics and biological effect of insulin in rat adipocytes. J. Biol. Chem. 256, 6250-6255. Sbrensen, S. S., Christensen, F., and Clausen, T. (1980). The relationship between the transport of glucose and cations across cell membranes in isolated tissues. X . Effect of glucose transport stimulation on the efflux of isotopically labelled calcium and 3-0-methylglucose from soleus muscles and epididymal fiat pads of the rat. Biochim. Bivphys. Acta 602, 433-445. Suzuki, K . . and Kono, T. (1980). Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site. Proc. Nail. Acad. Sci. U.S.A. 77, 2542-2545. Taylor, L. P., and Holman, G. D. (1981). Symmetrical kinetic parameters for 3-O-methyl-~-glucose transport in adipocytes in the presence and in the absence of insulin. Biochim. Biophys. Aria 642, 325-335. Thorsteinsson, B., Gliemann, J., and Vinten, J. (1976). The content of water and potassium in fat cells. Biochim. Biophys. Acta 428, 223-227. Uschkoreit, J . , Brandenburg. D., and Gliemann, J. (1981). Photoaffinity labelling of insulin receptors: Correlation of receptor occupancy and stirnulation of glucose transport in adipocytes. I n “Current Views on Insulin Receptors” (D. Andreani, ed.), pp. 317-322. Academic Press, New York.
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Vega. F. V., and Kono. T. (1978). Effects of insulin on the uptake of o-galactose by isolated rat epididymal fat cells. Biochim. Biophvs. Actu 512, 221-222. Vega. F. V . , and Kono, T. (1979). Sugar transport in fat cells. Effects of mechanical agitation cellbound insulin. and temperature. Arch. Biochem. Biophvs. 192, 120- 127. Vega. F. V., Key, R. J . , Jordan, J. E., and Kono. T. (1980). Reversal of insulin effects of fat cells may require energy for an activation of glucose transport but not for an activation of phosphodiesterase. Arch. Biochem. Biophys. 203, 167- 173. Vinten, J. (1978). Cytochalasin B inhibition and temperature dependence of 3-0-methylglucose transport in fat cells. Biochim. Biophys. Actu 511, 259-273. Vinten. J.. Gliemann, I., and Dsterlind. K. (1976). Exchange of 3-0-methylglucose in isolated fat cells. Concentration dependence and effect of insulin. J . Biol. Chem. 251, 794-800. Wardzdla, L. J., and Jeanrendud, B. (1981). Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. J . B i d . Chem. 256, 7090-7093. Wardzala, L. J.. Cushman, S. W.. and Salans, L. B. (1978). Mechanism of insulin action on glucose transport in the isolated rat adipose cell. J . B i d . Chem. 253, 8002-8005. Wheeler. T. 1.. and Hinkle, P. C. (1981). Kinetic properties of the reconstituted glucose transporter from human erythrocytes. J . Biol. Chem. 256, 8907-8914. Whitesell, R. R., and Gliemann, J. (1979). Kinetic parameters of transport of 3-0-methylglucose and glucose in adipocytes. 1. B i d . Chem. 254, 5276-5283. Whitesell, R. R . . Tarpley, H. L.. and Regen, D. M. (1977). Sugar transport kinetics of the rat thymocyte. Arch. Biochem. Biophvs. 181, 596-602. Wick. A. N . , Drury, D. R., Nakada, H . , a'nd Wolfe, J . B. (I951). Location of the primary metabolic block produced by 2-deoxy-o-glucose. J . B i d . Chem. 224, 963-969. Widdas, W. F. (1980). The asymmetry of the hexose transfer system in the human red cell membrane. Curr. Top. Membr. Transp. 14, 165-223. Wieringa, T. J., van Putten, J . P. M . , and Krans, H. M. J. (1981). Rapid phloretin-induced dephosphorylation of 2-deoxy-o-glucose-6-phosphatein rat adipocytes. Biochem. Biophys. Res. Commun. 103, 841-847. Wilbrandt, W. (1954). Secretion and transport of nonelectrolytes. Symp. SOC. Exp. Biol. 8, 136-162. Wright, E. M.. van Os, C. H., and Mircheff, A . K. (1980). Sugar uptake by intestinal basolateral membrane vesicles. Biochim. Biophvs. Acta 597, 112-124.
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 18
Epidermal Growth Factor Receptor and Mechanisms for Animal Cell Division MANJUSRl DAS Depurtmenr of Biochemistry and Biophysics University of Pennsylvania School of Medicine Philadelphia. Pennsylvunia
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Properties of E G F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The EGF Receptor . . .......... A. Identification o f t .......................................... B . Clustering, Internalization, and Degradation of EGF-Receptor Complexes . . . . . C. Protein Kinase Domain of the EGF Receptor and Its Relationship to the Oncogene Product, pp6oSrc . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . D. Antibodies Directed against the EGF Receptor.. . . . . . . . . . . . . . . . . . . . . . . . . . . E. Receptor Regulation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Studies on Location of the EGF-Receptor Gene.. ......................... G. lnsertion of Exogenous EGF Receptors into Receptor-Negative Variant Cells.. . IV. The Pathway to Nuclear DNA Replication.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Mitogenic Capability of EGF ............. B. The Mitogenic Pathway . . . . . . . . ............................. C. Biochemical Signals for Mitogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. A Family of EGF-like Polypeptides and Their Role in Animal Development and Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ... ........................................
1.
381 382 383 383 386 387 388 390 391 392 393 393 393 396 398 400
INTRODUCTION
One of the central problems in modern biology is the understanding of the mechanism by which extracellular molecules such as hormones, toxins, and neurotransmitters interact with surface receptors to regulate intracellular events. 381 Copyright ((1 l Y X 3 by A w k r n i c Pres. Inc All rights of reprodu~cmIn any forni rc\erved ISBN 0-12-1533 18-2
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Epidermal growth factor (EGF), a single chain polypeptide of -6000 daltons (Carpenter and Cohen, 1979), belongs to a new class of cytomodulatory factors that are hormone-like in their biological potencies. EGF stimulates DNA replication and cell division in a wide variety of cells including those of nonepidermal origin, and the EGF receptor, a cell surface polypeptide of 170,000- 180,000 daltons, has a wide tissue distribution. Among the various growth factors isolated to date, EGF is one of the most potent and best characterized as to its physical, chemical, and biological properties, and the EGF-receptor system has been an important stimulus to the development of new ideas on receptor action and mitogenesis. During the last decade there has been an exponential growth of literature and reviews on EGF (Carpenter and Cohen, 1979; Hollenberg, 1979; Adamson and Rees, 1981). The present article briefly recapitulates some of the earlier findings, and discusses in detail some of the more recent developments in this area.
II. PROPERTIES OF EGF EGF was first isolated from mouse submaxillary glands by Cohen (1962). It was described as an epidermal tissue stimulatory factor that caused precocious eyelid opening and tooth eruption in newborn mice, and was hence named epidermal growth factor. In an independent study, Gregory and his colleagues isolated a polypeptide from human urine that inhibited gastric acid secretion, and named it urogastrone (Gregory, 1975; Gregory and Willshire, 1975; Gregory and Preston, 1977). Comparison of amino acid sequences revealed regions of structural homology between the 53 residues long mouse and human polypeptides (Savage et al., 1972; Gregory, 1975). This led to a comparison of biological properties. It was found that both the mouse and human polypeptides shared tissue growth-stimulatory and gastric acid-inhibitory properties, and were capable of competing equally for the same receptor sites in a variety of animal tissues. This suggests that both peptides belong to a family of mitogenic, acid-inhibitory polypeptides that show some interspecies structural variations, but are probably near-identical in their active site regions that are responsible for receptor binding and biological activity. Normal plasma concentration of EGF in adults is 0.1-0.2 nM, and this is subject to hormonal modulations (Bynny et al., 1974; Barka et al., 1978). a-Adrenergic agents stimulate the release of EGF from submaxillary glands into plasma. Androgens increase the levels of EGF in submaxillary glands (Bynny et al., 1972; Barthe et a / . . 1974) but do not appear to stimulate its release into plasma. An in vivo role for the EGF-receptor system in embryonic and organ development is suggested by various studies on EGF binding and EGF action on embry-
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onic, amniotic, placental, and other developing tissues (Hassel, 1975; Hassel and Pratt, 1977; Ladda et al., 1979; Nexo et al., 1980). In adult animals, EGF in the gastrointestinal tract is likely to mediate regulation of gastric acid secretion (Gregory, 1975) and replenishment of the rapidly turning over intestinal epithelial cells (Forgue-Laffite et al., 1980). In addition, EGF in adults could mediate other vital processes, the nature of which is not yet known. Given the wide tissue distribution of the EGF receptor (O'Keefe et al., 1974), it is tempting to propose that EGF plays an important role as fundamental as other hormones in the well being and survival of animals.
111.
THE EGF RECEPTOR
A. Identification of the Receptor Specific and high-affinity receptors for EGF are present in a wide variety of cells including those of nonepidermal origin. Rapid and saturable binding of '251-labeled EGF has been demonstrated in fibroblasts (Hollenberg and Cuatrecasas, 1973; Carpenter et al., 1979, corneal cells (Frati et al., 1972; Gospodarowicz et al., 1977), lens cells (Hollenberg, 1975), kidney cells (Holley et al., 1977), intestinal epithelial cells (Forgue-Laffite et al., 1980), human glial cells (Westermark, 1977), 3T3 cells (Pruss and Henchman, 1977; Aharonov et al., 1978), granulosa cells (Vlodavsky et al., 1978), human epidermal carcinoma cells (Fabricant et d . , 1977), and human vascular endothelial cells (Gospodarowicz et al., 1978). Apparent dissociation constants for binding are in the range of 0.1-1 nM (Hollenberg and Cuatrecasas, 1973; Carpenter et al., 1975; Aharonov et al., 1978). The number of receptor sites per cell varies from 4- 10 x lo4 in fibroblastic cells (Hollenberg and Cuatrecasas, 1973; Carpenter et al., 1975; Das et al., 1977) to 1-2 X lo6 in human epidermal carcinoma cells (Haigler et al., 1978). The EGF receptor was first identified using a chemical cross-linking technique (Das et al., 1977; Das and Fox, 1978). Photoreactive derivatives of EGF were used to label and identify specifically the membrane receptor for EGF in murine 3T3 cells. Photoreactive arylazide derivatives of radioiodinated EGF were prepared using arylazide heterobifunctional cross-linking reagents, which are useful in identification of ligand-binding components in complex biological systems (Das and Fox, 1979). Photoactivable EGF, labeled with IZ5I, was incubated with 3T3 cells and then photolyzed in situ to generate a nitrene capable of reacting with a wide variety of chemical bonds. Analysis of the system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed besides the band of EGF, only one other major radioactive band at a position indicating an apparent molecular weight of 190,000. This band was absent when a nonresponsive and
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nonbinding variant of 3T3 was used. A direct proportionality between binding activity and cross-linked complex formation was demonstrated using a variety of binding conditions. The photoactivable derivative of EGF thus acted as a typical affinity label for its receptor, and there appeared to be only one protein (M, 184,000) involved in specific recognition and binding of EGF to 3T3 cells (Das et al., 1977) (Table I). This finding on receptor molecular weight was confirmed by Sahyoun et al. (1978) and Hock et al. (1979), who undertook the labeling of the 1251-labeled EGF binding components in placental and liver membranes by glutaraldehyde cross-linking followed by sodium borohydride reduction (Table I). In experiments with human placental membranes (Hock et al., 1979), two labeled components of M, 160,000 and 180,000 were observed. The latter value is in good accord with the value of M, 184,000 for the murine receptor (Das et ul., 1977). Same labeled components (M, 160,000 and 180,000) were observed after labeling human placental membranes with a photoaffinity analog of EGF. It was suggested that the two constituents observed in human placenta could be interrelated by either a biosynthetic or a degradative process. More recently, Cohen et ul. (1980, 1982) purified the human EGF receptor by using a procedure involving solubilization and affinity purification. Plasma membranes from human A-431 carcinoma cells which are exceptionally rich in EGF receptors were used in these studies. The receptor was solubilized with Triton X-100 and was purified by affinity chromatography on columns of agarose containing covalently bound EGF. Plasma membranes prepared using the procedure of Thom et al. (1977) yielded a receptor protein of M, 150,000 (Cohen et al., 1980). This receptor molecular weight is slightly smaller than the earlier reported molecular weights on the A-431 receptor (Wrann and Fox, 1979), the human placental receptor (Hock et al., 1979), and the murine 3T3 receptor (Das el al., 1977) (Table I). However, when plasma membrane vesicles were prepared using a rapid “hypotonic shedding procedure,” a higher receptor molecular weight (170,000) was observed (Cohen et al., 1982). It was suggested that the 170,000 M, protein is proteolytically degraded to a 150,000 form which retains its EGF binding function. A glycoprotein structure for the EGF receptor was proposed based on the ability of various lectins to inhibit reversibly the binding of ‘2sI-labeled EGF to human fibroblasts and to placental membranes (Carpenter and Cohen, 1977). In fact, lectin affinity columns have been useful in effecting considerable purification of the receptor (Hock ef al., 1980). Additional evidence for a glycoprotein structure comes from the finding that treatment of cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, results in a progressive loss of EGF-receptor activity (Bhargava and Makman, 1980). Also a mutant 3T3 cell line defective in protein glycosylating activity was found to be deficient in EGF binding activity (Pratt and Pastan, 1978).
TABLE I MOLECULAR WEIGHTDETERMINATIONS ON THE EGF RECEPTOR Source Murine 3T3 cells Human placental membranes Human placental membranes Human Munne Human Human
foreskin fibroblasts 3T3 cells A-431 carcinoma cells A-43 1 carcinoma cells
Method Specific labeling of the receptor with photoaffinity analogs of 1251-labeledEGF Glutaraldehyde cross-linking of 1251-labeled EGF to the receptor Specific labeling of the receptor with photoaffinity analogs of '251-labeled EGF Direct labeling of the receptor with 1251-labeled EGF Direct labeling of the receptor with 1251-labeled EGF Direct labeling of a receptor with ~251-labeledEGF Solubilization of the receptor from membranes followed by its affinity purification on EGF-agarose columns
Molecular weight 184,000 160,000 and 180,000 160,000 and 110,000 184,000 184,000 175.000 170,000
Reference Das er a/.(1977)
Hock et af. (1979) Hock er af. (1979) Baker el al. (1979) Linsley et a/.(1979) Wrann and Fox (1979) Cohen et a/.(1982)
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An interesting property of the EGF receptor is its ability to become covalently attached to bound EGF at 37°C (Baker et al., 1979; Linsley et al., 1979). This has allowed identification of the receptor in A-43 1 cells (Wrann and Fox, 1979). Only a portion of cell-bound EGF becomes covalently linked in this fashion, and known inhibitors of transglutaminase do not inhibit this reaction. The biological role or nature of this covalent linkage remains unclear.
B. Clustering, Internalization, and Degradation of EGF-Receptor Complexes The EGF-receptor system has been an important stimulus to the development of ideas on the mechanism of receptor-mediated ligand endocytosis. Carpenter and Cohen ( 1976a) showed that at 37°C cell bound 12sI-labeledEGF is degraded very rapidly with the appearance in the medium of [ 12sI]monoiodotyrosine.The degradation at 37°C is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol) and by a lysosomotropic agent (chloroquine). This suggests that cell surface bound 12sI-labeledEGF is rapidly internalized in an energy-dependent step, and then degraded within lysosomes (Carpenter and Cohen, 1976a). Direct visualization of the process of internalization has been reported by Gordon et af. (1978), Schlessinger et al. (1978), and Haigler et al. (1979). Different techniques were used in these studies, namely, electron microscope autoradiography of 12sI-labeledEGF, tracing of fluorescent derivatives of EGF, and visualizing EGF-ferritin conjugates by electron microscopy. In each case, binding of EGF to dispersed receptors on the cell surface was shown to be followed by surface aggregation and subsequent internalization of the EGF label into pinocytic vesicles or “receptosomes” (Pastan and Willingham, 198I), leading ultimately to its appearance in lysosome-like structures. The studies described above strongly indicate that EGF binding to dispersed receptors on the cell surface leads to receptor clustering and endocytosis. A direct study on the fate of the receptor was performed using the photoaffinity labeling approach outlined earlier (see Section 111,A). Murine 3T3 cells carrying in situ radiolabeled receptor (prepared using a photoreactive derivative of EGF) were incubated at 37°C for increasing time intervals (Das and Fox, 1978). There was a time-dependent reduction of radioactivity from the radiolabeled receptor band of M, 190,000, and the loss was accompanied by the appearance of three distinct low-molecular-weight bands of M, 62,000, 47,000, and 37,000. The radioactivity lost from the receptor band was recovered almost quantitatively from the low-molecular-weight bands, suggesting a precursor-product relationship between these proteins. Subcellular fractionation of cells containing the radiolabeled receptor and its degradation products revealed that the low-molecular-weight proteins banded in sucrose gradients with lysosomes, whereas the
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receptor cofractionated with the plasmalemmal fraction. These results suggest an endocytic degradative fate for the surface receptor after binding to EGF. It should be noted, however, that the rate of receptor processing/degradation in 3T3 cells is slow (about one-fifth) compared with the rate of EGF degradation. This suggests that most (about 80%) of the endocytosed receptors are perhaps not degraded, but recycled back to the plasma membrane.
C. Protein Kinase Domain of the EGF Receptor and Its Relationship to the Oncogene Product, pp6PrC Addition of EGF to A-43 1 plasma membranes results in a marked stimulation of cyclic nucleotide-independent phosphorylation of endogenous membrane proteins, including the EGF receptor (Carpenter er al., 1978, 1979; King et al., 1980b; Ushiro and Cohen, 1980; Cohen et al., 1980). The reaction was found to involve specific phosphorylation of tyrosine residues in substrate proteins, and that put it outside the common class of protein kinases which phosphorylate serine and threonine. Treatment of A-43 1 plasma membranes with Triton X-100 results in solubilization of both the EGF receptor and the kinase activity. Purification of the receptor on EGF-affinity columns results in a copurification of the kinase activity, suggesting a tight association between the receptor and the kinase (Cohen et al., 1980). More recently, it has been shown that antibodies directed against the EGF receptor can coprecipitate both the 170,000-dalton receptor protein and the kinase activity (Cohen et al., 1982). This strongly suggests that EGF binding activity and kinase activity are covalently linked, and that both activities may reside in the same 170,000-dalton polypeptide, in different domains within the same molecule. Linsley and Fox (1980) showed that EGF receptors on intact A-43 1 cells are autophosphorylated only when the cells are permeabilized with lysolecithin. More recently it has been shown that the purified 170,000-dalton receptor has a good capacity for autophosphorylation, but the degradation product of 150,000 daltons (which can bind EGF, but presumably has lost the tail end of the molecule that extends into the cytoplasm) is a poor substrate for autophosphorylation (Cohen et al., 1982). However, when challenged by exogenous substrates, the 150,000-dalton receptor is a better kinase compared with the 170,000-dalton receptor. This could be due to greater availability of phosphorylation sites in the 170,000-dalton receptor compared with the 150,000 form, which would account for the lower apparent kinase activity of the 170,000 form toward exogenous substrates (Cohen et al., 1982). Thus, the EGF receptor appears to be a multifunctional, multidomain protein, whose inherent kinase function is activated after EGF interacts with its external binding site. Cohen et al. (1982) compared the activation of receptodkinase by
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EGF "to the activation of ribonuclease S by S peptide, where peptide binds to the cleaved ribonuclease and converts an inactive enzyme to an active one." This raises the question of whether EGF and its receptors were parts of one functional protein whose functional domains became separate during evolution. In its specificity for tyrosine phosphorylation, the EGF receptodkinase resembles the tyrosine-specific kinase activities associated with the transforming proteins (oncogene products, pp60v-src) of several RNA tumor viruses (Hunter, 1980; Bishop, 1981; Erikson and Erikson, 1980); i.e., tyrosine-specific protein phosphorylation appears to be intimately associated with both virus-induced cell transformation and the action of a normal stimulant of cell division, EGF [recent studies (Ek et al., 1982) suggest that the action of platelet-derived growth factor, another normal stimulant of cell division, also involves tyrosine-specific phosphorylation]. Although oncogenes were first found in viruses, subsequent studies revealed the gene to be present in normal nontransformed cells. The product of cellular oncogene (named pp60c-src)was found to be indistinguishable (in terms of structure or activity) from the viral product p ~ 6 0 ~ -The " ~ ~version . of c-src found in fishes, birds, and mammals are all closely related to the viral gene v-src and to one another. The small amounts of pp60-src found in normal cells is not sufficient to induce cellular transformation, but it may well be required for the well being of the cells, as suggested by the survival of c-src through long periods of evolution. Perhaps cellular oncogenes (i.e., the genes for tyrosine-specific protein kinases) are part of a delicately balanced network of controls that regulate the growth and development of normal cells. Excessive activity of one of these genes might tip the balance of regulation toward incessant growth. The kinase domain of the EGF receptor (and perhaps of other growth factor receptors) may have evolved from an ancestral oncogene, and there may exist certain structural similarities between the oncogene product, pp6OC-"" or pp6OV-"' , and the mitogenic message transmitting kinase domain of the EGF receptor.
D. Antibodies Directed against the EGF Receptor In 1980, Haigler and Carpenter reported the preparation of an anti-EGFreceptor antiserum which was obtained after immunization of rabbits with human A-43 1 carcinoma cell membranes. The IgG fraction of this immune serum blocked 1251-labeledEGF binding to human and murine EGF receptors and also blocked the induction of DNA synthesis in quiescent fibroblasts by EGF. However, this antiserum was not receptor-specific and was capable of immunological interactions with nonreceptor proteins. Recently, however, three other groups have reported the preparation of specific anti-EGF-receptor antibodies (Schreiber et al., 1981a; Cohen et al., 1982; Carlin and Knowles, 1982). Schreiber et af. (1981a) have described the preparation of monoclonal murine
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antibodies against the human A-431 EGF receptor. The antibodies, of IgM type, were capable of inhibiting 1251-labeledEGF binding to both human and murine EGF receptors. In addition, these monoclonal IgM antibodies induced EGF-like biological effects. Like EGF, they enhanced protein phosphorylation in A-43 1 membranes, and stimulated DNA synthesis in human fibroblasts. In their ability to activate the EGF receptor, they resemble the anti-insulin-receptor antibodies that have been shown to exert potent insulin-like effects on cells (Kahn et a/., 1977). Although these monoclonal IgM antibodies are EGF-like, polyclonal IgG antibodies directed against the EGF receptor are incapable of producing any EGF-Iike biological effects (Haigler and Carpenter, 1980; Carlin and Knowles, 1982). Schreiber et a/. (1981a) suggest that this difference could be a consequence of the better receptor cross-linking ability of the decavalent IgM compared with the bivalent IgG antibodies. A different type of anti-EGF-receptor antibody was prepared by Cohen et a/. (1982). The affinity-purified A-431 EGF receptor was subjected to SDS-gel electrophoresis and the Coomassie blue-stained 170,000-dalton band in the gel was minced and used for immunizing rabbits. The antiserum obtained against the denatured receptor was capable of immunoprecipitating the solubilized EGF receptor and the associated kinase, but it did not inhibit the binding of 1251labeled EGF to the A-43 1 receptor, and did not inhibit basal or EGF-stimulated phosphorylation. Yet another type of anti-EGF-receptor antibody became available through a serendipitous route. It has been known for some time that specific IgG antibodies are produced against a M , 165,000 human protein when human-mouse somatic cell hybrids (containing chromosome 7 as the only human chromosome) are injected into syngeneic mice (Aden and Knowles, 1976; Ford et af., 1978). After it became known that the human EGF-receptor gene was associated with chromosome 7 (see Section II1,G) (Shimizu et a / . , 1980; Davies et al., 1980), the antibody was tested for anti-EGF-receptor activity, and it was found to be a potent inhibitor of EGF binding in human cells but not in murine cells (Carlin and Knowles, 1982). Immunoaffinity chromatography of human A-43 I cellular proteins on an antibody-agarose column resulted in the purification of a protein ( M , 175,000) which comigrated with the human EGF receptor during electrophoreses under reducing, denaturing conditions (Das et a/., 1982). The interaction of this antibody with the human EGF receptor was studied in further detail using affinity-purified 1251-labeledantibodies (Das et al., 1982). The IgG fraction of antisera was labeled with 1251. The 1251-labeledantireceptor antibody, which initially represented about 0.2% of total I2'I-labeled IgG, was enriched by selective adsorption to and subsequent elution from human WI-38 cells which contain EGF receptors. The purified I 251-labeled antireceptor antibody bound to human tissue culture cells (A-43 1 and human fibroblasts) and to human placental membranes in a time-, temperature-, and concentration-dependent manner. No
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binding was observed with the murine EGF receptor in 3T3 cells or in hepatic membranes. The binding of antibody to human cells was inhibited by unlabeled antibody and EGF, but not by nonimmune mouse IgG or hormones such as insulin and fibroblastic growth factor (FGF). For human EGF receptors of diverse origin (fibroblasts, A-43 1, and placenta) the ratios of 1251-labeledantibody binding activity to 12sI-labeledEGF binding activity were about the same, suggesting a close molecular similarity between these receptors from different sources. The preparation of high specific activity It51-labeled antireceptor antibodies by cytoadsorption and elution thus provides a sensitive method for detection and characterization of receptors.
E. Receptor Regulation 1. EGF-INDUCED REGULATION
A “down-regulation” phenomenon, quite similar to other receptor-macromolecule interactions such as antigenic modulation, has been observed with the EGF-receptor system (Carpenter and Cohen, 1976a; Aharonov et al., 1978; Das and Fox, 1978). It has been suggested that the hormone-induced loss of receptors is due to endocytic remova: of surface EGF-receptor complexes, without the concomitant production of new receptors. It could also be partly due to an EGFinduced drastic increase in receptor affinity leading to the formation of nondissociable EGF-receptor complexes. Down-regulated cells can be stimulated to regain receptors by removal of EGF and addition of serum (Carpenter and Cohen, 1976a). A 100% regain of receptors can be achieved within 9 hours. This recovery process is inhibited by cycloheximide or actinomycin D. This suggests the involvement of de now synthesis of receptors or of nonreceptor labile proteins which play a crucial role in receptor recycling. 2. REGULATIONBY STRUCTURALLY UNRELATEDAGENTS Lee and Weinstein ( 1978) demonstrated that tumor-promoting phorbol esters caused a rapid and marked inhibition of EGF binding to its receptors. It was initially thought that the reduction in binding was due to a decrease in the number of available EGF-receptor sites; but later it was shown that the number of EGFreceptor sites was not reduced, only the binding affinity was markedly decreased (Lee and Weinstein, 1979; Brown et al., 1979). The inhibitory effect was reversed upon the removal of phorbol esters from the medium. The effect of phorbol esters is EGF receptor-specific, because other non-EGF-
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receptor activities are unaffected. The inhibitory effect of phorbol esters is temperature sensitive. It is inhibitory at 37°C but not at 4°C. More recently, it has been shown that vitamin K, (a quinone) and vasopressin (a neurohypophyseal nonapeptide hormone) also markedly reduce the affinity of EGF receptors for '251-labeled EGF in a time- and temperature-dependent fashion (Shoyab and Todaro, 1980; Rozengurt e t a l . , 1981a). The properties of the inhibition of EGF binding by these agents have many similarities with those of the phorbol ester family. The inhibition of EGF binding by these agents does not require protein synthesis or degradation, but is completely blocked by reducing the temperature to 4°C. Such findings have led investigators to suggest that these inhibitory agents (vitamin K,, vasopressin, phorbol ester) bind to sites which are separate from the EGF receptor. (This is consistent with the absence of any structural analogy between these molecules.) It was proposed that the phorbol esterhasopressidvitamin K,-occupied receptors interact with the EGF-receptor sites in a diffusionally controlled, temperature-sensitive step, and thereby reduce the affinity of the EGF receptors for EGF.
F. Studies on Location of the EGF-Receptor Gene Somatic cell genetic techniques were used for studies on chromosomal location of the EGF-receptor structural gene (Shimizu et al., 1980; Davies et al., 1980). Fusion of mouse and human cells results in the formation of hybrids that usually retain all the mouse complement of chromosomes and a small random subset of human chromosomes. Because both the murine and human genes are usually functional, one can assay each hybrid clone for the presence of a given gene product. In these studies mouse cell mutants deficient in hypoxanthine phosphoribosyltransferase, and devoid of '251-labeled EGF binding activity were fused with human diploid cells, possessing EGF binding ability. The humanmouse cell hybrids were isolated after hypoxanthine/aminopterin/thymidineselection. Analysis of isozyme markers and chromosomes of a number of these human-mouse clones indicated that the expression of EGF binding ability is correlated with the presence of human chromosome 7. These results suggest that a gene on chromosome 7 could code for human EGF receptor or complement a deficiency in the mutant mouse cells. Immunologic analysis confirmed that the receptor in these human-mouse clones is nonmurine, and of human origin (Carlin and Knowles, 1982). A number of EGF-receptor-negative variants of murine 3T3 cells have been isolated by Pruss and Henchman (1977) using the colchicine selection technique. These variants have served as excellent specificity controls for studies on identification and insertion of the EGF receptor (Das et al., 1977; Bishayee et
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a / . , 1982). However, the genetic lesions that are responsible for the
loss of
receptor activity in these cells remain uncharacterized.
G. Insertion of Exogenous EGF Receptors into ReceptorNegative Variant Cells Polyethyleneglycol-mediated membrane fusion techniques have been used for putting foreign receptors into recipient cells, and for combining components from two different types of cells for studies on coupling between hormone receptor complexes and adenyl cyclase (Schramm e r a / ., 1977). It is intriguing to note that an exogenous EGF receptor can be inserted into a recipient cell (in a biologically active orientation) by a novel mechanism requiring no added fusogenic agent (Bishayee er al., 1982). A variant cell line NR-6, derived from mouse 3T3, can neither bind nor biologically respond to EGF (Pruss and Herschman, 1977). When these NR-6 cells were incubated with EGF-receptor-rich mouse hepatic membranes at 26°C for 6 hours in the absence of any added fusogen, there was a transfer of almost 20% of input EGF receptors to NR-6 cells, whereas only 1-2% of bulk hepatic proteins were transferred in a similar fashion. The results suggest a preferential insertion of the EGF receptor over the other hepatic proteins. Experiments with cycloheximide and tunicamycin suggest that the receptor gain by the NR-6 cells was not due to an activation of endogenous protein synthesis or to a glycosylation-induced activation of preexisting aglyco-receptors in NR-6 cells. The inserted receptor bound 12s1-labeledEGF with high affinity, and EGF was found to stimulate D N A synthesis (fourfold maximally) and cell division (twofold maximally) in these membrane-treated NR-6 cells in a concentration-dependent manner (this biological stimulation is dependent upon the quality of the membranes and is not always reproducible). In contrast, NR-6 cells not treated with hepatic membranes were totally unresponsive to EGF. These studies suggest the existence of a natural (affinity-mediated’?) mechanism for specific receptor transfer. Since EGF receptor is an integral membrane protein (detergents are required for its solubilization) it is not easy to visualize a mechanism for its insertion in the absence of added fusogens. It is possible that the preferential insertion of EGF receptors over other hepatic proteins is due to a specific NR-6 membrane protein which possesses a high affinity for the receptor and sequesters the receptor from the hepatic membrane by an unknown mechanism. Such a protein with a tendency to associate with the receptor may also be involved in the biological message transmission mechanism. Therefore, it is of interest to examine the existence of such a protein using purified EGF receptor as a probe.
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IV. THE PATHWAY TO NUCLEAR DNA REPLICATION A. The Mitogenic Capability of EGF A variety of cell types display a DNA replication response to EGF. This included fibroblasts (Armelin, 1973; Hollenberg and Cuatrecasas, 1973; Carpenter and Cohen, 1976b; Rose etal., 1976; Das and Fox, 1978), glia (Westermark, 1977). lens epithelial cells (Stoker et a l . , 1976). endothelial cells (Gospodarowicz et al., 1978), and kidney cells (Holley et a / . , 1977). It is known that EGF must be continuously present in cell medium for 5 hours for even a small level of DNA synthesis, and for the elicitation of a near-maximal DNA synthetic response a 12- to 15-hour exposure is required (Carpenter and Cohen, I976b). A comparison with other systems (those involving synergistic interactions between different mitogens) reveals the following. It has been shown that in quiescent responsive cells, a transient exposure to platelet-derived growth factor (PDGF) followed by a later exposure to plasma results in G I -+S transi1979; Vogel er a / . , 1978). This led to the suggestion that tion (Stiles et d., PDGF serves to mediate only the earlier events in the mitogenic pathway, and other factors such as those present in plasma are needed for progression through the rest of the pathway (Stiles et a / . , 1979). although Dicker and Rozengurt ( I98 1 ) suggest that a tight and stable association between PDGF and its receptor could make it possible to act at later stages in the mitogenic pathway despite early removal of PDGF from the culture medium. In any event, in the EGF system, both the early and late events in the mitogenic pathway appear to require the presence of EGF in the medium. This suggests that in this system, the mitogen-receptor functional complex is relatively labile, and that one (or more) of the EGF-receptor-generated biochemical signals for transit through the later stages of the G , -+ S pathway is labile. Thus, both early and late events in the mitogenic pathway may be dependent upon the presence of critical concentrations of appropriate signals generated by the EGF-receptor complex.
6. The Mitogenic Pathway Despite the apparent diversity of different mitogenic systems, it appears likely that the cellular protein components other than the receptor that are involved in the expression of mitogenic responses (hormone-induced or otherwise) are very similar in different species of normal and tumor cells. The G I phase of the cell cycle has been shown to be specifically lengthened in the presence of cycloheximide, whereas the remainder of the cell cycle (S, G,, and M ) is only slightly
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1971; Rossow et a/., 1979). These results lengthened (Schneiderman et d., suggest the existence of cycloheximide-sensitive, rapidly turning over G I proteins that can regulate entry into S phase. Considering these, it is of interest to identify the EGF-induced rapidly turning over proteins whose synthesis is markedly enhanced in the presence of EGF, and whose decay occurs abnormally fast in the absence of any mitogenic stimulation. 1. PROTEINS PARTICIPATING I N THE EGF-INDUCED PATHWAY
Some EGF-induced protein factor in cytoplasmic extracts of EGF-treated cell has been shown to stimulate DNA synthesis in sensitive (cell-free) nuclei (Das, 1980). Stationary density-inhibited cultures of 3T3 cells contain only insignificant amounts of the activator of DNA replication as measured by the cell-free assay, However, addition of increasing amounts of EGF to these contact-inhibited 3T3 cells results in an increasing intracellular production of the activator of DNA replication. This EGF-induced increase in activity is inhibited by cycloheximide, suggesting that the increase is mostly due to an enhanced rate of biosynthesis. The concentration of EGF required for half-maximal induction of the activator substance in quiescent 3T3 cells is about 0.1 nM,which is very similar to that required for half-maximal mitogenic response in 3T3 and other animal cells, suggesting a functionally important role for this factor in the initiation of growth and proliferation. The activity is trypsin-sensitive and nondialyzable, and sucrose density gradient centrifugal analysis reveals three peaks of activity corresponding to molecular weights of 46,000, 110,000, and 270,000 (Das, 1980). Activities very similar to the EGF-induced activity described above have been reported to be present in embryonic and tumor cells (Jazwinski et a / . , 1979; Benblow and Ford, 1973, and in concanavalin A-stimulated lymphocytes (J. Gutokowski and S . Cohen, personal communication). As previously suggested the EGF-induced pathway to mitogenesis may be very similar to that induced during tumorigenesis and other mitogenic stimulations. Other proteins that appear to be induced during EGF stimulation include a family of secreted glycoproteins of 34,000 daltons (Nilsen-Hamilton et a / ., 1980). The appearance of these glycoproteins in 3T3 cell medium closely correlates with the DNA synthetic response to EGF and other mitogens such as FGF. The biological function of these secreted proteins remains unclear at present. Recently it has been shown that EGF can stimulate poly(ADP-ribosylation) in 3T3 cells (presumably through induction of the appropriate proteins), and the stimulation appears to be temporally correlated with the cells’ entry into DNA synthesis (Shimizu and Shimizu, 1981). Poly(ADP-ribosylation) has been shown to occur on nuclear proteins including histones and nonhistone proteins (Sugimura, 1973; Hayaishi and Ueda, 1977; Burzio et al., 1979), and it is of interest
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EPIDERMAL GROWTH FACTOR RECEPTOR
to examine whether this brings about any alteration in chromatin structure and DNA replicative potential.
2. COMMITMENT TO DNA REPLICATION The mitogenic pathway, despite its enormous complexity and multicomponency, leads ultimately to the formation of a “committed” prereplicative cellular state (Temin, 1971) that appears to be an innate inherent property of the cell type independent of the external mitogen used for achieving commitment to replicate DNA (Das, 1981). It was observed that decay of the induced DNA synthetic ability induced with EGF, serum, or other mitogens is an exponential, first-order process. Internal commitment produced with either EGF or serum had identical half lives and the half-time was the same irrespective of the initial degree of commitment (Das, 198 1). These suggest the production of a preprogrammed common internal state in response to varying levels of diverse stimuli. Left to itself under normal conditions, the committed state would lead to a complete round of DNA replication accompanied perhaps by its own dissolution; but in the presence of inhibitors of DNA synthesis (e.g., hydroxyurea), the state decays in a single step. Thus commitment represents a distinctive state within the cell, a global property of the whole cell. Achieving this end-state must be a multistep
@ t t T r
DNA replication
I
t
zt t t
r
FIG. I . Commiited (C*) state model. The ultimate committed (C*) state appears to be a unit or global property o f the whole cell rather than. for example, a critical concentration of some active induced moleculc. The committed state i s perhaps coded into the configuration of some cellular macrostructure ruch as nuclear membrane or a part of the genome. which might require surpassing a critical concentration of active molecules earlier in the pathway. but the state i t s e l f represents a yes/ no whole-cell deciwn. Normally. attainment of the C* state would lead to DNA replication and suhsequent cell division; but. in the presence o f inhibitors (hydroxyureakxcess thymidine). DNA replication i s blocked and the state decays. ESI. external stimulant (EGF)-induced commitment pathway.
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process, and perhaps requires surpassing of critical concentrations of several protein factors (e.g., the EGF-induced factor described above) earlier in the pathway, but the state itself appears to decay in a single step (Fig. I ) . No presupposition is yet possible on the molecular nature of this global state. It could be coded into the configuration of some cellular macrostructure such as the nuclear membrane or a part of the genome, and it might be of interest to correlate decay of commitment with decay of individual proteins or coordinate decay of a number of different proteins.
C. Biochemical Signals for Mitogenesis Addition of EGF to quiescent cells results in an array of rapid biological changes that include activation of the protein kinase moiety within the receptor (see Section III,D), receptor clustering on the plasma membrane (see Section IlI,C), degradation of EGF within lysosomes (see Section lll,C), activation of Na+-K+-ATPase in the membrane (Rozengurt and Heppel, 1975), and increases in the active transport of nutrients such as amino acids (Hollenberg and Cuatrecasas, 1974) and glucose (Barnes and Colowick, 1976). An examination of these various candidates for signal generation reveals the following. 1 . EGF DEGRADATION
Cellular degradation of EGF is inhibited by various agents including the lysosomotrophic agent chloroquine (Carpenter and Cohen, 1976a), methylamine (Michael et ul., 1980; King er ul., 1980a), the microtubule disrupter colchicine (Brown et al., 1980), and leupeptin and antipain, both cathepsin B inhibitors (Savion ef a/., 1980). Some of these inhibitors (chloroquine, methylamine) can suppress EGF-induced DNA replication, but their suppressive effect could be related to their general cytotoxicity. Other inhibitors (colchicine, leupeptin, and antipain) are relatively nontoxic, and these compounds do not suppress EGFstimulated mitogenic responses. In fact, they somewhat potentiate EGF action. This strongly suggests a noninvolvement of EGF degradation in the signalgenerating mechanism. On the other hand, EGF degradation could serve a regulatory function by effectively reducing the amount of intact receptor-bound EGF.
2. RECEPTOR CLUSTERING AND INTERNALIZATION Earlier studies had shown that both receptor internalization and DNA synthetic stimulation had similar EGF requirements (Das and Fox, 1978; Fox and Das, 1979). Both processes were half-maximally stimulated in 3T3 cells at a EGF concentration (-0.1 nM) that caused 10% receptor occupancy. This suggested
EPIDERMAL GROWTH FACTOR RECEPTOR
397
that possibility that both events have the same limiting step. A more recent study (Schechter et nl.. 1979) using the cyanogen bromide (CNBr) cleaved analog of EGF (Holladay et d . . 1976) supports those observations. Murine EGF has a single methionine residue, and treatment with CNBr results in the production of two fragments which are disulfide linked. This analog can bind to EGF-receptor (although with less affinity), but it does not induce receptor clustering and is devoid of mitogenic activity. However, the addition of anti-EGF antibody to cells containing bound CNBr-EGF results in the restoration of both activities, namely, surface receptor clustering, and nuclear DNA replication. This strongly suggests that at least one of the biochemical signals necessary for induction for DNA synthesis is generated during the various stages of clusteringiendocytosis. It should be noted however, that certain cells are mitogenically unresponsive to EGF, but they can bind and internalize EGF, and are capable of receptor down regulation (Vlodavsky er a/., 1978). This suggests that clustering or endocytosis (and binding) may be necessary but not sufficient alone for induction of commitment to DNA replication. 3. EGF-ACTIVATED TYROSINE-SPECIFIC PROTEINKINASE The EGF-activated protein kinase within the receptor polypeptide autophosphorylates the EGF receptor and phosphorylates a number of other cellular proteins as well (Cohen e t a / . , 1982). An intriguing aspect of the EGF-receptor/ kinase is that similar tyrosine-specific protein kinase activities are associated with the transforming proteins ( ~ ~ 6) of 0 "several ~ ~ RNA tumor viruses (Hunter, 1980; Erikson and Erikson, 1980; Bishop, 1981). It is therefore tempting to propose that EGF-activated protein phosphorylation could serve as a second messenger in growth stimulation. However, it has been reported by Schreiber et a / . (1981b) that the cyanogen bromide-cleaved analog of EGF is as potent as EGF (at similar receptor occupancy) in enhancing protein phosphorylation, but it fails to induce DNA synthesis (and receptor clustering),. This suggests that even if EGF-induced protein phosphorylation is a necessary initiatory event, it is not a sufficient signal for the induction of DNA synthesis. Also, this shows that receptor clustering is not required for activation of the kinase. 4. N a + ENTRYA N D ACTIVATION OF Na+-K+-ATPAsE
EGF and a number of other growth factors enhance the activity of a plasma membrane-associated Na+ -K pump, as measured by an increased (ouabaininhibitable) XhRb+influx (Rozengurt and Heppel, 1975). The activation of glycolysis that is observed during EGF stimulation (Schneider et d., 1978) could be related to an alteration of this ion pump activity (Racker, 1976). It has been suggested that the stimulation of pump activity is due to an excessive entry of Na+ into the cell. Agents other than conventional growth factors, such as +
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MANJUSRI DAS
mellitin (an amphipathic polypeptide), can also enhance Na+ influx and increase the activity of the Na+-K+ pump; and at concentrations that promote ion fluxes, mellitin stimulates DNA synthesis in quiescent cells acting synergistically with EGF or insulin (Rozengurt et al.. 1981b). These results suggest indirectly that ion fluxes may provide at least one of the signals necessary for initiation of mitogenesis. 5. MULTIPLESIGNALS I N EGF ACTION
In summary, it appears that a simple single-hit (single-signal) model involving receptor-EGF interaction with a single transducer cannot explain the complexities involved in mitogenic hormone action. It seems likely that multiple signals are necessary, and these are generated by the EGF-receptor complex at various points. The signals are needed at both the early and late stages of the G , + S transition in EGF action (see Section IV,A). Thus, clustering may generate one of these signals and another signal may be generated by protein phosphorylation, but none of these alone is sufficient for entry into the DNA synthetic phase. It is hoped that further exploration of the EGF-receptor system will lead to a better understanding of the biochemical nature and mechanism of induction of these signals and their role in the regulation of mitogenesis.
V.
A FAMILY OF EGF-LIKE POLYPEPTIDES AND THEIR ROLE IN ANIMAL DEVELOPMENT AND GROWTH
An interesting property of the EGF receptor is its ability to bind to certain transforming polypeptides that are produced by tumor cells and sarcomas (Roberts et al., 1980; Todaro et al.. 1980). A group of sarcoma- and tumor cellderived transforming polypeptides (EGF-like or MSA-like) appears to interact with either the EGF receptor or the MSA receptor (Todaro et al.. 1981), while there are other transforming factors which appear nor to compete with either EGF or MSA for the receptor sites, but whose transforming action is clearly potentiated by EGF (Colbum and Gindhurt, 1981; Guinivan and Ladda, 1979; Roberts et al., 1982). The EGF-like factors can compete with EGF for the receptor sites, but they cannot compete with EGF for the anti-EGF antibodies in radioimmunoassays. Thus these factors are antigenically different from EGF. Like EGF, these factors can induce DNA replication and cell division in normal EGF-receptor-containing responsive cells, and, in addition (and unlike EGF), they can induce transformation-specific anchorage-independent growth of cells in soft agar (Todaro et al., 1981). An EGF-like transforming factor (hTGF,), isolated from conditioned medium
EPIDERMAL GROWTH FACTOR RECEPTOR
399
of human metastatic melanoma cells. was found to be a single chain polypeptide of 7400 daltons (Marquardt and Todaro, 1982). Like EGF, it contains three intrachain disulfide bridges, and no free sulfhydryl groups. However, the amino acid composition of hTGF, is unique, and, unlike human or murine EGF, it lacks tyrosine and methionine, and contains three phenylalanine residues. Despite this lack of structural/antigenic resemblance, hTGF, competed equally with EGF in radioreceptor assays, and completely displaced i2sI-labeled EGF binding to the human A-43 I EGF receptors, suggesting a close similarity in the receptor binding sites of hTGF, and EGF. It has been suggested that the EGF-like polypeptides (hTGF, and sarcoma growth factor) (Marquardt and Todaro, 1982; DeLarco et al.. 1980) may be produced during tumorigenesis by reactivation of genes which are expressed only during development of the embryo (Todaro et a / . , 1981). Search for EGF-like substances in mouse embryos has revealed a discrepancy between the results of radioreceptor assay and radioimmunoassay (Nexo et a/., 1980). Only negligible quantities of EGF were detected using the radioimmunoassay, but the radioreceptor assay revealed the presence of substantial quantities of an EGF-like substance which competed with 12s1-labeled EGF for the receptor sites. This suggests the presence in embryos of a substance that can interact with the EGF receptor, but which is structurallyiantigenically different from adult EGF. Thus a variety of sarcoma- and tumor cell-released polypeptides, and substances present in embryonic tissues, are capable of interacting with the EGF receptor, although they bear no antigenic resemblance to EGF. Also the sarcoma- and tumor cellreleased factors do not appear to be viral gene products (Todaro et a / . , 1981). Thus, clearly. an animal cell has the potential for making a variety of EGFreceptor-reactive polypeptides, and it does so presumably through processes involving gene rearrangements and transpositions. The system may be analogous to the insulin-like growth factor (IGF) system where a family of partially homologous polypeptides shares the same receptor (Blundell and Humbel, 1980). What could be the biological significance of such a diversity in EGF-like substances? The answer lies perhaps in the small and subtle differences in their biological properties and in their capacities for differential receptor activation. It is hoped that further explorations of these “EGF-like polypeptides” will provide vital clues regarding the in vivo role of this most fascinating polypeptide system in animal growth and survival. ACKNOWLEDGMENTS I wish to thank Dr. Subal Bishayee for his useful suggestions. and Mr. Larry Hyland for his help in the preparation of this article. I am also grateful to Dr. Stanley Cohen (Vanderbilt University) and to Dr. George Todaro (NIH) for providing manuscripts before publication. The support of NIH research grants (AM-258 19 and AM-25724) and Research Career Development Award (AM-00693) is acknowledged.
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Carpenter, G . , and Cohen, S. (1977). Influences of lectins on the binding of 12'I-labeled EGF to human fibroblasts. Eiochem. Eiophys. Res. Commitn. 79, 545-552. Carpenter, G., and Cohen. S. (1979). Epidermal growth factor. Annu. Rev. Eiorhern. 48, 193-216. Carpenter, G . , Lembach, K. J., Morrison, M. M.. and Cohen, S. (1975). Characterization of a binding of 12sI-labeled epidermal growth factor to human fibroblasts. J. Eiol. Chem. 250, 4297-4304. Carpenter, G . , King, L., and Cohen, S. (1978). Epidermal growth factor stimulates phosphorylation in membrane preparations in v i m . Nature (London) 276, 409-4 10. Carpenter, G . , King. L., and Cohen, S. ( 1979). Rapid enhancement of protein phosphorylation in A 43 I cell membrane preparations by epidermal growth factor. J. B i d . Chem. 254, 4884-489 I . Cohen. S. (1962). Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J . B i d . Chem. 237, 1555-1562. Cohen, S., and Carpenter. G. (1975). Human epidermal growth factor: Isolation and chemical and biological properties. Proc. Natl. Acad. Sci. U.S.A. 72, 1317- 132 I . Cohen, S., and Stasny, M. (1968). Epidermal growth factor. 111. The stimulation of polysome formation in chick embryo epidermis. Eiochim. Eiophys. Acta 166, 427-437. Cohen, S., Carpenter. G., and King, L. (1980). Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylating activity. J. Eiol. Chem. 255, 4834-4842. Cohen, S., Ushiro, H., Stoscheck, C., and Chenkers, M. (1982). A native 170,000 epidermal growth factor receptor-kinase complex from shad plasma membrane vesicles. J. Eiol. Chem. 257, 1523- 1539. Colburn, N. H., and Gindhurt, T. D. (1981). Specific binding of transforming growth factor correlates with promotion of anchorage independence in EGF-receptor less mouse JB6 cells. Eiochem. Eiophys. Res. Commun. 102, 799-807. Das, M. (1 980). Mitogenic hormone-induced intracellular message: Assay and partial characterizdtion of an activator of DNA replication induced by epidermal growth factor. Proc. Narl. Acad. Sci. U.S.A. 77, 112-1 16. Das. M. (1981). Initiation of nuclear DNA replication: Evidence for formation of a committed prereplicative cellular state. Proc. Narl. Acud. Sci. U.S.A. 78, 5677-5681. Das, M., and Fox, C. F. (1978). Molecular mechanism of mitogen action: Processing of receptor induced by epidermal growth factor. Proc. Natl. Accid. Sci. U.S.A. 75, 2644-2648. Das, M., and Fox, C. F. (1979). Chemical cross-linking in biology. Annu. Rev. Eiophvs. Eioeng. 8, 165- 193. Das, M., Miyakawa. T., Fox, C. F., Pruss, R. M., Aharonov, A., and Herschman. H. R. (1977). Specific radiolabeling of a cell surface receptor for epidermal growth factor. Proc. Natl. Acad. Sci. U.S.A. 74, 2790-2794. Das, M., Hyland, L., and Bishayee, S. (1982). Anti-EGF-receptor antibody: Binding and biological properties. In preparation. Davies. R. L., Grosse, V. A , , Kucherlapati, R., and Bothwell, M. (1980). Genetic analysis of epidermal growth factor action: Assignment of human epidermal growth factor receptor gene to chromosome 7. Proc. Natl. Acad. Sci. U . S . A . 77, 4188-4192. DeLarco, J. E.. Reynolds, R., Carlberg, K., Engle. C., and Todaro, G. (1980). Sarcoma growth factor from mouse sarcoma virus transformed cells: Purification by binding and elution from epidermal growth factor receptor-rich cells. J. Eiol. Chem. 255, 3685-3690. Dicker, P., and Rozengurt, P. (1981). Stimulation of DNA synthesis by transient exposure of cell cultures to TPA or polypeptide mitogens: Induction of competence or incomplete removal. J. Cell. Physiol. 109, 99-109. Ek, B., Westermark, B.. Wasteson, A . , and Heldin, C. H. (1982). Stirnulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature (London) 295, 4 19-420.
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Erikson, E., and Erikson, R . L. (1980). Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus transforming gene product. Cell 21, 829-836. Fabricant, R . N.. DeLarco, J . E., and Todaro. (7. J. (1977). Nerve growth factor receptors on human melanoma cells in culture. Proc. Null. Acad. Sci. U.S.A. 74, 565-569. Ford, S. R.. Aden, D. P., Mausner, R.. Trinchieri, G.. and Knowles. B. B. (1978). Partial characterization of cell-surface protein coded for by human chromosome 7. lmmunogenctics 6, 293-300. Forgue-Lafitte, M. E., Laburthe, M., Chemblier. M. C., Moody, A . J., and Rosselin, G. (1980). Demonstration of specific receptors for EGF-urogastrone in isolated rat intestinal epithelial cells. FEBS Lett. 114, 243-246. Fox, C. F., and Das, M. (1979). Internalization and processing of the EGF-receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis. J . Supramol. Srrircr. 10, 199-214. Frati. L.. Daniele, S . , Delogu, A., and Covelli, I . (1972). Selective binding of the epidermal growth factor and its specific effects on the epithelial cells of the cornea. ESP. Eve Res. 14, 135- 141. Gordon, P., Carpentier, J . , Cohen, S . , and Orci, L. (1978). Epidermal growth factor: Morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc. Narl. Acad. Sci. U.S.A 75, 5025-5029. Gospodarowicz, D., Mescher, A . L., Brown, K. D., and Birdwell, C. R. (1977). The role of fibroblast growth factor and epidermal growth factor in the proliferative response of the corneal and lens epithelium. Exp. Eye Res. 25, 631-649. Gospodarowicz, D., Brown, K. D., Birdwell. C. B.. and a l t e r , B. R . (1978). Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical fibroblast growth factor, epidermal growth factor and thrombin. J . Cell Biol. 77, 774-788. Gregory, H. (1975). Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature (London) 257, 325-327. Gregory. H., and Preston. B. M. (1977). The primary structure of human urogastrone. Int. J . Peptide Prorein Res. 9, 107-1 18. Gregory, H., and Willshire, I. R. (1975). The isolation of urogastrones-Inhibitors of gastric acid secretion from human urine. Hoppe-Seylers Z . Physiol. Chem. 356, 1765- 1774. Guinivan. P., and Lddda. R . L. (1979). Decrease in epidermal growth factor receptor levels and production of material enhancing epidermal growth factor binding accompany the temperaturedependent changes from normal to transformed phenotype. Proc. Natl. Acad. Sci. U.S.A. 76, 3377-338 I . Haigler, H., and Carpenter, C. (1980). Production and characterization of antibody blocking epidermal growth factor receptor interactions. Biochirn. Biophys. Acta 598, 3 14-325. Haigler. H.. Ash. J . F., Singer, S. J . , and Cohen. S . (1978). Visualization by fluorescence ofthe binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc. Nail. Acad. Sci. U.S.A. 75, 3317-3321. Haigler. H. T., McKanna. J . A., Cohen. S . (1979). Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J . Cell Biol. 81, 382-395. Hassel, J. R. (1975). The development of rat palatal shelves in virro: An ultrastructural analysis of the inhibition of epithelial cell death and palate fusion by the epidermal growth factor. DPY. B i d . 45, 90-102. Hassel, J . R., and Pratt, R. M . (1977). Elevated levels of CAMPalters the effect of epidermal growth factor in vitro on programmed cell death in the secondary palatal epithelium. Dcv. B i d . 106, 55-62. Hayaishi, 0.. and Ueda, K. (1977). Poly (ADP-ribose) and ADP-ribosylation of protein. Am. Rev. Biochem. 46, 95- I 16.
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Hock. R . A . . Nexo, E., Hollenberg. M. D. (1979). Isolation of the human placenta receptor for epidermal growth factor-urogastrone. Nurura (London) 277, 403-405. Hock. R. A., Nexo, E.. and Hollenberg, M. D. (1980). Solubilization and isolation of the human placenta receptor for epidermal growth factor-urogastrone. J . B i d . Chem. 255, 10737- 10743, Holladdy, L. A., Savage, C. R.. Cohen, S . , and Puett. D. (1976). Conformation and unfolding thermodynamics of epidermal growth factor and derivatives. Biochemistry 15, 2624-2633. Hollenberg, M. D. (1975). Receptors for insulin and epidermal growth factor: Relation to synthesis of DNA in cultured rabbit lens epithelium. Arch. Biochem. Biophw. 171, 371-377. Hollenberg, M. D. ( 1979). Epidermal growth factor-urogastrone, a polypeptide acquiring hormonal status. Vitcrm. Horm. 37, 69- 110. Hollenberg. M. D., and Cuatrecasas, P. (1973). Epidermal growth factor: Receptors in human fibroblasts and modulation of action by cholera toxin. Proc. Nut/. Acad. Sci. U.S.A. 70, 2964-2968, Hollenberg. M. D., and Cuatrecasas. P. (1975). Insulin and epidermal growth factor: Human fibroblast receptors related to DNA synthesis and amino acid uptake. J . Biol. Chem. 250, 3845-3853. Holley. R. W., Armour, R., Baldwin, J . A,. Brown, K. D.. and Yeh, Y. (1977). Density-dependent regulation of growth of BSC-I cells in cell culture: Control of growth by serum factors. Proc. Natl. Acad. Sci. U.S.A. 74, 5046-5050. Hunter, T. (1980). Proteins phosphorylated by the RSV transforming function. Cell 22, 647-648. Jazwinski, S. M . , Wang. J . L.. and Edelman. G . M. (1976).Initiation of replication inchromosomal DNA induced by extracts from proliferating cells. Proc.. Nut/. Acad. Sci. U.S.A. 73, 22312235. Kahn, C. R., Baird. K.. Flier, J. S., and Jarrett, D. B. (1977). Effect of auto antibodies to the insulin receptor on isolated adipocytes: Studies on insulin binding and insulin action. J . Clin. Invest. 60, 1094-1 106. King. A. C., Hernaez, L. I . , and Cuatrecasas. P. (1980a). Lysosomotropic amines cause intracellular accumulation of receptors for epidermal growth factor. Pror. N d . Acad. Sri. U.S.A. 77, 3238-3287. King. L. E., Carpenter. G., and Cohen, S. (1980b). Characterization by electrophoresis of epidermal growth factor stimulated phosphorylation using A-43 I membranes. Biochemistrji 19, I 524- 1528, Ladda. R. L.. Bullock, L. P., Glanopoulas. T., and McCormick, L. (1979). Radioreceptor assay for epidermal growth factor. Anal. Biochem. 93, 286-294. Lee, L. S . , and Weinstein. I . B. (1978). Tumor-promoting phorbol esters inhibit binding of epidermal growth factor to cellular receptors. Science 202, 313-315. Lee, L. S., and Weinstein, 1. B. (1979). Mechanism of tumor promotor inhibition of cellular binciing of epidermal growth factor. Proc. Nut/. Acad. Sci. U.S.A. 76, 5168-5172. Linsley. P. S . , and Fox, C. F. (1980). Controlled proteolysis of EGF receptors: Evidence for transmembrane distribution of the EGF binding site and the phosphate acceptor site. 1.Suprumol. Struct. 14, 461 -471. Linsley, P. S.. Blifeld, C. B., Wrann, M., and Fox, C. F. (1979). Direct linkage of epidermal growth factor to its receptor. Narure (London) 278, 745-748. Marquardt, H., and Todaro, G. H. (1982). Human transforming growth factors: Production by a melanoma cell line, purification and initial characterization. J . Biol. Chem. 257, 5220-5225. Michael. H., Bishayee, S . , and Das, M. (1980). Effect of methylamine on internalization, processing, and biological activation of epidermal growth factor receptor. FEBS Lett. 117, 125131. Nexo. E.. Hollenberg. M. D.. Figueroa, A,. and Pratt. R . M. (1980). Detection of epidermal growth
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factor-urogastrone and its receptor during fetal mouse development. Proe. Nut/. Arud. Sci. U.S.A. 77, 2782-2785. Nilsen-Hamilton, M., Shapiro, J . M., Massoglia, S. L., and Hamilton, R. T. (1980). Selective stimulation by mitogens of incorporation of Yhnethionine into a family of proteins released into the medium by 3T3 cells. Cell 20, 19-28. O’Keefe, E. J . , Hollenberg, M. D.. and Cuatrecasas. P. (1974). Epidermal growth factor: Characteristics of specific binding in membranes from liver, placenta and other target tissues. Arch. Biochem. Biophys. 164, 5 18-526. Pastan, I . , and Willingham. M. C. (1981). Journey to the center of the cell: Role of the receptoromc. Science 214, 504-509. Pratt, R . M . , and Pastan, I . (1978). Decreased binding of epidermal growth factor to BALBic 3T3 mutant cells defective in glycoprotein synthesis. Nature (London) 272, 68-70. Pruss. R. M., and Herschmann, H. R. (1977). Variants of 3T3 cells lacking mitogenic response to epidermal growth factor. Prac. Natl. Acad. Sci. U.S.A. 74, 3918-3921. Racker, E. (1976). Why do tumor cells have a high aerobic glycolysis. J . Cell. Physictl. 89, 697-700. Roberts, A. B., Lamb, L. C., Newton, D. L.. Sporn. M. B., DeLarco, J . E., and Todaro, G. J . ( 1980). Transforming growth factors: Isolation of polypeptides from virally and chemically transformed cells by acidiethanol extraction. Proc. Natl. Acad. Sci. U.S.A. 77, 349443498, Roberts, A. B . , Anzane, M. A.. Lamb, L. C., Smith, J. M., Frolik. C. A,, Marguardt, H., Todaro. G. J . , and Sporn, M. B. (1982). Isolation from murinc sarcoma cells of novel transforming growth factor potentiated by EGG. Nature (London 295, 417-419. Rose. S. P., Pruss, R. M., and Herschman, H. R. (1976). Initiation of 3T3 fibroblast cell division by epidermal growth factor. J . Cell. Phvsid. 86, 593-598. Rossow. P. W . , Riddle, V. 0. H., and Pardee, A. B. (1979). Synthesis of labile, serum-dependent protein in early G I controls animal cell growth. Proc. Natl. Acad. Sci. U.S.A.77, 3494-3498. Rozengurt, E., and Heppel, L. A. (1975). Serum rapidly stimulates ouabain-sensitive XhRbf influx in quiescent 3T3 cells. Proc. Nail. Acad. Sci. U.S.A. 72, 4492-4495, Rozengurt. E., Brown. K. D., and Pettican, P. (1981a). Vasopressin inhibition of epidermal growth factor binding to cultured mouse cells. J . B i d . Chem. 256, 716-722. Rozengurt, E., Gelehrter, T. D., Legg, A . , and Pettican, P. (1981b). Mellitin stimulates Na+ entry. Na-K pump activity and DNA synthesis in quiescent cultures of mouse cells. Cell 23, 78 1-788. Sahyoun, N., Hock. R. A., and Hollenberg, M. D. (1978). Insulin and epidermal growth factor-urogastrome: Affinity crosslinking to specific binding sites in rat liver membranes. Proc. Nail. Acad. Sci. U.S.A. 75, 1675-1679. Savage, C. R., Inaganii, J.. and Cohen, S . (1972).The primary structure of epidermal growth factor. J . B i d . Chem. 247, 7612-7621. Savion, N., Vlodavsky, I., and Gospodarowicz, D. (1980). Role of the degradation process in the mitogenic effect of epidermal growth factor. Proc. Nutl. Acad. Sri. U.S.A. 77, 1466-1470. Schechter, Y., Hernaez, L., Schlessinger, J . , and Cuatrecasas, P. (1979). Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor. Narure (London) 278, 835-838. Schlessinger, J., Shechter, Y., Willingham, M. C., and Pastan, I. (1978). Direct visualization of binding, aggregation and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc. Nail. Acad. Sci. U.S.A. 75, 2659-2663. Schneider, J. A., Diamond, I . , and Rozengurt, E. (1978). Glycolysis in quiescent cultures of 3T3 cells. Addition of serum, epidermal growth factor, and insulin increases the activity of phosphofructokinase in a protein synthesis independent manner. J . B i d . Chem. 253, 872-877. Schneiderman, M. H., Dewey, W. C., and Highfield (1971). Inhibition of DNA synthesis in synchronized Chinese hamster cells treated in G , with cycloheximide. Exp. Cell. Res. 67, 147- 155.
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Schramm, M. (1979). Transfer of glucagon receptor from liver membranes to a foreign adenylate cyclase by a membrane fusion procedure. Proc. Nail. Acad. Sci. U.S.A. 76, 1174-1178. Schramm, M., Orly, J . , Eimerl. S . . and Korner. M. (1977). Coupling of hormone receptors to adenylate cyclase of different cells by fusion. Nutitre (London) 268, 310-313. Schreiber, A. B., Lax, I . , Yarden, Y . , Eslhar. Z . , and Schlessinger, J . (1981a). Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor. Proc. Narl. Acad. Sci. U.S.A. 78, 7535-7539. Schreiber, A. B., Yarden, Y.,and Schlessinger. J . (1981b). A nonmitogenic analog of epidermal growth factor enhances the phosphorylation of endogenous membrane proteins. Biochem. Biophvs. Res. Commun. 101, 517-523. Shimizu, N., Behzadian, M. A., and Shimizu. Y . (1980). Genetics of cell surface receptors for bioactive polypeptides: Binding of epidermal growth-factor is associated with the presence of human chromosome 7 in human-mouse cell hybrids. Proc. Nut/. Acad. Sci. U.S.A. 77, 3600-3604. Shimizu, Y., and Shimizu, N. (1981). Insulin and epidermal growth factor stimulate poly ADPribosylation. Biochem. Biophvs. Res. Commun. 99, 536-542. Shoyab. M., and Todaro, G. J. (1980). Vitamin K 3 (menadione) and related quinones, like tumorpromoting phorbol esters, alter the affinity of epidermal growth factor for its membrane receptors. J . Biol. Chem. 255, 8735-8739. Stiles. C. D., Capone, G . T., Scher, C. D., Antoniades, H. N., Van Wyk. J. J . , and Pledger, W. J. (1979). Dual control of cell growth by somatomedin and platelet-derived growth factor. Proc. Nail. Acad. Sci. U.S.A. 76, 1279- 1283. Stoker, M. G. P., Pigott, D., and Taylor-Papadimitriou. J. (1976). Response to epidermal growth factors of cultured human mammary epithelial cells from benign tumors. Nature (London) 264, 764-767. Sugimura, T. (1973). Poly(adenosine diphosphate ribose). Prog. Nucleic Acid Res. Mol. Biol. 13, 127-15 I . Temin, H. M. (1971). Stimulation by serum of multiplication of stationary chicken cells. J . Cell. Physiol. 78, 161-170. Thorn. D., Powell, A. 1.. Lloyd, C. W . , and Rees, D. A. (1977). Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem. J . 168, 187-194. Todaro, G. J . , Fryling, C., and DeLarco, J . E. (1980). Transforming growth factors produced by certain human tumor cells: Polypeptides that interact with epidermal growth factor receptors. Proc. Nut/. Acad. Sri. U.S.A. 77, 5258-5262. Todaro, G. J., Marquardt, G . , DeLarco, J. E., Fryling, C. M.. Reynolds, F. H., and Stephenson, J. R. (1981). Transforming growth factors produced by human tumor cells: Interaction with EGF membrane receptors. I n “Cellular Responses to Molecular Modulation” (L. W. Mozes, J. Schultz, W . A. Scott, and R. Werner, eds.). Academic Press, New York. Ushiro, H., and Cohen, S. (1980). Identification of phosphotyrosine as a product of epidermal growth factor activated protein kinase in A-431 cell membranes. J . Biol. Chem. 255, 8363-8365. Vlodavsky, I . , Brown, K. D., and Gospodarowciz, D. (1978). A comparison of the binding of epidermal growth factor to cultured granulosa and h e a l cells. J . Biol. Chem. 253, 3744-3750. Vogel, A., Raines. E., Kariya, B., Rivest, M. J., and Ross. R. (1978). Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components. Proc. Natl. Acad. Sci. U.S.A. 75, 2810-2814. Westermark, B. ( 1977). Local starvation for epidermal growth factor cannot explain density-dependent inhibition of normal human glial cells. Proc. Nut/. A d . Sci. U.S.A. 74, 1619-1621. Wrann, M., and Fox, C. F. (1979). Identification of epidermal growth factor receptors in a hyperproducing human epidermoid carcinoma cell line. J . Biol. Chem. 254, 8083-8086.
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME I R
The Linkage between Ligand OccuDation and ResDonse of the NicoGnic Acety Ichol ine Recepto r PALMER TAYLOR, ROBERT DALE BROWN, AND DAVID A . JOHNSON Divisicm of Phurmucologv Department of Medicine Universiry of Culijorniu. Sun Diego La follu. Culijorniu
Introduction. . . .............. ................................ Structure of the Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Biophysical Properties of the Receptor Channel, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Behavior of Partial Agonists, Antagonists. and Anesthetics in Relation to Channel Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Desensitization of the Receptor. ............................. VI. Ligand Occupation and Transitions in Receptor State . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Other Ligands Affecting Receptor Function .................................. ... VIII. Analysis of Receptor Activation the IX . Toward the Understanding of Co Permeability Response . . . . . . . . .............................. X . Occupation and Activation by A ..................................... XI. Association of Antagonists with the Receptor and Functional Antagonism . . . . . . . . . ....... XII. Quantitation of Antagonist Occupation and Functional Antagonism XIII. Structural Implications and Arrangement of Subunits . . . . . . . . . . . . . . . . . . . . . . . . . . ............................. XIV . Analysis of the Bound Ligand States References ..................... .......................... 1.
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I. INTRODUCTION
Over a decade has passed since Changeux er al. ( 1970) employed the snake a-toxins to identify and monitor isolation of the nicotinic acetylcholine receptor from the electric eel, Electrophorus electricus. This successful initial application of a biochemical approach to characterizing neurotransmitter receptors relied 407
Copyrighl 0 1983 by A d c m i c Pre$a Inc All right5 of rcprodu~iionin m y form rcservcd ISBN 0 I2 153318-2
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heavily on earlier observations of Lee and Chang (1963), who found that the elapid a-toxins exhibited an essentially irreversible block of neurotransmission and that antagonism could be localized to a postsynaptic site. Perhaps the most definitive corroborative evidence that the macromolecule purified by monitoring a-toxin binding was the acetylcholine receptor was provided by the finding that the purified receptor contained a 40,000-dalton subunit which could be labeled by treatment of intact cells with the irreversible antagonist p-[3HH]maleimidobenzyltrimethylammonium (MBTA) (Reiter et al., 1972; Karlin and Colburn, 1973). Thus, two labeling techniques which relied on different recognition characteristics of the receptor identified the same macromolecule or component of it. Since then, the biochemical and structural properties of the nicotinic acetylcholine receptor have been examined in considerable detail, its immunochemical properties have been assessed, and in recent studies rec-onstitutionof its functional properties has been demonstrated. Owing to the specific peptide a-toxins and an abundant source of receptor in the electric fish, the acetylcholine receptor is our most thoroughly characterized pharmacologic receptor. Moreover, the a toxins have also facilitated studies of the biosynthesis and turnover of the receptor as a specialized membrane protein, and progress is emerging on the synthesis of the receptor under in vitro conditions. Details on these topics may be found in a number of authoritative reviews (Changeux, 1981; Karlin, 1980; Adams, 1981; Steinbach, 1980). The primary emphasis of this article is devoted to a still-emerging area of study on this receptor, that of the relationship between receptor occupation by ligands and the physiologic or pharmacologic response. It is, of course, the linkage between occupation and response which confers the unique transduction capacities to receptor molecules as well as dictates their pharmacologic specificity for agonists, partial agonists, and antagonists. Since agonists can induce both an open channel and a desensitized state of the receptor and combinations of ligands can alter the character of the open channel state, analysis of the response itself is complex. It is evident that the receptor can exist in various states which differ in their affinity for ligands, conductance behavior, and conformation. Hence our analysis should include a comparison of ligand binding functions with functions of receptor state. With our knowledge of receptor structure and the understanding of its channel properties in biophysical terms, both developing to a relatively advanced stage, it is becoming possible to relate functional behavior to molecular details on structure. Thus, it would be appropriate at the outset to proceed with considerations of receptor structure.
II. STRUCTURE OF THE ISOLATED RECEPTOR Fortunately, in the past 2 years there has been a convergence among investigators regarding the interpretation of the biochemical data on the receptor mole-
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cule. The isolated receptor obtained by solubilization and subsequent purification by affinity chromatography contains four subunits of molecular weights 40,000 (a), 49,000 (p), 60,000 (y), and 67,000 (6) (Weill et al., 1974; Raftery et al., 1975). A similar composition of subunits is found for membrane fragments purified for enrichment in receptor molecules (Reed et a/., 1974). A 43,000-dalton peptide found in the membranes may be removed by alkaline treatment without loss of receptor function (Neubig et al., 1979). Although the 43,000-dalton peptide was thought originally by some to be an integral component of the receptor, this peptide or family of peptides appears to be localized at the cytoplasmic face of the postsynaptic membrane (Cartaud et al., 1981; St. John et al., 1982). Its removal results in a disordering of receptor molecules within the membrane. Thus, this peptide likely plays a role in the alignment and orientation of the receptor molecules in the subsynaptic areas. Based on N-terminal analyses, the subunits in the receptor from Torpedo are present in the stoichiometric ratio of az,p, y, 6 (Raftery et al., 1980). Preparations with gel profiles showing a predominance of the a subunit greatly exceeding the above stoichiometry have now been shown to arise from proteolysis of the p, y, and 6 subunits. The a2Py6 stoichiometry (Reynolds and Karlin, 1978; Lindstrom e t a / . , 1979) would nominally sum to a molecular weight of 250,000, which is close to the value obtained from careful hydrodynamic analysis of the monomeric form of the receptor (Reynolds and Karlin, 1978). Molecular weight estimates by a variety of methods have yielded values between 230,000 and 350,000 (cf. Changeux, 1981; Karlin, 1980). This variance reflects the inherent inaccuracy of the methods for estimation of molecular weight of membrane proteins, neglect of the contribution of dimers within the preparation, and an incomplete or unsatisfactory accounting of the bound detergent. Most, if not all, of the subunits found in the Torpedo preparations appear as corresponding subunits in Electrophorus and mammalian receptors (Lindstrom et al., 1980a; Shorr et al., 1981; Nathanson and Hall, 1979). However, achieving sufficient quantities of receptor devoid of proteolysis from mammalian muscle presents a far more formidable problem. The receptor in Torpedo appears largely as a dimeric (13 S) form in which disulfide association is found between the 6 subunits (Chang and Bock, 1977; Hamilton et a l . , 1977; Hucho et al., 1978; Weiland et al., 1979). Reduction results in cleavage of the disulfide bond with the formation of the 9 S monomer upon solubilization. Cleavage of this interchain disulfide, however, does not affect either the organization in the membrane or the functional properties of the receptor (Kistler and Stroud, 1981; Anholt et al., 1980). The tendency to form dimeric units does not appear to be shared by the mammalian or Electrophorus receptor. A functional role for only the a-chain has been clearly delineated. Initial studies showed that following disulfide bond reduction ['HIMBTA reacted with the a-subunit as did the alkylating agonist, bromoacetylcholine (Damle et al.,
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1978). The predominant sites of a-toxin attachment following bifunctional crosslinking with a-toxin also appear on the a-subunit (Witzemann and Raftery, 1978; Nathanson and Hall, 1980). In the absence of reduction, the irreversible antagonist [ 3H]trimethylbenzenediazonium fluoroborate (TDF) reacts selectively with the a-subunit (Weiland et al., 1979). Thus, it appears that the a-subunit bears the agonist-antagonist recognition site as well as the major surface with which the -7000-dalton a-toxin peptide associates. The 6 subunit is the site of labeling of a covalent analog, 5-azido-trimethisoquin, of the local anesthetic trimethisoquin (Saitoh et al., 1980). The latter compound is a noncompetitive blocker of receptor function whose affinity for the receptor is actually enhanced by agonists. Chlorpromazine exhibits rather similar binding behavior to trimethisoquin, yet, following photoactivation, it reacts with all four subunits (Oswald and Changeux, 1981). Sequence work has been initiated on the individual subunits, and of particular interest is the degree of homology which exists among the peptides. Based on Nterminal sequences 30-50% homology exists in the N-terminal54 residues of the four peptide chains, suggesting their divergences from a common ancestral gene (Raftery et al., 1980). Probable homologous regions have also been detected by monoclonal antibody cross-reactivity (cf. Gullick et ul., 1981). Specific activities of the solubilized and purified receptor preparations in various laboratories have ranged between 5 and I0 pmole/g of protein (cf. Karlin, 1980; Changeux, 198I). The more homogeneous membrane preparations have yielded specific activities of 5040% of this range. Assuming an a*,p, y, 6 stoichiometry, a molecular weight of 250,000, and one a-toxin site per asubunit, we would not expect specific activities to exceed 8.0 pmole/g of protein unless portions of the receptor were lost through proteolysis or additional sites were detected by a-toxin binding. Since a-toxin binding prevents agonist and antagonist association and the latter ligands inhibit the initial rate of a-toxin binding in a competitive fashion, stoichiometry of association of the smaller reversible ligands can be related to a-toxin sites, thus obviating the variance inherent in quantitating protein content and assessing complete homogeneity of the preparation. Weber and Changeux (1974) initially found 1: 1 stoichiometry between acetylcholine and a-toxin binding, and this stoichiometry has been confirmed for a large number of ligands in membrane-associated and solubilized receptor preparations; a spin-labeled analog of decamethonium by electron spin resonance (Weiland et a[., 1976), a dansylacylcholine by fluorescence (Heidmann and Changeux, 1979), and radiolabeled acetylcholine and d-tubocurarine by equilibrium dialysis (Neubig and Cohen, 1979). Other investigators have found stoichiometric ratios of 0.5 for acetylcholine to a-toxin and carbamylcholine to a-toxin; however, in some cases these estimates have recently been revised (cf. Raftery et (11.. 1975; Schimerlik et al., 1979; Dunn et al., 1980). For irreversible antagonists, ratios of 0.5 equivalent of conjugated MBTA
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
41 1
and bromoacetylcholine per a-toxin site have been found following disulfide bond reduction (Damle and Karlin, 1978). The relationship of a-toxin occupation to the exclusion of [3H]MBTA labeling would suggest an arrangement of nonequivalent sites confined to the same receptor oligomer (Damle and Karlin, 1978). By altering the conditions of bromoacetylcholine labeling, Wolosin ef a f . , ( 1980) were able to achieve labeling of one bromoacetylcholine molecule per atoxin site in both Torpedo and the mammalian receptor. Despite 1: 1 stoichiometry for the alkylating acetylcholine analog the rate of the reaction was not uniform, suggesting that the sites of labeling do not show complete equivalence. TDF conjugation proceeds to 1: 1 stoichiometry (Weiland et a/., 1979). However, this ligand reacts with different residues; disulfide bond reduction is not required prior to TDF labeling. The difference in behavior of a-subunits which is revealed in an apparent chemical inequivalence of certan reactive groups will become important in future considerations. There is no evidence to date that the a-subunits differ in their primary structure. The existence of membrane patches containing high densities of receptor ( I 0 4 / ~ m 2has ) also facilitated ultrastructural characterization of the macromolecule. Electron microscopy following staining or freeze fracture and etching reveals that the receptor traverses the membrane bilayer and extends some 5.0-5.5 nm on the extracellular side. Its overall shape is roughly cylindrical with a long axis of 1 I .O nm and an average diameter of 8.0 nm (Cartaud eta/., 1978; Heuser and Salpeter, 1979; Allen and Potter, 1977; Klymkowski and Stroud, 1979). X-Ray diffraction (Kistler and Stroud, 1981 ) and neutron scattering (Wise et al., 1981a) have yielded further refinements in structure where the molecule is found to be funnel shaped, the widest diameter and largest protrusion (5.5 nm) appearing on the extracellular face. The extension through the cytoplasmic face is only 1 .O- 1.5 nm. In addition, the structure that encircles the central pit does not show three- or sixfold symmetry with respect to an axis perpendicular to the membrane; rather, three nonequivalent maxima can be identified (Kistler and Stroud, 1981). The simplest interpretation is that the three maxima constitute the larger of the subunits, p, y, and 6, with minima residing with the a-subunits, but further data are required before the subunits can be definitively placed. Image reconstruction and electron microscopy of receptor, biotinyl toxin-avidin complexes indicate that the two a-subunits are separated by 100" and hence are not contiguous (Holtzman et al., 1982; Kistler et al. 1982). Moreover, an angle of 45-85' separates the disulfide link in the &-subunitfrom one of the a-subunits. In face view subunit arrangements of 6aPay or 6ayap are most consistent with these data (Wise et a f . , 1981b). Selective labeling at a single membrane surface shows that all of the subunits span the membrane bilayer (Wennogle and Changeux, 1980; Strader and Raftery, 1980) and, hence, as transmembrane peptides, each subunit has both an intracellular and extracellular exposure. Based on the ultrastructure of the receptor and its biochemical composition, a
41 2
PALMER TAYLOR ET AL.
provisional model consistent with the large body of data can be developed (Fig. 1) (see also Karlin, 1980; Changeux, 1981). With the individual receptor subunits of a pentameric molecule traversing the membrane, it should not contain an axis of symmetry perpendicular to the membrane bilayer. Yet sequence homology between subunits may be sufficient to dictate the intersubunit associations within a pentameric structure. If we use the structural details of bacteriorhodopsin and cytochrome oxidase as model structures of channel-containing membrane proteins (cf. Stoeckenius and Bogomolni, 1982), the functional channel is not likely to be harbored within a single subunit; rather the perimeter of the channel would be formed by about seven a-helical segments contributed by most or all of the subunits. In this arrangement the two a-subunits will not have equivalent intersubunit contacts and hence we might anticipate that they will not exhibit precise functional equivalence. A number of examples of dimeric enzymes possessing identical sequences but lacking a twofold axis of symmetry are known (cf. Degani and Degani, 1980). In the case of the receptor, the inequivalence in activity of a-subunits is imposed simply by the necessity of including three additional subunits in the pentameric structure.
111.
BIOPHYSICAL PROPERTIES OF THE RECEPTOR CHANNEL
Although biophysical methods have yet to be applied to the rapid detection of ligand occupation on individual endplates or functional cells, techniques for measuring potential or conductance changes associated with transmitter activation have been available for decades. A more recent innovation has been the use of transient methods such as single channel and noise analyses to detect individual steps in the activation process. At low agonist concentrations and under voltage clamp the number of open
I I
I
B
x I6
I
49.000 60,000 67,000
FIG. 1. A plausible structural organization of subunits, a, @, y. 6, would allow for the nonequivalence of both agonist and antagonist binding in each oligomer, the positive cooperativity, and presumed concerted interaction required for receptor activation and desensitization. See text for details. The ligand binding sites are found on the a-subunits and all five subunits form the perimeter of the ion channel which is interior to the subunits. Each of the subunits has an intracellular and extracellular exposure.
LIGAND OCCUPATIONAND RESPONSE OF THE CHOLINERGIC RECEPTOR
413
channels varies as the square of the agonist concentration, and typically halfmaximal responses are obtained at carbamylcholine concentrations from 100 to 400 p l 4 and acetylcholine between 5 and 30 pkl (Adams, 1976; Sheridan and Lester, 1977; Dionne et al., 1978). A simple scheme commonly employed to explain activation data is kl
A f RR
A
k-,
-
vl kl P ARR ARRA-AR*R*A (closed) Z P - , (closed) u (open)
(1)
Thus, the channel exhibits a high probability of opening only when the receptor is in a doubly liganded state. At high agonist concentrations the emergence of desensitization and the loss of linearity of the response upon saturation limit the kinetic analyses that can be accomplished by voltage clamp measurements. The monitoring of individual transients was first carried out by Katz and Miledi (1972) who noted that statistical fluctuations in potential could be interpreted in terms of an average channel conductance and a lifetime of the open channel. Hence information may be obtained on the isomerization step in the above scheme:
P’
closed states
AR*R*A
a
The value of a appeared dependent on the agonist; for example a = 1 msec for acetylcholine whereas for suberyldicholine, a = 10 msec (Colquhoun, 1979). Also, a is much more sensitive to potential than is p’, decreasing approximately e-fold with a negative 70-80 mV change. While a is independent of agonist concentration, an examination of p’ over a range of agonist concentrations shows that it approaches a limiting value (Sakmann and Adams, 1978). Thus it appears possible to select low and high agonist concentrations where the bimolecular step ( k , ) and the isomerization (p) each becomes rate limiting [see Eq. ( l) ]. A comparison of channel opening rates for covalently bound (i.e., tethered) and reversibly associating agonists also indicates that at high agonist concentrations channel opening can become rate limiting (Lester et af., 1980). The development of patch electrodes which cover small areas of the muscle surface and confer large seal resistances has enabled measurements of conductance changes associated with the opening and closing of a single channel to be monitored rather than fluctuations occurring from a family of channels (Neher and Sakmann, 1976). The current pulses have a square wave shape of constant amplitude; their durations are exponentially distributed around defined lifetimes. Both the single channel conductance and the time constants showed good agreement with data derived from fluctuation analysis. Single channel measurements have allowed the determination of both p and a with a higher level of precision. The opening rate constant p is greater than a; thus at saturating agonist con-
414
PALMER TAYLOR ET AL.
centrations the open channel state would be a dominant species (Dionne et a l . , 1978; Dionne and Liebowitz, 1982). Single channel opening events may also contain closed intervals of very short duration and are detectable when electrode capacitance is low (Colquhoun and Hawkes, 1981; Colquhoun and Sakmann, 1981). More recently, the stochastic properties of intervals of successive opening and closing events have been analyzed to estimate channel opening p and ligand dissociation rates k . The probability analysis shows a 500 sec - I , p 750 sec-I, and k - I to be -4700 sec-' for acetylcholine (Dionne and Liebowitz, 1982). From these data and an equilibrium constant, k , is estimated to be 1.5 X lo8M - sec- I , a value in close accord with estimates from the reaction kinetics (cf. Section VI). Conductances for agonist activation are approximately 25 pS and would be equivalent to an ion flux rate of lo4 ions/msec. Since this value exceeds the capacity of a rotating carrier system, current appears to be carried by a channel which allows cations to move in accord with their concentration gradients (cf. Karlin, 1980). Reversal potential measurements reveal that cation selectivity depends primarily on ionic radius; cations with diameters up to 0.65 nm permeate freely (Dwyer et al., 1980). ~
,
-
-
IV. THE BEHAVIOR OF PARTIAL AGONISTS, ANTAGONISTS, AND ANESTHETICS IN RELATION TO CHANNEL ACTIVATION Compared to full agonists, partial agonists show saturation of their doseresponse relationships when a considerably smaller fraction of channels are open. Hence their actions might be explained simply on the basis of a small p/a ratio in Eq. (1). However, such compounds may also exhibit less than maximal responses by a secondary channel blocking action (Adams and Sakmann, 1978). Classic antagonists such as d-tubocurarine, gallamine, and pancuronium show competitive antagonism with agonists which is typically reflected as a parallel shift of the dose-response curve (Jenkinson, 1960; Colquhoun et al., 1979). Consistent with this competitive antagonism is the finding that antagonists decrease the frequency of channel opening events elicited b y an agonist without influencing their conductance or duration of opening. However, analyses of functional antagonism over a wide range of concentrations reveal a noncompetitive component of antagonism. The noncompetitive antagonism may reflect direct channel occlusion; it appears to be far more sensitive to membrane potential than competitive antagonism, becoming manifest at larger negative potential (Colquhoun et ul., 1979). The noncompetitive antagonism is also more evident with d-tubocurarine antagonism than with gallamine or pancuronium (Colquhoun and Sheridan, 1981; Katz and Miledi, 1978). Many local anesthetics or related compounds which noncompetitively block
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
415
steady-state responses directly modify channel conductance properties. This was originally detected by comparing miniature endplate currents following quanta1 release of acetylcholine (Steinbach, 1968). The decay of endplate currents shows a time course virtually identical to the time constant for the average lifetime of single channels opened by acetylcholine. Thus decay rates of miniature endplate currents also give a measure of a.In the presence of certain local anesthetics the normally exponential decay reveals at least two components. The dependences on agonist concentration and membrane potential suggest that the local anesthetic blocks the functional channel primarily while in an open configuration. Although it is an attractive possibility, these observations do not establish that the anesthetic directly enters and physically occludes the channel. Rather, the local anesthetic may simply favor binding to the open channel state of the receptor. Corroborative evidence favoring block of open channels comes from the analysis of single channels where local anesthetics serve to chop the square wave pulses into bursts of pulses of shorter duration (Neher and Steinbach, 1978). Such flickering can be interpreted in terms of repetitive blocking and unblocking of open channels. The anesthetics are a structurally disparate series of compounds, and it would indeed be surprising if a class of compounds with such diverse effects on membrane structure had a unitary mechanism of noncompetitive antagonism. Anesthetics such as QX222, procaine, and tetracaine may act primarily by noncompetitive block of conduction through association with the open pore state of the receptor. The less polar anesthetics such as dibucaine, chlorpromazine, and benzocaine do not exhibit such a marked voltage dependence in their noncompetitive block and hence may affect receptor function by altering annular lipid or associating at a hydrophobic site on the lipid-protein interface (Koblin and Lester, 1979). These agents enhance the rate and extent of desensitization resulting from exposure to agonists (Sine and Taylor, 1982). An inhibitory action which is related but not identical to the local anesthetics is observed for the noncompetitive antagonist histrionicotoxin and its hydrogenated analogs (Albuquerque et al., 1973; Burgermeister el al., 1977).
V.
DESENSITIZATION OF THE RECEPTOR
Upon continuous application of an agonist to the receptor the conductance response, rather than remaining constant, slowly decreases and this loss of responsivity resulting from prior exposure to agonist is termed desensitization. Kinetics of desensitization were initially examined by Katz and Thesleff ( 1957) who found that the onset is dependent on agonist concentration (up to a limiting concentration) and recovery is a slow unimolecular process that was independent of the agonist and its concentration used to promote desensitization. These workers suggested that a cyclic, two-state model best described the kinetics for onset
416
PALMER TAYLOR ET AL.
and recovery of desensitization: KR
AR
A+RM
A
1 1
+ R’+
M = R’/R
(3)
AR’ KR
In the absence of agonist the receptor is primarily in the R state (M < 1); the slow conversion to AR’ simply reflects the higher affinity of the R’ state for the agonist A (i.e., the dissociation constant K,. << K R ) . Removal of the agonist results in its rapid dissociation followed by a slower isomerization of R’ to R (see also Rang and Ritter, 1970). Thus, exposure to agonist not only leads to the rapid state transition of activation or channel opening but also gives rise to a slower transition to a desensitized state. In the functioning neuromuscular junction, an appropriate question is whether desensitization would be significant in physiologic responses where quanta1 release and rapid destruction of acetylcholine prevails. However, under conditions in which acetylcholine transients are likely to reside for slightly longer durations (such as with acetylcholinesterase inhibition) substantial desensitization is observed following repetitive nerve stimulation (Magleby and Pallota, 1981). Receptor states resembling the desensitized state can be produced in situ by other depolarizing agonists which are refractory to acetylcholinesterase hydrolysis (cf. Taylor, 1980). Parameters affecting desensitization have been extensively examined in the past decade and the onset is enhanced by membrane potential (Magazanik and Vyskocil, 1976) and Ca2+ concentrations (Fieckers et al., 1980; Magazanik and Vyskocil; 1976; Miledi, 1980). Additional complexities appear in the kinetics where ionophoretic application of agonists often yields more rapid rates of desensitization onset and recovery than bath application (cf. Adams, 1976).lonophoretic application is likely to give rise to high local concentrations of transmitter where a second component of desensitization may become manifest. Single channel studies also point to a multiphasic desensitization process where temporal distributions of open channel events change with prolonged agonist exposure (Sakmann et a(., 1980). With the decrease in frequency of channel openings seen with desensitization, bursts of single channel currents are observed at regular intervals. The onset of bursts might reflect an initial phase of desensitization where the efficacy of channel opening is altered. A subsequent slow phase results in the receptor channel becoming totally refractory. VI.
LIGAND OCCUPATION AND TRANSITIONS IN RECEPTOR STATE
Since the association of agonists and reversible antagonists appears competitive with the binding of a-toxin and a-toxin binding is essentially irreversible
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
41 7
when examined over short time intervals, measurement of competition with the initial rate of a-toxin binding by the ligand provides a convenient means for ascertaining occupation of the reversible competing ligand (Weber and Changeux, 1974). But early binding measurements with soluble receptor did not lend themselves to obvious correlations with the potencies of agonists deduced from electrophysiologic studies. Solubilization of the receptor resulted in alterations in agonist affinities and the affinity appeared to be affected by both the detergent and the particular purification procedure. Even the membrane-associated receptor possessed a sufficiently high affinity for acetylcholine that, assuming a diffusion-controlled association rate. the calculated rate of acetylcholine dissociation was far slower than the decay rate of the miniature endplate potential. A slow rate of acetylcholine dissociation would make it difficult to explain the rapid decay of endplate currents and repetitive activation of individual channels. An indication that dissociation constants for agonists ascertained following prolonged exposure to the agonist do not reflect binding to the active state of the receptor came from a study of Weber et al. ( 1975). These investigators found that exposure of the receptor to agonists slowly increases the apparent affinity of the complex. Exposure of the receptor to agonist did not affect the binding kinetics of a-toxin, only the capacity of agonist to compete with a-toxin binding (Weber et ul., 1975; Weiland et al.. 1976). Thus, the agonist itself induced a slow transition in receptor state such that agonist affinity increased over an interval of seconds to minutes. Reversion to the original state and binding affinity was also a slow process, but completely reversible. By using a nitroxide analog of decamethonium (a partial agonist itself) the increase in affinity monitored from initial rates of a-toxin binding was found to parallel closely the increase determined from quantitating the changes in free and bound ligand from the ESR spectrum (Weiland et al., 1976). Similarly. direct measurements of (3H]acetylcholinebinding by fast filtration methods have provided an additional means of monitoring the receptor transition from a low- to a high-affinity state (Boyd and Cohen, 1980a). Promoting the transition to the high-affinity state is a property shared by agonists, but for antagonists this property is markedly diminished (Quast ef al., 1978; Neubig and Cohen, 1979) or absent (Weiland et al., 1977; Weiland and Taylor, 1979) (cf. Table I ) . An exception lies with the metaphilic antagonists (Weiland and Taylor, 1979). These agents have been shown in the intact neuromuscular junction to exhibit greater antagonism of receptors which had been exposed to agonists and hence are desensitized (Rang and Ritter, 1969). Accordingly, metaphilic antagonists would be expected to bind with higher affinity to receptors in the desensitized state and in themselves to promote the transition to the high-affinity state. If the slow change in receptor affinity for agonists seen in Torpedo membrane fragments reflects receptor desensitization in situ. then the kinetics of conversion between states should be subject to the same constraints of the cyclic scheme developed for receptor desensitization (see Section V). Various groups have
41 8
PALMER TAYLOR ET AL.
TABLE I DISSOCIATION CONSTANTS FOR AGONISTSA N D ANTAGONISTS WITH ASSOClATtD Torpedo R E C ~ I T O R ~
THE
MtMHRANE-
Agonists Suberyldicholine Acetylcholine Carbamylcholine Phenyltrimethylammonium Nicotine
1.3 x 10-7 6 . 1 x 10-7 1.5 x 3.5 x 1 0 - 5 6.4 x
4.3 x lo-') 2.4 x 4.8 x 1 0 - 7 1 . 6x 10-6 2.5 x
4.0 x 2.3 x 4.5 x 10-8 1.5 x 10-7 2.4 x lo-'
Partial agonists Decamethonium Decamethonium mononitroxide
4.7 x 10-6 4.1 x
8.3 x 10-7 9.2 x 1 0 - 7
9.0 x 1.1 x
Metaphilic antagonists Dinaphthyldecamethonium Diphenyldecamethoniurn
6.2 x 10-7 1.5 x IO-"
3.0 x 1 0 - 7 3.9 x 1 0 - 7
4.9 x 10-8 4.6 x 10-8
12.7 32.6
Antagonists d-Tubocurarine AH-8 165 Dimethyl d-tubocurarine Gallamine
3.1 3.9 8.5 9.8
3.8 x 10-7 3.5 x 10-7
-
<4.0 <4.0 C4.0 c4.0
x 10-7 x 10-7
x lo-' x 10-7
8.5 x lo-' 1 . 1 x 10-6
325 269 34 I 267 23 I
10-8
52
10-7
37
-
From Weiland ct a / . ( 1979). All measurements were made in 0. I M NaCl containing 0.01 M Na phobphate, pH 7.4. at 22°C.
analyzed the kinetics of the state transitions in the receptor from Torpedo rnembranes (Weiland ct af., 1977; Quast et a/., 1978; Barrantes, 1978; Heidmann and Changeux, 1979; Boyd and Cohen, 1980a,b) and in intact muscle cells (Sine and Taylor, 1979) and have shown that the constraints of the cyclic scheme [Eq. (3)j are largely met. For the overall scheme KR
A+R+AR 1
A
1 1 A,,
I
+ R'-
*I
M
=
R'/R
(4)
AR' KR
the kinetic constants obtained by various procedures for Torpedo are in notably close accord (Table 11). In the absence of agonist the low-affinity (or activatable) state of the receptor is favored, i.e., M = 0.1-0.2. The R' state of the receptor possesses an affinity that is three orders of magnitude greater than the R state. Some differences in kinetic constants might be anticipated for different buffer conditions since Ca2 is known to accelerate desensitization (see Section V) and +
TABLE 11 KINETICAND EQUILIBRIUM CONSTANTS DETERMINED FOR A TWO-STATE SCHEME FOR DESENSITIZATION [EQ. (4)] Carbamylcholine (Weiland er a / . . l977)<' O.OOO4 sec - I 0.004 sec-I 0.1 2 x 10-5M S x 10-8M 0.012 sec-I 0.0003 sec-) 5 x 10-7 M
Carbamylcholine (Quast et a / . .1978)b
0.0019 sec-I 0.004 sec-I 0.48 4 x 10--5M 1.2 x 1 0 - 7 ~ 0.14 sec I 0.006 sec - I 1.7 x 1 0 - h M -
Suberyldicholine (Barrantes. 1978)<
Carbamylcholine (Boyd and Cohen, 1980b)d
0.008 sec - I 0.017 sec I 0.47 1.0 x I O - 6 M 6 x 10-"M 1.5 sec - I 0.006 sec I -
-
in 0. I M NaCI, 10 mM sodium phosphate, pH 7.4, at 20°C. in Torpedo Ringer's at 25°C; a departure from the two-state cyclic scheme was noted. in Torpedo Ringer's at 20°C. in Torpedo Ringer's at 4°C.
1
0.0005 sec - I 0.0023 sec - I 0.22 5 x 1 0 - 7 ~ 1.4 x 10-9 M 0.06 sec - 1 0.0008 sec - I 8 x lO-9M
0.0005 sec-' 0.0023 sec I 0.22 3 x 1 0 - 5 ~ 2.5 x 1 0 - X M 0. IS sec - I 0.00054 sec I 1.2 x 10-7 M -
-
~~
Determined Determined Determined Determined
Acetylcholine (Boyd and Cohen. 1980b)d
..
__
420
PALMER TAYLOR ET AL.
the rate of conversion to the high-affinity state (Lee et al., 1977). Binding at equilibrium should be reflected by a smooth hyperbolic function; the overall equilibrium constant, K,,, reflects the dissociation constants for both states, K, and K,,, and the equilibrium distribution between states, M :
Independent procedures are required to assign these individual constants. For example, by exposing the receptor to a high concentration of agonist, we should drive the major fraction into the R' state; subsequent dilution will allow the receptor to revert to the original R or low-affinity state. A measurement of ligand binding following conversion to the R' state, but before complete reversion to the R state occurs, has yielded the expected two components of the ligand binding curve. From these data both K , and K,. for the two detectable receptor states can be directly ascertained (Weiland et al., 1979). A second objective of these in vitro kinetic studies would be to establish that receptor activation proceeded from a low-affinity state and that conversion to a high-affinity state resulted in a receptor with a refractory channel. Popot et ul. ( 1976) have shown that exposure of receptor-containing vesicles from Torpedo to agonist results in a conversion in state and a diminution of the permeability response which occur over the same time interval. However, the rapid rate of ion influx in these vesicles precluded quantitative correlations owing to equilibration of the tracer ion. In intact clonal muscle cells, the quantitation could be improved since the larger internal volume to membrane surface area permits initial rates of Na+ influx to be measured (Sine and Taylor, 1979). At high agonist concentrations, rates of desensitization exceed the temporal resolution of the measurements. Nevertheless, at lower agonist concentrations the onset of desensitization could be demonstrated to parallel the conversion from the low- to high-affinity state. Correspondingly, the recovery of the response at various intervals following exposure to agonist parallels the return to the low affinity state. Although the ligand binding function at equilibrium (Y)and a state function for desensitization ( d )differ, they are both expressions of the same three constants: K,, K R r ,and M. For Eq. (4) in the absence of cooperativity
p=
AR
AR + AR' + AR' + R + R'
A
-
+ M) ( l / K R + MIK,.) (1
A
+
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
42 1
When two subunits are involved and the responses are positively cooperative, then h.n
A t RR
y = (A/K,)(l
(I
ZK,
ARR
/
AURA
+ A/K,) + M(AIK,.)(I + AIK,.) + A/K,)2 + M ( l + AIK,,)’
(9)
Hence, by determining the binding function and its component constants, the experimentally measured state function for desensitization can be described (Sine and Taylor, 1979. 1982). The receptors from Torpedo membranes and mammalian cells have similar ratios of K,/K,.; interestingly M = 0.1-0.2 in Torpedo whereas on the intact mammalian BC3H-I cell M = 1 x ]OW4 (Sine and Taylor, 1982). This difference in the fraction of receptor in the desensitized state in the absence of ligand, i.e. (R’/R), is reflected in the following observations: ( 1 ) K,, for the receptor in BC3H- I cells differs from K, only by a factor of 2-3 while this factor is 30 in Torpedo receptors. In Eq. (6), when M becomes very small, K,, approaches K,. Similar limits also emerge in Eq. (9). (2) Agents such as local anesthetics which increase M decrease the apparent equilibrium binding constants for agonists by a factor between 3 and 10 in Torpedo (Taylor et d . , 1980; Cohen et al., 1980) while in the mammalian cells, the decrease is greater than 30-fold (Sine and Taylor, 1982). This also would be predicted from Eqs. ( 5 ) , (6), and (9) since in the limit as M becomes large K,, = Kk. (3) The extent of desensitization following exposure to agonist also appears larger in the Torpedo system where essentially complete antagonism of the ion. flux can be achieved (Sugiyama et a / . , 1976). In BC3H-I cells, the maximal permeability response cannot be determined accurately and hence is likely underestimated; nevertheless, residual permeability following desensitization is 12-20% of the permeability response obtained in the absence of agonist exposure (Sine and Taylor, 1980). The state functions for desensitization [Eqs. (7) and (lo)] show that at high ligand concentrations the extent of desensitization will be inversely proportional to M . A second difference in the two systems is revealed in the extent of cooperativity found in the agonist concentration dependence for occupation. Both the muscle cells and the isolated Torpedo vesicles show Hill coefficients for activation between 1.5 and 2.0 (Cash et al., 1980; Neubig and Cohen, 1979; Sine and Taylor, 1979, 1980). However, Hill coefficients for receptor occupation in Torpedo appear close to 1 .0 (Weiland et u l . , 1977; Quast et ul., 1978; Boyd and
422
PALMER TAYLOR ET AL.
Cohen, 1980b), whereas positive cooperativity, n = 1.3-1.7, can be detected for receptor occupation in the BC3H-I cells following either instantaneous or prolonged exposure to agonists (Sine and Taylor, 1979, 1980). The differences in extent of cooperativity for occupation and activation can be interpreted in terms of an overall scheme which includes three states of the receptor: open channel, closed channel (activatable), and desensitized. In the case of activation the population of open channel states in relation to total agonist concentration is being monitored while occupation measurements assess the population of all bound species to total agonist concentration. In the case of receptor occupation at equilibrium, the cooperativity for occupation will depend on the value of M ;the Hill coefficient will approach the theoretical limit of the number of subunits when M is of similar magnitude to K,.IK,. As M approaches unity, as it does with the Torpedo receptor (cf. Table II), we would anticipate diminished apparent cooperativity for occupation. The monitoring of the association of acetylcholine and dansylhexylcholine with the receptor by rapid flow methods has enabled investigators to assign kinetic constants to the overall scheme in Eq. 4 (Table 111) (Boyd and Cohen, 1980b; Heidmann and Changeux, 1979). A rapid component of binding occurred with approximately 20% of the total receptor population. This value is in accord with previous estimates of M and could be increased by prior exposure of the receptor to agonist followed by subsequent dilution. Thus, this rapid step reflected binding to the high affinity R' state of the receptor. Association rates were of the order of lo8 M-' sec-' with dissociation occurring with rate TABLE 111 ESTIMATES OF KINETIC CONSTANTS POR LIGAND ASSOCIATION W I T H THE ACETYLCHOLINE RECEPTOR Acetylcholine (Boyd and Cohen. 1980a)" (23°C)
DNS-Cb-Chol. (Heidrnann and Changeux, 1979)" 9.5
X 107
M - I sec-l
0.30 sec - I
0.22 -
3.5 x 10sM-1 sec-I 0.29 sec - I 0.009 sec - I 0.036 sec - I 0.25 0.18 sec-1 0.00027 sec - I
'' Fast filtration of acetylcholine. Fluorescence detection of [ I ,5-(dimethylaminonaphthalene)suIfonarnide] n-hexanoic acid P-N-trimethylammonium bromide ethyl ester. Data are obtained in Torpedo physiologic saline solution (250 mM NaCI, 5 mM KCI, 4 mM CaCIZ, 2 mM MgCIZ, 5 mM sodium phosphate, pH 7).
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
423
’
constants of 0.15 sec- I for acetylcholine and 0.3 sec- for the dansylcholine analog. A slow step was also detected which reached a limiting rate at high agonist concentrations. This step was likely the result of the transition between the low- and high-affinity states. In addition, a discrete kinetic step of intermediate rate was evident; its concentration dependence suggested that a transition to a third state may be involved. Since this transition is detected in a concentration range below that required for activation it is possible that this transition results in an initial stage of desensitization where the receptor converts to a state of diminished responsivity (see also Neubig and Cohen, 1980). The existence of an intermediate state is also consistent with recent observations that desensitization occurs with two distinct kinetic components (cf. Section V).
VII.
OTHER LIGANDS AFFECTING RECEPTOR FUNCTION
Early electrophysiologic studies demonstrated that a large series of agents block the functional response to agonists noncompetitively (cf. Steinbach, 1968). Much of the early work employed local anesthetics but the list of noncompetitive inhibitors encompasses anesthetics, certain naturally occurring toxins, peptide antibiotics, and assorted compounds of rather ill-described pharmacologic activity that possess spectroscopic behavior enabling their detection. Since the binding of a-toxin is competitive with agonist, it would be predicted that these agents should not influence a-toxin binding. Indeed, there is a rather large window between concentrations blocking the permeability response and those inhibiting a-toxin binding kinetics (Weber and Changeux, 1974). More surprising is that within this concentration window many of these compounds enhance the apparent affinity of the receptor for agonists (Cohen et al., 1974; Kato and Changeux, 1976; Weiland et a / . , 1977). Since this behavior is characteristic of allosteric activators or inhibitors of regulatory enzymes, a mode of action of these compounds can be incorporated into a two- or multiple-state scheme. By simply altering the equilibrium ratio of R to R’, these agents can influence the affinity of the agonist for the receptor and yet be noncompetitive with agonist binding. The conversion in receptor state has been studied by agonist binding in the presence of local anesthetics (Weiland et at., 1977; Krodel et al., 1979; Sine and Taylor, 1982), general anesthetics (Young et al., 1978), peptide antibiotics (Brown and Taylor, 1982), and histrionicotoxin (Kato and Changeux, 1976; Burgermeister et al., 1977). In addition, electrophysiological studies have shown that quinacrine possesses local anesthetic-like properties (Adanis and Feltz, 1977); on the isolated receptor its enhanced binding in the presence of agonists could be directly monitored by fluorescence and the signal of the bound species eliminated by nonfluorescent local anesthetics and histrionicotoxin (Grunhagen and Changeux, 1976). The continuing analysis of activity of these ligands has revealed some interesting differences. Most of the local anesthetics enhance the rate of conversion to the R’ state that is induced by
424
PALMER TAYLOR ET AL.
agonists (Weiland et ul., 1977; Cohen et a / . , 1980) and, in fact, the local anesthetic will affect the ratio of R to R’ in the absence of agonist (Cohen et al., 1980; Taylor et al., 1980). However, certain other anesthetics, while increasing conversion to the R’ state, actually do so in the face of a reduced rate of conversion (Blanchard et al., 1979). In Torpedo, histrionicotoxin increases the affinity of agonists only to a limited extent (Cohen et al., 1980) and, analogously, prolonged exposure to agonists only slightly enhances the binding of histrionicotoxin (Elliott and Raftery, 1979; Aronstam et a / . , 1981). In the intact mammalian cells, histrionicotoxin and the local anesthetic both cause an essentially complete conversion of the receptor to the high-affinity state which reflects a quantitatively different action of histrionicotoxin on the receptor from two different species (Sine and Taylor, 1982). Short exposure to agonist is more effective in enhancing histrionicotoxin binding rates in Torpedo than is longer exposure to agonist (Aronstam et al., 1981). Since the data suggest that anesthetics affect the distribution of receptor among its various states, it is worthwhile to consider their action within a more quantitative framework. If the anesthetics simply increase M and do not create an additional receptor state, then the apparent dissociation constant for agonist occupation K,, in the presence of anesthetic should not become smaller than K R , . In cases where K,. has been independently estimated, it has been found to be smaller than the agonist dissociation constants measured in the presence of anesthetic (Cohen et a / . . 1980; Taylor et a / . , 1980; Sine and Taylor, 1982). Second, the rate of recovery from the R’ to R state following removal of either anesthetic or agonist should be similar (Cohen e t a / . , 1980). Third, the extent of anesthetic-induced shift of the equilibrium binding profile for agonists should depend on M as well as the concentration of local anesthetic. Thus, the greater shift in K,, by local anesthetics that is seen in receptors from BC3H-I cells relative to Torpedo membranes can be explained by the smaller M value for receptors from the mammalian cells (Sine and Taylor, 1982). If we define M‘ as the apparent equilibrium distribution of receptor in the presence of local anesthetic or other heterotropic ligand, H, then
+ where Mo is the allosteric constant in the absence of heterotropic ligand, and KR.Hand KR,,Hare dissociation constants for binding of heterotropic ligand to the R or R’ state. The modulation of M by heterotropic ligand should be reflected in both the agonist equilibrium binding function [Eq. (9)] and the state function for desensitization [ Eq. (lo)]. At saturating concentrations of heterotropic ligand the allosteric constant is increased in proportion to the selectivity of the ligand for the R‘ state, e.g., M’IM, = ( K R , H / K R r , H ) n . Thus a smaller enhancement of agonist binding in Torpedo receptors achieved
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
425
by histrionicotoxin in comparison with the local anesthetics (Cohen et al., 1980) can be explained by a smaller ratio of K R t , Hto KR,Hfor histrionicotoxin than for the local anesthetics. The effect of the heterotropic ligand in modifying M, to become M' should be reflected in the state function for desensitization [Eq. (lo)] as well as the equilibrium binding function [Eq. (911. The fact that the action of the noncompetitive inhibitors can be put into a generalized framework does not imply that they all share a common site or induce the same conformational change in the receptor. As considered earlier, the noncompetitive antagonism elicited by local anesthetics is likely manifested by association with more than one type of binding site.
VIII.
ANALYSIS OF RECEPTOR ACTIVATION
Considerable progress has been made in recent years in the measurement of receptor activation in various simplified cellular systems and reconstituted membranes. That a solubilized and purified receptor can be reconstituted to replicate its original responses in situ has been demonstrated convincingly in recent studies. Desensitization of the response and the reproduction of antagonist sensitivity was shown in initial studies (Epstein and Racker, 1980) and more recent work with single channel measurements in reconstituted bilayers shows that the responding channels exhibit kinetic behavior closely resembling the native channel (Bohein et al., 1981; Nelson er al., 1980, Schindler and Quast, 1980). A second objective of developing assays for activation in defined systems is to examine the relationship between ligand occupation and the response elicited. To accomplish this the response in a population of receptors identical to those in which occupation is measured is monitored. Early studies with vesicles obtained from homogenates of electroplaques established that agonists can enhance cation permeabilities in sealed vesicles, but the relatively high densities of receptors coupled with the larger surface to entrapped volume of a vesicle of a few microns in diameter preclude the measurement of the initial rate of influx. To relate permeability to receptor number, initial rates should be measured in the entire population of vesicles and a wide size distribution would further complicate the analysis. The problem of initial rate measurements has been obviated in part by the development of quench-flow methods which enable the measurement of ion flux over periods of 10-20 msec (Cash et at., 1980, 1981; Neubig and Cohen, 1980). The latter group has been successful in measuring both response and occupation within a time frame of 100 msec. Desensitization kinetics has been monitored by these procedures and in Torpedo vesicles a rapid desensitization step occurring in the subsecond to second time frame has been detected (Cash et al., 1981). Thus, the two phases of desensitization detected in single channel measurements are apparent in the rapid ion flux assays. Prior to desensitization
426
PALMER TAYLOR ET AL.
flux rates approaching lo7 ions sec- receptor- are found for acetylcholineand carbamylcholine-elicited responses. This rate compares well to values found for single channel measurements (see Section Ill).
IX. TOWARD THE UNDERSTANDING OF COUPLING BETWEEN OCCUPATION OF THE RECEPTOR AND THE PERMEABILITY RESPONSE To approach the question of the relationship between occupation and the functional response, simultaneous measures of both parameters are required. At present, two systems seem suited for this purpose. The first is the isolated and sealed vesicle isolated from homogenates of Torpedo sp. electric organs (Neubig and Cohen, 1980; Cash et al., 1980). This system carries the advantages that measurements are made on the same receptor in which structural characterization is most complete and the system can be studied in suspension which permits measurements of Permeability and occupation in a time frame approaching several milliseconds (Neubig and Cohen, 1980). Vesicles are formed and purified in the presence or absence of tracer ion (*6Rb+ or **Na+) and influx or efflux of the tracer cation is measured following rapid mixing with agonist. Ion flux is quenched by rapid addition of antagonist at a prescribed interval after agonist addition. The relative nonselectivity of the channel enables efflux to be measured; however, with such measurements it is difficult to measure desensitization and its recovery from prior agonist exposure. A variation of this procedure is to incorporate a fluorescent dye into the vesicles and monitor influx of a cation such as T1+ which quenches the dye's fluorescence by a collisional mechanism (Wilson et at., 1981). A second system for measuring Iigand occupation and the elicited response is the cultured muscle cell, which, when grown in monolayer, allows for equivalent exposure of agonists to surface receptors (Catterall, 1975; Sine and Taylor, 1979). In clonal cells such as the BC3H-I cells, each cell is the daughter of a single progenitor and permeability changes can be monitored in cells of comparatively uniform size and shape. The cell line also carries the advantages that measurements can be conducted on mammalian receptors in intact cells and that flux in the appropriate inward direction is ascertained for the major currentcarrying ion involved in agonist-elicited depolarization of the membrane, Na+ . For analysis of permeability by ion fluxes, it is important not to violate initial rate conditions for the entire population of cells or organelles. Thus one strives to achieve a relatively narrow size distribution of enclosed volumes. The relatively small surface-to-internal volume of intact cells compared to membrane vesicles enables experiments to be carried out over a more extended time frame. Desensitization can be minimized by working at 3"C, but desensitization steps occurring
427
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
in the subsecond time frame will not be detected in the flux assay on intact cells. To ensure that permeability is not influenced by changes in membrane potential occurring during the measurements of unidirectional flux, potential is adjusted to near zero by balancing external and internal Na+ and K + .
X. OCCUPATION AND ACTIVATION BY AGONISTS Under conditions in which we are able to eliminate a certain fraction of binding sites, let us consider how the residual permeability response will be affected. Since the a-toxins bind with high affinity to the sites on the two asubunits and for the purposes of these experiments their binding is irreversible, they offer an ideal means for fractionally inactivating the surface binding sites on the intact cell. Moreover, since the association rate of a-toxin exhibits bimolecularity, no selectivity for either of the two sites or cooperativity in binding is evident (cf. Sine and Taylor, 1979). Accordingly, with 50% overall occupation of the sites the species of receptor-toxin complexes should exhibit a binomial distribution in which 25% of the receptors are totally unoccupied, 25% are fully occupied by two a-toxin molecules, and the remaining 50% are essentially "hybrid" species carrying a vacant site and one bound a-toxin molecule at the other site. Upon addition of agonist, should the hybrids be nonfunctional, overall occupation by a-toxin of 50% of the sites leads to a residual response of 25% (cf. Fig. 2). The more general equations for the relationship between prior a-toxin occupation and the residual response can be calculated from the fraction of active
Species
"IA
nIB
"0
"IA "IB -+T 2
#-Toxin Occupation=y ( 0 5 ) Carbamylcholine occupotionofter In - t o m = m Carbomylcholine activation = &/kGo
t "2
Sn
em
@ IZ @
@
lr
"0 +%+% 2 2 m= kG/kGo'
En
"O En
i
=I-y
(I-v)~
FIG.2. The relationship between prior a-toxin (aT)occupation and carbamylcholine (C) occupation and activation of the acetylcholine receptor. The receptor is depicted as a nonequivalent dimer of binding sites. a-Toxin association does not distinguish between sites.
428
PALMER TAYLOR ET AL.
species present and the linkage relation for ligand association to a dimer of binding sites. These equations are shown in Fig. 3. The relationship between atoxin occupation, y , and the fractional permeability kG/kG, is best described by a parabolic function of the form
Thus, at least two bound agonist molecules are required for receptor activation and hybrid species carrying a bound a-toxin molecule and the agonist (i.e., carbamylcholine) remain mute with respect to ion permeability (Sine and Taylor, 1980). By using fast flow methods with native Torpedo membrane vesicles, Neubig and Cohen (1980) found that after a-toxin occupation of 70% of the sites, the residual permeability response was 16% of the original permeability, a value that lies close to the 9% calculation from the parabolic function. Also, these workers compared integral permeability responses to a partial agonist conducted in the presence and absence of fractional receptor blockade with a-toxin. Analysis by the null method indicated that antagonism was greater than that predicted by a linear relationship between a-toxin occupation and functional sites. Using vesicles to which purified acetylcholine receptor had been added, Lindstrom er al. ( 1980b) found an apparent linear relationship between the fraction of sites occupied by a-toxin and the fractional reduction in response. These workers proposed a rather unique model for oligomeric proteins in which one of the asubunits plays an obligatory role for activation while the other is subservient in only augmenting the cooperativity of the response. However, half-maximal responses were occurring in the range of 1 carbamylcholine, which is a considerably lower concentration than those found for half-maximal activation by fast flow methods (Neubig and Cohen, 1980; Cash, et al., 1981) or bath application of agonists in other systems (Adams, 1976; Dionne et af., 1978; Dreyer et al., 1978; Lester et af., 1978). Departures from the true relationship could arise if wide distribution of surface receptor density to internal volumes exists in the vesicle population. Under such circumstances initial rates of influx will not be measured in the entire population of vesicles. A similar problem is likely to exist in the interpretations of Delegeane and McNamee (1980). These workers observed an apparent maximal response in permeability following complete reaction with MBTA. Since MBTA alkylates one-half of the a-bungarotoxin binding sites under these conditions, these workers proposed that each of the carbamylcholine binding sites could independently activate the receptor. More recent studies by Lindstrom and colleagues (Anholt et a/., 1982) reveal that the relationship described by Eq. (1 2 ) is approached when vesicles of larger internal volume and lower density are employed. Should the parabolic relationship of Eq. (12) hold, two supporting observations are necessary to complete the argument. First, since only the doubly unoccupied receptor species will respond to subsequent agonist exposure with a
*
429
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
f
FRACTIONAL OCCUPANCY BY a.TOXlN
P
.40
.
20* .40 .60 O .80 1.00 b
L
20 .40 6 0 .80 1.00 FRACTIONAL OCCUPANCY BY "'I-Q-TOXIN
FIG. 3 . Reduction of the permeability increase. kG, elicited by carbamylcholine resulting from progressive occupancy of receptors by tz51-labeled a-toxin. (A) Theoretical relationships for the fractional permeability response versus fractional occupancy of receptors by a-toxin generated from Model 3, - - - -,Model 4; --, Model 5 . Model Models I to 5 : .-., Model I ; ...., Model 2; -* I : Functional receptor contains two binding sites and full activation results when one or two binding sites are occupied under saturating agonist concentrations. Occupation of both sites by toxin is required to block function.
... kc;
kG,,(I - V 2 )
Model 2: Functional receptor contains two binding sites and activation results when one or two binding sites are occupied under saturating agonist concentrations. Occupation of each site by a-toxin reduces the response capacity of that receptor by one-half.
Model 3: Functional receptor contains two binding sites and activation requires occupation of both binding sites by agonist. Occupation of either site by a-toxin will completely block function.
:. ka
=
k(j,,(l
- V)2
Models 4 and 5 represent a tetramer of binding sitcs where occupation of one and two sites, respectively, by a-toxin will block function. Details are given in Sine and Taylor (1980). (B-D) Experimentally determined fractional kG following progressive degrees of occupancy by 12s1-labeled a-toxin. (B) Permeability increase, determined from the rate of sodium influx elicited by 30 carbamylcholine in a 75-second interval. (C) Permeability increase resulting from activation by 60 pJ4 carbamylcholine measured in a 30-second interval. (D) Permeability increase resulting from activation by 100 pJ4 carbamylcholine measured in a 15-second interval. In each panel, the solid line corresponds to the function kG = kc,(l - y)2 resulting from Model 3, where v is the fractional occupancy of receptors by a-toxin.
430
PALMER TAYLOR ET AL.
change in permeability, fractional titration of sites with a-toxin should not alter the concentration dependence or the cooperativity for agonist activation. The Hill coefficient for activation of the residual sites was found to be unaffected by fractional prior a-toxin occupation, nor did a-toxin occupation affect the agonist concentration dependence for achieving desensitization of the residual response following exposure to agonist (Sine and Taylor, 1980). Alterations in cooperativity for receptor activation following fractional a-toxin occupation have not been investigated for the Torpedo vesicle system. The linear relationship between a-toxin occupation and the fractional reduction in permeability suggested in some studies (Delegeane and McNamee, 1980; Lindstrom et al., 1980b) would be expected to give rise to a loss of cooperativity of the residual permeability response with fractional a-toxin blockade. Second, with a concerted interaction between subunits involved in activation, we would anticipate that prior a-toxin occupation would influence the cooperativity of carbamylcholine occupation of the neighbor of paired sites in the dimer. Thus, upon prior titration with a-toxin, the hybrid species containing one of the two sites occupied by a-toxin will begin to predominate over the doubly unoccupied species by the ratio 2y( 1 - y ) / ( 1 - y)’ and the positive cooperativity for occupation by agonist should diminish. This can be readily tested in the intact cell since occupation of the receptors by agonist displays positive cooperativity. Interestingly, not only is the Hill coefficient (n,) for carbamylcholine occupation reduced, but, as y increases, a,, goes through unity and approaches a limiting value of 0.65 (Sine and Taylor, 1980). This observation holds for association with the low-affinity states (which measures binding to the activatable, active, and perhaps the partially desensitized states of the receptor) as well as for association at equilibrium (which also includes binding to the desensitized state of the receptor). The fact that a limiting Hill coefficient of less than unity is achieved indicates that the two a-subunits are not functionally equivalent and the concerted transition is superimposed on an intrinsic binding inequivalence of the two a-subunits.
XI.
ASSOCIATION OF ANTAGONISTS WITH THE RECEPTOR AND FUNCTIONAL ANTAGONISM
Antagonists are found to exhibit Hill coefficients of less than unity for their association with Torpedo and mammalian receptors (Weiland et al., 1979, Neubig and Cohen, 1979; Sine and Taylor, 1979). Neubig and Cohen (1979) have measured antagonist binding to Torpedo receptors by equilibrium dialysis and by competition with the initial rate of a-toxin association. Both procedures yielded Hill coefficients less than unity and evidence for two classes of binding sites for antagonist occupation. However, the estimated dissociation constants differed for the two methods, the basis of which is still unexplained. Classic antagonists,
431
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
of course, do not promote the transition associated with activation or channel opening and show little or no propensity for driving the receptor into its desensitized state (Weiland et al., 1979). Hill coefficients less than unity for antagonists could be due to two possibilities. The least likely is true negative cooperativity in which antagonist binding to one of the two sites diminishes the affinity of the neighboring site for this ligand. This can be ruled out by the observation that prior a-toxin occupation does not alter the concentration dependence of reversible antagonist binding to the residual sites (Sine and Taylor, 1980). In the negative cooperativity model, convergence to a Hill coefficient of 1.O would be expected as hybrid species are formed. When hybrid species predominate antagonist binding would not be influenced by prior binding of the reversible antagonist at neighboring sites. The alternative for the low Hill coefficient is based on a nonequivalence of binding sites and here we can develop two limiting cases: either the two nonequivalent sites, A and B, are confined to a single oligomer (i.e., AB oligomers), or two discrete types of receptor oligomers each containing identically paired subunits are present (AA and BB oligomers). If the two types of receptors (AA and BB) were present in near-equal populations, binding experiments alone would not distinguish between these two cases. In both cases fractional occupation
=
0.5(XA) + 0.5(XB)
(13)
=
0.5L/(L + K A )
(14)
+ 0 . 5 L / ( L + KB)
where L is the ligand concentration, X , and X , are the fractional occupations of the A and B sites, respectively, and K , and K , are the respective dissociation constants. It is to be expected that reversible antagonists, like a-toxin, will block an agonist-elicited response by binding to a single site. Then, if the binding sites are confined to a single oligorner:
kc/kG,
= (1 -
XA)(l - X,)
= [K,/(L
f
(15)
K A ) ] [ K B / ( L+ K B ) ]
(16)
If the nonequivalent sites exist on separate oligomers each containing identically paired subunits, then k,/k,,
+ 0 . 3 1 - X,)’
=
0 . 3 1 - X,)’
=
0.5( L A K , )+2 0.5( L
+
(17)
A)’ + K,
When K , -+ K , , Eqs. (16) and (18) converge, whereas when K , and K , differ, the overall functions differ substantially. Data for the association of five classic nicotinic antagonists reveal Hill coefficients less than one (Table IV) and the binding curves all can be fit to a two-site model in which there are equal populations of sites with differing dissociation constants (Fig. 4). Within this
TABLE I V PARAMETERS FOR
$
N
Alcuronium Pancuronium AH8165 Gallamine Dimethyl-dtubocurarine
ANTAGONIST COMPETITION
4.2 X 10-8 2.3 X 10-8 6.0 x 1 0 - 7 1.5 x 1 0 - 5 3.1 x 10-6
0.87 0.86 0.78 0.70 0.51
5 5
2 ? 5
WITH
0.03 0.02 0.01 0.03 0.03
U-TOXIN BINDING
A N D INHIBITION OF THE PERMtABILITY INCREASE E L i r i T m B Y CARBAMYLCHOLINE
2.14 X 9.11 X lo-" 1.83 X l o p 7 3.70 x 10-6 3.09 X lo-'
8.68 X 6.93 X 1.84 X 5.50 x 2.75 X lo-'
4. I 7.6 10.3 14.8 89.0
1.3 7.4 2.1 3.7 4.7
x 10-8 x 10-9 x x x 10-7
0.99 t 0.02 1.16 t 0.07 1.08 2 0.03 1.00 2 0.06 0.85 t 0.05
3.3 3.2 2.9 4.0 6.9
0 Kp is the concentration of antagonist which decreases the initial rate of toxin binding. RT. by 5 0 F , and n is the associated Hill coefficient. K, and n are calculated by fitting the Hiil equation to the data. Hill coefficients are the slopes of the best fit line 2 the standard error of the mean. b KA and K, are the best fit high- and low-affinity intrinsic dissociation constants resulting from the fit of Eq. (14) to the experimental data. K,,, is the concentration of antagonist which diminishes the permeability increase elicited by 30 p M carbamylcholine by 50%. and n is the associated Hill coefficient (Sine and Taylor, 1981).
1OOr
I t-
x
-I
0
:I
2o
0'
-100
-
a0
60
- 40
' I
' 10-0
I
10.'
I
10-7
[Alcuronium]
16-8
10-B
I
lo-'
lo-'
(Pancuronium]
-
2o
10-8
1
O9 J
n
P
0 0
[Gallamine]
[Dimethyl d-Tubocurarine]
FIG. 4. Concentration dependence for antagonist inhibition of the initial rate of '2sII-labeleda-toxin binding and inhibition of the carbamylcholine-mediated permeability increase to 2zNa+ . Cells were covered with buffer, cooled slowly (30 minutes) to 3.5"C. and incubated for 20 minutes in the presence of the specified concentrations of antagonist. Antagonist occupation was then measured by its competition with the initial rate of a-toxin binding which was determined in a 120-second interval (squares) and is expressed relative to the control rate, kT determined in the absence of antagonist. Functional antagonism was measured by replacing the prior incubation solution with an identical solution supplemented with 30 ph4 carbamylcholine and zzNa+,after which the initial rate of tracer sodium uptake was monitored in a 75-second interval (circles). The resulting permeability change, k G . is calculated in terms of a first-order exchange of isotope and is expressed relative to kc,,measured in the absence of antagonist. Each experimental point is the mean of duplicate determinations. The solid curve associated with the squares is the best fit of Eq. ( 14) to measurements of antagonist binding. and best fit values of K A and K , are listed in Table IV. The remaining solid and dotted curves are, respectively, the predictions for functional antagonism in terms of Eqs. (16) and (18), using the values K A and K , , which are derived independently from analysis of antagonist binding (Table IV). (From Sine and Taylor. 198 I. )
434
PALMER TAYLOR ET AL.
series of antagonists K,IK, ranges between 4 for alcuronium and 89 for dimethyl-d-tubocurarine. By obtaining dissociation constants from the occupation curves [Eq. (14)] one can compare the concentration dependence for functional antagonism with the predictions of Eqs. (16) and (18). The experimental observations in the mammalian cells clearly show a close correspondence with Eq. (16) or the limiting case where the two sites are confined to a single oligomer (Sine and Taylor, 1981). Another approach allows one to corroborate this model. Cobra a-toxin association rates do not reveal selectivity for the a-toxin sites on the a-subunits. However, if cobra a-toxin association were allowed to occur in the presence of an antagonist (such as dimethyl-d-tubocurarine) the high affinity or A sites would be protected with a-toxin being retained on the B sites. It can then be demonstrated that dimethyl-d-tubocurarine binding to the vacant sites approaches the behavior of binding to a single class of sites of high affinity. A Hill coefficient approaching 1 .O and a dissociation constant approaching K , are found (Sine and Taylor, 1981). Thus, with a means for selectively directing a-toxin to one of the two sites, let us consider the relationship between fractional permeability (k,lk,J and a-toxin occupation, y . If the two sites are confined to a single oligomer (AB oligomers), then kclk,,, = ( 1 - Y,)(l
- YR)
(19)
while if two different oligomers with identically paired subunits (AA and BB) exist: kclkG,) = 0 3 1
- Y,)~
+ 0 . 31 - Y , ) ~
(20)
Since ( 1 - y J I - y s ) < ( I - yI2 < O S ( 1 - vA)2 + O S ( 1 - y s ) 2 , a comparison of the fractional permeability following selective and nonselective direction of a-toxin to its sites should distinguish between limiting cases. We find that a greater reduction in permeability results with selective direction of atoxin than random direction, again consistent with the nonequivalent sites being confined to one oligomer (Sine and Taylor, 1981). Intuitively, in the AB oligomer model we would expect the greater fractional reduction of permeability when a-toxin sites are labeled in the presence of a reversible antagonist, since the abundance of hybrid species would exceed that predicted by a binomial distribution.
XII. QUANTITATION OF ANTAGONIST OCCUPATION AND FUNCTIONAL ANTAGONISM A comparison of the concentration dependences for antagonist occupation and functional antagonism reveals certain salient features in the analysis of doseresponse curves (Fig. 4 and Table IV). First, the concentration dependences for
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
435
antagonist occupation and antagonism of the response elicited at low agonist concentration are not identical as we can see in the two extremes. When K , << K,, K,,, = K,, whereas when K , + K , then
Second, Hill coefficients for the concentration dependences of functional antagonism are predicted to be larger than the corresponding values for receptor occupation. For functional antagonism, when K , and K , differ, nH = 1 .O, whereas in the limit of K , + K,, nH = 1.2. In the pharmacologic quantitation of competitive antagonism, a null method or Schild plot (Schild, 1949) is also employed to determine the dissociation constant for the antagonist. For oligomeric receptors such as in the situation above, an essentially parallel shift of the dose-response curve will still be achieved in the presence of Competitive antagonists. In the null method antagonist potency is assessed through direct competition with agonist for occupied sites rather than blockade of unoccupied sites. Hence the dissociation constant for antagonists calculated from the null method for a receptor with nonequivalent sites will differ from that obtained from functional antagonism at low agonist concentrations and also will not equal the overall apparent dissociation constant for occupation of the receptor by the antagonist. Hence, for the various receptors, an understanding of the mechanism of antagonism is needed to relate quantitatively the dissociation constant obtained from functional antagonism to the true dissociation constant(s) obtained from ligand occupation.
XIII. STRUCTURAL IMPLICATIONS AND ARRANGEMENT OF SUBUNITS While the data are consistent with the major population of receptors existing as AB oligomers rather than AA and BB pairs, it becomes more difficult to dis-
tinguish this limiting model from more complex arrangements of subunits. For example, three or more distinct oligomers could arise through associations of the subunits (i.e., AA, AB, BB). For the overall azpyS composition, such arrangements require either microscopic differences in subunit compositions within the individual oligomers or permutations in the subunit arrangements. Since up to 85% of the Torpedo receptor is disulfide associated as dimers and this linkage occurs through the 6-subunit (Hamilton et af., 1977), differences of subunit compositions of individual monomers giving rise to an overall a#$ stoichiometry seem unlikely. The possibility of permutations in subunit arrangements in the individual molecules is a more difficult possibility to discard. For example, if the positions of the two a-subunits were fixed, permutations in the arrangements of p, -y, and 6 would lead to three pentameric species differing
436
PALMER TAYLOR ET AL.
only in subunit arrangement. While more complex models introduce additional free parameters and hence can provide a closer correspondence with the experimental data, experiments in intact cells lack the precision to distinguish the small quantitative differences between complex models. The AB oligomer scheme is defined simply by a single receptor species while all other arrangements dictate that multiple oligomeric receptor species are present. The nicotinic acetylcholine receptor can be described most economically in terms of a functionally asymmetric oligomer in which the two sites within the oligomer do not exhibit equal binding affinities in the case of both agonists and reversible antagonists. For antagonist association this is reflected in Hill coefficients less than I .O. Agonists, however, initiate state transitions both for activation and desensitization. Positive cooperativity is evident and thus at least two agonist molecules are required to effect a transition to the activated (open channel) and to the desensitized state. Analyses of both the binding and state functions for desensitization would suggest that a concerted or symmetry-driven mechanism (cf. Changeux, 1981) best describes these transitions (Sine and Taylor, 1982). Receptor hybrids that form containing bound a-toxin and agonist or reversible antagonists and agonists do not appear to convert to the open channel state nor do they show a positively cooperative transition to the desensitized state. It is only when the hybrid species predominates and the concerted transition is unable to occur, that the inequivalence in the two agonist sites is revealed. When two agonists in equieffective concentrations are applied to the neuromuscular junction the predominant channel open time corresponds to that elicited by neither of the two agonists alone but rather a hybrid value. This result suggests that receptor oligomers occupied with different agonists at each site will not only be active but produce a unique channel opening duration (Trautmann and Feltz, 1980). Although the two a-subunits in Torpedo receptors appear chemically identical on the basis of available N-terminal sequences (Raftery et al., 1980), their functional inequivalence is not unexpected. Nonsymmetric arrangements and functional inequivalence would be expected for a pentamer in which the p-, y-, and &subunits are associated with two a-subunits. Since most and possibly all of the subunits traverse the membrane (Wennogle and Changeux, 1980) and all display considerable sequence homology (Raftery et al., 1980), it is likely that similar segments in the linear sequence of each subunit form the presumed ahelical domains of some 25-30 residues that traverse the membrane bilayer. Arranging the subunits as in Fig. I necessitates that the intersubunit contacts for the two a-subunits will not be identical. Accordingly, it is not possible to place a twofold axis of symmetry perpendicular to the membrane. Thus, a structural basis exists for the functional inequivalence as well as for differences in sulfhydry1 reactivity (Damle and Karlin, 1978; Wolosin et al., 1980). For the receptor in BC3H-1 cells, details on the structure of the individual subunits are lacking,
LIGAND OCCUPATION AND RESPONSE OF THE CHOLINERGIC RECEPTOR
437
yet the ci2PyS arrangement is consistent with the subunits detected from Coomassie blue staining of the purified receptor (Boulter and Patrick, 1977).
XIV.
ANALYSIS OF THE BOUND LlGAND STATES
If we accept that the receptor behaves as a nonequivalent dimer of binding sites we can represent the overall scheme for transitions between the activatable R,R,, desensitized RARL, and open channel form of the receptor RZR; as follows:
A +
A +
With nonequivalent binding sites for the subunits we also might consider the possible species which can form when both agonist, A, and antagonist, T, can simultaneously combine with R,R,:
/RaARbT\
RaTRbT,
J'
,
RaRb /Ramb\
/RaARbA
(23)
RaTRbZ / R ,TRbA
RaRbA
An examination of Eqs. (22) and (23) shows the complexities intrinsic to modeling of all the species involved in receptor activation and inhibition. Moreover, Eq. (22) accounts for only a single desensitized state and it is likely that two steps occur in the desensitization process (Sakmann er al., 1980). Since no technique possesses both the requisite temporal resolution and discrimination in detection of all of the individual states, analyses are constrained to consideration of transitions within a particular time frame or to those transitions coupled to steps in which a change in signal can be monitored. Under such circumstances transitions are measured between composites of receptor states. Nevertheless, inroads have been made into the linkages between ligand occupation and the responsive and nonresponsive receptor states.
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PALMER TAYLOR ET AL.
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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 1X
The I 1te raction of Cholera Toxi n with Gang iosides and the Cell Membrane SIMON VAN HEYNINGEN Departmen! of Biochemistry Universiry of Edinburgh Edinburgh, Scorland
Structure and Action of Cholera Toxin . . . . . . . . . ....... . A. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . ................. ................................. B. The Nature of the Toxin.. . . . . . C. Protein Chemistry.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Role of Each Subunit.. . . . . . . . . . . . . . . . . . . . . . . E. The Catalytic Action of the Al Peptide ... 11. The Role of Ganglioside GMI as a Cell-Surface Receptor.. .................... A. The Nature of the Gangliosides . ........................... B. The Binding of Cholera Toxin to Cells.. . . . . . . . . . . . . . . . C. The Evidence That the Receptor Is Ganglioside GMI . . . . . D. The Distribution of Ganglioside GM I in the Membrane .................... 111. The Nature of the Reaction between Ganglioside and Toxin.. . . . . . . . . . . . . . . . . . . . A. Detection of Ganglioside Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Ganglioside Binding in Purification and Detection of Toxin .. C. Role of Subunit B in Binding the Ganglioside.. . . . . . . . . . . . . . . . . . . . . . . . . . . D. Specificity for the Ganglioside.. ............................. E. Stoichiometry and Kinetics of Toxin-Ganglioside Binding. F. Conformational Change following the Binding of Gangliosi IV. Transport of Cholera Toxin across the Cell Membrane and the Role of Binding to Ganglioside . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Penetration of the Membrane by Peptide A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Function of the Gdnglioside . . . . . . . . . ..... ...... C. The A1 Peptide by Itself . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Possible Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Other Compounds That Bind to Gangliosides. ...... .... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Copyright 0 1983 by Academic Press, Inc. All rights of reprcduction In any forni resewed. ISBN 0-12-I533 18-2
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1.
STRUCTURE AND ACTION OF CHOLERA TOXIN
A. Introduction Although cholera toxin is the means by which Vibrio cholerae causes a disease that kills thousands of people every year, its biochemical action is very similar to that of a hormone: it is a powerful activator of adenylate cyclase in almost all types of eukaryotic cells. Its mechanism of action is now better understood than that of most hormones. In particular, the receptor for cholera toxin on the cell surface, a ganglioside molecule, was one of the earliest receptors to be identified and remains about the best characterized. However, as 1 shall describe; it is perhaps not a receptor after all in the strict sense, even though the toxin certainly binds to it before activating the cyclase. In this article, 1 propose to outline briefly the general mechanism and structure of the toxin, and to discuss how it activates the cyclase. I shall pay more attention to the binding to the cell membrane and the subsequent events triggered by this binding. Much early work is described more fully in earlier reviews (e.g., van Heyningen, 1977a; Gill, 1977, 1978; Moss and Vaughan 1979; Lai, 1980).
B. The Nature of the Toxin Cholera is a simple though unpleasant disease; its most obvious symptom is a massive diarrhea in which the patient can excrete more than a liter of fluid every hour. This leads to dehydration of the surrounding tissues and an upset in the salt balance, and is often fatal. The disease is caused by Vibrio cholerae which grows in the gut and secretes the protein toxin in to its environment. The action of the toxin is to increase the rate of water transport across the gut wall. How it does this is beyond the scope of this article, but it turns out to be due at root to the activation of adenylate cyclase in the brush border cells. Subsequent experiments showed that the toxin can activate adenylate cyclase not only in the brush border cells, but also in essentially every eukaryotic cell that has been tried from pigeon erythrocytes to Drosophila and even including the archaebacteria. The high specific activity and the effect on adenylate cyclase are what makes cholera toxin seem like a hormone. However, the absence of any tissue specificity distinguishes it sharply from hormones. Studies on the action of the toxin with cells (see below and summarized in van Heyningen, 1977a) have shown that its first action is to bind to the cells tightly and irreversibly. There is then a lag varying from about 15 to 90 minutes depending on the type of cell before the intracellular adenylate cyclase activity begins to rise. The first notion that cholera toxin might bind to a ganglioside came from the
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observation (van Heyningen et ul., 1971) that preincubation of the toxin with mixed gangliosides inhibited its activity in the bioassay closest to cholera itself, in which the flow of fluid into ligated portions of the gut of a living animal is measured. Further experiments showed that the effect of ganglioside is also found when the toxin is assayed directly for its ability to activate adenylate cyclase, but only when this is done with intact cells. When the adenylate cyclase is directly available to the toxin in broken cell preparations, ganglioside has no effect and there is no lag phase. Gangliosides also have no direct effect on adenylate cyclase, so presumably they must be involved in the original binding to the cell surface. The inhibitory effect of gangliosides on cholera toxin is now universally accepted. In fact, ganglioside has even been used in clinical trials as a treatment for cholera although without a great deal of success (see Holmgren, 1981).
C. Protein Chemistry Cholera toxin (M,82,000) is a simple protein, easily purified from culture filtrates of V . cholerue. It has no detectable lipid or carbohydrate. It is a very stable protein that can refold to an active configuration after exposure to 1% (wh) sodium dodecyl sulfate or 6 M guanidine hydrochloride. Crystals have been grown and X-ray diffraction studies are under way. The toxin has a complicated subunit structure. There are two different types of subunit. Each toxin molecule has five B subunits (M,= 11,500) and one A subunit. The 5 subunits have been sequenced; there is one intrachain disulfide bond. An aggregate of the five subunits B alone without any subunit A is secreted into its medium by V . cholerae along with the toxin. It is known as “choleragenoid” and thought of as “natural toxoid” because it is immunologically almost indistinguishable from whole toxin. Most of the antigenicity of whole toxin is in the 5 subunits. Subunit A is originally synthesized by the bacterium as a single polypeptide chain, but this is rapidly “nicked” by a proteolytic enzyme into two chains-A I (M,= 22,000) and A2 (M,= 5000)which remain covalently linked by a disulfide bond. Much of the sequence of subunit A has also been determined (and even more of the probably very similar sequence of subunit A from the toxin of Escherichia coli, Spicer et al., 1981). Subunit A is in some ways also a very stable protein: it can be boiled in sodium dodecyl sulfate (SDS). However it is awkward to work with since it tends to come out of solution at protein concentrations greater than about 1 mg/ml and to be irreversible absorbed to glasswear and chromatographic media. Cross-linking experiments and theoretical considerations have led to an idea of the structure of the whole molecule which is supported by electron micrographs
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and preliminary X-ray diffraction data. The B subunits probably form a symmetrical ring with subunit A sitting in the middle joined to one or more of them through the A2 peptide (but presumably not to all of them since the peptide is not big enough), leaving the A1 peptide on the outside of the molecule. This is shown in Fig. 1.
D. Role of Each Subunit Once the basic structure of the toxin was known it was demonstrated almost immediately that each subunit plays a very different role. The B subunits can bind the ganglioside receptor, but they have no other activity (see Section 111,C). Subunit A does not bind ganglioside significantly, but the A1 peptide alone can activate adenylate cyclase in cell-free extracts (where the cell membrane is no longer intact). Adenylate cyclase is always found on the inside surface of the cell membrane, so these experiments (and others to be described below) lead to the idea that the first action of the toxin was binding to the cell membrane through the B subunits, and that this in some way allowed or at least facilitated the entry of the A1 peptide to the interior of the cell where it could interact with its substrates. This general model of the toxin action (shown in Fig. 2) has been widely accepted, perhaps not least because it has several attractive similarities to the action of certain other toxins such as diphtheria toxin.
E. The Catalytic Action of the A1 Peptide Intense work in recent years has led to a good idea of how the A1 peptide works (see van Heyningen, 1980a). it will be very briefly summarized here as being outside the scope of this article.
Plan FIG.
I.
Elevation
An impression of the structure of cholera toxin. (From van Heyningen, 1977a.)
449
CHOLERA TOXIN AND GANGLIOSIDES
/
I
Slow dissociation and entry
FIG.2. A widely accepted idea of how peptidc A l might enter the cell As discussed in the text, it is not certain that the whole of the peptide passes through the membrane, or that it all binds to the GTP-binding protein.
Studies with purified membranes and with isolated proteins from them have shown that the action of A l is to catalyze the ADP-ribosylation of a GTP-binding protein that is one of the components of the adenylate cyclase complex. The reaction is NAD
+
+ protein
adeninc-ribose-phosphate-phosphateribose-protein
+ nicotinamide + H +
There is some disagreement as to whether there is only one such protein substrate or whether there are in fact several. The ADP-ribosylation of this protein is irreversible under physiological conditions, and this leads to the more-or-less permanent activation of the cyclase. It is because cholera toxin is a catalyst of this reaction that it is so active, and only a very few molecules of A l per cell are needed to produce a maximum effect. Why the ADP-ribosylation of this peptide leads to the activation of the cyclase is still a subject of some discussion. The simplest hypothesis is that of Cassel and Selinger ( 1977) who suppose that the complex of cyclase and GTP-binding protein can exist in two states which are in equilibrium with each other: an active state when GTP is bound and an inactive state when GDP is bound. Normally the binding protein itself having a GTPase activity hydrolyzes the bound GTP and so converts the complex into an inactive state. However the ADP-ribosylation inactivates this GTPase activity, keeping the complex in the active, GTP-binding state. This is shown in Fig. 3. There are numerous other theories more complex than this, but they retain the basic notion.
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SIMON VAN HEYNINGEN ACTNE
ADPR
Cyelase
f
Protein
\
GDP
Hormones
v
/
4
GTP
jJqJ
Protein
ACTNE
No toxin
i
INACTIVE
F] Protein
FIG. 3. The activation of adenylate cyclase by cholera toxin
II. THE ROLE OF GANGLIOSIDE GM1 AS A CELL-SURFACE RECEPTOR A. The Nature of the Gangliosides The gangliosides are a family of complex lipids that are found in the plasma membrane of essentially all eukaryotic cells (for reviews, see Fishman and Brady, 1976; van Heyningen, 1974a; Svennerholm et al., 1980). Their most important property is their amphiphilicity: they all have a long hydrophobic ceramide portion and a negatively charged hydrophilic portion containing sugars and N-acetylneuraminic acid residues. This structure makes the gangliosides very good cell surface receptors. The hydrophobic region dissolves in the fluid plasma membrane lipids and can move freely in the plane of the membrane; the hydrophilic region remains in the aqueous environment of the cell where it can react with water-soluble ligands. In aqueous solution, gangliosides cluster into micelles that can interact with the water. Although the gangliosides are so widespread, there is no clear idea as yet of their normal physiological function. There are a large number of gangliosides that differ from each other principally in the carbohydrate region, and especially in the number and position of
CHOLERA TOXIN AND GANGLIOSIDES
45 1
the N-acetylneuraminic acid residues. Studies (as described below) with different purified gangliosides have shown that cholera binds to the one whose structure is given in Fig. 4 and is generally known as ganglioside GMI . It has only one Nacetylneuraminic acid residue [see the IUPAC-IUB Commission on Biochemical Nomenclature ( 1 977) for details of the nomenclature of gangliosides and their abbreviations].
B. The Binding of Cholera Toxin to Cells The binding of cholera toxin has generally been studied quantitatively using 1251-labeledtoxin and assuming that the labeling does not affect the toxin. Such studies (reviewed in van Heyningen, 1977a) have shown that the binding is complete in a few minutes at most, and cannot be reversed even at low concentrations except at very early stages. The number of binding sites varies considerably with the type of cell, e.g., erythrocytes bind only a few thousand molecules (Gill and King, 1975), fat cells about 20,000 (Cuatrecasas, 1973a), and some mucosal cells more than two million (Holmgren et al., 1975). However with all these cells the dissociation constant remains at around lod9 M , tight binding by most standards. (Such constants are, of course, measured by very indirect means such as concentration of ligand at half saturation and so cannot be very accurate.) The fact that these values are so similar supports the idea that the receptor is the same in different types of cell.
C. The Evidence That the Receptor Is Ganglioside GM1 Since these early results there has been a lot of confirmatory work on the idea of gangliosides as the receptor for the toxin, and, for several years now, there has been more or less total agreement that ganglioside GMI is what cholera toxin binds to at least in the first instance, although there is some evidence discussed below that the true in vivo receptor may sometimes be rather more complicated. The first direct evidence for a role for ganglioside was the observation that adding exogenous ganglioside to cells could increase the binding of toxin and the activation of cyclase. When ganglioside molecules in solution are incubated with cells, their hydrophobic portions insert into the lipid membrane and they presumably become indistinguishable from endogenous ganglioside. Early studies by Cuatrecasas (1973b) showed that the response of fat cells to toxin was increased 10 times when the cells were preincubated with ganglioside and that this was not an increase in the potential maximum activity, but rather in the sensitivity. Pigeon erythrocytes were also made more responsive to toxin by preincubation with ganglioside (Gill and King, 1975; King er al., 1976). In these
0
N-acetylneuraminic acid
0
II
0-CH
[
/c,
HCNH OH D-galactose
I I
H-C-OH CH \CH
I cy2 ,“ya
OH N-acetylgalactosamine
CH2
D-galactose Hydrophilic ponion
OH D -glucose
/cq CH2 /cpCH2 /cy2 /cq / c v /cp ,CQ CH2 CH2 CH2 CH,
CH2
sugars
,cp CH2 /Cyz /cq ,CHI CH2 CH2
/ y 2
CH2
CH2
Hydrophobic portion
FIG. 4. The structure of ganglioside GMI. (From van Heyningen, 1977a.)
ceramide
,“Q
CHZ
CHJ
CHOLERA TOXIN AND GANGLIOSIDES
453
experiments, incorporation of tritiated ganglioside into the membrane was shown directly and correlated with the toxin binding. There was a considerable amount of nonproductive binding. Holmgren et al. (1975) allowed nature to do the experiment for them; they compared the binding of toxin to intestinal mucosa from different species and found a good correlation between binding and the content of ganglioside GMI. There were also reports that treating cells with neuraminidase, which increases their GMl content at the expense of other gangliosides by removing N-acetylneuraminic acid residues, increased sensitivity to toxin (King and van Heyningen, 1973; Haksar et a / . , 1974; Staerk el al., 19741, but not everyone agreed with this (e.g., Holmgren et al., 1974a). A more sophisticated approach and the one that has produced the most convincing evidence of all is to use cells in culture whose ganglioside content can be measured and altered artificially. Hollenberg et a/. (1974) looked at the binding of toxin to three different mouse cell lines transformed with viruses and found a correlation between ganglioside GMI content, toxin binding, and activation of the cyclase. They reasoned that cells with no detectable GM1 in the membrane would still bind toxin, but it appeared that the chemical methods were a less sensitive assay for GMI than was the ability to bind toxin. A cell line from transformed mouse fibroblasts (NCTC 2071) that did not need serum (which may contain some free gangliosides) for growth and contained no GM 1 and very little other gangliosides (but which did have an adenylate cyclase that responded to cholera toxin after lysis of the cells) would not normally respond to toxin when intact. However, it would take up as much as lo5 molecules of tritiated GMl per cell and then respond to toxin. The ganglioside was not changed in the incorporation. Other gangliosides were also incorporated, but they did not make the cells sensitive to toxin. GMI in these membranes can be tritiated in situ by oxidation (e.g., with galactose oxidase) followed by reduction with NaB3H,, but the GMl in cells that had been preincubated with toxin was not available for this reaction, presumably because access was blocked by the bound toxin (Moss et al.. 1976a, 1977a; Mullin et al., 1976; Fishman et al., 1976). Such cells could even be sensitized by exposure to fetal calf serum because of the minute amount of GMI present in the serum (Fishman et al., 1978a). Similar results were obtained with human fibroblasts in culture (Fishman et ul., 19771, and in rat adipocytes where the reaction with cholera toxin could be used for detection of ganglioside GMI (Pacuszka et al., 1978) [although earlier workers (Kanfer et al., 1976) had claimed that these fat cells did not have ganglioside GMI even though they did respond to toxin]. When 3T3-LI preadipocytes in culture differentiate to adipocytes they lose about 80% of their ganglioside GM I content and they also lose response to and binding of cholera toxin concomitantly (Reed et al.. 1980). Another way of increasing the GMI content of cells is to treat them with short-chain carboxylic acids, especially butyric acid. With both HeLa cells and Friend erythroleukemic cells, such treat-
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SIMON VAN HEYNINGEN
ment increased both the GM 1 content and the binding of cholera toxin (Fishman and Atikkan, 1979). An alternative and more direct way of investigating the nature of the receptor was introduced by Critchley and his group (Critchley et al., 1979). They first labeled galactose residues in their cells (mouse fibroblasts and a mouse lymphoid cell line) with tritium using galactose oxidase, and then incubated the cells with toxin. Then they solubilized the membrane with a mild detergent (NP40), and treated the extract with antibodies to cholera toxin. Immune complexes containing toxin were then precipitated using protein A from Staphylococcus aureus and investigated chromatographically. It was clear that ganglioside GM 1 was by far the major component of the toxin receptor complexes, but there was also evidence for some involvement of galactoproteins and even perhaps of larger complexes composed of ganglioside, phospholipid, and galactoprotein. This is not the only evidence that the true receptor in vivo could be something a bit more complicated than a ganglioside alone. King and van Heyningen (1975) found that ganglioside reacted with toxin differently in the presence and absence of other lipids. More recently, Morita el al. (1980) have found at least five different glycoproteins in rat intestinal microvillus membranes that are capable of binding the toxin as judged by experiments using tritiated galactose residues and iodinated toxin. There was evidence that these glycoproteins might have a carbohydrate portion similar to that of the gangliosides. However Critchley e f al. (1981) have challenged these findings. Their very thorough experiments showed that the membranes had some high-affinity binding sites for toxin, which were resistant to protease and neuraminidase but could be extracted with organic solvents. These binding sites had all the properties of ganglioside GM1. They found no evidence for binding of toxin by glycoproteins. These experiments (and the evidence discussed in Section Ill for specific reactions between ganglioside GM I and the toxin protein) are universally accepted as showing that ganglioside GM 1 is at least a major receptor for the toxin. It still seems likely that, at least in some cells, certain other compounds such as glycoproteins can also act as receptors. There is nothing very surprising about this observation. Many of the theories for the function of the toxin-receptor binding in the action of the toxin (discussed in Section IV,D) depend only on the toxin binding to something on the cell surface and not on what that something is. There are cases in which although binding is inhibited by simple gangliosides the true receptor is almost certainly much more complicated. An example is thyrotropin, which will bind ganglioside GDlb, but whose true receptor is probably a glycopeptide (Ledley e f al., 1977).
D. The Distribution of Ganglioside GM1 in the Membrane On the assumption that GMI is the receptor, labeled cholera toxin has been used in several studies of the distribution of GMI in cells and tissues. For
CHOLERA TOXIN AND GANGLIOSIDES
455
example, conjugates of cholera toxin and peroxidase were used in an investigation of the surface distribution of GMI in brain cells (Manuelidis and Manuelidis, 1976); there was evidence of a gradual loss of GMl from the surface when these cells were maintained in monolayer culture. Hansson er al. (1977) incubated cells with toxin and then localized the bound toxin with toxin-specific peroxidase-conjugated antibody. They used this method (whose validity they had demonstrated in an artificially controlled system) to show that, in the central nervous system, GM 1 is concentrated in the pre- and postsynaptic membranes of the synaptic terminals. Ackerman er al. ( 1 980) used colloidal-gold-labeled IgGF(ab’) and cholera antitoxin for their immunocytochemical studies of the distribution of ganglioside GMl on human blood cells, and showed distinct differences in the extent of surface labeling with various types of blood cell. Electron microscopy has also given information about what happens to the bound toxin. Thus studies using fluorescent toxin or a “sandwich” technique with fluorescent antitoxin have shown patching and capping of the toxin on the surface of lymphocytes (Holmgren et al., 1974a; Revesz and Greaves, 1975; Craig and Cuatrecasas, 1975, 1976). The capping is temperature dependent and is inhibited by compounds such as cytochalasin B and colchicine. This has also been demonstrated in an interesting indirect way by Sedlacek er al. (1976), who put the fluorescent label not on the toxin but on the ganglioside, which became incorporated into the membrane and could be shown to cap when toxin was added. This of course constitutes yet another piece of evidence that ganglioside GM I is indeed a receptor for the toxin. Whether patching and capping have any significance in the action of the toxin or whether they merely reflect the fact that one molecule of toxin can bind several molecules of ganglioside and all such ligands behave in this way is an open question.
111.
THE NATURE OF THE REACTION BETWEEN GANGLIOSIDE AND TOXIN
A. Detection of Ganglioside Binding The earliest evidence for an interaction between toxin and ganglioside came from the observation described above that incubation with ganglioside destroyed the biological activity of the toxin. However, there are a number of ways of demonstrating the interaction more directly, and thus of at least trying to get closer to a quantitative measurement. Most of these ways depend on the fact that the toxin is multivalent for ganglioside and that ganglioside is functionally multivalent for toxin because of the micelles normally present. This means that the toxin-ganglioside complexes soon become very large and precipitate out of solution (van Heyningen et al., 1971; Holmgren et al., 1973; Staerk et al., 1974). This complex is rather like the precipitating complex formed between multivalent antibodies and antigens, and can be demonstrated by the diffusion
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SIMON VAN HEYNINGEN
toward each other of a solution of ganglioside and a solution of toxin: a sharp insoluble white line is formed where the solutions meet, very like the line seen in the Ouchterlony immunodiffusion experiment (Holmgren et al., 1973). Another way is to use ganglioside that has been insolubilized either by complexing it to the insoluble lipid cerebroside or by making covalent complexes with some insoluble chromatographic medium such as agarose or polystyrene beads. The toxin is adsorbed to the complex and the activity remaining in solution can be measured (see King and van Heyningen, 1975; Cuatrecasas et al., 1973). A more sensitive variant of this method (Holmgren, 1973) uses an enzyme-linked assay. A better way of measuring the interaction with ganglioside in free solution would be by equilibrium dialysis or some such dynamic method, but these are all very difficult because of the micelle formation (see Sections 11,D and E for a fuller discussion).
6. Ganglioside Binding in Purification and Detection of Toxin This ability of toxin to bind tightly to insolubilized ganglioside has been used to advantage in several ways. For example, it has been used for an amazingly large scale purification of the toxin in which 1000 liters of culture medium was absorbed onto insolubilized ganglioside GM1 and eluted with buffer at pH 2.8. In this way 20 g of toxin was purified in little more than a single step (Tayot et al., 1981). Similarly mutants of V . cholerae altered in the production of toxin have been detected using “affinity filters”~ellulosefilter disks to which ganglioside albumin conjugates have been attached (Mekalanos ef al., 1978). lZ5ilabeled toxin will bind directly to gangliosides on thin-layer chromatograms (Magnani et al., 1980).
C. Role of Subunit B in Binding the Ganglioside Very soon after the subunit structure of the toxin was determined, it became clear that each subunit has a different function. The initial observation (van Heyningen, 1974b) was that when toxin was bound to insolubilized ganglioside and the complex was treated with 8 M urea, only subunit A was released. Subunit B remained bound to the ganglioside and could be released only with a more powerful chaotropic agent, 6 M guanidine hydrochloride. Subsequent experiments in several laboratories showed clearly that purified subunit B would bind ganglioside (Holmgren and Lonnroth, 1975; Sattler et al., 1975). Subunit A does not bind ganglioside in the same way; there is some binding, but no more than there is between almost all protein and gangliosides, presumably because of hydrophobic interactions.
CHOLERA TOXIN AND GANGLfOSlDES
457
These observations can be extended to binding to cells, where again subunit B has been shown to bind, but not subunit A (references as in Section 11,B). This result explained an earlier observation that choleragenoid, the naturally occurring aggregate of subunit B , would protect cells and even intact loops of gut (Pierce, 1973) from subsequent challenge with toxin (Holmgren et al., 1974a; Gill and King, 1975; Cuatrecasas, 1973a); the available ganglioside binding sites are all occupied with subunit B and so are not available for reaction with toxin.
D. Specificity for the Ganglioside GMl is the only ganglioside that binds cholera toxin significantly; other gangliosides bind very much less and do not inhibit (see King and van Heyningen, 1973; Cuatrecasas, 1973b; Holmgren et al., 1973, 1980; Staerk et al., 1974). There have been several studies designed to show which parts of the molecule are important for this specificity. It is immediately apparent that much of the specificity must lie in the carbohydrate portion of the molecule, since gangliosides differing only in this portion (for example, by having more than one N-acetylneuraminic acid residue) do not bind. Indeed the carbohydrate region by itself (GM I-oligosaccharide, monosialogangliotetraose) will inhibit precipitation of the toxin by GM 1 (Holmgren et al., 1974b). Equilibrium dialysis experiments with the oligosaccharide (possible since it does not form micelles) also showed that it bound to toxin or subunit B more tightly than whole ganglioside (Sattler et al., 1977; and see Section 111,E). However, if the terminal galactose residue was removed, or the free carboxyl group of the N-acetylneuraminic acid residue was reduced to alcohol, then most of the binding was lost. Fishman et al. (1980) used their GMl-deficient mouse fibroblasts (Section lI,C), which are insensitive to toxin until receptor is added, and found that oxidation of the GMl (with galactose oxidase or sodium periodate) or replacing N-acetylneuraminic acid with N-glycolylneuraminic acid did not destroy the binding ability of the ganglioside. The hydrophobic part of the ganglioside is also important. Experiments using artificial analogs of the gangliosides (gangliosidoides) have shown that the lipophilic chains must be at least 14 carbon atoms long if a toxin-ganglioside precipitate is to form (Wiegandt et al., 1976). However, this may have something to do with the reduced ability of the gangliosidoides to form micelles rather than with any difference in the ability to act as a receptor. Using their GMldeficient cells, Fishman et al. (1980) found that GM 1 , in which the lipid had been altered by replacing the long chain fatty acid with an acyl group, retained the ability to act as a receptor. It bound as much toxin as native GM1 and actually increased the efficiency of bound toxin in activation of the adenylate cyclase. Perhaps it is a more efficient promotor of whatever process is needed to get the A1 peptide inside the cell.
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SIMON VAN HEYNINGEN
E. Stoichiometry and Kinetics of Toxin-Ganglioside Binding Determining the amount of ganglioside that binds to toxin and the tightness of this binding is very difficult, principally because of the great complication that is introduced by the self-association of the ganglioside monomers into micelles. There has been disagreement in the literature about the critical micelle concentrations: estimates vary from as low as 10- l o M or less (Formisano et uf., 1979) to M (e.g., Yohe and Rosenberg, 1972), which is the most widely about accepted value. This means that it is very difficult to choose experimental conditions in which the ganglioside molecules are present entirely as monomers, or even conditions in which their state of aggregation can be accurately forecast. This is a particular problem in equilibrium dialysis experiments which depend on the free passage of a ligand through a membrane impermeable to the protein. Ganglioside molecules cannot penetrate such a membrane. Two groups have circumvented this problem by measuring the binding not of the whole ganglioside molecule, but of the isolated carbohydrate part which does penetrate dialysis membranes and can reasonably be assumed to bind in the same way. Fishman et al. (1978b) used tritiated GM1-oligosaccharide and showed that the toxin became saturated with carbohydrate when the carbohydrate-to-protein ratio was greater than about 4.8. Their data could by analyzed more quantitatively in the usual way (e.g., by a Scatchard plot), but since such plots were not linear they did not attempt to calculate association constants. Sattler et al. (1977) also used oligosaccharide in equilibrium dialysis studies and obtained nonlinear Scatchard plots, but, from their direct binding isotherms, they found half-saturation at about 65 nM carbohydrate and a ratio of about 3.9 at saturation. In a later paper (Sattler et af., 1978) they showed evidence for cooperativity in the binding of ganglioside by the B subunits (which would explain the nonlinearity of the Scatchard plots) and found Hill coefficients around 1.2. Fishman et af. (1978b) also investigated the binding ratio by looking at the amount of ganglioside that produced the greatest conformational change in the B subunits (see Section III,F), and by gel permeation chromatography on BioGel P-60. These experiments all gave values of around 5, not surprising as there are five B subunits in the toxin molecule. The dissociation constant for the reaction between ganglioside and toxin found by these equilibrium dialysis experiments and by certain competition experiments is rather lower than the values that have been found for the binding of toxin to cells (see Section 11,B). Much has been made of the different dissociation constants by some authors, but it is doubtful whether any of them are really accurate enough to justify detailed analysis.
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F. Conformational Change following the Binding of Ganglioside When a protein binds a ligand it nearly always alters its conformation, if only in the neighborhood of the ligand-binding site. Direct evidence for such conformational change when cholera toxin binds ganglioside has come from several sources. The first was the observation of a blue shift in the intrinsic protein fluorescence spectrum following reaction of whole toxin or subunit B with ganglioside (Mullin et al., 1976; Moss et al., 1977c) or with the carbohydrate portion (Fishman et al., 1978b). This change occurred only when the ganglioside was GM 1 : controls with other gangliosides that do not bind specifically to the toxin produced no comparable change. The shift reached a maximum when &he ratio of oligosaccharide to toxin was 5.6. Since the intrinsic fluorescence of proteins is due almost entirely to tryptophan residues, these results suggest a change in the environment of at least one such residue. The circular dichroism spectrum of the B subunits around 230 nm (a particularly sensitive indicator of conformation) was also affected by the oligosaccharide in a specific way. These experiments suggesting a role for some tryptophan residue in the ganglioside binding have been supported by others. Modification of tryptophan residues with specific reagents completely destroys the ability of the B subunits to bind ganglioside or to aggregate to the usual pentamers (de Wolf et al., 1981a). These modifications may have produced quite large changes in the structure of the subunits. Solute quenching of fluorescence and fluorescence transfer experiments (de Wolf et al., 198lb) suggested that a single tryptophan residue in each subunit (number 88 in the sequence) was involved, and that the residue was very near the ganglioside binding site, perhaps even interacting directly with its oligosaccharide moiety. Another very sensitive probe for protein conformation is I3C nuclear magnetic NMR spectrum of resonance. They are some resolved resonances in the cholera toxin, and one of these is probably due to the €2 carbon of tryptophan residues. When the oligosaccharide of ganglioside GMl is added to the protein, there is a broadening of the resonance or a movement upfield suggesting that there has been a conformational change in the environment of the tryptophan residues (Sillerud et al., 1981). So these results are compatible with those of the fluorescence and modification studies. Whatever conformational changes there are in the molecule need not be extensive; many of the more general ways of looking for conformational change that have been used in these experiments are involved with tryptophan residues and thus they may all be examining the same rather minor changes in one part of the molecule alone. Fluorescent probes that react with free amino groups such as dimethylaminonaphthalene sulfonyl chloride (dansyl chloride) (de Wolf et al.,
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198I b) and 4-chloro-7-nitrobenzofuran (Nbf-chloride, van Heyningen, 1982) do not have their spectrum altered by ganglioside, so these residues are presumably not in an area of the protein in which there is a lot of change. There is, however, one piece of evidence for a more significant alteration in the conformation of subunit B: a conformational change that is transmitted to the A subunit. It is not surprising that binding ganglioside has some effect on the subunit to which it binds: it is more surprising that it also affects the conformation of the other subunit. This was shown by experiments (van Heyningen, 1982) in which ganglioside GM 1 (but not other gangliosides) altered the spectrum of a fluorescent probe (Nbf-chloride) bound to subunit A only in an intact toxin molecule containing unlabeled B subunits. Furthermore this conformational change reached a maximum when only one ganglioside molecule had bound per molecule of toxin, i.e., when the full potential of the B subunits was not yet satisfied. This suggests that a conformational change (altering the environment of the probe bound probably to a free lysine residue) is transmitted from subunit B to subunit A and that an alteration in only one of the B subunits is enough for the full effect to be found.
IV. TRANSPORT OF CHOLERA TOXIN ACROSS THE CELL MEMBRANE AND THE ROLE OF BINDING TO GANGLIOSIDE A. Penetration of the Membrane by Peptide A1 As discussed in Section I,C, it is now almost universally accepted that at least some part of the active A1 peptide of the toxin has to penetrate the cell membrane to gain access to the adenylate cyclase bound to the inner face of the membrane. The characteristic lag phase between binding of the toxin to the cell surface and the subsequent increase in cyclase activity is abolished when the cells are lysed and active peptide can therefore get directly to the cyclase without any involvement of the membrane. This suggests immediately that the lag phase represents the time taken to penetrate the membrane. It has generally been assumed that subunit B would not have to penetrate the membrane as well, but there is some evidence that it does. For example, Zinnaka and Ohtomo (1981) have reported finding both subunits inside the cell using immunocytochemical techniques. Furthermore, toxin in which subunit B, peptide A l , and peptide A2 are all covalently cross-linked and so could not come apart remains active with intact pigeon erythrocytes (van Heyningen, 1977b). It is also known that peptide A1 can retain in vitro enzymic activity while remaining bound to the rest of the toxin (Mekalanos er al., 1979). There is also some evidence from cytochemical work for endocytosis of the whole toxin (see Section IV,D). However, photoaffinity labeling experiments show that only subunit A penetrates the membrane (Wisnieski and Bramhall, 1981; and see Section IV,C).
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Another important possibility (and one that could account for the activity of the cross-linked toxin) is that only some part of the A 1 peptide needs to cross the membrane. This could be a proteolytic fragment [and such fragments are undoubtedly active and are formed in vivo although perhaps not in large enough amounts to account for the activity (Matuo et al.. 1976; van Heyningen and Tait, 1980)l. It could also be that the A1 peptide is long enough to pass through the membrane so that the active part is inside the cell while another enzymically inactive part remains bound to the B subunits outside the cell. But whatever happens some part of the A subunit must get inside the cell.
B. Function of the Ganglioside A simple experiment on the likely effect of ganglioside in membrane binding to a protein is to make artificial lipid bilayers containing ganglioside and see what happens. There is evidence that ganglioside molecules in such bilayers (Delmelle et al., 1980) and in membranes (Sharom and Grant, 1977) aggregate themselves into clusters. Such bilayers bind toxin tightly (Tosteson et a l . , 1980) and this seems to give rise to conducting channels (Tosteson and Tosteson, 1978). In similar experiments (Moss ef al., 1976b, 1977b) toxin has been shown to bind specifically to liposomes containing ganglioside GM 1, and this leads to release of glucose trapped inside the liposomes. This effect is specific for subunit B; subunit A did not release the trapped glucose. However, subunit A must have penetrated the membrane to some extent by itself, since exposure to subunit A (but not subunit B) followed by antitoxin and complement did release glucose. There is one heretical observation similar to this that was made with a system closer to real cells (Bavros et al., 1981). Toxin was found to have a direct effect on the ion permeability of brush border membrane vesicles prepared from rabbit small intestine. This was shown by measuring differences in sodium-dependent glucose transport by the vesicles stimulated by membrane potential, as well as by measuring the accumulation of a lipophilic cation. There were similar effects when intact brush border was treated with toxin before the vesicles were made. These vesicles did not contain any active adenylate cyclase so that cholera toxin cannot have been exerting its effect via an increase in cyclic AMP concentration as it is normally supposed to. This system is not necessarily very closely related to the sort of changes in permeability that occur during cholera itself, but it may be a rather similar one to the artificial membranes just described. These experiments then suggest that at least in these simple systems, the movement of ganglioside induced by binding to subunit B could by itself create channels that might make an entry for subunit A. However, the cell membrane is a much more complicated thing, and the observed patching and capping of the toxin-ganglioside complex (see Section II,D), even if it has no other significance, does show that some binding to proteins and to the cytoskeleton must take
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place. A more definite piece of evidence for some protein involvement is the observation that treatment of macrophages with inhibitors of protein synthesis (such as cycloheximide and puromycin) blocks the activation of cyclase although it does not affect the concentration of GMl or of cyclase (Hagmann and Fishman, 1981). The production of channels and the patching and capping are phenomena that are presumably dependent on one molecule binding several molecules of ganglioside at the same time; a satisfactory model for the action of the toxin should explain why cholera toxin has five binding sites for ganglioside. There is evidence (Fishman and Atikkan, 1980) that the toxin affects the adenylate cyclase of intact cells only when it is binding several ganglioside molecules at the same time. The crux of this evidence comes from measuring the inhibition of the action of the toxin by the GMI-oligosaccharide which can bind the toxin (Section II,D), but is not incorporated into the membrane. With HeLa cells which have only about 15,000 toxin-binding sites per cell, the oligosaccharide can inhibit by more than 90%. In the presence of 100 pA4 oligosaccharide, 5% of the maximum possible toxin was still bound to HeLa cells, but the activation of cyclase was less than 10% (although in principle that should still have been quite enough bound toxin to produce the maximum effect). Oligosaccharide reduced the binding to human lymphocytes to 20%,but there was no activation of cyclase at all. In the crucial experiment, when the oligosaccharide was removed, the activation increased even though no more toxin had been added. The suggestion was that toxin binds initially to a single ganglioside molecule, but that this binding is nonproductive. Not until the same toxin molecule has bound to more ganglioside molecules can it activate the cyclase. In the presence of oligosaccharide the toxin molecules are bound to the cells through a single ganglioside; they cannot bind any more because the binding sites on the B subunits are occupied with oligosaccharide. The lag phase also seems to be related to the GMI concentration in the cells (Fishman, 1980); increasing the concentration decreases the lag.
C. The A1 Peptide by Itself Subunit A is active with at least some whole intact cells by itself without any subunit B (e.g., pigeon erythrocytes, van Heyningen and King, 1975; WodnarFilipowicz and Lai, 1976). Although the specific activity is less than that of native toxin, the time lag is the same, but pretreatment of the cells with ganglioside or with subunit B has no effect. So at least in this system, the role of subunit B is not an absolutely critical one. Furthermore, since the A1 peptide activates the cyclase enzymically, very few molecules need to enter the cell for activation to be complete: since many thousand molecules can bind to the cell
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surface, entry does not need to be a very likely or frequent event. This is one reason why increasing the binding to the cell does not always increase the maximum activation (Section 11,C). This sort of experiment suggests that subunit A can penetrate the lipid membrane directly and by itself. However, this is a difficult thing for a protein to do (Waksman et al., 1980) unless it has very unusual properties, which neither subunit A nor B have: charge shift experiments and calculations based on amino acid composition show neither to have important hydrophobic areas on their surface (Ward et af., 1981). It is possible that the A1 chain could unfold itself into some quite different conformations while penetrating the membrane; it is a very stable protein (active after treatment with boiling SDS) that could refold to the native conformation in the aqueous environment of the cytoplasm. It has also been suggested that the A2 peptide might act analogously to the leader sequence peptides that some proteins use when being exported from a cell (see Waksman et af., 1980); there may be no great mechanistic difference between transport into or out of a cell. There is one very useful experiment which shows clearly that when the toxin reacts with a cell the A1 peptide can be found inside the membrane (Wisnieski and Bramhall, 1981). A photoactivated label could be shown by electron spin resonance to be buried in a model membrane system, the envelope of Newcastle disease virus. This probe labels relatively nonspecifically only those residues which are in the outer monolayer of the membrane. When toxin was incubated with the viral membranes subunit B did not become labeled even after some time, but the A1 peptide was labeled within seconds of binding to the viral membrane. Within the first minute of incubation at 37°C greater labeling of the A1 peptide occurred, but in the next 2 minutes, the amount of labeling decreased. This suggests rapid penetration of A 1 through the membrane. If direct transport of A1 peptide across the membrane is an important mechanism then one would expect that membrane fluidity would be important, and indeed procedures that increases that fluidity do decrease the lag time, e.g., temperature (Fishman, 1980) or treatment with benzyl alcohol (Houslay and Elliott, 1979) or with liposomes so that the membranes become partially depleted of cholesterol (S. van Heyningen, unpublished). Decreasing the temperature can inactivate the toxin completely (e.g., Bennet and Cuatrecasas, 1975; Lonnroth and Lonnroth, 1977).
D. Possible Mechanisms There remain three basic ideas about how the membrane might be penetrated. The one that seems to have been taken up by most textbooks is that of Gill (1976) who suggested that the five B subunits bound flat to the membrane and dissolved
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in it, forming a hydrophilic tunnel through which the A1 peptide could pass. This is an attractive idea, but there is no direct evidence for it and the photoaffinity labeling experiments described above seem to rule it out at least in this form. A modification of this theory which is gaining wide acceptance (but for which there is still no direct evidence) is that the B subunits might combine directly with a protein already present in the membrane and so be able to diffuse through it. Such a combination between subunit B and a preexisting carrier protein might be able to form the required channel (Gill, 1978). This theory accounts for the evidence that some sort of membrane protein is required, and fits in with the observed conformational change in subunit A following the binding of ganglioside to subunit B (Section 111,F). A second theory (van Heyningen and King, 1975), based primarily on the observation that subunit A is active even by itself, is that there is no specific mechanisms for entry, but that the binding to ganglioside serves chiefly to increase the local concentration of peptide A1 at the cell surface thereby increasing the chance of its entering by some almost random method such as transient openings in the dynamic cell surface or by dissolving in the membrane. The conformational change in subunit A could lead to its release from the B subunits, and, once it had entered the cell, the disulfide bond linking peptides Al and A2 could be cleaved at the intracellular redox potential or by the thio1:protein disulfide oxidoreductase enzyme which is found associated with membranes (Moss et al., 1980). There is evidence that this enzyme could be involved in the reduction of subunit A I . This theory implies that the binding to a receptor, although it clearly happens and must have some importance, has a nonspecific rather than a specific function. There is some indirect evidence for such nonspecific action by cell-surface binding components in the “hybrid” or “chimeric” toxins in which the active component of one toxin is artificially linked to the binding component of another. For example, diphtheria toxin fragment A carries the enzymic activity (the ADP-ribosylation of elongated factor 2) that is essential for the toxicity of the toxin. It works inside the cell, and generally gets there following the binding of diphtheria toxin fragment B (to which it is linked by a disulfide bond) to the outer cell surface. An artificial hybrid in which diphtheria fragment A was joined by disulfide bond to cholera subunit B was toxic to cells and this toxicity was dependent on the action of both components (Mannhalter et al., 1980). In this case the binding of cholera B to the cell was clearly aiding the entry of fragment A, but this could not have been in a specific manner since the hybrid was an artificial one. Many other such hybrids have been prepared in the last few years (Olsnes, 1981). The chief argument against this hypothesis must be that proteins, especially hydrophilic ones, are not thought to cross cell membranes by themselves enough to account for the observed activity of the toxin.
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A final possibility that has recently been much more widely discussed is that entry into the cell is by endocytosis, another relatively nonspecific process that occurs in a great number of cells. However, endocytosis by itself is not enough, since the endocytosed protein is still separated from the cytoplasm by a membrane. There is little direct evidence for endocytosis of cholera toxin, although membrane-bound toxin has been shown to be partly endocytosed (Manuelidis and Manuelidis, 1976; Hansson ef at., 19771, and toxin labeled with horseradish peroxidase is taken by endocytosis into neuroblastoma cells where it enters the Golgi/endoplasmic reticulurdlysosome (GERL) system (Joseph et a/. , 1978). However, toxin taken up in this way does not seem to be active with intact cells. The problem with interpretation of these experiments is that one would expect a protein binding tightly and in large amounts to the cell surface to be endocytosed at least to some extent. It is much harder to show that the endocytosis is a prerequisite for activity. People who work on these toxins sometimes have great difficulty in persuading themselves that what is true for one toxin is not true for them all. A great deal of good evidence that diphtheria toxin is internalized by endocytosis followed by fusion of the vesicles with lysosomes and subsequent release of fragment A from the acid environment of the lysosomes has accumulated very recently (Uchida, 1982; van Heyningen, 1981). This has led to the thought that similar mechanisms may be important with cholera toxin, and there is some evidence for this. Inhibitors of receptor internalization and lysosomal processing such as methylamine, ammonium chloride, chloroquine, and dansyl cadaverine all inhibit the action of the toxin in HeLa cells (Lin and Taniuchi, 1980) and in hepatocytes (Houslay and Elliott, 198 1). They do not affect the initial binding to the cell surface or the activation of adenylate cyclase in broken cells. Further work on this idea would be useful and is doubtless being done. Whatever the mechanism of entry, it is worth pointing out that ganglioside is not a “receptor” for cholera toxin in the true sense. Binding to the ganglioside is a prerequisite for its action in the activation of adenylate cyclase, but it does not in itself bring that activation about. Binding to true receptors such as that for adrenaline is more directly coupled with the physiological effect of the ligand.
V.
OTHER COMPOUNDS THAT BIND TO GANGLIOSIDES
The amphiphilic structure of the gangliosides make them very suitable to be receptor molecules on the surface of cells and a variety of different kinds of molecule has been found to bind. Most of these are molecules, like cholera toxin, not involved in the normal life of a cell; a physiological role for the gangliosides compatible with their ubiquity and the wide range of different types found in
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many membranes has yet to be discovered. Many people have suggested that they are involved in cell-cell recognition, but there is little direct evidence for this. A protein toxin produced by some strains of E . cofi produces a toxin very similar to cholera toxin in subunit structure, amino acid sequence, and mechanism of action (Spicer et al., 1981). The B subunit of this toxin reacts with ganglioside, but the reaction is considerably weaker than it is with cholera toxin and it is not clear whether it is physiologically significant (see van Heyningen, 1977a). Tetanus toxin has a two-component structure reminiscent of cholera toxin: there is an H-chain (M, = 100,000) linked by a disulfide bond to an Lchain (M,= 50,000) (van Heyningen, 1980b). The H-chain and the whole toxin binds ganglioside (not GMl) and this binding leads to some internalization of the toxin into rat cerebral neurons (Yavin et al., 1981). It is not yet known what the function of the L-chain is or what the physiological significance of the binding might be. There is evidence for some similarities in the receptor for tetanus toxin and for the hormone thyrotrophin, although the hormone receptor is probably really something rather more complex (Ledley et a/., 1977). Interferon seems also to bind a ganglioside, and its binding is inhibited by both cholera and tetanus toxins (see van Heyningen, 1977a). The properties of the gangliosides have enabled them to bind a curious collection of molecules. ACKNOWLEDGMENTS I am grateful to the Medical Research Council for grants in suppon of my own research on cholera and tetanus toxins. REFERENCES Ackerman, G. A,, Wolken. K. W., and Gelder. F. B. (1980). Surface distribution of monosialoganglioside GMI on human blood cells and the effect of exogenous GMI and neuraminidase on cholera toxin surface labelling. J. Hisrochem. Cyrochem. 28, 1100-1 112. Bavros. F., Del Le PeAa, P., Gascon, S.. Ramos, S . , and Lam. P. S. (198 I ) . Cholera toxin induces changes in the ion permeability of intestinal brush border membranes. Biochim. Biophys. Acra 644, 143-146. Bcnnett, V . , and Cuatrecasas, P. (1975). Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. J. Memhr. Biol. 22, 29-52, Cassell, D.. and Selinger, 2. (1977). Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site. Proc. Natl. Acad. Sci. U.S.A. 74, 3307-331 1 . Craig, S. W., and Cuatrecasas, P. (1975). Mobility of cholera toxin receptors on rat lymphocyte membranes. Pror. Natl. Acad. Sci. U.S.A. 72, 3844-3848. Craig. S. W . , and Cuatrecasas, P. (1976). Immunological probes into the mechanism of cholera toxin action. Immunol. Commun. 5 , 387-400. Critchley, D. R., Ansell, S . , Perkins, R., Dilks, S., and Ingram, J. (1979). Isolation ofcholera toxin receptors from a mouse fibroblast and lymphoid cell line by immune precipitation. J . Suprumol. Strucr. 12, 273-291.
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Critchley, D. R.. Magnani. J . L.. and Fishman. P. H. (1981). Interaction of cholera toxin with rat intestinal brush border membranes. J . B i d . Chem. 256, 8724-8731. Cuatrecasas, P. ( 1973a). Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochernistn; 12, 3547-3558. Cuatrecasas. P. ( I973b). Gangliosides and membrane receptors for cholera toxin. Biochemistry 12, 3558-3566. Cuatrecasas, P.. Parikh, 1.. and Hollenberg, M. D. (1973). Affinity chromatography and StI’UCtUrdl analysis of Vibrio enterotoxin-ganglioside agarose and the biological effects of gangliosidecontaining soluble polymers. Biochemistn; 12, 3577-3581. Delmelle, M.. Dufrane. S. P., Brasseur. R . , and Ruysschaert, J . M. (1980). Clustering of gangliosidea in phospholipid bilayers. FEBS Leu. 121, 11-14, De Wolf, M. J. S . , Fridkin, M.. Epstein, M.. and Kohn. L. D. (1981 a). Structure function studies of cholera toxin and its A and B protomers. J . B i d . Chem. 256, 5481-5488. De Wolf, M. J . S., Fridkin, M.. and Kohn. L. D. (1981b). Tryptophan residues of cholera toxin and its A and B protomers. J . Biol. Chem. 256, 5489-5496. Fishman. P. H. ( 1980). Mechanism of action of cholera toxin: Studies on the lag period. J . Membr. Biol. 54, 61-72. Fishman, P. H., and Atikkan, E. E. (1979). Induction of cholera toxin receptors in cultured cells by butyric acid. J . B i d . Chem. 254, 4342-4344. Fishman. P. H.. and Atikkan. E. E. (1980). Mechanism of action of cholera toxin: Effect of receptor density and multivalent binding on activation of adenylate cyclase. J . Membr. B i d . 54, 5 1-60. Fishman, P. H., and Brady, R. 0. (1976). Biosynthesis and function of gangliosides. Science 194, 906-9 15. Fishman, P. H., Moss, J., and Vaughan. M. (1976). Uptake and metabolism of gangliosides in transformed mouse fibroblasts. J. B i d . Clzem. 251, 4490-4494. Fishman, P. H.. Moss, J . , and Manganiello. V. C. (1977). Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts. Biochemistry 16, 187 1- 1875. Fishman, P.H., Bradley. R. M.. Moss, J.. and Manganiello, V . C. (1978a). Effect of serum on ganglioside uptake and choleragen responsiveness of transformed mouse fibroblasts. J . Lipid Res. 19, 77-81. Fishman, P. H . . Moss. J . , and Osbome, J . C. (1978b). Interaction ofcholeragen with the oligosaccharide of ganglioside GM I : Evidence for multiple oligosaccharide binding sites. Biochemistry 17, 71 1-716. Fishman, P. H.. Pacuszaa. T., Hom. B.. and Moss, J . (1980). Modification of ganglioside GMI. J . Biol. Chem. 255, 7657-7664. Formisano, S.. Johnson, M. L., Lee, G.. Aloj, S. M., and Edelhoch, H. (1979). Critical micelle concentration of gangliosides. Biochemistry 18, I 1 19-1 124. Gill. D. M. (1976). The arrangement of subunits in cholera toxin. Biochemistry 15, 1242-1248. Gill. D. M. (1977). The mechanism of action of cholera toxin. Ad)’. Cyclic Nuclcotide Res. 8, 85-1 18. Gill, D. M. (1978). Seven toxic peptides that cross cell membranes. In “Bacterial Toxins and Cell Membranes” (J. Jeljaszewicz and T. Wadstrom. eds.). pp. 291-332. Academic Press, New York Gill, D. M.. and King. C. A. (1975). The mechanism of action of cholera toxin in pigeon erythrocytes lysates. J . Biol. Chem. 250, 6424-6432. Hagmann, J . . and Fishman, P. H. (1981). Inhibitors of protein synthesis block action of cholera toxin. Biochrm. Biophvs. Res. Commun. 98, 677-684. Haksar, A., Maudsley, D. B., and Peron, F. (1974). Neuraminidase treatment of adrenal cells increases their response to cholera enterotoxin. Nu/ure (London) 251, 5 14-515
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Hansson, H.-A., Holmgren, J., and Svennerholm, L. (1977). Ultrastructural localization of cell membrane GMI ganglioside by cholera toxin. Proc. Nail. Acad. Sci. U.S.A. 74, 3782-3786. Hollenberg, M. D., Fishman, P. H., Bennett, V., and Cuatrecasas, P. (1974). Cholera toxin and cell growth: Role of membrane gangliosides. Proc. Narl. Acad. Sci. U.S.A. 71, 4224-4228. Holmgren, J. (1973). Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8, 85 1-859. Holmgren, J. (1981). Action of cholera toxin and the prevention and treatment of cholera. Nature (London) 292, 413-417. Holmgren. J., and Lonnroth, 1. (1975). Oligomeric structure of cholera toxin: Characteristics of H and L subunits. J . Gen. Microbiol. 86, 49-65. Holmgren. J., Lonnroth, I., and Svennerholm, L. (1973). Tissue receptor for cholera exotoxin: Postulated structure from studies with GM I ganglioside and related glycolipids. Infect, Immun. 8, 208-214. Holrngren, J.. Lindholm, L., and Lonnroth. I. (1974a). Interaction of cholera toxin and toxin derivatives with lymphocytes. J. Exp. Mad. 139, 801-819. Holmgren, J., Mansson, J. E . , and Svennerholm, L. (1974b). Tissue receptor for cholera exotoxin: Structural requirements of GI1 ganglioside in toxin binding and inactivation. Med. Eiol. 52, 229-233. Holmgren, J., Lonnroth, I . , Mansson, 3 . E., and Svennerholm, L. (1975). Interaction of cholera toxin and membrane GMI ganglioside of small intestine. Proc. Narl. Acad. Sci. U.S.A. 72, 2520-2524. Holrngren, J., Elwing. H., Fredman, P., and Svennerholm. L. (1980). Polystyrene-adsorbed gangliosides for investigation of the structure of the tetanus-toxin receptor. Eur. J. Eiochem. 106, 37 1-379. Houslay, M. D., and Elliott, K. R. F. (1979). Cholera toxin mediated activation of adenylate cyclase in intact rat hepatocytes. FEES Leu. 104, 359-363. Houslay, M. D., and Elliott, K. R. F. (1981). Is the receptor-mediated endocytosis ofcholera toxin a prerequisite for its activation of adenylate cyclase in intact rat hepatocytes? FEBS Lett. 128, 289-292. IUPAC-IUB Commission on Biochemical Nomenclature (1977). The nomenclature of lipids. Eur. J . Eiochem. 79, 11-21, Joseph, K. C., Kim, S. U . , Steiner, A., and Gonatas, N. K . (1978). Endocytosis of cholera toxin into neuronal GERL. Proc. Natl. Acad. Sci. U.S.A. 75, 2815-2819. Kanfer, J. N., Carter, T. P., and Katzen, H. M. (1976). Lipolytic action of cholera toxin on fat cells. J . Eiol. Chem. 251, 7610-7619. King, C. A., and van Heyningen, W. E. (1973). Deactivation of cholera toxin by a siahdase-resistant monosialosyl-ganglioside.J . Infect. Dis. 127, 639-647. King, C. A., and van Heyningen, W. E. (1975). Evidence for the complex nature of the ganglioside receptor for cholera toxin. J. Infect. Dis. 131, 643-648. King, C. A., van Heyningen, W. E., and Gascoyne, N. (1976). Aspects of the interaction of Vibrio cholerae toxin with the pigeon red cell membrane. J. Infect. Dis. 133, S75-S8 I . Lai, C.-Y. (1980). The chemistry and biology of cholera toxin. Crit. Rev. Eiochem. 9, 171-206. Ledley, F. D., Lee, G . , Kohn, L. D., Habig, W. H., and Hardegree, M. C. (1977). Tetanus toxin interactions with thyroid plasma membranes. J. Biol. Chem. 252, 4049-4055. Lin, M. C., and Taniuchi, M. (1980). Inhibition of cholera toxin activation of the adenylate cyclase system in intact HeLa cells. J. Cyclic Nurleotide Res. 6 , 359-367. Unnroth, I., and Lonnroth, C. (1977). Interaction of cholera toxin and its subunits with lymphocytes. Exp. Cell Res. 104, 15-24. Magnani, J. L., Smith, D. F., and Ginsburg, V. (1980). Detection of gangliosides that bind cholera
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Index
A
inhibition GTP-dependent, 70-71 role of GTP hydrolysis and, 75-76 involvement in CAMP in hormone action, 149- 15 I membrane fluidity as regulator of activity, 185-186 benzyl alcohol, 186- 192 cholesterol, 202-207 fatty acids as modulators, 192-194 hormone-mediated alterations in membrane fluidity, 207-208 manipulation of phospholipid species, 201-202 temperature effects, 194-201 model of hormone action, 241-245 evaluation in relation to results of other approaches, 248-253 phospholipid head group composition and, 223-225 protein-protein interactions in regulation of activation by GTP-binding protein, 128- I3 I general considerations, 127- 128 regulation of G/F by receptor and hormone, 131-136 rate of activation, guanylnucleotides and, 20-22 of rat liver plasma membrane, application of target size analysis to, 239-241 receptor binding of inhibitory iigdnd and, 73-75 regulation by glycoprotein hormones important features of system, 151- 152 effect of guanine nucleotides, I52 nature of hormones, 144-145 nature of receptors, 145- 149 relationship between N, and Ni, 76-77
Adenylate cyclase adrenergic receptor coupling and characterization of detergent-solubilized receptors, 57-63 development of radioligands specific for receptors, 47-49 study of receptors in membranes, 49-57 applying kinetic theory to data generated by turkey erythrocytes methodological considerations, 22-23 role of GppNHp as modulator, 35-4 I slow activation by sequential mechanism, 29-35 slow activation versus rapid equilibrium, 23-29 catecholamine-induced changes in, 93-95 desensitization and down-regulation by homologous hormone, 152- 154 effect of GTP on isolated plasma membrane, 161-165 of LH-responsive Leydig cell tumor, 154-161 possible mechanisms involved, 170-172 site of lesion in LH-desensitized Leydig cell tumors, 165-170 disease states and. 225-226 dually regulated, cholera toxin and, 77-78 effects of fluoride, 246-248 hormone-sensitive catalytic component, 115-121 cell surface receptors, 125- I27 identities of proteins of, I 10- I I I properties of, 288 resolution of catalytic and regulatory proteins, 112-1 15 stimulatory GTP-binding regulatory protein, 121-125 473
474 Adenylate cyclase (cont.) selective modulation by asymmetric perturbation of membrane bilayer, 208 calcium. 219-222 mitogenic agents. 223 positively and negatively charged local anesthetics. 209-2 19 stimulation of, 68-70 structural studies on dually regulated systems calmodulin, 78 cholera toxin, 77-78 receptors, 77 ultrastructural studies, 78 Adenylate cyclase-hormone receptor. sttuctural aspects of interaction, 183-185 Adenylate cyclase system(s) bimodally regulated, 71-73 hormone receptors and, historical overview, 3-8 Adipocyte(s) differentiation. hormone receptors and, 290-294 human hexose transport system in. 371 insulin sensitive hexose transport system in critical steps in methodology cell preparation, 342-343 measurement of fluxes, 344-347 historical background, 340-342 kinetic approach to study equilibrium exchange experiments, 349-35 I general concepts, 348-349 infinite cis experiments, 353-354 infinite trans experiments, 354-355 zero trans experiments, 35 1-353 mechanism of ability of insulin to increase V,,,,, 367-370 modulation of transport system by glucose metabolites, 366-367 nontransported competitive inhibitors of transport, 360-362 in obesity and diabetes, 371-372 reconstitution of transporter, 372-373 requirements for D-glucose binding, 359-360 sugars which are both transported and phosphorylated-rate-limiting steps. 362-366 summary of present status, 339-340
INDEX
transport of nonmetabolizable sugars and sugar analogs and, 355-359 Adrenergic receptor(s) catecholamine-induced changes in binding, 93-95 detergent-solubilized. characterization of, 57-63 development of radioligands specific for, 47-49 differential expression during growth of 1321Nl cells, 100 down-regulation and recovery of lost receptors, 100-103 in membranes. study of, 49-57 native and desensitized, separation of. 95-97 Astrocytoma cell line 1321Nl differential expression of PAR during growth, 100 isoproterenol-induced changes in agonist binding properties of intact cells, 103-104 origin and characteristics of, 88-90
B Benzyl alcohol, membrane fluidity and, 186- I92 Blood vessels, vasopressin isoreceptors in, 263-265
C Calcium. modulation of adenylate cyclase and, 2 19-222 Calmodulin. dually regulated adenylate cyclase and, 78 Catalytic component, of adenylate cyclase, 115-121 Catecholamine changes in adenylate cyclase and PAR binding properties, 93-95 desensitization of intact cells and analysis of rates of synthesis and degradation of CAMP in whole cells, 91-93 origin and characteristics of human astrocytoma cell line 1321N1, 88-90 Cell cultures, use for' study of differentiation process, 289
INDEX
475
Cholera toxin binding to cells. 451 dually regulated adenylate cyclase and, 77-78 nature of reaction with ganglioside conformational change and, 459-460 detection of binding. 455-456 purification and detection of toxin and, 456 role of subunit B in binding, 456-457 specificty of ganglioside, 457 stoichiometry and kinetics of binding, 458 structure and action catalytic action of A1 peptide, 448-449 nature of toxin, 446-447 protein chemistry. 447-448 role of each subunit, 448 transport across cell membrane A1 peptide by itself, 462-463 function of ganglioside, 461-462 penetration by peptide A l , 460-461 possible mechanisms, 463-465 Cholesterol. membrane fluidity and. 202-207 Cyclic adenosine monophosphate analysis of rates of synthesis and degradation in whole cells, 91-93 involvement in hormone action on adenylate cyclase. 149-151
E Endocytosis as mechanism for agonist-induced receptor desenitization, 97-98 receptor-mediated, of glycoconjugates, 327-335 Epidermal growth factor family of similar polypeptides and their role in animal development and growth, 398-399 pathway to nuclear DNA replication biochemical signals for mitogenesis, 396-398 mitogenic capability of EGF, 393 mitogenic pathway, 393-396 properties of, 382-383 receptor antibodies against, 388-390 clustering. internalization, and degradation of EGF-receptor complex, 386-387 identification of. 383-386 insertion of exogenous receptors into receptor-negative variant cells. 392 protein kinase domain and relationship to oncogene product pp60Src. 387-388 regulation of, 390-391 studies on location of EGF-receptor gene, 391-392
D Deoxyribonucleic acid, replication, pathway ot EGF to, 393-398 Desensitization, agonist-induced kinetic model for, 98-100 receptor endocytosis as mechanism, 97-98 Diabetes. adipocyte hexose transport system in, 371-372 Differentiation chemical induction of, 289-290 model systems granulosa cells. 300-303 liver cells. 294-300 Madin-Darby canine kidney cells, 303-306 3T3-LI adipocytes, 290-294 use of cell cultures for study of, 289 Disease states, adenylate cyclase and, 225-226
F Fatty acids, as modulators of adenylate cyclase activity, 192-194 Fluoride, effects on adenylate cyclase, 246-248
G Canglioside(s), other compounds that bind to, 465-466 Ganglioside GMI nature of reaction with colera toxin conformational change and, 459-460 detection of binding, 455-456 purification and detection of toxin and, 456
INDEX
476 Ganglioside GMI (conr.) role of subunit B in binding, 456-457 specificity of ganglioside, 457 stoichiometry and kinetics of binding, 458 role as cell-surface receptor binding of cholera toxin to cells, 451 distribution of ganglioside GMI in membrane, 454-455 evidence that receptor is ganglioside GMI, 451-454 nature of gangliosides, 450-45 I role in transport of cholera toxin across cell membrane A1 peptide by itself, 462-463 function of ganglioside, 461-462 penetration of peptide AI, 460-461 possible mechanisms, 463-465 Glucose metabolites, insulin-sensitive hexose transport system and, 366-367 requirements for binding to adipocyte hexose transport system, 359-360 Glycoconjugates, receptor-mediated endocytosis of, 327-330 cycling and recycling receptors, 334-335 receptor recycling and weak bases, 330-334 Glycoprotein(s), extracellular, role of oligosaccharide moiety in recognition of, 323-324 Glycoprotein hormones involvement of CAMP in action of, 149- 15 1 regulation of adenylate cyclase by nature of hormones, 144-145 nature of receptors, 145-149 Granulosa cells, differentiation, hormone receptors and, 300-303 Guanosine triphosphate desensitizing effect on isolated plasma membrane, 161-165 inhibition of adenylate cyclase and, 70-71 role of GTP hydrolysis in, 75-76 Guanylnucleotides, rate of activation of adenylate cyclase and, 20-22 Guanylnucleotide subunit, position in activation pathway of adenylate cyclase, 35-41
H Hexose transport, insulin-sensitive, see Adipocytes
Hormone(s), see also Glycoprotein hormones alterations in lipid fluidity and, 207-208 Hormone receptor(s) biological significance, 309-3 10 induction biochemical mechanisms, 307-309 general requirement for, 307 Hormone receptor-adenylate cyclase, structural aspects of interaction, 183-185 Hormone responsiveness, as differentiated function, 288-289 Hydrolases, newly synthesized, transfer to lysosomes, 321-322
I Insulin-sensitive hexose transport, see Adipocytes Irradiation inactivation analysis of data, 237-239 general considerations, 233-235 studies on membranes, practical considerations, 236-237 lsoproterenol, changes in agonist binding properties of intact 1321N1 cells and, 103-104
K Kidney differentiation of cells, hormone receptors and, 303-306 membranes and cells, vasopressin binding to, 259-262 vasopressin receptors effect of nucleotides and other putative effectors on, 272 transduction mechanisms. 265-266
L Leydig cell tumor LH-desensitized, determination of site of lesion in, 165-170 LH-responsive, desensitization and downregulation of, 154-161
477
INDEX
Liver differentiation of cells, hormone receptors and, 294-300 membranes and isolated hepatocytes, vasopressin binding to, 262-263 vasopressin receptors effect of nucleotides and other putative effectors on, 272-274 transduction mechanisms, 266-270 Local anesthetics, positively and negatively charged, modulation of adenylate cyclase and, 209-2 I9 Lysosomal enzymes extracellular, role of oligosaccharide moiety in recognition of, 323-324 mannosyl recognition system macrophages and, 324-326 mannose-binding protein, 326-327 phosphomannosyl recognition pathway biosynthesis of mannose 6-phosphate marker, 319-321 packaging through alternate pathway, 322 transfer of newly synthesized hydrolases to lysosomes, 321-322
coupling between occupation of receptor and permeability response, 426-427 desensitization of, 415-416 isolated, structure of, 408-412 ligand occupation and transitions in receptor state, 416-423 occupation and activation by agonists, 427-430 other ligands affecting receptor function, 423-425 quantitation of antagonist occupation and functional antagonism, 434-435 structural implications and arrangement of subunits, 435-437
0 Obesity, adipocyte hexose transport system in, 371-372 Oligosaccharide moiety, role in recognition of extracellular lysosomal enzymes and glycoproteins, 323-324
P Y Macrophages, mannosyl recognition system and, 324-326 Mannose-binding protein, lysosomal enyzmes and, 326-321 MembraneW. biological. structure of, 180- 183 Mitogenic agents, modulation of adenylate cyclase and, 223
N Nicotinic acetylcholine receptor analysis of activation, 425-426 analysis of bound ligand states, 437-438 association of antagonists and functional antagonism, 430-434 behavior of partial agonists, antagonists and anesthetics in relation to channel activation, 414-415 biophysical properties of receptor channel, 41 2-414
Phospholipid(s) headgroup composition, adenylate cyclase and, 223-225 manipulation, membrane fluidity and, 20 1-202 Polypeptides, EGF-like, role in development and growth, 398-399 Protein(s), components of hormone-sensitive adenylate cyclase catalytic component, 115-121 cell surface receptors, 125- 127 identities of proteins, 1 10- 1 I 1 resolution of catalytic and regulatory proteins, 112-1 15 stimulatory GTP-binding regulatory protein, 121-125
R Receptors dually regulated adenylate cyclase and, 77 endocytosis as mechanism for agonistinduced desensitization, 97-98 kinetic model for. 98- 100
478
INDEX
Receptor theory, signal-response coupling and basic kinetic formulation: one-step model, 13-15 guanylnucleotides and rate of activation of adenylate cyclase, 20-22 separation of sensation and function, 15-20
S Signal-response coupling, receptor theory and basic kinetic formulation: one-step model, 13-15 guanylnucleotides and rate of activation of adenylate cyclase, 20-22 separation of sensation and function, 15-20 Sugars, nonmetabolizable, transport in adipocyte, 355-359
T Target size analysis, application to rat liver plasma membrane adenylate cyclase. 239-241 Temperature, effects on adenylate cyclase activity, 194-201
V Vascular smooth muscle cells, vasopressin receptors in, 270 Vasopressin isoreceptors methodological basis for characterization, 256-259 recognition patterns of, 275-279 Vasopressin receptors effects of nucleotides and other putative effectors on in kidney, 272 in liver, 272-274 kinetics of hormone binding to in blood vessels, 263-265 kidney membranes and cells, 259-262 liver membranes and isolated hepatocytes, 262-263 solubilized, physiochemical characteristics of, 274-275 transduction mechanisms triggered by in kidney, 265-266 in liver, 266-270 in other responsive cells, 270-272 in vascular smooth muscle cells, 270