Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing (Molecular Biology Intelligence Unit)

M OLECULAR BIOLOGY I N T E L L I G E N C E U N I T

2

Vivian Y.H. Hook

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

R.G. LANDES C O M P A N Y

MOLECULAR BIOLOGY INTELLIGENCE UNIT

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing Vivian Y.H. Hook Department of Medicine University of California, San Diego La Jolla, California, U.S.A.

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MOLECULAR BIOLOGY INTELLIGENCE UNIT Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1998 R.G. Landes Company All rights reserved. No part of this book 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. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-553-4

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Proteolytic and cellular mechanisms in prohormone processing / [edited by] Vivian Y.H. Hook. p. cm. -- (Molecular biology intelligence unit) ISBN 1-57059-553-4 (alk. paper) 1. Peptide hormones--Metabolism. 2. Proteolytic enzymes. 3. Peptide hormones-Physiological transport. 4. Protein precursors. 5. Post-translational modifications. I. Hook, Vivian Yuan-Hen Ho, 1953- II. Series. QP572.P4P767 1998 572'.76--dc21 98-28730 CIP

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CONTENTS 1. Targeting and Activation of Peptide Hormones in the Secretory Pathway ........................................................................... 1 Ken Teter and Hsiao-Ping H. Moore Introduction ............................................................................................. 1 Trafficking and Modification of Peptide Hormone Precursors ........... 3 Prohormone Sorting Mechanisms ......................................................... 8 Site of Prohormone Sorting .................................................................. 10 Prohormone Activation ........................................................................ 12 Summary and Future Perspectives ....................................................... 15 2. The Mechanism of Sorting Proopiomelanocortin to Secretory Granules and Its Processing by Aspartic and PC Enzymes................... 29 Niamh X. Cawley, David R. Cool, Emmanuel Normant, Fu-Sheng Shen, Vicki Olsen and Y. Peng Loh General Introduction ............................................................................ 29 Mechanism of Sorting POMC to the Regulated Secretory Pathway ................................................. 31 Endoproteolytic Processing of Proopiomelanocortin ......................... 34 Future Directions ................................................................................... 42 3. The Mammalian Precursor Convertases: Paralogs of the Subtilisin/ Kexin Family of Calcium-Dependent Serine Proteinases ..................... 49 Nabil G. Seidah, Majambu Mbikay, Mieczyslaw Marcinkiewicz, Michel Chrétien Introduction ........................................................................................... 49 Subtilisin/Kexin-like Precursor Convertases (PCs): Structural and Cellular Considerations ............................................ 51 Ontogeny, Tissue Expression and Subcellular Localization ................ 59 Structure, Loci, and Evolution of PC Genes ........................................ 62 Antisense Transgene Inhibition ............................................................ 63 Heritable Deficiency of PC in Human and Mouse .............................. 65 Inhibitors of PCs .................................................................................... 66 Enzymatic Cascades: ADAM Family and PCs ..................................... 67 Conclusions ........................................................................................... 68 4. The Neuroendocrine Prohormone Convertases PC1, PC2 and PC5 ... 77 Margery C. Beinfeld Introduction ........................................................................................... 77 The Discovery of the Subtilisin Family of Prohormone Convertases ............................................................. 77 Distribution of PC1, PC2 and PC5 ....................................................... 79 Biosynthesis and Activation of PC1, PC2 and PC5 ............................. 79 Regulation of PC Expression ................................................................ 80 Experimental Systems Used to Study Processing ................................ 80 Enzymatic Activity of PC1, PC2, and PC5 ........................................... 81

Antisense PC1 and PC2 Strategies to Study Proneuropeptide Processing .............................................. Endoproteases in CCK Processing, a Case in Point ............................. Processing Enzyme Knockouts and Mutations ................................... Future Challenges ..................................................................................

81 82 82 83

5. ‘Prohormone Thiol Protease’ (PTP), a Novel Cysteine Protease for Proenkephalin and Prohormone Processing ................................... 89 Vivian Y.H. Hook, Yuan-Hsu Kang, Martin Schiller, Nikolaos Tezapsidis, Jane M. Johnston and Ada Azaryan Introduction ........................................................................................... 89 The Novel ‘Prohormone Thiol Protease’ (PTP): A Major Proenkephalin Processing Enzyme in Chromaffin Granules ......... 92 Participation of PC1/3 and PC2 Subtilisin-Like Proteases, and 70 kDa Aspartyl Protease (PCE) in Proenkephalin Processing in Chromaffin Granules ............................................... 100 Conclusions ......................................................................................... 100 6. Regulation of Prohormone Conversion by Coordinated Control of Processing Endopeptidase Biosynthesis with That of the Prohormone Substrate ............................................................... 105 Terence P. Herbert, Cristina Alarcon, Robert H. Skelly, L. Cornelius Bollheimer, George T. Schuppin and Christopher J. Rhodes Introduction ......................................................................................... 105 Coordinated Regulation of Prohormone and Processing Enzyme mRNA Levels ........................................... 106 Coordinated Translational Regulation of Specific Prohormone and Processing Enzyme Biosynthesis ............................................. 110 7. Carboxypeptidase and Aminopeptidase Proteases in Proneuropeptide Processing ............................................................ 121 Vivian Y.H. Hook and Sukkid Yasothornsrikul Introduction ......................................................................................... 121 Neuroendocrine-specific Carboxypeptidase E/H .............................. 122 Molecular Genetic Analysis of Mutant Carboxypeptidase E/H in fat/fat Obese Mice: Effects of Inactive CPE/H on Prohormone Processing ............................................................ 129 Mutant CPE/H in fat/fat Mice Leads to Discovery of Novel Carboxypeptidase D and Carboxypeptidase Z ............... 130 Evidence for CPE/H as a Sorting Receptor for the Intracellular Routing of POMC and Possibly Other Prohormones to the Secretory Vesicle ................................................................... 132 Aminopeptidase(s) for Prohormone Processing ............................... 133 Conclusions and Future Perspectives ................................................. 134

8. The Neuroendocrine Polypeptide 7B2 as a Molecular Chaperone and Naturally Occurring Inhibitor of Prohormone Convertase PC2 .......................................................... 141 A. Martin Van Horssen and Gerard J.M. Martens Introduction ......................................................................................... 141 History of 7B2 ...................................................................................... 141 The 7B2 Gene and Its Regulation ....................................................... 142 Evolutionary Aspects ........................................................................... 144 7B2 is a Neuroendocrine-Specific Polypeptide .................................. 144 Biochemical Characteristics of 7B2 .................................................... 145 Posttranslational Modifications of 7B2 .............................................. 145 Regulated Secretion of 7B2 ................................................................. 146 The Quest for the Role of 7B2 ............................................................. 146 Model of the Interaction Between 7B2 and PC2 ............................... 149 Implications and Future Prospects ..................................................... 151 9. Neuroendocrine α1-Antichymotrypsin as a Possible Regulator of Prohormone and Neuropeptide Precursor Processing .................. 159 Shin-Rong Hwang and Vivian Y.H. Hook Introduction ......................................................................................... 159 Biochemical Evidence for α1-Antichymotrypsin (ACT) as an Endogenous Regulator of the ‘Prohormone Thiol Protease’ (PTP) and Other Prohormone Processing Proteases .................... 160 Molecular Cloning Reveals Multiple Isoforms of Bovine ACT Expressed in Neuroendocrine Tissues ........................................... 164 10. Proteolytic Inactivation of Secreted Neuropeptides ........................... 173 Eva Csuhai, Afshin Safavi, Michael W. Thompson and Louis B. Hersh Introduction ......................................................................................... 173 Neprilysin ............................................................................................. 174 Aminopeptidases ................................................................................. 176 Angiotensin Converting Enzyme ........................................................ 178 Pyroglutamyl Peptidase II ................................................................... 178 Proline Specific Peptidases .................................................................. 179 Soluble Neuropeptidases ..................................................................... 180 Endopeptidase 24.15 and Endopeptidase 24.16. ................................ 181 Summary .............................................................................................. 182 11. Stimulation of Peptidergic Receptors by Peptide Hormones and Neurotransmitters: Studies of Opioid Receptors ......................... 191 George Bot, Allan D. Blake and Terry Reisine Introduction ......................................................................................... 191 Opioid Receptor Types ........................................................................ 191 Endogenous Opioids ........................................................................... 192 Endogenous Peptide Receptor Selectivity .......................................... 192 Opioid Ligands .................................................................................... 194

Opioid Cellular Activity ...................................................................... 195 Opioid Receptor Cloning .................................................................... 196 ORL1 and Nociceptin/Orphanin FQ .................................................. 196 Structure-Function Analysis of Cloned Opioid Receptors ............... 198 µ Receptor Knockout Mice Model ..................................................... 201 Agonist Regulation of Cloned Opioid Receptors .............................. 201 G Protein Role in Differential Agonist Activity ................................. 204 Conclusion ........................................................................................... 204 Index ................................................................................................................ 213

EDITORS Vivian Y.H. Hook Department of Medicine University of California, San Diego La Jolla, California, U.S.A. Chapters 5, 7, 9

CONTRIBUTORS Cristina Alarcon Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6

Cornelius Bollheimer Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6

Ada Azaryan Department of Pharmacology Uniformed Services University of the Health Sciences Bethesda, Maryland, U.S.A. Chapter 5

George Bot Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. Chapter 11

Margery C. Beinfeld Department of Pharmacology and Experimental Therapeutics Tufts University School of Medicine Boston, Massachusetts, U.S.A. Chapter 4

Niamh X. Cawley Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2

Allan D. Blake Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. Chapter 11

Michel Chrétien J.A. De Sève Laboratories of Molecular Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3

David R. Cool Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Eva Csuhai Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10 Terence P. Herbert Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6 Louis B. Hersh Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10 Shin-Rong Hwang Department of Medicine University of California, San Diego La Jolla, California, U.S.A. Chapter 9 Jane M. Johnston Department of Neurological Surgery Albert Einstein College of Medicine Bronx, New York, U.S.A. Chapter 5

Yuan-Hsu Kang Naval Medical Research Institute Bethesda, Maryland, U.S.A. Chapter 5 Y. Peng Loh Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Mieczyslaw Marcinkiewicz J.A. De Sève Laboratories of Molecular Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3 Gerard J.M. Martens Department of Animal Physiology University of Nijmegen, Nijmegen Toernooiveld, The Netherlands Chapter 8 Majambu Mbikay J.A. De Sève Laboratories of Molecular Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3 Hsiao-Ping H. Moore University of California at Berkeley Department of Molecular and Cell Biology Berkeley, California, U.S.A. Chapter 1

Emmanuel Normant Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Vicki Olsen Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Terry Reisine Department of Pharmacology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, U.S.A. Chapter 11 Christopher J. Rhodes Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6 Afshin Safavi Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10

Martin Schiller Department of Neuroscience Johns Hopkins University Baltimore, Maryland, U.S.A. Chapter 5 George T. Schuppin Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6 Nabil G. Seidah J.A. De Sève Laboratories of Biochemical Neuroendocrinology Clinical Research Institute of Montreal Montreal, Quebec, Canada Chapter 3 Fu-Sheng Shen Section on Cellular Neurobiology Laboratory of Developmental Neurobiology National Institutes of Child Health and Human Development National Institutes of Health Bethesda, Maryland, U.S.A. Chapter 2 Robert H. Skelly Gifford Laboratories for Diabetes Research Department of of Internal Medicine and Pharmacology University of Texas Southwestern Medical Center Dallas, Texas, U.S.A. Chapter 6

Ken Teter University of California at Berkeley Department of Molecular and Cell Biology Berkeley, California, U.S.A. Chapter 1 Nikolaos Tezapsidis Department of Psychiatry Mt. Sinai Medical Center New York, New York, U.S.A. Chapter 5 Michael W. Thompson Department of Biochemistry College of Medicine University of Kentucky Lexington, Kentucky, U.S.A. Chapter 10

A. Martin Van Horssen Department of Animal Physiology University of Nijmegen, Nijmegen Toernooiveld, The Netherlands Chapter 8 Sukkid Yasothornsrikul Department of Medicine University of California, San Diego La Jolla, California, U.S.A. Chapter 7

PREFACE

P

roteolysis of proneuropeptides is key for the production of bioactive neuropeptides that mediate cell-cell communication in the endocrine and nervous systems. The conversion of inactive precursor to active peptide hormone or neurotransmitter is required for neuroendocrine functions. This text is designed to provide the reader with an understanding of current knowledge concerning proteases involved in prohormone processing, and cellular aspects that must be considered for proper processing, storage, and secretion of bioactive peptides. It is well known that prohormone processing occurs in well-defined subcellular compartments of the regulated secretory pathway. Knowledge of the cell biology of prohormone processing is required in the search for processing enzymes that are colocalized with prohormone substrates and neuropeptide products. Therefore, important cellular aspects of the targeting and activation of peptide hormones in the secretory pathway are discussed in the first two chapters. The next several chapters (chapters 2-6) present evidence for endoproteases that have been demonstrated to be involved in prohormone processing. These endoproteases include the large family of subtilisin/kexin prohormone convertases, a novel cysteine protease known as ‘prohormone thiol protease’ (PTP), and an aspartyl protease that has been termed “POMC converting enzyme” (PCE). These studies provide evidence for three different mechanistic classes of endoproteases that participate in prohormone processing. Subsequent to the actions of endoproteases, carboxypeptidase and aminopeptidase enzymes (discussed in chapter 7) that remove basic amino acids from the COOH- and NH2-termini of peptide intermediates are needed to complete the proteolytic processing of precursors into peptide forms. Moreover, recent molecular genetic studies illustrate the role of prohormone convertases 1 and 2, as well as the carboxypeptidase E/H, in obesity and conditions related to diabetes. The processing pathway is critical for generating active peptides. Therefore, it is likely that endogenous regulators exist that control the prohormone processing pathway. Evidence for the 7B2 polypeptide as a molecular chaperone and inhibitor of a prohormone convertase is discussed in chapter 8. Also, the role of endogenous isoforms of the protease inhibitor α1-antichymotrypsin in regulating prohormone processing enzymes is presented in chapter 9. Upon secretion of neuropeptides into the extracellular environment, the actions of these active peptides can be terminated by proteolytic inactivation. Therefore, chapter 10 discusses extracellular proteases involved in inactivation of secreted peptides. However, before the extracellular proteolysis is complete, the essential function of the released peptide is to activate its specific receptor on the target cell to initiate certain physiological responses. Thus, chapter 11 presents the manner in which peptide hormones and neurotransmitters stimulate peptidergic receptors, with discussion of the opioid receptors as the main example.

The authors have presented the latest developments in this field. A wealth of knowledge has been gained over the last few years concerning the identity, regulation, and molecular and cell biology of proteases and protease inhibitors involved in prohormone processing. However, there are still many open areas to investigate in this field. It is likely that there are still, as yet, unknown processing proteases to be discovered. Importantly, future knowledge of the key proteases and regulatory components required in prohormone and proneuropeptide processing may provide future design of clinical therapeutics that modify the processing pathway in health and disease.

ACKNOWLEDGMENTS I wish to thank the authors who participated in this volume for their expertise and enthusiasm in providing discussions of the current status of knowledge in the prohormone and proneuropeptide processing field. In addition, support from the National Institutes of Health is appreciated. Finally, this book is dedicated to my family, who have shared with me the excitement of science and the continuous effort that has allowed this scientific endeavor to be achieved.

CHAPTER 1

Targeting and Activation of Peptide Hormones in the Secretory Pathway Ken Teter and Hsiao-Ping H. Moore

Introduction

P

rofessional secretory cells—generally cells of neuronal, endocrine, or exocrine origin— utilize two divergent secretory pathways with distinct temporal and spatial characteristics (reviewed in refs. 1-4). The first pathway of constitutive secretion mediates the continual and unstimulated transport of lipids, membrane proteins, and soluble cargo to the cell surface. This pathway thus provides the plasma membrane with a steady supply of protein and lipid components while it simultaneously releases secretory proteins into the extracellular space. Most cells are capable of constitutive secretion, but it was commonly thought that the second pathway of regulated secretion was limited to professional secretory cells. Only these cells were believed to express the specialized machinery required to sort and store specific cargo into a subpopulation of vesicles which accumulate intracellularly until a secretagogue triggers release. Yet recent evidence suggests that many other cell types utilize this process as well, and the mechanism of regulated exocytosis may in fact be used for such additional purposes as membrane repair during wound healing and the regulation of membrane permeabilities to water, ions and nutrients. As summarized in Table 1.1, regulated exocytosis has been detected by a number of techniques in many cell types which have previously been considered to possess only the constitutive secretory pathway. Insights gained from the study of peptide hormone trafficking can thus be generalized to similar events occurring in a variety of cells. Regulated secretory vesicles can be divided into two classes based on morphology, origin and content: Dense core secretory granules (SGs) arise from the trans-Golgi network (TGN) and contain an electron dense peptide hormone aggregate packaged in a 100-200 nm vesicle,5-8 whereas the synaptic vesicles are electron translucent 50 nm structures which derive from the endosomal system and carry nonpeptide neurotransmitters as cargo. After budding from the TGN, SGs are transported to the cell periphery in a microtubule-dependent process.8,9 Peptide hormone precursors are converted to a bioactive state en route by a family of enzymes called prohormone convertases (PCs, reviewed in refs. 10-13) and are incorporated into a highly condensed protein aggregate. Because the soluble constituents of SGs are delivered via the secretory pathway, this route of regulated secretion is referred to as the biosynthetic pathway. Synaptic vesicles, which recruit membrane proteins from the endosomal system and soluble cargo from the cytoplasm, utilize what has been termed the recycling pathway for biogenesis. The function and generation of synaptic vesicles has been reviewed elsewhere. 4,14-17 This chapter will focus on the sorting and activation of Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

stimulated release of lysosomal enzymes and pre-internalized BSA-gold complex

membrane wound/disruption

perfusion of cytosol with Ca2+

Ca2+ ionophore

Bovine endothelial cells

CHO fibroblasts

NRK fibroblasts

stimulated release of sulfated GAG chains

Ca2+ ionophore

L cells; CHO fibroblasts

229

227, 228

226

225

224

223

221, 222

Mechanisms of regulated secretion are widespread in animal cells. Although regulated secretion has historically been viewed as a specialized property of professional secretory cells, this phenomenon has recently been characterized in other cell types and may be used for a variety of physiological processes. One such purpose appears to be the transient modification of cell surface permeability. This facilitates the uptake of water, ions, or nutrients and is accomplished by the stimulated translocation of membrane channels and pumps from a specialized intracellular pool to the plasma membrane. The same general mechanism may be widely used for membrane repair during wound healing.

release of pre-loaded acetylcholine detected by patch clamp measurements of an abutting myocyte

Ca2+ influx; membrane depolarization

targeting of transfected Glut4 to a unique vesicle population, presumably a storage organelle similar to the Glut4-containing vesicle in adipocytes and muscles

vesicle accumulation and microvilli formation at wound site visualized by electron microscopy; heightened release of pre-internalized dye in wounded cells; increase in cell size

induction of numerous exocytic ‘pores’ visualized with fluorescent dyes and confocal microscopy; heightened release of pre-loaded dye in wounded cells

Amphibian myocytes and fibroblasts; CHO fibroblasts

CHO fibroblasts; not determined 3T3 fibroblasts

increase in membrane capacitance, reflecting increased cell surface area

membrane wound/disruption

Sea urchin eggs and embryos; NIH fibroblasts

220

mobilization of H+/K+-ATPase to the cell surface

histamine

Gastric cells

15, 218, 219

15, 217

References

mobilization of Glut4 glucose transporter to the cell surface

mobilization of water channels to the cell surface

vasopressin

insulin

Kidney ductules

Observation/Method of Detection

Adipocytes

Stimulus

Cell Type

Table 1.1. Nonclassical regulated secretion in animal cells

2 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Targeting and Activation of Peptide Hormones in the Secretory Pathway

3

prohormones and prohormone converting enzymes along the biosynthetic regulated secretory pathway.

Trafficking and Modification of Peptide Hormone Precursors Proteins destined for either regulated or constitutive release are transported and modified together as they migrate from the ER, through the Golgi and to the TGN. Targeting to the regulated pathway then begins in the TGN with the preferential packaging of materials into a budding immature secretory granule (ISG),18,19 the intermediate vesicle that is biochemically distinct from the mature SG.7,20-25 Sorting appears to continue during the period of granule maturation, since the lysosomal enzymes and constitutively secreted proteins that are initially incorporated into the nascent SG are found in substantially reduced quantities in the mature SG. As such, the ISG has sometimes been regarded as the functional extension of the TGN by completing the sorting function that is initiated at that site (reviewed in ref. 26). In most cases, prohormone conversion begins in the TGN but takes place predominantly in the ISG. This organelle therefore represents a key intermediate station for prohormone sorting and cleavage (Table 1.2). The extent of prohormone sorting and activation occurring in the TGN vs. the ISG appears to vary for different proteins and cell types, but in all cases the maturation process eventually generates a homogeneous SG vesicle population with highly concentrated bioactive peptides. The production of mature peptide hormones thus involves numerous sorting and processing events.

Role of ER and Golgi Prohormone sorting and modification begins in the ER. Peptide hormones enter the secretory pathway by virtue of a hydrophobic signal sequence which directs cotranslational passage through a ‘translocon’ complex into the ER lumen (reviewed in refs. 27-29). Once positioned in the ER, preprohormones are modified by a series of reactions: The signal sequence and/or prosequence are cleaved, N-linked core carbohydrates are added, and the prohormones are transiently associated with molecular chaperones. In the case of thyroglobulin, sequential interactions with the BiP and calnexin chaperones are required for its proper folding.30 BiP also prevents proinsulin degradation during folding and dimerization.31 This process, as well as proinsulin disulfide bond formation, is facilitated by the oxidizing lumenal environment of the ER.32 Since proper folding is a requisite for ER export, the time required to complete these modifications determines, in part, the exit rate for each protein (reviewed in ref. 33). Until recently, secretory proteins were thought to leave the ER by default. This ‘bulk flow’ mechanism would allow transport and constitutive secretion of proteins with no targeting information other than the initial signal sequence (reviewed in refs. 34, 35). According to this model, only residents of organelles would require specific targeting signals in order to be retained within their respective compartments, and a variety of organelle targeting motifs have indeed been identified (Table 1.3). However, another prediction of the bulk flow model—lack of sorting and concentration of migrant proteins upon exit from the ER—is not supported by recent observations. Many yeast proteins, for instance, are concentrated in ER-derived carrier vesicles (reviewed in refs. 36, 37). Quantitative immunoelectron microscopy has also established that albumin and VSV G are concentrated in ER-derived vesicles in mammalian cells.38,39 These observations indicate that exit of migrant proteins is facilitated by an active sorting mechanism. In the case of VSV G, this process requires a cytoplasmic, di-acidic anterograde targeting signal.40 A phenylalanine-containing anterograde transport signal has also been found on two putative ER cargo sorting receptors, p24 and ERGIC-53.41-45 Although cognate transport signals in prohormones have yet to be

tubulo-cisternal

+++

yes

mildly acidic/neutral

yes

limited

no

no

morphology

lysosomal enzymes

clathrin coat

pH

sorting compartment

processing compartment

stimulated release

unstimulated release yes

yes

yes

yes

5.0-7.0 average 5.7-6.3

partial

++

vesicles of irregular shape and size, 80 nm average diameter

perinuclear to peripheral

ISG

limited

yes

limited

no

5.0-5.5

no

+/–

100-200 nm vesicles

peripheral

SG

23, 82, 118, 153, 155

8, 118, 148, 153, 155

20, 82, 107, 108, 121, 153, 174, 178-180, 182, 183, 188, 200, 201, 230-234

18, 19, 24, 118, 145, 146, 149, 154, 157, 158

9, 108, 188, 200-202, 204-206

7, 20-22, 24

149, 158, 159

6, 8, 22

22, 24, 118

References

Serving as an intermediate between the TGN and SG, the ISG shares characteristics of both organelles. Like the TGN, the ISG is decorated with γ-adaptin and clathrin, contains lysosomal enzymes, maintains an average pH of approximately 6.2, and acts as sorting station for routing secretory traffic to multiple sites. Prohormone processing also takes place in both the TGN and ISG, although in most cases cleavage in the TGN is fairly limited. Another major difference between the two organelles is the response to secretagogues: Only the ISG is capable of stimulated exocytosis. The ISG resembles the mature SG in this respect, although stimulation usually results in the preferential exocytosis of young granules. In addition, the contents of newly formed ISGs exhibit a higher rate of unstimulated release. In some cases, this secretion has been shown to result from the budding of transport vesicles from ISGs. With time, however, the maturation process places release from ISGs under tight regulation and eventually transforms the vesicles into a uniform population of SGs. Note that the characteristics summarized in the table above are derived from numerous studies involving different cell types, marker proteins and model systems. This variability may account for some of the inconsistencies present in the literature.

perinuclear

location

TGN

Table 1.2. Relationship between the TGN, ISG and SG

4 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Targeting and Activation of Peptide Hormones in the Secretory Pathway

5

identified, trafficking of these proteins may also involve ER export signals and cargo sorting receptors. By contrast, bulk flow may apply to intra-Golgi transport since further concentration of migrant proteins does not occur as trafficking continues across the Golgi stacks.38,39,46 While the exact mechanism of intra-Golgi transport is currently under debate,47-49 it is clear that a number of protein modifications occur at this site. Proinsulin hexamerization is initiated in an early Golgi compartment by the addition of zinc,32,50 and a subset of regulated secretory proteins are phosphorylated in the trans-Golgi.51 A prevalent modification is the addition of N- and O-linked carbohydrate chains, generated by the coordinate action of a characteristic set of enzymes in each Golgi cisternae (reviewed in refs. 52,53). The multiple Golgi cisternae also appear to act as a “molecular sieve”54 which allows repeated opportunities for the capture and return of missorted ER resident proteins. This retrieval process is mediated by a set of carrier vesicles bearing the coat complex COPI (reviewed in refs. 36,55,56). Proteins destined for secretion appear to be excluded from these retrograde transport carriers, as recent studies indicate that proinsulin and VSV G protein are segregated into distinct vesicles from those containing the ER retrieval KDEL receptor.46 Interestingly, the proinsulin-containing vesicles are also COPI-coated. The possibility that COPI functions in both anterograde and retrograde transport remains an issue that requires further clarification.56-59

Role of TGN, ISG and SG The TGN serves as a major sorting station for secretory traffic, diverting proteins to regulated, constitutive, lysosomal, and (in polarized cells) apical or basolateral destinations (reviewed in refs. 60, 61). Recent studies indicate that in nonpolarized mammalian cells and in yeast, different classes of constitutive vesicles, each with a distinct set of cargo, are also generated by the TGN.62-64 Its central role in these trafficking events has made the TGN a subject of numerous studies. Originally defined as the site at which newly synthesized plasma membrane proteins accumulate at 20°C, the TGN could be visualized by electron microscopy as a tubulocisternal network directly apposed to the trans-Golgi cisternae.65-67 Later, it was defined by two biochemical reactions—sialation and tyrosine sulfation—which occur at this site.68,69 The intracellular distributions of TGN38, furin and the mannose 6-phosphate receptor have also been used to delineate the compartment.70-74 Thus, over time the TGN has been defined by many different parameters and markers. These definitions have been used interchangeably, but a close examination of the literature reveals considerable inconsistencies regarding the response of the TGN to drug treatment (Table 1.4). This raises the possibility that the organelle known as the “TGN” may in fact be referring to more than one compartment. Because models for prohormone sorting and activation rely critically on the exact locations in which these events occur, the definition of the TGN needs to be revisited in order to avoid confusion caused by inconsistent usage of the term “TGN”. In addition to sulfation and sialation, one other major prohormone modification— processing to a bioactive state—is initiated in the TGN. Functional peptide hormones are usually quite small but are often synthesized as larger, inactive precursors which are eventually cleaved by the PC enzymes. Each prohormone convertase recognizes different dibasic consensus sequences, so the generation of a specific bioactive peptide relies upon the activity of one or more PCs. As a result, the same prohormone can yield different products depending on which PC(s) it encounters.75-78 Prohormone cleavage also exposes C-terminal basic amino acids which are recognized and removed by another modifying enzyme, carboxypeptidase E (CPE, reviewed in ref. 79). In some instances, alpha-amidation may also follow prohormone cleavage (reviewed in ref. 80).

241 241 144, 242, 243 244 242, 243

intercalates into glycolipid ‘rafts’ destined for apical membrane interaction with the lectin-like, carbohydrate binding receptor VIP36 unknown mechanism

238 166, 167, 239 166, 167, 240

unknown retention mechanism;may involve “kin recognition” or an interaction with the Golgi lipid bilayer unknown retention mechanism unknown retention mechanism;involves phosophorylation state retrieval mechanism most likely involving cytoplasmic coat (clathrin and AP-2) proteins addition of phosphate to a mannose residue in the cis Golgi pH-dependent sorting mechanism involving a receptor in the TGN

55, 237

236

235

36, 55, 56

36, 55

References

Protein targeting requires specific localization signals. Organelle residence is established by a combination of retention and retrieval. The retention mechanism prevents most resident proteins from continuing along the secretory pathway, while an auxillary retrieval mechanism captures errant proteins in distal secretory compartments and returns them to the proper organelle. Targeting is most often due to either a unique physical property of the protein or to a receptor/motif interaction. Variations on these two themes are used throughout the secretory pathway, including diversion to the SG, lysosome, or polarized plasma membrane.

transmembrane domain (TGN38) cytoplasmic acidic cluster (furin) cytoplasmic tyrosine tight-turn motif (TGN38 and furin) Lysosomes specific conformational motif mannose 6-phosphate residue Epithelial plasma membrane apical surface GPI lipid anchor, transmembrane domain N-linked glycans basolateral surface cytoplasmic tyrosine tight turn motif or dileucine sequence

TGN

transmembrane domain

unknown retrieval mechanism

cytoplasmic N-terminal RR motif

Golgi

unknown retention mechanism

transmembrane domain

type II membrane proteins

retrieval mechanism effected by cytoplasmic coat (COPI) proteins

pH-dependent retrieval mechanism involving a KDEL receptor

TargetingMechanism

cytoplasmic C-terminal KK motif

lumenal C-terminal KDEL tag

Targeting Determinant

type I membrane proteins

ER soluble proteins

Destination

Table 1.3. Targeting signals for organelles along the secretory path

6 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

large vesicular structures ER ER cofractionates with TGN38 ER not determined MTOC MTOC MTOC

20° block site VSV G viral protein SFV viral proteins secreted GFP fusion construct radiolabeled proinsulin Sialyltransferase compartment Site for sulfation TGN38 compartment Furin compartment MPR compartment blocked blocked blocked blocked not applicable blocked enhanced not inhibited not inhibited

Export in the Presence of BfA

245 246 247 248 249-252 82, 148, 182, 245, 253 254, 255 73, 256 257

References

Serving as a major sorting station for secretory traffic, the TGN has been the subject of numerous studies and can be defined by a number of parameters. Yet a survey of the literature reveals that these parameters produce conflicting results when subjected to Brefeldin A (BfA) treatment. In the presence of BfA, a fungal metabolite which induces the redistribution of Golgi residents to the ER,258 the TGN (as defined by TGN38, furin, or MPR) did not collapse to the ER but was instead found at a tubularized perinuclear site which also contained internalized transferrin. This established the TGN as a functionally and physically distinct organelle, separate from the trans-Golgi. However, the TGN does redistribute to the ER when it is defined by other criteria (i.e., the 20° block site or the sialyltransferase compartment). This inconsistency and the differential effect of BfA on TGN transport leave open the possibility that two distinct compartments are both being defined as the TGN. Further examination of the nature of the TGN and the relationship between the various definitions of the compartment may thus help to clarify some of the discrepancies regarding the site of prohormone sorting and activation.

Location in the Presence of BfA

Definition

Table 1.4. Definitions of the TGN and responses to Brefeldin A

Targeting and Activation of Peptide Hormones in the Secretory Pathway 7

8

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Prohormone cleavage and activation continues in the ISG, the vesicular intermediate linking the TGN and SG. Concomitant with this event are other steps of granule maturation: Translocation to the cell periphery, progressive lumenal acidification, homotypic fusion between two or more ISGs, and loss of the clathrin coat with the simultaneous removal of missorted proteins and excess membrane (reviewed in refs. 25,26,81). Many of the missorted proteins are lysosomal and constitutive secretory proteins, but membrane proteins of the constitutive fusion machinery may also be incorporated into the budding ISG. Mistargeting of components of this fusion machinery could explain the high level of unstimulated release of ISGs early in the maturation process.82 In contrast, other proteins required for regulated exocytosis are apparently added to the ISG. The SG membrane protein VAMP-2, for example, is delivered to the nascent SG from a post-Golgi site (B. Eaton and H.-P. Moore, manuscript in preparation). The delayed delivery of components of the regulated fusion machinery could also account for the short lag period in which newly formed ISGs are refractory to stimulated release in pituitary AtT20 cells (M. Haugwitz and H.-P. Moore, manuscript in preparation). SG biogenesis is thus a multi-step process involving numerous budding and fusion events which serve to alter both the soluble and membrane composition of an ISG.

Prohormone Sorting Mechanisms The mechanisms for targeting peptides to the regulated secretory pathway appear to have been conserved within the family of professional secretory cells: Regulated proteins which are not endogenously expressed by a particular professional secretory cell are still recognized and stored in SGs when introduced into that cell by DNA transfection.83 This indicates that the regulated sorting machinery recognizes common determinants present in many secretory proteins and explains why multiple secretory products can often be found within the same granule.18,84-88 As with other intracellular targeting events, there are two conceptually distinct mechanisms that may explain sorting to the regulated pathway. Sorting may be accomplished by a receptor which recognizes a targeting motif on specified proteins and in turn delivers them to the proper compartment. Typically, the receptor would cycle between the sorting and target compartments to mediate multiple rounds of binding and dissociation. An alternative mechanism involves the formation of specific macromolecular complexes between molecules destined for the same organelle. This would entail a conformational transition that initiates the formation of these molecular aggregates at the site of sorting. This mechanism thus allows multiple components to be sorted synchronously without the need of a cycling receptor. Numerous studies in recent years have uncovered specific interactions between various protein components of the regulated secretory pathway. To date, a cycling receptor that mediates multiple rounds of binding and dissociation has not been found. Instead, the available data support a model in which the formation of specific macromolecular aggregates of granule components dictates their coordinate sorting into regulated secretory granules. These interactions can be divided into two types: content-to-content interactions, and content-to-membrane interactions.

Interactions between SG Contents Homotypic interactions Proteins targeted to the regulated pathway are highly concentrated in the mature SG (reviewed in refs. 1, 2, 89). Electron microscopic studies have shown that the concentration process begins at the dilated rim of the trans-Golgi (reviewed in refs. 89, 180), thus leading to the hypothesis that selective aggregation of soluble SG content plays a key role in sorting

Targeting and Activation of Peptide Hormones in the Secretory Pathway

9

to the regulated pathway.2,4 In support of this, the aggregation and condensation of regulated secretory proteins can be triggered in vitro by a combination of high (1-10 mM) Ca2+ and low (<6.2) pH,90-100 conditions thought to approximate the ionic milieu of the TGN (discussed below). Aggregation can also be induced in ER-derived microsomes when the physiological conditions are altered to a high Ca2+, low pH environment.93 Resident ER proteins and constitutively secreted proteins are not efficiently incorporated into the aggregates,90,93,99,100 and thus a simple sorting model based on the differential solubilities of regulated and constitutive proteins can be envisioned: The condensed aggregate of regulated proteins partitions into one vesicle population, while the remaining soluble proteins flow into another. In some instances, heterogeneity in SG populations has been described; the individual subpopulation not only contains distinct cargo but also exhibits differential secretagogue sensitivities (reviewed in refs. 101,102). Sorting in these cases may be achieved by the varying efficiency of aggregation of different regulated secretory products. Missorting of regulated secretory products under alkalinizing conditions103-108 could also be explained by the lack of aggregate formation at neutral pH. Heterotypic interactions Aggregation is a concentration dependent phenomenon,92,97,98,100 but not all regulated proteins are present in sufficient quantity to aggregate.109 In addition, some regulated proteins such as proinsulin do not self-aggregate.99,109 Several proteins incapable of homotypic aggregation can, however, undergo condensation when combined with other granule proteins.99 Heterotypic interactions between certain exocrine and endocrine products110,111 may also be the basis for specific SG targeting of these exocrine proteins in endocrine cells.85 Rosa et al105 have furthermore shown that a constitutively secreted protein can be diverted to the regulated pathway when bound to chromogranin B (CgB), a major constituent of the neuroendocrine SG (for reviews of granin structure and function, see refs. 112-114). It has been proposed that CgB and the related protein chromogranin A serve to increase SG sorting efficiency by promoting heterotypic aggregation of regulated secretory proteins.100,115,116

Interactions Between Granule Content and Membrane Aggregation alone is not sufficient for diversion of secretory proteins to the regulated pathway. Aggregates of salivary proline-rich protein or recombinant fibronectin are not efficiently stored in the AtT20 SG,117,118 and a DTT-reduced, aggregated form of CgB is constitutively secreted by PC12 cells.119,120 In addition, granule targeting is not always correlated with a protein’s aggregation state. Insulin condensation, for example, does not begin until the protein has entered the ISG, presumably after some degree of sorting has already taken place at the TGN.121 These results imply that another SG targeting mechanism is at work, one conceivably involving interactions with membrane-associated components. Although the term ‘receptor’ has been used to describe such a membrane component, it should be noted that ‘receptor’ in this case may be nothing more than a membrane constituent of the macromolecular complexes described in the previous section, and as such should be distinguished from the classical cycling receptors (e.g., the lysosomal sorting mannose 6phosphate receptor and the ER retrieval KDEL receptor). Membrane interaction is presumably mediated by specific sorting sequences on prohormones, and the nature of these sequences has been addressed by examining the targeting efficiency of recombinant proteins transfected into cultured cells. Localization of a human growth hormone/soluble VSV G hybrid protein to the AtT20 SG initially demonstrated that regulated proteins contain positive sorting information,122 and subsequent work has found that the propeptides of several hormone precursors contain SG targeting signals.123-128 However, deletional analysis of proopiomelanocortin (POMC), protrypsinogen

10

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

and proinsulin has indicated that the prosequences of these hormones are not necessary for proper targeting.86,129-131 These conflicting results are most likely explained by the presence of redundant localization signals in the prohormone structure.132 One targeting signal present in the prosequence of POMC has been identified as an Nterminal amphipathic loop stabilized by a single disulfide bond.133 A similar motif is also found in other prosequences,120,133,134 and in the case of CgB the integrity of the disulfide loop has been shown to be necessary for proper localization of the aggregated protein.119,120 Identification of the amphipathic loop on POMC has thus led to the hypothesis that this structural domain may mediate sorting by binding to a putative membrane receptor. In support of this model, Tam et al127 have shown that the POMC N-terminal peptide binds to Golgi and SG membranes in a pH dependent fashion. Carboxypeptidase E (CPE) has recently been reported to mediate this interaction.135 Loss of CPE activity in a mutant mouse strain leads to loss of regulated release of mature ACTH135 and growth hormone,136 and these observations were taken to support CpE’s functional role as a prohormone sorting receptor. However, proinsulin sorting is normal in the pancreas of the mutant mice,137 ruling out the possibility that CPE serves as a general sorting receptor for all prohormones. Interestingly, lack of CPE activity dramatically inhibits prohormone processing even though the PC activities appear to be normal.135,137,138 The exact role of CPE in prohormone processing warrants further investigation. Another candidate for a membrane anchor for sorting into the regulated pathway is CgB. Up to 10% of total cellular CgB in the neuroendocrine PC12 cell line was found to be membrane-associated, and this observation has led to a model in which membrane-associated CgB binds to soluble CgB and directs CgB to the regulated pathway.139 CPE and CgB are not expressed in exocrine cells, but another protein may perform the same function in this cell type. GP-2, a major component of exocrine granules which is present in both soluble and GPI-linked (i.e., membrane-bound) forms,140,141 undergoes pH- and Ca2+-dependent aggregation.94,95,142 Its ability to coaggregate with other regulated proteins could therefore allow the GPI-linked variant of the protein to act as a membrane anchor. GP-2 is not sorted to the regulated secretory pathway of neuroendocrine cells, indicating that the SG targeting of membrane proteins may, in some instances, involve cell type specific sorting mechanisms.142,143 CPE, CgB and GP-2 have been considered as putative SG sorting receptors, but there is no evidence to suggest that they mediate multiple rounds of sorting as per a classical cycling receptor. Their role in sorting may therefore simply be to act as membrane anchors which promote the association of granule content with the appropriate membranes. It remains to be determined if these proteins associate with additional components to form ‘raft’ microdomains which facilitate targeting in a manner similar to those found in polarized cells (reviewed in ref. 144).

Site of Prohormone Sorting The mechanisms used for SG targeting may be triggered in either the TGN or ISG. Models supporting both possibilities have been proposed.4,26,135 Sorting may occur in the TGN as a membrane anchor/receptor binds either individual or aggregated regulated secretory proteins and is subsequently incorporated into a budding ISG. Exclusion of constitutive proteins from the nascent ISG would then result in the selective packaging of regulated secretory products into dense-core granules. This model is termed “sorting for entry”, as the emphasis here is placed on selective targeting of hormones to the budding ISG. Alternately, both classes of proteins could be packaged into an ISG. Sorting within the ISG would then be accomplished by the removal of constitutive proteins which had not undergone aggregation and selective association with the granule membrane. This model is termed “sorting by

Targeting and Activation of Peptide Hormones in the Secretory Pathway

11

retention”. These two concepts are not mutually exclusive, and the exact site of sorting may in fact differ for different proteins.

Sorting for Entry Before ISG formation, regulated and constitutive proteins can be found together throughout the Golgi. Only after exit from the TGN are the two classes of proteins segregated into distinct vesicle populations. This was initially documented by electron microscopy18,19 and was later reconstituted in a cell-free system using sulfate labeling as an indicator of TGN residency.145,146 Constitutive trafficking from the TGN involves a population of light, 100-300 nm vesicles which rapidly (t1/2 = 10 min) fuse with the plasma membrane.18,116,145-147 In contrast, the ISGs consist of a heterogeneous collection of dense vesicles which remain in the vicinity of the TGN for an extended period of time.22,145,146 The generation of an ISG thus represents the initial sorting event in targeting proteins to the regulated pathway. Interaction of aggregated granule content with a membrane-associated factor(s) such as CPE, CgB or GP-2 may facilitate this process.

Sorting by Retention Some amount of mistargeting occurs as proteins are sorted from the TGN into the constitutive and regulated secretory pathways. This results in the constitutive release of a fraction of newly synthesized prohormones83,116,133 and also in the packaging of some constitutive proteins and lysosomal precursors into the regulated secretory granules.118,146,148,149 In some instances the extent of missorting can be quite substantial, but the missorted proteins are generally removed effectively from the ISG. The ISG therefore represents a major sorting compartment in addition to the TGN. The mechanism which operates to remove missorted constitutive secretory proteins from the ISG involves the formation of transport vesicles which are capable of what has been termed “constitutive-like secretion” (CLS). CLS takes place over a sustained period of time, resulting in the unstimulated release of up to 15% of granule content in primary cultured cells,23,24,104,150-153 and 50% in tumor cell lines.82,118 The removal of content is not a homogenous process, however, as the efficiency of incorporation into constitutive-like vesicles varies for different proteins.24,104,146,154 For example, the C-peptide produced from insulin processing is released by CLS in molar excess of mature insulin.153,155,156 Likewise, salivary amylase and proline-rich protein are preferentially removed from the ISG of transfected AtT20 cells.118 Thus, as maturation proceeds, the selective removal of contaminating proteins by CLS results in a decreasing ratio of constitutive:regulated proteins in the maturing SG. Carrier vesicles mediating CLS have not been directly isolated, and the pathway of CLS remains to be established. With a transport rate of t1/2 = 1.5-2.5 hours, it is possible that the vesicles transit to an intermediate site(s) such as the endosomes before traveling to the cell surface.23,153 Lysosomal enzymes can also be found in the ISG.149,157,158 The majority of these hydrolyases are routed to the lysosomes, although some are released via CLS and others end up in the SG.149,158,159 Recognition of missorted hydrolyases is mediated by the mannose 6-phosphate receptor and could involve the pool of clathrin and gamma-adaptin associated with the ISG membrane.149,158,160 The relationship between CLS and lysosomal sorting within the ISG remains to be established, but the possibility that the two pathways originate from the same vesicle population is supported by work documenting the lysosomal degradation of insulin C-peptide.156 The above observations support aspects of both the sorting for entry (TGN) and sorting by retention (ISG) models of SG targeting. Sorting begins in the TGN with the budding of two distinct transport carriers, the ISG and constitutive vesicle. The relative partitioning of cargo into each vesicle type appears to vary significantly for different proteins and cell

12

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

types.118,145,146,148 “Sorting for entry” enriches the ISG with regulated secretory proteins, but does not efficiently exclude constitutive secretory proteins or lysosomal enzymes. These missorted proteins are later removed by vesicles budding from the ISG as “sorting by retention” maintains the granule association of condensing regulated secretory products. Although aggregation may increase the efficiency of sorting, this alone is not sufficient for proper targeting both at the TGN and within the ISG. Rather, membrane association is most likely necessary for localization of a condensing aggregate to the maturing SG. Remnants of this association seem to persist even after exocytosis, as a significant fraction of CgB can be detected on the plasma membrane following SG fusion.139

Prohormone Activation Active peptide hormones are generated by the processing of prohormone precursors at specified dibasic cleavage sites. Proteolysis occurs at a late stage in the pathway, so prohormones which are missorted and constitutively secreted do not undergo complete processing. This occurs in spite of the cotransport of prohormones and PCs through the early stages of the secretory pathway. PC activity must therefore be restricted to a late stage of the pathway, much as the activity of lysosomal hydrolyases is limited to the late endosomes and lysosomes. The regulation of PC localization and activity thus dictates the site of prohormone activation. These regulatory mechanisms are best understood for furin, PC1 and PC2.

Targeting and Processing of Prohormone Convertases Furin Furin is expressed ubiquitously and is one of the few members of the PC family which contains a membrane-spanning segment. Initially synthesized as a 104 kDa proenzyme, it is quickly converted to a 98 kDa form in the ER via autocatalytic removal of the N-terminal pro-region.73,161-164 Synthesis and cleavage of the propeptide appear to be essential for proper folding, trafficking and activation of furin since unprocessed pro-furin is retained in the ER and has no substrate processing activity.73,163,164 The cleaved propeptide remains bound to the mature protein and serves as a trans-acting inhibitor.165 The enzyme therefore remains inactive in the proximal part of the secretory pathway until it reaches the TGN, where the trans-acting inhibitor is further processed and dissociates from the complex to allow full activation of furin. In contrast to the SG localization of PC1 and PC2, furin is targeted to the TGN.73,74 This is accomplished by two complementary processes of retention and retrieval, with an acidic cytoplasmic cluster serving as a retention signal and a tyrosine-based tight turn motif acting as a retrieval signal.166,167 Furin retention is inefficient, so retrieval from the cell surface via endosomal intermediates is required to maintain a steady state distribution within the TGN.73,74,168 Continual cycling between the TGN and cell surface contributes to the turnover of furin, either by eventual lysosomal targeting74 or by secretion of a soluble, processed furin peptide.162,163,169 PC1 Like furin, the N-terminal propeptide of PC1 is rapidly removed in the ER by an autocatalytic mechanism.170-176 This event generates the 87 kDa form of PC1, which exhibits enzymatic activity in vitro.171,173,177 However, full substrate processing activity is not attained until PC1 reaches the ISG. As a result, many regulated proteins remain largely unprocessed by PC1 until ISG formation.82,121,153,174,178-183 Processing activity is triggered in the ISG after removal of the PC1 C-terminal peptide generates the mature, 66 kDa form of

Targeting and Activation of Peptide Hormones in the Secretory Pathway

13

PC1.170,174,175,182,184 The C-terminal domain thus acts as an autoinhibitor by silencing the activity of the 87 kDa enzyme. For this reason, C-terminal truncations of PC1 exhibit enhanced processing activity in both fibroblasts and professional secretory cells.185-187 Whether this mechanism alone is sufficient to completely inhibit PC1 activity in the proximal part of the secretory pathway is unclear. It has been postulated that PC1 activity may also be regulated by the N-terminal propeptide in a manner similar to furin.165 The inherent instability of mature PC1173 may also place a limitation on PC1 activity; rapid denaturation of the mature, fully active PC1 could restrict processing to a short window of time and may explain the presence of a small but significant percentage of unprocessed prohormone in the mature SG.132,178,179 PC2 PC2 is also synthesized as a proenzyme, but in this case the propeptide is not removed until the protein reaches the TGN and ISG.176,184,188 Conversion mainly occurs in the ISG184,188 and follows proteolysis of an associated protein, the 25 kDa trans-inhibitor 7B2.189-192 This protein binds to proPC2 in the ER with 1:1 stiochiometry and therein acts as a molecular chaperone by facilitating the maturation of proPC2.190,191 However, unlike other chaperones 7B2 remains bound to its target, inhibiting enzyme activity until processing in the TGN generates an 18 kDa proteolytic remnant of 7B2.189,190,192 Slow, intermolecular autocatalytic removal of the propeptide then generates the active form of PC2.176,193-196 Continued association with the processed 7B2 fragment may stabilize PC2 activity within the ISG and SG.196 PC1, PC2 and CPE all contain a C-terminal amphiphilic alpha-helix which may be involved with membrane association and SG targeting.79,197,198 However, preliminary work indicates that the helix is not required for proper SG localization. Furthermore, PC1 and PC2 are differentially partitioned within the SGs of pancreatic islets: PC1 and PC2 colocalize in one SG subpopulation, but a second subpopulation contains only PC2.199 This indicates that an additional sorting signal is involved in PC1 and/or PC2 localization. One such signal could be the selective aggregation of proPC2 under low pH/high Ca2+ conditions.96

Role of Organelle Physiology in PC Activation PC activation is ultimately controlled by the termination of inhibitor action in a specific processing compartment. In principle, this could be accomplished in at least two ways. One possibility is that the inhibitors are physically removed from the processing compartment. This would entail an exclusion or removal of the inhibitors during biogenesis of a processing-competent organelle. Alternatively, the inhibitors enter the processing compartment but their actions are terminated by the specific ionic milieu of the organelle; the lumenal environment may either trigger inhibitor dissociation or induce proteolytic cleavages to eliminate the inhibitors. By this model, the formation of processing-competent organelles would involve the sorting or activation of specific ion transporters in order to establish the unique ionic milieu required for processing. Evidence supporting the ionic milieu model came from the finding that proteolytic cleavages of POMC could be induced within the ER or TGN by experimentally acidifying these compartments to below pH 6.0.182 A similar finding has recently been documented for furin: Exposure of a soluble, ER-localized furin construct to an acidic environment resulted in the activation of this endoprotease.165 These results thus support an attractive hypothesis for PC activation: The near-neutral pH of the ER and Golgi allows inhibitor association and hence suppression of PC activity until the acidic lumen of the target compartment inactivates the inhibitor and allows full expression of PC activity.

14

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Compartmental pH and Ca2+ levels thus play a pivotal role in trafficking within the regulated pathway, influencing: 1. formation of macromolecular aggregates; 2. membrane association of regulated proteins such as CPE; and 3. PC activation. Yet surprisingly little is known about the regulatory mechanisms which set the ionic milieu of individual organelles. These conditions have themselves just recently been examined in a quantitative manner and are of considerable interest since the standard model of prohormone sorting and activation necessitates significant changes in compartmental physiology as proteins transit from the trans-Golgi, through the TGN and to the ISG. SG and ISG pH The pH of mature SGs has been studied with DAMP, a weak base which accumulates in acidic compartments and can be visualized with specific antibodies. Since the level of accumulation is proportional to the acidity of the compartment, the relative labeling efficiency of DAMP can be used as a measure of compartmental pH. Antibody quantitation by electron microscopy thus recorded a SG pH within the range of 5.0-5.5.200,201 These results complemented previous work which estimated a SG pH between 5-6 using isolated organelles.202 The ability to measure single granule pH has also revealed the relatively uniform pH of mature SGs. The minor variations in pH among individual mature SGs stands in contrast to the heterogeneous pH found in the ISGs. DAMP quantitation documented an ISG pH ranging from 5.0-7.0 with an average of 5.7-6.3, thus reflecting a milder acidic interior and a wider distribution of individual ISG values.108,200,201 In addition, a correlation between ISG pH and processing was observed. ISGs with the highest pH also had the highest content of unprocessed prohormones, whereas little or no prohormone remained in ISGs with low pH.108,201 pH-dependent processing of SgII has also been observed and was used to calculate an average ISG pH of 6.3.188 These results and the block of prohormone processing which follows H+-ATPase inhibition182,203 suggest that activation of prohormone processing is dependent on the progressive acidification of an ISG. TGN, Golgi and ER pH As predicted by the prohormone sorting models, initial studies with DAMP found the TGN to be acidic. These observations, however, apply to fibroblasts and epithelial cells.204,205 DAMP does not significantly accumulate in the TGN of professional secretory cells.108,179,200,206 By targeting pH sensitive dyes to specific organelles of living cells, we have found that the TGN of AtT20 cells is only mildly acidic (>6.5) (G. Giorgi, S. Lin, G. Chandy, H.-P. Moore and T. Machen, manuscript in preparation). The near neutral pH of the TGN raises important questions about whether peptide hormone sorting can indeed occur at this site. DAMP does not accumulate in the Golgi apparatus of either fibroblasts or professional secretory cells, suggesting that the Golgi cisternae may be neutral.108,179,200,204,206 However, refined in vivo techniques using retrograde transport of labeled verotoxin have found the average Golgi pH to be mildly acidic, in the vicinity of pH 6.5.207 A liposome fusion method which targeted pH-sensitive dyes to the trans-Golgi was used to obtain a pH of 6.2-6.3,208,209 although these studies have been widely misquoted as TGN pH. It remains to be established whether the trans-Golgi of peptide hormone secreting cells is also acidic, and whether the level of acidity in the trans-Golgi is sufficiently low to promote aggregation of hormones. Measurements of cis- and medial-Golgi pH have yet to be reported. The in vivo pH of the ER has been determined via specific targeting of a pH-sensitive dye; in this case, an avidinKDEL fusion construct was used to generate a resident ER marker which could then target

Targeting and Activation of Peptide Hormones in the Secretory Pathway

15

a membrane-permeant biotin derivative to this site. J. Llopes, M. Wu, K. Teter, R.Y. Tsien, T. Machen and H.-P. Moore (manuscript in preparation) were thus able to record an ER pH of ~7.6 in living HeLa cells. Compartmental Ca2+ Levels According to the models of prohormone sorting and activation, lumenal pH should drop and free Ca2+ levels should rise as prohormones pass through the distal organelles of the secretory pathway. Thus far, this prediction is only supported in part by actual experimental evidence. The ER indeed maintains a neutral pH and contains low levels of free Ca2+. The free Ca2+ concentration of the ER has been determined in vivo with the use of ERtargeted fusion constructs, initially with the Ca2+-activated photoprotein aqueorin210,211 and more recently with Ca2+-sensing derivatives of green fluorescent protein “cameleons”.212 A free Ca2+ concentration of 0.3-400 µM has been recorded for this compartment. Thus far, estimates of Golgi Ca2+ have been limited to total rather than free Ca2+ levels. These measurements, obtained with fixed cells, have shown that the Golgi apparatus contains a significant store of total intracellular Ca2+.213-215 Surprisingly, the Ca2+ concentration within the TGN of PC12 cells is below the threshold of detection.215 Professional secretory cells do, however, maintain a major repository of organellar Ca2+ within the ISG and SG,213,215,216 and a free Ca2+ concentration of 24 µM has been estimated for the mature SG.216 Further application of the improved techniques for measuring free Ca2+ and pH in specific compartments of living cells is needed to clarify the pH and free Ca2+ concentrations of the compartment(s) in which prohormone sorting occurs.

Summary and Future Perspectives Prohormone sorting and modification is a continuous process which occurs throughout the secretory pathway. Proper folding and core glycosylation are executed in the ER, after which prohormones are likely to be sorted and concentrated in COPII transport vesicles. Posttranslational modifications continue as hormone precursors move through the Golgi stack. Endoproteolytic cleavage and homo- or heterotypic aggregation then begin at the TGN as (pro)hormones are sorted and concentrated in ISGs by a “sorting for entry” process. Sorting continues as the “sorting by retention” mechanism retains regulated secretory proteins in the ISG while CLS removes missorted constitutive proteins and lysosomal enzymes. Efficient prohormone processing is concomitantly facilitated by the progressive acidification of an ISG, and the granule membrane undergoes significant remodeling during maturation to attain a regulated exocytotic state. The molecular details of each of the aforementioned steps require further investigation. It will be important to determine if active sorting occurs during export of prohormones from the ER, and to identify such ER export signals and the cognate transport receptor. Identification of putative membrane attachment proteins has already generated a number of interesting questions: 1. Does the membrane association occur at the TGN or ISG? 2. How is the membrane anchor itself targeted to the SG? 3. How does a receptor like CPE perform both sorting and enzymatic functions? 4. Are there multiple membrane anchors? and 5. Does the membrane anchor associate with other components to form a raft? The process of CLS also deserves further attention. Is the main function of CLS to remove missorted proteins, or is this process a byproduct of granule membrane remodeling during the transformation of ISG to a functional SG? The exocytotic state of an ISG exhibits characteristics of both constitutive and regulated secretory vesicles, and it is possible that the multiple fusion and fission events which accompany granule maturation serve to modify

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

16

the composition of the granule fusion machinery. Budding of constitutive-like vesicles may help to remove protein components of the constitutive fusion machinery from the ISG. Finally, given the role of pH and Ca2+ in prohormone sorting and activation, an understanding of how an individual organelle establishes and maintains precise pH and Ca2+ levels will be a major challenge in the future.

Acknowledgment The authors thank Michael Haugwitz, Ben Eaton and other members of the Moore lab for critical reading of the manuscript. This work was supported by NIH grant GM (35239) and a grant from the Cystic Fibrosis Foundation to HPM. KT was supported by the MCB training grant.

Abbreviations BfA CgB CLS CPE DTT ER GFP ISG MPR MTOC PC POMC SG TGN VSV

Brefeldin A chromogranin B constitutive-like secretion carboxypeptidase E dithiothreitol endoplasmic reticulum green fluorescent protein) immature secretory granules mannose 6-phosphate receptor microtubule organization center prohormone convertase proopiomelanocortin secretory granules trans-Golgi network vesicular stomatitis virus

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152. Carroll RJ, Hammer RE, Chan SJ et al. A mutant human proinsulin is secreted from islets of Langerhans in increased amounts via an unregulated pathway. Proc Natl Acad Sci USA 1988; 85:8943-8947. 153. Kuliawat R, Arvan P. Protein targeting via the “constitutive-like” secretory pathway in isolated pancreatic islets: Passive sorting in the immature granule compartment. J Cell Biol 1992; 118(3):521-529. 154. De Lisle RC, Bansal R. Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pacreatic acinar cells. Eur J Cell Biol 1996; 71:62-71. 155. Arvan P, Kuliawat R, Prabakaran D et al. Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. J Biol Chem 1991; 266(22):14171-14174. 156. Neerman-Arbez M, Halban PA. Novel, non-crinophagic, degradation of connecting peptide in transformed pancreatic beta cells. J Biol Chem 1993; 268(22):16248-16252. 157. Sossin WS, Fisher JM, Scheller RH. Sorting within the regulated secretory pathway occurs in the trans-golgi network. J Cell Biol 1990; 110(1):1-12. 158. Kuliawat R, Arvan P. Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells. J Cell Biol 1994; 126(1):77-86. 159. Tooze J, Hollinshead M, Hensel G et al. Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells. Euro J Cell Bio 1991; 56:187-200. 160. Dittie AS, Hajibagheri N, Tooze SA. The AP-1 adaptor complex binds to immature secretory granules from PC12 cells, and is regulated by ADP-ribosylation factor. J Cell Biol 1996; 132:523-536. 161. Leduc R, Molloy SS, Thorne BA et al. Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage. J Biol Chem 1992; 267(20):14304-14308. 162. Rehemtulla A, Dorner AJ, Kaufman RJ. Regulation of PACE propeptide-processing activity: Requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. Proc Natl Acad Sci USA 1992; 89:8235-8239. 163. Vey M, Schaefer W, Berghofer S et al. Maturation of the trans-Golgi network protease furin: Compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J Cell Biol 1994; 127(6, part 2):1829-1842. 164. Creemers JWM, Vey M, Schafer W et al. Endoproetolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J Biol Chem 1995; 270(6):2695-2702. 165. Anderson ED, Vanslyke JK, Thulin CD et al. Activation of the furin endoprotease is a multiple-step process: Requirements for acidfication and internal propeptide cleavage. EMBO J 1997; 16(7):1508-1518. 166. Voorhees P, Deignan E, van Donselaar E et al. An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J 1995; 14(20):4961-75. 167. Schafer W, Stroh A, Berghofer S et al. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin. EMBO J 1995; 14(11):2424-35. 168. Chapman RE, Munro S. Retrieval of TGN proteins from the cell surface requires endosomal acidification. EMBO J 1994; 13(10):2305-12. 169. Wise RJ, Barr PJ, Wong PA et al. Expression of a human proprotein processing enzyme: Correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site. Proc Natl Acad Sci USA 1990; 87:9378-9382. 170. Vindrola O, Lindberg I. Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells. Molecular Endocrinology 1992; 6(7):1088-1094. 171. Zhou Y, Lindberg I. Purification and characterization of the prohormone convertase PC1 (PC3). J Bio Chem 1993; 268(8):5615-5623. 172. Benjannet S, Rondeau N, Paquet L et al. Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases

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PC1 and PC2: Glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2. Biochem J 1993; 294(3):735-743. 173. Zhou Y, Lindberg I. Enzymatic properties of carboxyl-terminally truncated prohormone convertase 1 (PC1/SPC3) and evidence for autocatalytic conversion. J Biol Chem 1994; 269(28):18408-18413. 174. Milgram SL, Mains RE. Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins. J Cell Sci 1994; 107:737-745. 175. Vindrola O. Rapid cleavage of the endogenous PC3 prosegment and slow conversion to 74 kDa and 66 kDa proteins in AtT-20 cells. Neuropeptides 1994; 27(2):109-120. 176. Shennan KIJ, Taylor NA, Jermany JL et al. Differences in pH optima and calcium requirements for maturation of the prohormone convertases PC2 and PC3 indicates different intracellular locations for these events. J Biol Chem 1995; 270(3):1402-1407. 177. Jean F, Basak A, Rondeau N et al. Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells. Biochem J 1993; 292(3):891-900. 178. Orci L, Ravazzola M, Amherdt M et al. Direct identification of prohormone conversion site in insulin-secreting cells. Cell 1985; 42:671-681. 179. Orci L, Ravazzola M, Storch M-J et al. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987; 49(6):865-868. 180. Schnabel E, Mains RE, Farquhar MG. Proteolytic processing of pro-ACTH endorphin begins in the golgi complex of pituitary corticotropes and AtT-20 cells. Molecular Endocrinology 1989; 3(8):1223-1235. 181. Song L, Fricker L. Processing of procarboxypeptidase E into carboxypeptidase E occurs in secretory vesicles. J Neurochem 1995; 65(1):444-53. 182. Schmidt WK, Moore HPH. Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells. Mol Biol Cell 1995; 6:1271-1285. 183. Paquet L, Zhou A, Chang EY et al. Peptide biosynthetic processing: distinguishing prohormone convertases PC1 and PC2. Mol Cell Endocrinol 1996; 120(2):161-8. 184. Zhou A, Mains RE. Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2. J Biol Chem 1994; 269(26):17440-17447. 185. Zhou Y, Rovere C, Kitabgi P et al. Mutational analysis of PC1 (SPC3) in PC12 cells. 66 kDa PC1 is fully functional. J Biol Chem 1995; 270(42):24702-24706. 186. Zhou A, Paquet L, Mains RE. Structural elements that direct specific processing of different subtilisin-like prohormone convertases. J Biol Chem 1995; 270(37):21509-21516. 187. Jutras I, Seidah NG, Reudelhuber TL et al. Two activation states of the prohormone convertase PC1 in the secretory pathway. J Biol Chem 1997; 272(24):15184-15188. 188. Urbe S, Dittie AS, Tooze SA. pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules. Biochem J 1997; 321:65-74. 189. Martens GJ, Braks JA, Eib DW et al. The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2. Proc Natl Acad Sci USA 1994; 91(13):5784-5787. 190. Braks JA, Martens GJ. 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell 1994; 78(2):263-273. 191. Zhu X, Lindberg I. 7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity. J Cell Biol 1995; 129(6):1641-1650. 192. Van Horssen AM, Van den Hurk WH, Bailyes EM et al. Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2. J Biol Chem 1995; 270(24):14292-14296. 193. Davidson HW, Rhodes CJ, Hutton JC. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic beta cell via two distinct site-specific endopeptidases. Nature 1988; 333:93-96. 194. Shennan KIJ, Smeekens SP, Steiner DF et al. Characterization of PC2, a mammalian Kex2 homolog, following expression of the cDNA in microinjected Xenopus oocytes. FEBS Lett 1991; 284:277-280.

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195. Matthews G, Shennan KIJ, Seal AJ et al. Autocatalytic maturation of the prohormone convertase PC2. J Biol Chem 1994; 269(1):588-592. 196. Lamango NS, Zhu X, Lindberg I. Purification and enzymatic characterization of recombinant prohormone convertase 2: Stabilization of activity by 21 kD 7B2. Arch Biochem Biophys 1996; 330(2):238-250. 197. Seidah NG, Gaspar L, Mion P et al. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: Tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA Cell Biol 1990; 9(6):415-424. 198. Smeekens SP, Steiner DF. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast diabasic processing protease Kex2. J Biol Chem 1990; 265(6):2997-3000. 199. Tanaka S, Kurabuchi S, Mochida H et al. Immunocytochemical localization of prohormone convertases PC1/PC3 and PC2 in rat pancreatic islets. Arch Histol Cytol 1996; 59(3):261-271. 200. Orci L, Ravazzola M, Amherdt M et al. Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles. J Cell Biol 1986; 103(6 Pt 1):2273-2281. 201. Orci L, Halban P, Perrelet A et al. pH independent and -dependent cleavage of proinsulin in the same secretory vesicle. J Cell Biol 1994; 126:1149-1156. 202. Hutton JC. The internal pH and membrane potential of the insulin-secretory granule. Biochem J 1982; 204(1):171-8. 203. Rhodes CJ, Lucas CA, Mutkoski RL et al. Stimulation by ATP of proinsulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump. J Biol Chem 1987; 262(22):10712-10717. 204. Anderson RGW, Pathak RK. Vesicles and cisternae in the trans-Golgi apparatus of human fibroblasts are acidic compartments. Cell 1985; 40(3):635-643. 205. Barasch J, Kiss B, Prince A et al. Defective acidification of intracellular organelles in cystic fibrosis. Nature 1991; 352(6330):70-73. 206. Anderson RG, Orci L. A view of acidic intracellular compartments. J Cell Biol 1988; 106(3):539-543. 207. Kim JH, Lingwood CA, Williams DB et al. Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor. J Cell Biol 1996; 134(6):1387-99. 208. Seksek O, Biwersi J, Verkman AS. Direct measurement of trans-Golgi pH in living cells and regulation by second messengers. J Biol Chem 1995; 270(10):4967-70. 209. Seksek O, Biwersi J, Verkman AS. Evidence against defective trans-Golgi acidification in cystic fibrosis. J Biol Chem 1996; 271(26):15542-15548. 210. Kendall JM, Badminton MN, Dormer RL et al. Changes in free calcium in the endoplasmic reticulum of living cells detected using targeted aequorin. Analyt Biochem 1994; 221:173-181. 211. Button D, Eidsath A. Aequorin targeted to the endoplasmic reticulum reveals heterogeneity in luminal Ca2+ concentration and reports agonist- or IP3-induced release of Ca2+. Mol Biol Cell 1996; 7(3):419-34. 212. Miyawaki A, Llopis J, Heim R et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin [see comments]. Nature 1997; 388(6645):882-7. 213. Roos N. A possible site of calcium regulation in rat exocrine pancreas cells: An X-ray microanalytical study. Scan Micr 1988; 2(1):323-329. 214. Chandra S, Kable EPW, Morrison GH et al. Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy. J Cell Sci 1991; 100:747-752. 215. Pezzati R, Bossi M, Podini P et al. High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Mol Biol Cell 1997; 8(8):1501-1512. 216. Bulenda D, Gratzl M. Matrix free Ca2+ in isolated chromaffin vesicles. Biochemistry 1985; 24:7760-7765. 217. Verkman AS, van Hoek AN, Ma T et al. Water transport across mammalian cell membranes. Am J Physiol 1996; 270(1 Pt. 1):C12-30.

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218. Slot JW, Geuze HJ, Gigengack S et al. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 1991; 113(1):123-135. 219. Smith RM, Charron MJ, Shah N et al. Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxy-terminal epitope of intracellular GLUT4. Proc Natl Acad Sci 1991; 88:6893-6897. 220. Forte JG, Hanzel DK, Urushidani T et al. Pumps and pathways for gastric HCl secretion. Ann NY Acad Sci 1989; 574:145-158. 221. Steinhardt RA, Guoqiang B, Alderton JM. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science 1994; 263:390-393. 222. Bi GQ, Alderton JM, Steinhardt RA. Calcium-regulated exocytosis is required for cell membrane resealing. J Cell Biol 1995; 131(6 Pt 2):1747-58. 223. Miyake K, McNeil PL. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J Cell Biol 1995; 131(6 Pt 2):1737-45. 224. Coorssen JR, Schmitt H, Almers W. Ca2+ triggers massive exocytosis in Chinese hamster ovary cells. Embo J 1996; 15(15):3787-91. 225. Rodriguez A, Webster P, Ortego J et al. Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J Cell Biol 1997; 137(1):93-104. 226. Herman GA, Bonzelius F, Cieutat AM et al. A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4. Proc Natl Acad Sci USA 1994; 91(26):12750-4. 227. Girod R, Popov S, Alder J et al. Spontaneous quantal transmitter secretion from myocytes and fibroblasts: Comparison with neuronal secretion. J Neurosci 1995; 15(4):2826-38. 228. Morimoto T, Popov S, Buckley KM et al. Calcium-dependent transmitter secretion from fibroblasts: Modulation by synaptotagmin I. Neuron 1995; 15(3):689-96. 229. Chavez RA, Miller SG, Moore HPH. A biosynthetic regulated pathway in constitutive secretory cells. J Cell Biol 1996; 133:1177-1191. 230. Tooze J, Hollinshead M, Frank R et al. An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage. J Cell Biol 1987; 105(1):155-162. 231. Steiner DF, Michael J, Houghten R et al. Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: Demonstration of cleavage within clathrin-coated (pro)secretory vesicles. Proc Natl Acad Sci USA 1987; 84:6184-6188. 232. Tanaka S, Nomizu M, Kurosumi K. Intracellular sites of proteolytic processing of proopiomelanocortin in melanotrophs and corticotrophs in rat pituitary. J Histochem Cytochem 1991; 39(6):809-821. 233. Tanaka S, Kurosumi K. A certain step of proteolytic processing of proopiomelanocortin occurs during the transition between two distinct stages of secretory granule maturation in rat anterior pituitary corticotrophs. Endocrinol 1992; 131(2):779-786. 234. Xu H, Shields D. Prohormone processing in the trans-Golgi network: Endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells. J Biol Chem 1993; 122(6):1169-1184. 235. Tang BL, Low SH, Hong W. Endoplasmic reticulum retention mediated by the transmembrane domain of type II membrane proteins Sec12p and glucosidase I. Eur J Cell Biol 1997; 73(2):98-104. 236. Schutze M, Peterson PA, Jackson MR. An N-terminal double-arginine motif maintains type II membrane proteins in the endoplasmic reticulum. EMBO J 1994; 13(7):1696-1705. 237. Bretscher MS, Munro S. Cholesterol and the Golgi apparatus. Science 1993; 261(5126): 1280-1281. 238. Ponnambalam S, Rabouille C, Luzio JP et al. The TGN38 glycoprotein contains two nonoverlapping signals that mediate localization to the trans-Golgi network. J Cell Biol 1994; 125(2):253-68.

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239. Jones BG, Thomas L, Molloy SS et al. Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. EMBO J 1995; 14(23):5869-5883. 240. Luzio JP, Banting G. Eukaryotic membrane traffic: Retrieval and retention mechanisms to achieve organelle residence. Trends Biochem Sci 1993;18(10):395-8. 241. Kornfeld S, Mellman I. The biogenesis of lysosomes. Ann Rev of Cell Biol 1989; 5:483-525. 242. Mostov KE, Cardone MH. Regulation of protein traffic in polarized epithelial cells. BioEssays 1995; 17(2):129-138. 243. Matter K, Mellman I. Mechanisms of cell polarity: Sorting and transport in epithelial cells. Curr Op Cell Biol 1994; 6:545-554. 244. Fiedler K, Simons K. The role of N-glycans in the secretory pathway. Cell 1995; 81(3):309-12. 245. Miller SG, Carnell L, Moore HPH. Post-Golgi membrane traffic: Brefeldin A inhibits export from the distal Golgi compartment to the cell surface but not recycling. J Cell Biol 1992; 118(2):267-283. 246. Sariola M, Saraste J, Kuismanen E. Communication of post-Golgi elements with early endocytic pathway: regulation of endoproteolytic cleavage of Semliki forest virus p62 precursor. J Cell Sci 1995; 108(Pt 6)):2465-75. 247. Teter K, Hacker J, Moore HPH. Unpublished observations. 248. Huang XF, Arvan P. Formation of the insulin-containing secretory granule core occurs within immature beta-granules. J Biol Chem 1994; 269(33):20838-44. 249. Tang BL, Wong SH, Qi X et al. Golgi-localized beta-galactoside alpha 2,6-sialyltransferase in transfected CHO cells is redistributed into the endoplasmic reticulum by brefeldin A. Eur J Cell Biol 1992; 59(1):228-31. 250. Berger EG, Burger P, Hille A et al. Comparative localization of mannose-6-phosphate receptor with alpha-2,6-sialyltransferase in HepG2 cells: An analysis by confocal double immunofluorescence microscopy. Eur J Cell Biol 1995; 67(2):106-11. 251. Strous GJ, van Kerkhof P, van Meer G et al. Differential effects of brefeldin A on transport of secretory and lysosomal proteins. J Biol Chem 1993; 268(4):2341-7. 252. Nakamura N, Rabouille C, Watson R et al. Characterization of a cis-Golgi matrix protein, GM130. J Cell Biol 1995; 131(6 Pt 2):1715-26. 253. Rosa P, Barr FA, Stinchcombe JC et al. Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules for the trans-Golgi network. Euro J Cell Biol 1992; 59(2):265-274. 254. Ladinsky MS, Howell KE. The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A. Eur J Cell Biol 1992; 59(1):92-105. 255. Reaves B, Banting G. Perturbation of the morphology of the trans-Golgi network following Brefeldin A treatment: Redistribution of a TGN-specific integral membrane protein, TGN38. J Cell Biol 1992; 116(1):85-94. 256. Lin S, Moore HPH. Unpublished observations. 257. Wood SA, Park JE, Brown WJ. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 1991; 67(3):591-600. 258. Klausner RD, Donaldson JG, Lippincott-Schwartz J. Brefeldin A: Insights into the control of membrane traffic and organelle structure. J Cell Biol 1992; 116(5):1071-80.

CHAPTER 2

The Mechanism of Sorting Proopiomelanocortin to Secretory Granules and Its Processing by Aspartic and PC Enzymes Niamh X. Cawley, David R. Cool, Emmanuel Normant, Fu-Sheng Shen, Vicki Olsen and Y. Peng Loh

General Introduction

P

roopiomelanocortin (POMC, Fig. 2.1) is a 31 kDa precursor of a number of hormones/ neuropeptides, which include adrenocorticotropin (ACTH), β-endorphin (β-END), α-melanocyte stimulating hormone (α-MSH), β-lipotropin (β-LPH) and N-POMC1-48.1-3 These POMC-derived peptide products have different functions such as opiate-like activity (β-END), mitogenic activity (N-POMC1-48) and steroidogenic activity (ACTH). The precursor is expressed abundantly in the melanotrophs of the intermediate lobe of the pituitary, the site of α-MSH and β-END synthesis and in the corticotrophs of the anterior lobe of the pituitary where the main products generated are ACTH and β-LPH.3,4 In addition, POMC is expressed in the arcuate nucleus of the hypothalamus where POMC products are similar to that of the melanotrophs.5,6 POMC gene expression is under the negative control of dopamine in the intermediate lobe, and under the positive control of corticotropin releasing hormone (CRH) and negative control of glucocorticoids in the anterior lobe.7 Similar to other precursors, POMC is processed at paired basic residues by various endoproteases, e.g., prohormone convertases, PC1, PC2 and proopiomelanocortin converting enzyme (PCE), to yield the active peptides (Fig. 2.1). These cleaved peptides are then subsequently modified by the removal of basic residues at the N and/or C-terminus by amino-8 and carboxypeptidases (carboxypeptidase H/E)9,10 respectively, followed by C-terminal amidation, and/or N-terminal acetylation for α-MSH and β-END peptides. The biosynthesis and processing of POMC involves routing of the prohormone from the rough endoplasmic reticulum (RER), the site of synthesis, to the trans-Golgi network (TGN), similar to that of other secretory proteins (Fig. 2.2). At the TGN, POMC is segregated from other proteins, such as lysosomal enzymes, plasma membrane proteins and constitutively secreted proteins, which have traversed a common pathway thus far.11,12 POMC, like other prohormones, is specifically sorted at the TGN and packaged into immature Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

Fig. 2.1. Schematic diagram of bovine pro-opiomelanocortin (POMC). This figure illustrates the POMC domains that are delineated by dibasic cleavage sites. These sites are cleaved in a tissue specific manner to produce a variety of peptide hormones depending on the complement of processing enzymes present. ACTH and β-LPH are primarily found in the anterior pituitary while the smaller POMC derived peptides, N-POMC1-48, γ3-MSH, α-MSH and β-END, are primarily found in the intermediate lobe of the pituitary and brain. ACTH, adrenocorticotropin hormone; LPH, lipotropin hormone; MSH, melanocyte stimulating hormone; END, endorphin. and represent N- and O-linked glycosylation sites, respectively.

30 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Sorting Proopiomelanocortin to Secretory Granules and Its Processing

31

Fig. 2.2. Schematic diagram illustrating the presence of the regulated and constitutive secretory pathways in endocrine cells. Immature secretory granules (ISG) of the regulated secretory pathway bud from the TGN. Prohormones (open stars) in the ISGs are processed and the mature secretory granule (MSG) appears to have a dense core. MSGs are stored in this region, awaiting an extracellular stimulus (secretagogue) that will cause them to fuse with the plasma membrane and release their contents of peptide hormone (closed stars) to the blood. In the constitutive secretory pathway, smaller vesicles containing proteins to be secreted, such as serum albumin or immunoglobulins (open arrowheads), receptors, ion channels, membrane transporters and other plasma membrane proteins, are rapidly segregated away from the regulated secretory proteins and shunted to the plasma membrane, where they fuse without the need for a secretagogue and release their contents to the blood. Reprinted with permission from Loh YP et al, Trends in Endocrinology and Metabolism 1997; 8:130.

secretory granules of the regulated secretory pathway and processed within these organelles.13 The processed POMC derived peptides in the granules form a dense core, from which, upon stimulation, they are released by exocytosis in a calcium-dependent manner into the circulation or extracellular space.

Mechanism of Sorting POMC to the Regulated Secretory Pathway Introduction The mechanism by which prohormones, proneuropeptides and other secretory proteins are sorted at the trans-Golgi network into the regulated secretory pathway has been debated for a long time. Two hypotheses have been raised since the 1980s.14,15 One is the sorting signal ligand-receptor active sorting hypothesis in which a sorting signal in the prohormone binds to the receptor located in the lumen of the TGN, followed by budding of the TGN and formation of an immature granule encapsulating the proteins destined for the regulated secretory pathway. The other is an aggregation passive sorting hypothesis in which the proteins, by forming a condensing aggregate, are trapped and packaged into the granules.

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Attempts have been made to search for consensus sorting signals in the primary sequence of regulated secretory pathway proteins but none were found that were proven experimentally. 16,17 On the other hand, evidence accumulated that various hormones, 18 and chromogranins19 aggregate at the TGN under conditions of acidic pH and high Ca2+. Hence, the aggregation hypothesis became more plausible. However, in 1993, with the identification of a sorting signal domain in chromogranin A20 and chromogranin B,21 the ACTH/ endorphin prohormone, proopiomelanocortin (POMC),22 and proenkephalin,23 the tide began to turn. Recent studies have provided strong evidence in support of a sorting signalreceptor mediated mechanism for targeting POMC to the regulated secretory pathway. However, passive sorting of other hormones and regulated secretory pathway proteins cannot be discounted at this point.

Identification of the POMC Sorting Signal Deletion and mutagenesis studies have identified a POMC sorting signal for the regulated secretory pathway within the N-terminus of the prohormone.24 This signal consists of a loop between residues Cys8-Cys20 (Fig. 2.3). Proof that this sorting signal is sufficient and necessary for sorting POMC to the regulated pathway came from two lines of evidence: 1. transfection experiments in Neuro2a cells, an endocrine cell line, showing that deletion of N-POMC1-26, which contains the sorting signal, resulted in the constitutive secretion of POMC;25 and 2. fusion of N-POMC1-26 to a reporter protein, chloramphenicol acetyltransferase (CAT), caused the transport of this bacterial protein to the regulated secretory granules.22 Molecular modeling studies have identified the POMC sorting signal motif as an amphipathic loop structure comprised of residues Cys8-Cys20 (Fig. 2.3). The disulfide bridge between Cys8-Cys20 is essential to stabilize the loop.24 A key feature in this motif is the exposure of two aliphatic hydrophobic residues (Leu11 and Leu18) and two acidic residues (Asp10 and Glu14), with specific molecular distances apart, at the surface of the amphipathic loop (Fig. 2.3). Similar sorting signal motifs have also been identified in proenkephalin and proinsulin in modeling studies but not in chromogranin A, another regulated secretory pathway protein (Snell and Loh, unpublished data). Future studies will determine the commonality of the motif found in POMC for prohormone sorting to the regulated secretory pathway.

Identification of the POMC Sorting Receptor Identification of the regulated secretory pathway sorting signal in POMC led to the search for a sorting receptor. Using N-POMC1-26, which contains the POMC sorting signal (N-POMC8-20), as the ligand in binding studies, a sorting receptor has recently been demonstrated in bovine pituitary secretory granule and Golgi-enriched membranes.26 In crosslinking studies followed by purification, amino acid sequencing and Western blot, the receptor has been identified as the membrane form of Carboxypeptidase E (CPE, also known as Carboxypeptidase H). The optimum pH of binding the POMC sorting signal to CPE was between 5.5-6.5, consistent with the pH of the trans-Golgi network where sorting occurs.27 Equilibrium binding studies and Scatchard analysis revealed a Kd = 6 µM, IC50 = 65 µM and a Bmax = 580pmoles/mg protein,26 characteristic of low affinity first-order kinetics of binding, similar to that of enzyme-substrate interactions. Proinsulin and proenkephalin also bound CPE-containing vesicle membranes which were displaced by N-POMC1-26.26,28 This indicated that CPE is a sorting receptor for other prohormones and proneuropeptides as well. The N-POMC1-26 binding was highly specific for the lumenal side of the secretory granule membranes, since the POMC sorting signal did not bind to plasma membranes and bound minimally to Golgi membranes from L cells, a nonendocrine fibroblast cell line.

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Fig. 2.3. Schematic diagram illustrating the sorting signal of human proopiomelanocortin. The three-dimensional computer model of the N-terminal region shows the predicted conformation of the sorting signal (POMC8-20) stabilized by a disulfide bridge. The conserved charged or hydrophobic amino acids exposed to the surface are shown (Asp10, Leu11, Glu14, Leu18). Paired- and tetra-basic cleavage sites are shown with the single amino acid abbreviations K=Lys, R=Arg. ± indicates that these sites are not always glycosylated. Reprinted with permission from Loh YP et al, Trends in Endocrinology and Metabolism 1997; 8:130.

Moreover, only full length POMC, but not mutant POMC with the sorting signal or other domains of the POMC molecule deleted, bound to CPE in the secretory granule membranes.26 Also, constitutively secreted proteins such as immunoglobulins and proalbumin did not bind to CPE. A role for CPE as a sorting receptor was further supported by the finding that membrane CPE was tightly associated with secretory granule membranes and was resistant to depletion by high salt and detergent.26 CPE in pituitary secretory granules is primarily membrane-associated and exhibits poor enzymatic activity.29 Interaction of the POMC sorting signal with CPE appears to be at a different site than the active site, since the specific inhibitor for CPE, guanidino-ethylmercaptosuccinic acid (GEMSA) did not inhibit binding (Cool, D.R. unpublished).

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Evidence that CPE is a sorting receptor in vivo came from studies in the neuroendocrine cell line, Neuro2a, which was downregulated in the expression of CPE by CPE antisense RNA.26 POMC transfected into these cells was secreted unprocessed, through the constitutive pathway. Also, immunocytochemistry showed no POMC-containing secretory granules in these cells. The role of CPE as a regulated secretory pathway sorting receptor in vivo was further demonstrated in a mouse model, Cpefat/Cpefat, which has a natural mutation in the Cpe gene.30 CPE in these mice has a Ser202 → Pro202 substitution in the enzyme, rendering it unstable, resulting in rapid degradation after synthesis.31 Secretion studies showed that intact POMC was the major component released from primary cultures of intermediate and anterior pituitary cells of Cpefat mice.26,32 Moreover, it was secreted at a high basal level, constitutively, in an unregulated manner. There was very little ACTH1-39 released from anterior pituitary cells in the Cpefat mice, and most of the ACTH were C-terminal basic residue-extended forms.32 These data are consistent with the misrouting of POMC to the constitutive pathway in the pituitary of Cpefat mice. CPE performs a dual role, as a membrane bound sorting receptor for the regulated secretory pathway and as an exopeptidase to remove basic residues from the C-terminal of peptide hormones subsequent to cleavage from the prohormone9,10(see also chapter 7). Recently, CPE has been implicated as a regulated secretory pathway sorting receptor for a number of other prohormones as well, including proinsulin, proenkephalin and growth hormone.26,28,32

Summary Current studies have provided strong evidence in support of a receptor-mediated mechanism for the sorting of POMC to the regulated secretory pathway, although aggregation also plays a role in enhancing the efficiency of sorting. Thus, we propose a regulated secretory pathway sorting mechanism for POMC which involves aggregation of POMC18 as a concentration step, followed by binding of the aggregate via the sorting signal to the receptor, CPE, at the TGN. Subsequently, the granule buds off from the TGN to form an immature granule in which processing takes place (Fig. 2.4).

Endoproteolytic Processing of Proopiomelanocortin Introduction During transport through the Golgi to mature secretory granules, posttranslational modifications of the secretory proteins occur, including glycosylation, phosphorylation, sulfation and endoproteolytic processing. POMC is processed by specific endoproteases that recognize paired-basic cleavage sites (e.g., Lys-Arg), and it is likely that the variety of bioactive peptides generated in the observed tissue-specific manner depends on the complement of enzymes present. POMC therefore represents a complicated prohormone to study in light of its mechanism of tissue-specific proteolytic processing and multiple levels of regulation. The search for the physiologically relevant enzymes capable of processing POMC and other prohormones in vivo has been a long and ongoing endeavor. It is the goal of this section to give a historical perspective and to highlight the significant experiments up to the current time that have advanced our understanding of the endoproteolytic processing of POMC. For this purpose the section has been divided into two parts covering the processing of POMC by two classes of enzymes that have been most extensively studied: 1. the prohormone converting aspartic proteases; and 2. the prohormone convertases.

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Fig. 2.4. Working model for POMC sorting by binding to membrane associated carboxypeptidase E. In this model, POMC aggregates in the TGN and binds via its sorting signal to CPE (1). The TGN buds, carrying the CPE/POMC complex (2). The immature secretory granule becomes acidified; processing enzymes begin to cleave POMC and CPE (3). The more enzymatically active soluble CPE begins to process the C-terminal basic residues from the peptide cleavage products of POMC (4).

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Processing of POMC by Prohormone Converting Aspartic Proteases Proopiomelanocortin converting enzyme (PCE) The definition of a prohormone processing enzyme is one that can specifically cleave the prohormone substrate to the correct products under conditions similar to those that are found in vivo and is localized to the same cellular compartment where processing occurs. While knowledge of the cleavage sites33,34 predicted the processing enzymes to be trypsinlike, it was surprising to find that the first activity which fulfilled these criteria was characterized as an aspartic protease from bovine and rat pituitary secretory vesicles.35-37 The enzyme was subsequently purified from bovine pituitary intermediate lobe secretory vesicles, the site of POMC processing, and was capable of cleaving the Lys-Arg pairs of POMC in vitro under acidic conditions, i.e., at a pH consistent with the intragranular pH of dense core secretory vesicles38,39 to yield 21-23 kDa ACTH, β-LPH, 13 kDa and 4.5 kDa ACTH, β-END, and 16 kDa NH2-terminal glycopeptide (see Fig. 2.5).40 The enzyme was characterized as a 70,000 dalton glycoprotein with an acidic pH optimum. Its enzymatic activity was specifically inhibited by pepstatin A and diazoacetyl-norleucine, both aspartic protease inhibitors, but not by other class-specific inhibitors such as phenylmethanesulfonyl fluoride, p-chloromercuribenzoate and ethylenediamine tetraacetic acid.40 Its ability to cleave POMC specifically under conditions similar to those in vivo rendered this enzyme a candidate prohormone processing enzyme and was named proopiomelanocortin converting enzyme, PCE (EC 3.4.23.17). A similar enzyme was also purified from bovine pituitary neural lobe secretory vesicles. In addition to its ability to cleave mouse POMC in a similar manner to that of the intermediate lobe PCE, this enzyme was shown to cleave proinsulin and provasopressin to generate insulin and vasopressin-Lys-Arg, respectively.41,42 PCE exhibited coordinate secretion from the intermediate lobe with α-MSH and an aminopeptidase B-like enzyme.43 The role that PCE plays in POMC processing in vivo was assessed by analyzing the processing of POMC in mouse neuro-intermediate lobe cells in the presence of pepstatin A.44 POMC processing decreased by 36.4% in the pepstatin A treated cells, indicating a role for PCE in POMC processing in vivo. PCE was able to cleave not only the intact POMC molecule but also its fragments, specifically human β-LPH and bovine N-POMC1-77. Human β-LPH was cleaved first at the Lys-Lys junction within γ-LPH generating an intermediate of β-MSH/β-END1-31. This intermediate was further cleaved primarily between the Lys-Arg cleavage site to release βMSH and Arg-β-END1-31 (Fig. 2.5).45 Bovine N-POMC1-77 was also cleaved by purified soluble PCE and a membrane bound form of PCE from bovine intermediate lobe secretory vesicle membranes to generate N-POMC1-49 and Lyso-γ3-MSH.46 This cleavage, however, depended on the glycosylation state of Thr45 of POMC, which is an ‘O’-linked glycosylation site. Processing at this site by PCE occurred only when Thr45 was not glycosylated, or only partially glycosylated, and the sugar chains lacked the terminating sialic acid residues.47 It is thought that the sialic acid forms a salt bridge with the Arg49 of POMC, providing steric hindrance and thereby preventing cleavage by PCE. PCE did not cleave the Lys28-Lys29 of β-END1-31 or the tetrabasic residues Lys15-Lys16-Arg17-Arg18 of ACTH1-39 to generate des-acetyl-α-MSH.40,45 Chromaffin granule aspartic protease (CGAP) In addition to other mammalian aspartic proteases that have been characterized by their ability to cleave prohormone substrates,48,49 the recently characterized 70 kDa chromaffin granule aspartic protease (70 kDa CGAP) from bovine adrenal medulla50,51 was also shown to process POMC in vitro at similar sites to PCE (Fig. 2.5). The physical properties,

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Fig. 2.5. Schematic diagram of bovine proopiomelanocortin illustrating its potential peptide products. The basic residue cleavage sites are indicated by shaded and hatched bars representing lysine and arginine residues, respectively. The sites of POMC that can be cleaved by the aspartic and PC enzymes are summarized and the corresponding references where such processing was identified are listed. The asterisk indicates that the products were generated by endogenous convertases and have been included in the table within the context of the enzymes that were being studied.

inhibitor profile and specificity are very similar to PCE, rendering the designation of the 70 kDa CGAP as the adrenal form of pituitary PCE. Interestingly, this 70 kDa CGAP was also shown to cleave proenkephalin at three sites, one of which was identified by peptide microsequencing as being on the carboyxl side of Lys172-Arg173,50 making this enzyme a candidate processing enzyme of proenkephalin in vivo, along with the prohormone thiol protease (PTP) and the prohormone convertases PC1 and PC2 also present in these granules.52,53 Analysis of the relative cleaving activity of these enzymes for proenkephalin, proneuropeptide Y and POMC was carried out. It was found that PTP preferred proenkephalin while 70 kDa CGAP and the PCs preferred POMC; however, 70 kDa CGAP appeared to be more efficient than the PCs at cleaving POMC.54

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Yeast aspartic protease 3 (Yapsin 1) The first enzyme to be characterized biochemically and genetically as a prohormone processing enzyme was yeast Kex2, a subtilisin-like serine protease responsible for the processing of α-mating factor precursor at Lys-Arg cleavage sites in yeast.55-57 Since its discovery, a vast amount of information has been discovered about this class of enzymes which include the mammalian prohormone convertases (PCs), (see Seidah et al, this volume). However, it is of special interest in this section to note that not long after the discovery and characterization of Kex2, an aspartic protease was cloned in Kex2-deficient yeast.58 The aspartic protease was cloned on the basis of its ability to suppress the Kex2 deficient phenotype observed in these cells and was initially named yeast aspartic protease 3 (Yap3). Yap3 was also cloned independently a few years later based on its ability to cleave the heterologously expressed anglerfish prosomatostatin II at the monobasic cleavage site to generate somatostatin-28,59 a result that was supported by in vitro experiments.60 At that time Yap3p was proposed to be a homologue of the anglerfish somatostatin-28 generating enzyme which was also characterized as an aspartic protease.61 Since the initial characterization of PCE, efforts to clone the enzyme have been unsuccessful and, as a result, this class of prohormone converting aspartic proteases had remained somewhat obscure. However, with the cloning of yeast aspartic protease 3, now named yapsin 1,62 a new era in the characterization of prohormone processing aspartic proteases began. Yapsin 1 was overexpressed, purified and characterized with respect to its physical and chemical properties.63-65 It was initially characterized as a 68,000 dalton glycoprotein with an acidic pH optimum and demonstrated many similarities to bovine PCE. In fact, the antiyapsin 1 serum MW283 crossreacted with PCE on Western blot.66,67 The purified enzyme was able to cleave mouse POMC in vitro to generate ACTH and β-LPH63 (Fig. 2.5). It also cleaved human β-LPH to generate β-MSH and β-END1-31, and bovine N-POMC1-77 to generate Lyso-γ3-MSH and γ3-MSH. Differently from the action of PCE, yapsin 1 cleaved the Lys-Lys site of β-END1-31 to generate β-END1-28 and β-END1-29 and cleaved ACTH1-39 very efficiently to generate ACTH1-15, which is currently used as the standard assay of yapsin 1 activity.65 Recently, yapsin 1 was cotransfected with POMC into the rat pheochromocytoma cell line, PC12 cells, and was found to be correctly activated and targeted to the secretory pathway in these cells to generate ACTH1-39 and ACTH1-14, providing evidence that a yeast aspartic protease could be expressed and functionally active in the secretory pathway of mammalian cells.68 PCE: A mammalian homologue of Yapsin 1 PCE shares many properties with yapsin 166,69 which include size, specificity, inhibitor profile and localization to the secretory pathway. Since an antibody to yapsin 1 crossreacted with purified PCE,67 it is likely that PCE represents a mammalian homologue of yapsin 1. Based on these data, PCE has been renamed yapsin A to represent the first mammalian member of this novel subclass of prohormone converting aspartic proteases. Two yeast members currently include yapsin 1 and yapsin 2, previously called Yap358 and MKC7,70 respectively. To further explore the existence of mammalian homologues of yapsin 1, the antiserum MW283 was used for immunocytochemistry (ICC) in combination with in situ hybridization of peptide hormones.67 The results of this work showed a distinct distribution of yapsin 1-like immunoreactive cells in mammalian brain and pituitary. In the brain the immunostaining was distributed in neuropeptide-rich regions and was specifically colocalized with cholecystokinin mRNA in rat hippocampus and cortex and (Arg)vasopressin mRNA in the supraoptic nucleus of the hypothalamus. Yapsin 1-like immunoreactivity was also colocalized with POMC mRNA in the melanotrophs of the intermediate lobe by ICC in

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combination with in situ hybridization, and with POMC immunoreactivity in the corticotrophs of the anterior lobe by double ICC. These results support the hypothesis that mammalian prohormone converting aspartic proteases (mammalian yapsins) are likely to play a role in the processing of POMC, and potentially other prohormones or proneuropeptides, in vivo. The extent to which these mammalian yapsins function in this capacity has yet to be determined once their molecular structures have been elucidated.

Processing of POMC by Prohormone Convertases The explosion of information within the ten years following the cloning and characterization of Kex255-57 has slowly settled and has since been thoroughly reviewed.71-74 Of particular interest were the initial experiments of Thomas et al,75 who successfully cotransfected Kex2 and mouse POMC into a number of mammalian cell lines and documented the generation of β-LPH, γ-LPH and β-END1-31 (Fig. 2.5). These experiments were the first of their kind and further stimulated interest in the search and eventual cloning of the mammalian prohormone convertases. Mammalian prohormone convertases (PCs) were cloned using a PCR strategy with primers based on the predicted conserved homology of the active site of Kex2 and furin, a human gene product with homology to Kex2 and previously thought to be an oncogene.76 Since then, a family of PCs has emerged that have been shown to play important roles in the processing of proproteins (see Seidah et al, this volume). Two members of this family, PC177-79 (also called PC380) and PC277,81 were found to be expressed primarily in endocrine and neuroendocrine tissues,71,77,78,82-84 implicating a specialized function for these enzymes in prohormone and proneuropeptide processing. In particular, the expression of PC1 as the predominant PC in anterior pituitary, and the presence of PC2 and PC1 in the intermediate lobe of the pituitary82,85 implicated even further a role for PC1 and PC2 in the differential processing of POMC. Indeed, experiments demonstrating coordinate regulation of PC1 and PC2 mRNA with that of POMC mRNA by the dopamine agonist and antagonist, bromocryptine and haloperidol, in the rat pituitary intermediate lobe suggests a significant relationship between POMC and PC1 and PC2.85,86 Transfection experiments BSC-40 cells The ability of PC1 and PC2 to cleave POMC correctly was investigated primarily by cotransfection studies of their cDNAs using vaccinia virus expression vectors into cell lines from endocrine and nonendocrine origins. The POMC gene was expressed either alone or in conjunction with PC1 and/or PC2 cDNA in the African green monkey kidney epithelial cell line BSC-40, which does not contain a regulated secretory pathway. The transfected POMC alone was secreted in a constitutive manner in an unprocessed form,87-89 indicating the absence of endogenous convertase activity in this nonendocrine cell line. When cotransfected with PC1, POMC was processed primarily to 13 kDa ACTH and β-LPH88-90 and to a lesser extent γ-LPH and β-END1-3188,89 (Fig. 2.5). When PC2 was cotransfected with POMC in these cells, β-END1-31, but very little or no ACTH, was identified. However, an intermediate ACTH immunoreactive peak was identified by one group as JP-ACTH88 and as ACTH linked to γ-LPH by another group.89 Expression of PC2 together with PC1 resulted in the efficient conversion of β-LPH to γ-LPH and β-END1-31.89 Rin m5F cells In higher order mammalian species, there are two Lys-Lys cleavage sites present in the POMC molecule giving rise to β-MSH in one case and resulting in the formation of β-END1-26

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Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

and β-END1-27 in the other case. These latter two products are found in the intermediate pituitary and arcuate nucleus of the hypothalamus and act as potential antagonists91,92 of β-END1-31 in vivo. In the insulinoma cell line Rin m5F, which contains PC2 as the predominant PC enzyme,93 transfected mouse or monkey POMC were processed to γ-LPH and desacetyl β-END1-31 and secreted in a regulated manner.87,94 β-END1-27 was only observed when the Lys-Lys cleavage site of β-END was mutated to Lys-Arg.87 In fact when the Lys-Arg site between ACTH and β-LPH and the Lys-Lys site within β-END of mouse POMC were permutated to every variation of a paired-basic cleavage site, (e.g., Arg-Lys, Arg-Arg, LysLys, Lys-Arg), it was found that cleavage sites containing Lys as the second basic residue were not cleaved. This indicated a strict requirement by the endogenous processing enzymes for an Arg in this position.95 In contrast, however, monkey POMC was processed at the Lys-Lys site within γ-LPH to generate a significant amount of β-MSH,94 implicating either the existence of a specific Lys-Lys cleaving enzyme for this site in these cells or a significant role for the secondary structure surrounding these sites in the regulation of processing by PC2. BAM cells The specificity of endogenous processing enzymes was further assessed in primary cultures of bovine adrenomedullary chromaffin cells, which were shown to contain primarily PC1. Thorne et al95 transfected these cells with the wild type and the mutant POMC constructs mentioned above, and analyzed the products formed. These cells produced ACTH and β-LPH in addition to γ-LPH and β-END, a pattern similar to anterior pituitary corticotrophs. When cotransfected with PC2, the products generated were more of an intermediate lobe pattern, with the identification of α-MSH and the complete conversion of β-LPH to γ-LPH and β-END.89 The same order of cleavage preferences for the POMC mutants were observed as that of the Rin m5F cells, with inefficient processing of Lys-Lys and Arg-Lys sites. PC12 cells In the pheochromocytoma cell line PC12, cotransfection of mouse POMC (mPOMC) or porcine POMC (pPOMC) with PC1 or PC2 generated products similar to those observed when these molecules were coexpressed in BSC-40 cells. However, an additional product was identified as des-acetyl α-MSH from the PC2 cotransfected PC12 cells88 and N-POMC1-80 was tentatively identified from the PC1 cotransfected cells, indicating the release of joining peptide (JP) by PC190 (Fig. 2.5). N-POMC1-80 was not observed, nor was the ACTH/β-LPH site cleaved by PC2 cotransfected into these cells. In addition, N-POMC1-49 was not observed in either experiment, indicating that the Arg-Lys site of N-POMC1-49/γ3MSH was not a readily cleaved site by PC1 or PC2 in these cells90 (Fig. 2.5). Neuro2a cells When monkey POMC was transfected into Neuro2a cells, a cell line with no measurable PC1 mRNA and only low levels of PC2 mRNA, β-END immunoreactivity was observed to be released from POMC without the intermediate production of β-LPH,93 implying that PC2 cleaved β-END directly from POMC. What is interesting to note here is that while the Lys-Lys site of γ-LPH was not cleaved, a significant amount of β-END1-27 was generated in these cells, a result that is reversed in Rin m5F cells, which contain abundant PC2.93,94 This cell-specific differential processing of the Lys-Lys sites of monkey POMC suggests the existence of a different enzyme capable of processing these sites.

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AtT20 cells In AtT20 cells, a mouse corticotroph cell line, POMC is expressed endogenously and processed into ACTH biosynthetic intermediates (ABI), 13 kDa and 4.5 kDa ACTH and β-LPH. A significant amount of β-END was observed in these cells96 and may be a result of the high levels of expression of PC1 in these cells, estimated at a 1:5 ratio with that of POMC,97 or the low levels of PC2, estimated at a 1:20 ratio with that of PC1.88 When stably transfected with PC2, ACTH1-39 was converted to ACTH1-13(NH2) and β-LPH was converted to β-END which was shown to contain primarily β-END1-31.96 In addition, after a longer pulse/ chase paradigm, a Lyso-γ3-MSH peak was identified (see Fig. 2.5). The absence of significant amounts of β-END1-27 generated in these PC2 overexpressing cells supports further the existence of a different enzyme more suited for this type of cleavage site. It was also reported that JP levels remained the same as that in wild type cells, indicating that PC1 was responsible for these cleavages. In similar experiments with overexpression of rat PC1 in AtT20 cells, two observations were made: 1. the rate of synthesis of the normal cleavage products was enhanced; and 2. the endogenous ACTH decreased significantly with the corresponding increase of ACTH1-13(NH2), demonstrating that PC1 could cleave at the tetra-basic site within ACTH.97 The role that PC1 plays in POMC processing in vivo was further assessed by expression of its antisense mRNA in these cells. AtT20 cells stably expressing antisense PC1 were analyzed for the extent of processing of the endogenous POMC. The results showed a significant decrease in ACTH-containing cleavage products both intra- and extra-cellularly, with a concomitant increase of intact POMC being secreted.98 This demonstrated a significant correlation between PC1 expression and the early steps in POMC processing in AtT20 cells. GH3 cells The role that PC2 plays in the processing of POMC was investigated by transfection experiments in GH3 cells.99 In this cell line, PC2 but no PC1 was detected by RT-PCR. When bovine POMC was transfected into this cell line after secretory granules were induced to form by β-estradiol and insulin, the products generated were identified as predominantly ACTH1-15, β-END and Lyso-γ3-MSH (Fig. 2.5), a result that demonstrated the ability of PC2 to cleave POMC to the smaller peptides in the absence of PC1. In the GH3 cells stably expressing antisense PC2 RNA, a significant decrease of POMC products was observed in a dose dependent level of PC2 knockout.99 In vitro experiments In addition to cotransfection experiments, the in vitro processing of mouse POMC has been investigated. In the first case,100 a lysate of purified insulin secretory granules, a source of type I and type II proinsulin processing endopeptidases, was shown to contain an activity capable of cleaving mPOMC into products similar to those generated when Rin m5F cells were transfected with mPOMC87 and in BSC-40 cells cotransfected with mPOMC, PC1 and PC2.89 The proinsulin processing endopeptidases I and II were subsequently identified as PC1 and PC2, respectively.101,102 Recombinant PC1 was also shown to cleave mPOMC into 16 kDa N-POMC, ACTH and β-LPH, while other products were tentatively identified as N-POMC1-74 and JP-ACTH103 (see Fig. 2.5). When the partially purified PC1 was tested against bovine N-POMC1-77, Lyso-γ3-MSH and γ3-MSH were generated and ACTH1-15 was generated from ACTH1-39 (see Fig. 2.5). These last two processing events are not normally observed in corticotroph cells of the anterior pituitary but are biologically active peptides presumably generated by PC2 in the intermediate lobe. The ability of PC1 to generate Lyso-γ3-MSH in vitro is different from the results obtained from overexpression of PC1 in

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Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

AtT20 cells, since even when PC1 was in molar excess of POMC, no Lyso-γ3-MSH was seen,97 indicating cellular regulation by glycosylation in vivo. The significance of PC1’s ability to cleave this site is highlighted by the observation of mitogenic activity of N-POMC1-49 from anterior pituitary for adrenal regeneration.104

Summary From the present literature, it would seem that PC1, PC2 and the aspartic protease PCE play direct roles in the processing of POMC, as well as other prohormones in vivo (Fig. 2.5). The combined results of cotransfection and in vitro experiments have demonstrated the ability of PC1 to cleave POMC primarily at the dibasic cleavage sites flanking ACTH with some processing at the γ-LPH/β-END junction, a pattern found in anterior pituitary corticotrophs where PC1 is present. PC1 is also capable of cleaving the N-POMC/γ3-MSH junction and the γ3-MSH/JP junction. In addition, overexpression of PC1 in AtT20 cells and in vitro studies show that PC1 can cleave at the tetra-basic cleavage site of ACTH to generate α-MSH as well. PC2 generated Lyso-γ3-MSH, α-MSH and β-END1-31 and depending on the cell line β-END1-31 was further processed to β-END1-27. This pattern of POMC products are found in the intermediate lobe where PC2 is abundant and PC1 is present in significant amounts. PCE, found in anterior and intermediate lobes of the bovine and rat pituitary, appears to be able to cleave POMC at the ACTH/β-LPH junction as well as other sites cleaved by PC1 and PC2, with the exception of the tetra-basic site of ACTH and the Lys-Lys site of β-END1-31. Thus, there is redundancy in the enzymes capable of processing POMC. Future kinetic studies to determine the efficiency of cleavage of each of the sites of POMC by recombinant enzymes will determine which is likely to be the primary enzyme performing each of the cleavages in vivo. Other enzymes have also been found in bovine pituitary intermediate lobe secretory vesicles, which have a more specific function. This includes an acidic ACTH converting enzyme that generates ACTH1-17 from ACTH1-39105 and a mono- and dipeptidyl serine carboxypeptidase involved in the removal of Glu and Glu-Phe, respectively, from the carboxyl terminal of ACTH1-39,106 which may have significant effects on the steroidogenic properties of ACTH, an important neurotransmitter in the central nervous system107 in addition to being a source of Glu.

Future Directions While major advances have been made in the understanding of the mechanism of sorting POMC to the regulated secretory granules, how the processing enzymes are cosorted into these organelles remains an enigma. Sorting domains have been found in PC5A108 and PC2.109 Future studies on determining whether these sorting domains interact directly with POMC, which in turn is bound to CPE, the sorting receptor, or are sorted by directly binding to CPE, or other regulated secretory pathway proteins, will shed light on this very important question. With regards to the processing of POMC, much remains to be understood with respect to the interplay of the roles of PC1, PC2 and PCE, since these enzymes share some common cleavage specificity for the various cleavage sites of POMC. Kinetic studies using recombinant enzymes to evaluate the efficiency of each enzyme for the different POMC cleavage sites will further determine the redundancy of PC1, PC2 and PCE in POMC processing. Suggestions of redundancy were evident in the patient with hyperproinsulinemia who had a defect in the PC1 enzyme, yet showed significant processing of POMC to ACTH.110 The continued search and identification of patients with genetic defects in their prohormone processing enzymes will be a major focus of this field and lead to a better understanding of the level of redundancy of the enzymes for the processing of different prohormones.

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References 1. Nakanishi S, Inoue A, Kita T et al. Nucleotide sequence of cloned cDNA for bovine corticotropin-B-lipotropin precursor. Nature 1979; 278:423-427. 2. Crine P, Gossard F, Seidah NG et al. Concomitant synthesis of β-endorphin and αmelanotropin from two forms of pro-opiomelanocortin in the rat pars intermedia. Proc Natl Acad Sci USA 1979; 76:5085-5089. 3. Eipper B, Mains RE. Structure and biosynthesis of pro-adrenocorticotropin hormone. Endocrinol Rev 1980; 1:1-27. 4. Loh YP, Gritsch HA. Evidence for intragranular processing of pro-opiocortin in the mouse pituitary intermediate lobe. Eur J Cell Biol 1981; 26:177-183. 5. Krieger DT, Liotta AS, Brownstein MJ et al. ACTH, beta-lipotropin, and related peptides in brain, pituitary, and blood. Recent Prog Horm Res 1980; 36:277-344. 6. Liotta AS, Loudes C, McKelvy JF et al. Biosynthesis of precursor corticotropin/endorphin, corticotropin-, alpha-melanotropin-, beta-lipotropin-, and beta-endorphin-like material by cultured neonatal rat hypothalamic neurons. Proc Natl Acad Sci USA 1980; 77:1880-1884. 7. Autelitano DJ, Lundblad JR, Blum M et al. Hormonal regulation of POMC gene expression. Annu Rev Physiol 1989; 51:715-726. 8. Gainer H, Russell JT, Loh YP. An amino peptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin(60-65). FEBS Lett 1984; 175:135-139. 9. Fricker LD, Snyder SH. Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase. J Biol Chem 1983; 258:10950-10955. 10. Hook VYH, Loh YP. Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary. Proc Natl Acad Sci USA 1984; 81:2776-2780. 11. Griffiths G, Simons K. The trans Golgi network: Sorting at the exit of the Golgi complex. Science 1986; 234:438-443. 12. Sossin WS, Fisher JM, Scheller RH. Sorting within the regulated secretory pathway occurs in the trans-Golgi network. J Cell Biol 1990; 110:1-12. 13. Tooze J, Tooze SA, Fuller SD. Sorting of progeny coronavirus from condensed secretory proteins at the exit from the trans-Golgi network of AtT20 cells. J Cell Biol 1987; 105:1215-1226. 14. Kelly RB. Pathways of protein secretion in eukaryotes. Science 1985; 230:25-32. 15. Tooze SA, Chanat E, Tooze J et al. Secretory granule formation. In: Loh YP, ed. Mechanisms of Intracellular Trafficking and Processing of Proproteins. Boca Raton, FL: CRC Press, Inc., 1993:158-177. 16. Kizer JS, Tropsha A. A motif found in propeptides and prohormones that may target them to secretory vesicles. Biochem Biophys Res Comm 1991; 174:586-592. 17. Gorr S-U, Darling DS. An N-terminal hydrophobic peak is the sorting signal of regulated secretory proteins. FEBS Lett 1995; 361:8-12. 18. Colomer V, Kicska GA, Rindler MJ. Secretory granule content proteins and the luminal domains of granule membrane proteins aggregate in vitro at mildly acidic pH. J Biol Chem 1996; 271:48-55. 19. Chanat E, Huttner WB. Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network. J Cell Biol 1991; 115:1505-1519. 20. Parmer RJ, Xi X-P, Wu H-J et al. Secretory protein traffic: Chromogranin A contains a targeting signal for the regulated pathway. J Clin Invest 1993; 92:1042-1054. 21. Chanat E, Weiss U, Huttner WB et al. Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathway. EMBO J 1993; 12:2159-2168. 22. Tam WHH, Andreasson KA, Loh YP. The amino-terminal sequence of pro-opiomelanocortin directs intracellular targeting to the regulated secretory pathway. Eur J Cell Biol 1993; 62:294-306. 23. Bamberger AM, Cool DR, Snell CR et al. Identification of a common amphipathic loop motif in the N-terminal region of POMC and pro-enkephalin as a sorting signal for the

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regulated secretory pathway. In: Cell Biology Annual Meeting. San Fransisco, CA. 1994. Abstract #2598 24. Cool DR, Fenger M, Snell CR et al. Identification of the sorting signal motif within proopiomelanocortin for the regulated secretory pathway. J Biol Chem 1995; 270:8723-8729. 25. Cool DR, Loh YP. Identification of a sorting signal for the regulated secretory pathway at the N-terminus of pro-opiomelanocortin. Biochimie 1994; 76:265-270. 26. Cool DR, Normant E, Shen F-S et al. Carboxypeptidase E is a regulated secretory pathway sorting receptor and genetic obliteration leads to endocrine disorders in the Cpefat mouse. Cell 1997; 88:73-83. 27. Seksek O, Biwersi J, Verkman AS. Direct measurement of trans-Golgi pH in living cells and regulation by second messengers. J Biol Chem 1995; 270:4967-4970. 28. Cool DR, Loh YP. Parameters of binding the regulated secretory pathway sorting signal of pro-opiomelanocortin to the receptor, carboxypeptidase E, in secretory granule membranes. J Biol Chem 1997; under revision. 29. Hook YVY. Differential distribution of carboxypeptidase-processing enzyme activity and immunoreactivity in membrane and soluble components of chromaffin granules. J Neurochem 1985; 45:987-989. 30. Naggert JK, Fricker LD, Varlamov O et al. Hyperproinsulinemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nature Genetics 1995; 10:135-142. 31. Varlamov O, Leiter EH, Fricker L. Induced and spontaneous mutations at Ser202 of carboxypeptidase E. J Biol Chem 1996; 271:13981-13986. 32. Shen F-SS, Loh YP. Intracellular misrouting of pituitary hormones and endocrinological abnormalities in the Cpefat mouse associated with carboxypeptidase mutation. Proc Nat Acad Sci USA 1997; 94:5314-5319. 33. Steiner DF, Cunningham D, Spiegelman L et al. Insulin biosynthesis: Evidence for a precursor. Science 1967; 157:697-699. 34. Chretien M, Li CH. Isolation, purification and characterization of γ-lipotropic hormone from sheep pituitary glands. Ca J Biochem 1967; 45:1163-1174. 35. Chang T-L, Gainer H, Russell JT et al. Proopiocortin-converting enzyme activity in bovine neurosecretory granules. Endocrinology 1982; 111:1607-1614. 36. Loh YP, Chang TL. Pro-opiocortin converting activity in rat intermediate and neural lobe secretory granules. FEBS Lett 1982; 137:57-62. 37. Chang T-L, Loh YP. Characterization of proopiocortin converting activity in rat anterior pituitary secretory granules. Endocrinology 1983; 112:1832-1838. 38. Orci L, Ravazzola M, Storch M-J et al. Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987; 49:865-868. 39. Loh YP, Tam WWH, Russell JT. Measurement of δ-pH and membrane potential in secretory vesicles isolated form bovine pituitary intermediate lobe. J Biol Chem 1984; 259:8238-8245. 40. Loh YP, Parish DC, Tuteja R. Purification and characterization of a paired basic residuespecific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. J Biol Chem 1985; 260:7194-7205. 41. Parish DC, Tujeta R, Alstein M et al. Purification and characterization of a paired basic residue-specific prohormone-converting enzyme from bovine pituitary neural lobe secretory vesicles. J Biol Chem 1986; 261:14392-14397. 42. Loh YP, Birch NP, Castro MG. Pro-opiomelanocortin and pro-vasopressin converting enzyme in pituitary secretory vesicles. Biochimie 1988; 70:11-16. 43. Castro MG, Birch NP, Loh YP. Regulated secretion of pro-opiomelanocortin converting enzyme and an aminopeptidase B-like enzyme from dispersed bovine intermediate lobe pituitary cells. J Neurochem 1989; 52:1619-1628. 44. Loh YP. The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)converting enzyme, on POMC processing in mouse intermediate pituitary. FEBS Lett 1988; 238:142-146.

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45. Loh YP. Kinetic studies on the processing of human β-lipotropin by bovine pituitary intermediate lobe pro-opiomelanocortin-converting enzyme. J Biol Chem 1986; 261:11949-11955. 46. Estivariz FE, Birch NP, Loh YP. Generation of Lys-γ3-melanotropin from pro-opiomelanocortin1-77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme. J Biol Chem 1989; 264:17796-17801. 47. Birch NP, Estivariz FE, Bennett HP et al. Differential glycosylation of N-POMC1-77 regulates the production of γ3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing. FEBS Lett 1991; 290:191-194. 48. Krieger TJ, Hook VYH. Purification and characterization of a cathepsin D protease from bovine chromaffin granules. Biochemistry 1992; 31:4223-4231. 49. Toomin CS, Hook VYH. Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary. Biochem Biophys Res Commun 1992; 183:449-455. 50. Azaryan AV, Schiller M, Mende-Mueller L et al. Characteristics of the chromaffin granule aspartic proteinase involved in proenkephalin processing. J Neurochem 1995; 65:1771-1779. 51. Azaryan AV, Schiller MR, Hook VY. Chromaffin granule aspartic proteinase processes recombinant proopiomelanocortin (POMC). Biochem Biophys Res Commun 1995; 215:937-944. 52. Krieger TJ, Hook VY. Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor. J Biol Chem 1991; 266:8376-8383. 53. Azaryan AV, Krieger TJ, Hook VY. Purification and characteristics of the candidate prohormone processing proteases PC2 and PC1/3 from bovine adrenal medulla chromaffin granules. J Biol Chem 1995; 270:8201-8208. 54. Hook VYH, Schiller MR, Azaryan AV. The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preference among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. Arch Biochem Biophys 1996; 328:107-114. 55. Julius D, Brake A, Blair L et al. Isolation of the putative structural gene for the lysinearginine-cleaving endopeptidase required for processing of yeast prepro-α-factor. Cell 1984; 37:1075-1089. 56. Fuller R, Brake A, Thorner J. The Saccharomyces cerevisiae KEX2 gene, required for processing prepro-alpha-factor, encodes a calcium-dependent endopeptidase that cleaves after Lys-Arg and Arg-Arg sequences. In: Levine L, ed. Microbiology. Washington, D.C.: American Society for Microbiology, 1986:273-278. 57. Fuller RS, Brake A, Thorner J. Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc Natl Acad Sci USA 1989; 86:1434-1438. 58. Egel-Mitani M, Flygenring HP, Hansen MT. A novel aspartyl protease allowing KEX2-independent MFα propheromone processing in yeast. Yeast 1990; 6:127-137. 59. Bourbonnais Y, Ash J, Daigle M et al. Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: Cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites. EMBO J 1993; 12:285-294. 60. Cawley NX, Noe BD, Loh YP. Purified yeast aspartic protease 3 cleaves anglerfish prosomatostatin I and II at di- and monobasic sites to generate somatostatin-14 and -28. FEBS Lett 1993; 332:273-276. 61. Mackin RB, Noe BD, Spiess J. The anglerfish somatostatin-28-generating propeptide converting enzyme is an aspartyl protease. Endocrinology 1991; 129:1951-1957. 62. Cawley NX, Loh YP. Yapsin 1 (yeast aspartic protease 3). In: Barrett AJ, Rawlings ND, Woessner JF, eds. Handbook of Proteolytic Enzymes. London: Academic Press 905-907. 63. Azaryan AV, Wong M, Friedman TC et al. Purification and characterization of a paired basic residue-specific yeast aspartic protease encoded by the YAP3 gene. Similarity to the mammalian pro-opiomelanocortin-converting enzyme. J Biol Chem 1993; 268:11968-11975. 64. Cawley NX, Wong M, Pu L-P et al. Secretion of yeast aspartic protease 3 is regulated by its carboxy-terminal tail: Characterization of secreted YAP3p. Biochemistry 1995; 34:7430-7437.

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65. Cawley NX, Chen H-C, Beinfeld MC et al. Specificity and kinetic studies on the cleavage of various prohormone mono- and paired-basic residue sites by yeast aspartic protease 3. J Biol Chem 1996; 271:4168-4176. 66. Loh YP, Cawley NX, Friedman TC et al. Yeast and mammalian basic residue-specific aspartic proteases in prohormone conversion. Adv Exp Med Biol 1995; 362:519-527. 67. Cawley NX, Pu L-P, Loh YP. Immunological identification and localization of yeast aspartic protease 3-like prohormone processing enzymes in mammalian brain and pituitary. Endocrinology 1996; 137:5135-5143. 68. Cool DR, Louie DY, Loh YP. Yeast aspartic protease 3 (YAP3p) is sorted to secretory granules and activated to process pro-opiomelanocortin in PC12 cells. Endocrinology 1996; 137:5441-5446. 69. Loh YP, Cawley NX. Processing enzymes of the pepsin family: Yeast aspartic protease 3 and pro-opiomelanocortin converting enzyme. Meth Enzym 1995; 248:136-146. 70. Komano H, Fuller RS. Shared functions in vivo of a glycosyl-phosphatidylinositol-linked aspartyl protease, Mkc7, and the proprotein processing protease Kex2 in yeast. Proc Natl Acad Sci USA 1995; 92:10752-10756. 71. Seidah NG, Day R, Benjannnet S et al. The prohormone and proprotein processing enzymes PC1 and PC2: Structure, selective cleavage of mouse POMC and human renin at pairs of basic residues, cellular expression, tissue distribution, and mRNA regulation. Nida Res Monogr 1992; 126:132-150. 72. Van de Ven WJM, Van Duijnhoven HLP. Structure and function of eukaryotic proprotein processing enzymes of the subtilisin family of serine proteases. Crit Rev Oncogen 1993; 4:115-136. 73. Seidah NG, Chretien M, Day R. The family of subtilisin/kexin like pro-protein and prohormone convertases: Divergent or shared functions. Biochimie 1994; 76:197-209. 74. Rouille Y, Duguay SJ, Lund K et al. Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: The subtilisin-like proprotein convertases. Front Neuroendocrinol 1995; 16:322-361. 75. Thomas G, Thorne BA, Thomas L et al. Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells. Science 1988; 241:226-230. 76. van den Ouweland AMW, van Duijnhoven HLP, Keizer GD et al. Structural homology between the human fur gene product and the subtilisin-like protease encoded by yeast KEX2[published erratum appears in Nucleic Acids Res 1990 Mar 11;18(5):1332]. Nucleic Acids Res 1990; 18:664. 77. Seidah NG, Gaspar L, Mion P et al. cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: Tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases. DNA and Cell Biol 1990; 9:415-424. 78. Seidah NG, Marcinkiewicz M, Benjannet S et al. Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, furin, and Kex2: Distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol 1991; 5:111-122. 79. Nakayama K, Hosaka M, Hatsuzawa K et al. Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites. J Biochem 1991; 109:803-806. 80. Smeekens SP, Avruch AS, LaMendola J et al. Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans. Proc Natl Acad Sci USA 1991; 88:340-344. 81. Smeekens SP, Steiner DF. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J Biol Chem 1990; 265:2997-3000. 82. Hakes DJ, Birch NP, Mezey E et al. Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats. Endocrinol 1991; 129:3053-3063.

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83. Schafer MK-H, Day R, Cullinan WE et al. Gene expression of prohormone and proprotein convertases in the rat CNS: A comparative in situ hybridization analysis. J Neurosci 1993; 13:1258-1279. 84. Pu L-P, Ma W, Barker JL et al. Differential coexpression of genes encoding prothyrotropinreleasong hormone (pro-TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: Implications for differential processing of pro-TRH. Endocrinology 1996; 137:1233-1241. 85. Day R, Schafer MK-H, Watson SJ et al. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 1992; 6:485-497. 86. Birch NP, Tracer HL, Hakes DJ et al. Coordinate regulation of mRNA levels of proopiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in the rat pituitary intermediate lobe. Biochem Biophys Res Commun 1991; 179:1311-1319. 87. Thorne BA, Caton LW, Thomas G. Expression of mouse proopiomelanocortin in an insulinoma cell line. Requirements for β -endorphin processing. J Biol Chem 1989; 264:3545-3552. 88. Benjannet S, Rondeau N, Day R et al. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-3568. 89. Thomas L, Leduc R, Thorne BA et al. Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 1991; 88:5297-5301. 90. Seidah NG, Fournier H, Boileau G et al. The cDNA structure of the porcine pro-hormone convertase PC2 and the comparative processing by PC1 and PC2 of the N-terminal glycopeptide segment of porcine POMC. FEBS Lett 1992; 310:235-239. 91. Nicholas PR, Hammonds G, Li CC. β-Endorphin-induced analgesia is inhibited by synthetic analogs of β-endorphin. Proc Natl Acad Sci USA 1984; 81:3074-3077. 92. Bals-Kubik R, Herz A, Shippenberg TS. β-Endorphin(1-27) is a naturally occurring antagonist of the reinforcing effects of opioids. Naunyn Schmiedebergs Arch Pharmacol 1988; 338:392-396. 93. Day NC, Lin H, Ueda Y et al. Characterization of pro-opiomelanocortin processing in heterologous neuronal cells that express PC2 mRNA. Neuropeptides 1993; 24:253-262. 94. Lin H-L, Day NC, Ueda Y et al. Tissue-specific and substrate-specific endoproteolytic cleavage of monkey pro-opiomelanocortin in heterologous endocrine cells: Processing at LysLys dibasic pairs. Neuroendocrinology 1993; 58:94-105. 95. Thorne BA, Thomas G. An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes. J Biol Chem 1990; 265:8436-8443. 96. Zhou A, Bloomquist BT, Mains RE. The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in a strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 1993; 268:1763-1769. 97. Zhou A, Mains RE. Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 and 2. J Biol Chem 1994; 269:17440-17447. 98. Bloomquist BT, Eipper BA, Mains RE. Prohormone-converting enzymes: Regulation and evaluation of function using antisense RNA. Mol Endocrinol 1991; 5:2014-2024. 99. Friedman TC, Cool DR, Jayasvasti V et al. Processing of pro-opiomelanocortin in GH3 cells: Inhibition by prohormone convertase 2 (PC2) antisense mRNA. Mol Cell Endocrinol 1996; 116:89-96. 100. Rhodes CJ, Thorne BA, Lincoln B et al. Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases. J Biol Chem 1993; 268:4267-4275. 101. Bailyes EM, Shennan KIJ, Seal AJ et al. A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. Biochem J 1992; 285:391-394. 102. Bennett DL, Bailyes EM, Nielsen E et al. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J Biol Chem 1992; 267:15229-15236.

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103. Friedman TC, Loh YP, Birch NP. In vitro processing of proopiomelanocortin by recombinant PC1 (SPC3). Endocrinology 1994; 135:854-862. 104. Estivariz FE, Morano MI, Carino M et al. Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary. J Endocrinol 1988; 116:207-216. 105. Estivariz FE, Friedman TC, Chikuma T et al. Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. J Biol Chem 1992; 267:7456-7463. 106. Friedman TC, Chen H-C, Loh YP. Generation of (1-37) and (1-38) forms of adrenocorticotropin by mono- and di-peptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles. Endocrinol 1993; 133: 2951-2961. 107. Dani JA, Mayer ML. Structure and function of glutamate and nicotinic acetylcholine receptors. Curr Opin Neurobiol 1995; 5:310-317. 108. De Bie I, Marcinkiewicz M, Malide D et al. The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments. J Cell Biol 1996; 135:1261-1275. 109. Creemers JW, Usac EF, Bright NA et al. Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. J Biol Chem 1996; 271: 25284-25291. 110. O’Rahilly S, Gray H, Humphreys PJ et al. Brief report: Impaired processing of prohormones associated with abnormalities of glucose homeostasis and adrenal function. New Engl J Med 1995; 333:1386-1390. 111. Vieau D, Seidah NG, Mbikay M et al. Expression of the prohormone convertase PC2 correlates with the presence of corticotropin-like intermediate lobe peptide in human adrenocorticotropin-secreting tumors. J Clin Endocrinol Metab 1994; 79:1503-1506.

CHAPTER 3

The Mammalian Precursor Convertases: Paralogs of the Subtilisin/Kexin Family of Calcium-Dependent Serine Proteinases Nabil G. Seidah, Majambu Mbikay, Mieczyslaw Marcinkiewicz, Michel Chrétien

Introduction

O

ver the last 30 years,1-4 our understanding of the complex cellular processing of inactive secretory precursors into active polypeptides and proteins by limited proteolysis has greatly matured. It is now becoming clear that following removal of the signal peptide, precursor cleavage can occur either intracellularly, at the cell surface or within the extracellular milieu. The sites of cleavage are either composed of: 1. single or pairs of basic residues (K or R) within the general motif (R/K)-(X)n-(K/R)↓, where n = 0, 2, 4, or 6 (Table 3.1); 2. hydrophobic amino acids (e.g., Leu, Phe, Val or Met) (Table 3.2); or 3. small amino acid residues such as Ala or Thr (Table 3.2). The subdivision of precursors cleaved at basic residues into four types (Table 3.1), is based on the amino acids surrounding the cleavage site which could be critical for efficient processing. For example, cleavage of type I precursors within the motif R-X-(K/R)-R↓ is usually accomplished within the trans-Golgi network (TGN) by one or more resident enzymes, which often process precursors expressed in cells devoid of dense core secretory granules, the products(s) of which reach the cell surface by the constitutive secretory pathway.14 In contrast, cleavage of type II precursors at pairs of basic residues usually occurs within immature secretory granules and involves precursors whose products are stored in dense core granules and exit the cell via the regulated secretory pathway. Cleavage at monobasic residues (type III) and of type IV precursors can occur in either of the above secretory pathways. Some of the proteinases involved in intracellular endoproteolytic events resulting in cleavage at specific basic residues have recently been identified and molecularly characterized. They form a family of calcium-dependent serine proteinases of the subtilisin/kexintype, of which seven mammalian members (paralogs) are known so far (for recent reviews, see refs. 4, 15-18). The various names given to these “Precursor Convertases” (PCs) are presented in Table 3.3. For simplicity, throughout this chapter we will refer to the seven convertases as PC1, PC2, furin, PC4, PC5, PACE4, and PC7. In a combinatorial fashion, these enzymes determine the cell type and time at which biologically active products are derived from a given inactive precursor protein. Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Table 3.1. Precursor classification based on basic (B) amino acid cleavage motifs Precursor Protein

Cleavage Site Sequence

Type I precursors [R-X-(K/R)-R]

P6 P5 P4 P3 P2 P1 [X- X- R- X- K/R- R Thr-His-Arg-Ser-Lys-Arg Gln-Val-Arg-Glu-Lys-Arg Pro-Ile-Arg-Arg-Lys-Arg Leu-Ala-Arg-Gly-Arg-Arg Asn-Ser-Arg-Lys-Lys-Arg Val-Gln-Arg-Glu-Lys-Arg Gln-Ser-Arg-Met-Arg-Arg Val-Glu-Arg-Val-Lys-Arg His-Gln-Arg-Ala-Arg-Arg Asn-Leu-Arg-Met-Lys-Arg Arg-Asn-Arg-Gln-Lys-Arg Glu-Arg-Arg-Lys-Arg-Arg

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X- X] Ser-Ser Leu-Asp Ser-Ile Ser-Leu Glu-Ile Ala-Val Ala-Ala Arg-Ala Ser-Val Asp-Thr Phe-Val Ser-Val

P6 P5 P4 P3 P2 P1 [X- X- X- X- K/R- R Pro-Val-Gly-Lys-Lys-Arg Pro-Pro-Lys-Asp-Lys-Arg Thr-Pro-Lys-Thr-Arg-Arg Gly-Ser-Leu-Gln-Lys-Arg Ser-Gln-Pro-Met-Lys-Arg Ala-Pro-Leu-Thr-Lys-Arg His-Val-Ile-Ser-Lys-Arg Ser-Cys-Lys-Leu-Lys-Arg

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X- X] Arg-Pro Tyr-Gly Glu-Ala Gly-Ile Leu-Thr His-Ser Ser-Thr Arg-Gly

ProβNGF Leptin proreceptor ProPDGF-A ProPDGF-B Integrin α6 HIV-1 gp160 Profertilin α proTACE proKUZ (mouse)—site 1 ————————site 2 proStromelysin-3 Pro7B2 Type II precursors [(K/R)-(K/R)] POMC (α−MSH/CLIP) (γ−LPH/β−END) Proinsulin (B/C chain) (C/A chain) ProRenin PACAP-RP Integrin α4 Profertilin β Type III precursors [Single R]

P8 P7 P6 P5 P4 P3 P2 P1 (B)- X- (B)- X- (B)- X- X R Arg-Gln-Phe-Lys-Val-Val-Thr-Arg Arg-Ala-Leu-Leu-Thr-Ala-Pro-Arg Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg Leu-Lys-Pro-Thr-Lys-Ala-Ala-Arg Ala-Thr-Pro-Ala-Lys-Ser-Glu-Arg Glu-Asp-Gly-His-His-Leu-Asp-Arg Asp-Leu-Arg-Trp-Trp-Glu-Leu-Arg

↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X - X Ser-Gln Ser-Leu Ser-Ala Ser-Ile Asp-Val Asn-Ser His-Ala

P8 P7 P6 P5 P4 P3 P2 P1 (B)- X- (B)- X- (B)- X- (B)-R/K PACAP-RP Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg ProMullerian Inhibiting Substance Glu-Gly-Arg-Gly-Arg-Ala-Gly-Arg Proglucagon Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg ProIGF-II Glu-Ala-Phe-Arg-Glu-Ala-Lys-Arg ProPTPµ receptor Val-Glu-Glu-Glu-Arg-Pro-Arg-Arg

↓ ↓ ↓ ↓ ↓ ↓

P1' P2' X -R/K Tyr-Lys Ser-Lys Asn-Arg His-Arg Thr-Lys

Prodynorphin (C-peptide) ProANF Prosomatostatin (SS-28) ProIGF-I ProIGF-II ProEGF (N-terminal) ProEGF (C-terminal) Type IV precursors [P2'= R/K]

The precursors listed in this table have been reported previously [4]. The motif recognized in each precursor type is emphasized. This list represents only a limited repertoire of the known PC substrates.

The Mammalian Precursor Convertases

51

As an alternative to cleavage at basic residues, some precursors are processed intracellularly at single or paired hydrophobic amino acids (e.g., Leu, Val, Ile, Phe and Met and combinations thereof), or following small amino acids, usually Ala or Thr (Table 3.2). Although so far the proteinases responsible for such processing are not known, efforts towards their identification are underway in a number of laboratories, especially since they could play major roles in the regulation of cholesterol and fatty acid metabolism7 and in Alzheimer disease.12

Subtilisin/Kexin-like Precursor Convertases (PCs): Structural and Cellular Considerations A schematic representation of the primary structure and proposed domains of the known 7 mammalian convertases and their membrane-bound isoforms, as compared to yeast kexin and bacterial subtilisin BPN’ is shown in Figure 3.1. The degree of sequence identities between each member within each of the pro-, catalytic and P-domains are compared in Tables 3.4-3.6. In all three domains, the best sequence identity is between PACE4 and PC5, varying between 67%, 75% and 61%, respectively. So far, it is apparent that while furin15 and PC732-36 are always first synthesized as type I membrane-bound enzymes, the isoforms PACE4-E28 and PC5-B31,37 are also membrane-associated (Fig. 3.1). Downstream of the signal peptide (Fig. 3.1), the subtilases contain a highly basic prosegment which, by analogy to bacterial subtilisins, is thought to act as an intramolecular chaperone guiding their folding within the endoplasmic reticulum (ER). Once cleaved by an autocatalytic mechanism, the pro-segment becomes a potent inhibitor regulating the intracellular site of activation of the enzyme (for reviews see ref. 38) until it is disposed of via a secondary cleavage(s) during cellular transit to the trans-Golgi Network (TGN).4,18,39 Overall, PC7 exhibits the least conserved pro-segment among the mammalian PCs (Table 3.4). Intramolecular autocatalytic zymogen cleavage of the pro-segment occurs in the neutral pH environment of the ER for furin,40 PC1,41-44 PC5,37 PACE428,45-47 and PC7.36 For these convertases, it is likely that the cleaved pro-segment remains bound as a complex to the enzyme until it reaches the TGN, where it is thought that a secondary event(s), e.g., a second cleavage within the pro-segment,39 causes the disassembly of the complex and the generation of the active convertase, which can then process other precursors in trans. In contrast, removal of the pro-segment of PC2 occurs within the acidic milieu of the TGN/ immature secretory granules (ISG)42,44 and is highly facilitated by the presence of 7B2,48,49 a PC2-specific binding protein.16,50-53 Thus, PC2 is unique among the convertases in that it is activated late along the secretory pathway and requires the presence of 7B2 for optimal activation. The catalytic domain of each convertase which contains the Asp, His and Ser of the catalytic triad and the oxyanion hole Asn (except for PC2, where it is an Asp) is the most conserved segment (Table 3.5), emphasizing its critical role in the recognition of single or paired basic residues within substrates. The conserved oxyanion hole Asp+ in PC2 seems to be important for its interaction with the precursor of 7B2.53 Alignment of the catalytic domain of all the PCs reveals that, with the exception of PC2, within the 350 amino acid catalytic domain 9 residues are conserved in all the mammalian PCs (Fig. 3.2). In PC2, these include 3 conservative and 6 nonconservative substitutions of which 2 are not conserved in all species and, hence, only 4 are considered to be significant changes. All these amino acid substitutions occur between the PC2's active site •D166 and the oxyanion hole +D . They include: 309 1. the oxyanion hole +D309 itself (as opposed to N); 2. two in the segment S81-S-N-D-P-Y86-P-Y-P-R, in which the underlined S81 and Y86 are unique, as they are N and D respectively in all other PCs; and finally 3. a Q134 replacing the usual G in all other PCs.

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Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Table 3.2. Precursor classification based on hydrophobic (ϕ) and/or small (σ) amino acid cleavage motifs Precursor Protein

Cleavage Site Sequence P8 P7 P6 P5 P4 P3 P2 P1 P1' P2' ϕσ -X- Bϕσ ϕσ -X- Bϕσ ϕσ -X- ϕσ - ϕσ ↓ X- ϕσ Bϕσ

(r)proRelaxin (B-chain)

Ala-Ser-Val-Gly-Arg-Leu-Ala-Leu ↓ Ser-Gln

(h)SREBP-2 (h)SREBP-1a

Ser-Gly-Ser-Gly-Arg-Ser-Val-Leu ↓ Ser-Val His-Ser-Pro-Gly-Arg-Asn-Val-Leu ↓ Gly-Thr

(h) proCCK (CCK5)

Arg-Ile-Ser-Asp-Arg-Asp-Tyr-Met ↓ Gly-Trp

(r)α-Endorphin (r)γ-Endorphin

Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu ↓ Phe-Lys Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe ↓ Lys-Asn

(r)proAVP (CPP)

Gly-Pro-Ser-Gly-Ala-Leu-Leu-Leu ↓ Arg-Leu

(r)proRenin

Lys-Ser-Ser-Phe-Thr-Asn-Val-Thr ↓ Ser-Pro

(b)Chromogranin A (65↓66) (b)Chromogranin A (291↓292) (b)Chromogranin B (609↓610) (b)Chromogranin B (614↓615)

Leu-Leu-Lys-Glu-Leu-Gln-Asp-Leu ↓ Ala-Leu Met-Ala-Arg-Ala-Pro-Gln-Val-Leu ↓ Phe-Arg Glu-Leu-Glu-Asn-Leu-Ala-Ala-Met ↓ Asp-Leu Ala-Ala-Met-Asp-Leu-Glu-Leu-Gln ↓ Lys-Ile

(h)β-APP β-Secretase site β-Secretase site (Swedish) βε1-Secretase site βε2-Secretase site γ40-Secretase site γ42-Secretase site

Glu-Glu-Ile-Ser-Glu-Val-Lys-Met ↓ Asp-Ala Glu-Glu-Ile-Ser-Glu-Val-Asn-Leu ↓ Asp-Ala Ile-Ser-Glu-Val-Lys-Met-Asp-Ala ↓ Glu-Phe Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr ↓ Glu-Val Gly-Leu-Met-Val-Gly-Gly-Val-Val ↓ Ile-Ala Met-Val-Gly-Gly-Val-Val-Ile-Ala ↓ Thr-Val

The precursors listed in this table include those of rat relaxin [5], human cholescytokin to produce CCK5 [6] and human sterol responsive element binding proteins 1a and 2 [7], all of which involve cleavage at either a hydrophobic (ϕ) or small (σ) residue and exhibit the presence of a basic (B) R or K at P4 and either a σ or ϕ amino acid at P2' or a B/ϕ/σ at either P4 and/or P8. Some of these cleavages could involve an enzyme(s) recognizing the motif (R/K)-X-X-ϕ↓ [7]. Other cleavages occur at either single or pairs of hydrophobic residues such as in the production of α- and γ-Endorphin [8], the above mentioned chromogranin A (9) and chromogranin B (10) sites, vasopressin C-terminal glycopeptide fragment CPP 1-19 [11] and processing of the human amyloid precursor protein (β-APP) processed at either the native or Swedish mutation of the β-secretase site or at the γ-secretase-40 site [12]. Some precursors are also cleaved at σ amino acids, e.g., in the γ-secretase-42 site and rat proRenin. Often in such precursors we note the presence of a ϕ or σ amino acid at either P4, P6 or P8. In addition, in some precursors we also observe the presence of a Gly at P5. The βε1- and βε2-secretase sites have been recently reported to increase with age in the brain of Azheimer and Down syndrome patients (13).

The Mammalian Precursor Convertases

53

Table 3.3. Various names of Precursor Convertases (PCs) Adopted Convertase Name (Reference)

Alternative Name(s)(Reference)

Furin (19)

PACE (20) SPC1 (17)

PC1 (21,22)

PC3 (23) SPC3 (17)

PC2 (21,24)

SPC2 (17)

PC4 (25,26)

SPC4 (17)

PACE4 (27,28)

SPC5 (17)

PC5 (29)

PC6 (30,31) SPC6 (17)

PC7 (32)

LPC (33) PC8 (34) SPC7 (35)

Nomenclature of convertases with the original references shown in brackets.

Table 3.4. Sequence identities of the Pro-domains of PCs Convertases

hPC1

hPC2

rPC4

hfurin

hPACE4

hPC5

hPC7

hPC2 rPC4 hfurin hPACE4 hPC5 hPC7 ykexin

35% 35% 47% 50% 41% 35% 25%

31% 43% 41% 35% 31% 29%

53% 45% 44% 36% 26%

60% 58% 34% 29%

67% 35% 26%

30% 29%

23%

Table 3.5. Sequence identities of the catalytic domains of PCs Convertases

hPC1

hPC2

rPC4

hfurin

hPACE4 hPC5

hPC7

ykexin

hPC2 rPC4 hfurin hPACE4 hPC5 hPC7 ykexin subt. BPN’

55% 59% 64% 63% 60% 55% 50% 36%

56% 58% 54% 53% 51% 47% 35%

70% 62% 62% 53% 46% 34%

68% 67% 54% 49% 37%

75% 54% 48% 34%

48% 34%

34%

54% 48% 34%

54

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Fig. 3.1. Schematic representation of the primary structures of the PCs as compared to those of Subtilisin BPN’ and yeast kexin. The various domains are emphasized, together with the active sites Asp, His and Ser and the oxyanion hole Asn (Asp for PC2).

The Mammalian Precursor Convertases

55

Table 3.6. Sequence identities of the P-domains of PCs Convertases

hPC1

hPC2

rPC4

hfurin

hPACE4

hPC5

hPC7

hPC2 rPC4 hfurin hPACE4 hPC5 hPC7 ykexin

43% 42% 44% 43% 39% 37% 41%

43% 47% 52% 52% 42% 32%

55% 43% 43% 38% 33%

45% 51% 38% 32%

61% 45% 33%

43% 38%

32%

We have already obtained the D/N mutant of the oxyanion hole of PC2. This substitution abrogates the binding of proPC2 to pro7B2 in the ER but not that of PC2/7B2 in the TGN.53 These data suggest that the unique oxyanion hole of proPC2 is important for its binding to pro7B2 in the ER but is not needed for the predicted second binding site of 7B2. Finally, based on homology modeling to subtilisin, suggestions have been made identifying the PC residues potentially critical for substrate recognition.54 Resolving the crystal structure of one or more PCs should shed more light on the intricate details of their structures and their ability to discriminate between various substrates. The P-domain has been shown to be critical for zymogen cleavage and for enzyme secretion of prokexin55 proPACE4,46 proPC2,56 for the substrate cleavage activity of furin,57,58 and for the sorting of PC2.59 The N-terminus of the P-domain starts at the end of the catalytic domain, delimiting the end of the homologous subtilisin sequence. Its C-terminal border has been defined as that amino acid close to an L-X-(L/F)-X-G sequence18 (Fig. 3.3), beyond which further deletions would irreversibly abolish the enzymatic activity of the PC.46,55,57 Interestingly, this functional definition coincides with the endpoint of similarity among the PCs.18,55 It is noted, however, that the P-domain of PC7 extends beyond the L-X(L/F)-X-G sequence.36 In addition to the above consensus sequence at the end of the P-domain of mammalian PCs, this segment exhibits the presence of 20 other identical amino acids, 10 of which are absolutely conserved among all PC orthologues18 (residues bold and underlined in Fig. 3.3). Among these, and with the exception of PC7, the RRGDL sequence is conserved in the other mammalian PCs, and its RG dipeptide is always present in all PC orthologues reported. In view of the RGD sequence found in the PCs and in fibronectin, it was originally suggested that this motif may be important for the interaction of the PCs with cell adhesion integrins.21 Interestingly, three RGD copies exist in Hydra PC1. The PCs which do not contain an RGD sequence include mammalian (human, rat and mouse) PC7, Drosophila furin 2, Lymnaea stagnalis PC2, Aplysia californica PC1 and PC2 and yeast XPR6 (reviewed in ref. 18). The conservation of this motif suggested a structural or functional role which may be common to most mammalian convertases. Binding of fibronectin to its integrin receptor was shown to be dependent on the RGD sequence and to be abrogated when it was replaced by RGE.60 Recently, we addressed the question of the functional relevance of the RRGDL motif within the model enzyme mouse PC1.61 The parameters examined included its importance for POMC cleavage, precursor processing (pro-segment removal and C-terminal cleavage), stability of the enzyme and intracellular trafficking. The data revealed that the RRGDL motif is critical for the stability of PC1 and for its zymogen activation in the ER, as well as for the elaboration of maximal PC1 activity releasing β-LPH from POMC. In addition, since the resulting RGD/E mutant enzyme does not enter secretory

YFNDPiWsnm YFNDPKWPsm eptDPKFPQQ vptDPwFskQ lFNDPmWnQQ nmNDPlFtkQ hFNDPKYPQQ ---DP-----

NYDsyASYDv NYDalASCDv NYDPgASFDv NYDPlASYDF NYDPEASYDF NYnsDASYDF NYsPEgSYDL NY----S-D+ MLDGD.VTDV MLDGD.VTDm MLDGE.VTDa MLDGa.ITDI MLDGi.VTDa MLDqpfmTDI vLDGp.lTDs -LD----TD-

10 ...qaRsdSl dYdlsRaqSt ......dVyq .......slV .svprdsaln gYrdineIdI ........SI ---------70 IERNHPDLAp IERtHPDLmq IEkNHPDLAg IEkdHPDLwa lEWNHtDiya IDYlHPDLAy VEhtvqDiAp ------D--130 AYNAKIGGIR AFNAKIGGVR AYNArIGGVR AFNArIGGVR AYNsKVGGIR AYNsKVaGIR AYgsrIaGIR A------G-R

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus

150 VEAkSLgirP VEAkSvsYNP VEArSLgLNP VEAqSLsLqP IEAsSigFNP IEAsSishmP mEAvaFnkhy -EA-------

+ + 90 NgNDyDPsPR NgNDlDPmPR NDqDPDPqPR NDyDPDPqPR NDNDhDPfPR ssNDPyPyPR NsNDPDPmPh ---D--P-P-

30 WYMHCaDkNs WYMHCsDNth WYL.....sg WYM.....Nk WYLqdTrmta WYLfnTgqad WhL....NNr ----------

nyIDIYSASW QHVhIYSASW nHIhIYSASW QHIhIYSASW gHVDIYSASW QlIDIYSASW QinDIYScSW ----IYS-SW

• YDasNENKHG YDasNENKHG YtqmNDNrHG YtpndENrHG YDptNENKHG YtddwfNsHG pDeeNgNhHG ----N---HG

Rcrs.EMNVq pcqs.DMNIe vtqr.DLNVk eieq.DLNIl slPkLDLhVi gtPgLDLNVa RsPgrDiNVt W---------

+ 170 GPDDDGKTVD GPDDDGKTVD GPEDDGKTVD GPEDDGrTVD GPnDDGKTVE GPtDnGKTVD GPDDDGKTVD GP-D-G-TV-

110 TRCAGEVAAs TRCAGEVAAt TRCAGEVAAv TRCAGEVsAt TRCAGEIAmq TRCAGEVsAa TRCAGEIAAv TRCAGE----

50 aAWkRGYTGK gAWkRGYTGK eAWaqGFTGr kvWnqGLTGr pvWqkGiTGK eAWelGYTGK gvWeRnvTGr --W----TG-

GPGRLakQAF GPapLtrQAF GPaRLaeeAF GPGlLtqeAF GPGRLaqkAF GPreLtlQAM GPhqLgkaAL GP--L---A-

+ ANNSYCiVGI ANNShCtVGI ANNgvCGVGV ANNgFCGaGV ANNhkCGVGV AsNniCGVGV pNNSFCaVGV --N--C--G-

• nVVVTILDDG nIVVTILDDG GIVVsILDDG GVVVsILDDG GVVITVLDDG GVtIgIMDDG GVtVvVvDDG -------DDG

56 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

kTSRPAhLkA RTSRaghLNA qTSkPAhLNA RaSRPAqLqA WTSeydpLan lTSkrnqLhd FT..atqyed ----------

310 TWRDvQHLlV TWRDvQHvIV TWRDMQHLVV TWRDLQHLVV TWRDMQHLVV TWRDMQHLtV TWRDvQHiIV TWRD-QH--V

GhKVSHLYGF GFKVSHLYGF GrKVSHsYGY GrqVSHhYGY GLmVnsrFGF GLefnHLFGY GFshSHqhGF G-------G-

+ 350 GLvDAeALVl GLMDAeAMVm GLLDAGAMVa GLLDAGlLVd GLLnAkALVd GvLDAGAMVk GLLnAwrLVn G---A---V-

eA.. eAE. LA.. LA.. LADp MA.. aA.. -A--

ALALEANnqL ALALEANPfL ALtLEANknL ALALEANPlL ALALEANPnL ALALEANvdL ALmLqvrPcL AL-L-----L

hkPWYlEECa kkPWYlEECS nvPWYsEaCS RvPWYsEaCa lsPWYaEkCS RtaLYdEsCS RmPFYaEECa

Fig. 3.2. Alignment of the catalytic subunits of the 7 PCs. (•) = catalytic triad and the oxyanion hole. (+) = conserved residues, except for PC2 which is underlined.

330 s..DWKvNGA N..DWKTNaA N..DWaTNGv e..DWriNGv N.pgWKkNGA evhqWrrNGv hraDWlTNeA ----W--N--

290 vSAPMvAGII ASAPMAAGII ASAPLAAGII ASAPLAAGmI ASAPLAAGIf AaAPeAAGVf AaAPLAAGmI --AP--AG--

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus

+ • .CTDGHTGTS .CTDnHTGTS .CTEsHTGTS .CTDkHTGTS .CTEtHTGTS .CTlrHsGTS gCTEGHTGTS -CT--H-GTS

afyErk..IV EsyDkk..II nqnEkq..IV vvtDpq..IV Dytnqr..It rkrnpeagVa Dkmlr..sIV ----------

250 STLATTYSSG STLATTYSSG STLATTYSSG STFtTTFSSG STLATsYSSG STLAsTFSnG SmLAvTFSgG S------S-G

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus

270 TTDLRQr... TTDLRQr... TTDLRQk... TTDLhhq... saDLhnd... TTDLygn... TTDwdlqkgt --D-------

190 +/• 210 + 230 EyGIKKGRQG LGSIFVWASG NGGRegDhCs CDGYTNSIYT ISVSStTEnG EnGVrmGRrG LGSVFVWASG NGGRskDhCs CDGYTNSIYT ISISStaEsG frGVsqGRgG LGSIFVWASG NGGRehDsCN CDGYTNSIYT lSISSaTqfG rrGVtKGRQG LGtlFIWASG NGGlhyDnCN CDGYTNSIhT lSVgStTrqG EyGVKqGRQG kGSIFVWASG NGGRqgDyCd CDGYTdSICT ISISSasqqG adGVnKGRgG kGSIYVWASG dGG.syDdCN CDGYasSmWT ISInSaindG qhGVmaGRQG FGSIFVvASG NGGqhnDnCN YDGYaNSIYT VtIgavdEeG -G-GR-G -G--ASG -GG-D-C- -DGY-S-T ----G --Y-E-C-

rPACE rPC5 rfurin rpc4-a rpc1 rpc2 rpc7 Consensus

The Mammalian Precursor Convertases 57

ysRRGDLhVt atRRGDLnIn hPRRGDLqIh hPRRGDLaIy ynRRGDLaIh ysRRGDLeIf hPRRGsLelk --RRG-L---

vGtWTLkVtD RGtWTLEl.g eGEWTLEVqD aGDWvLEVyD sGEWvLEIEn qGlWTLglEn RGvYrLvIrD -G---L----

51 EHVQfeaTIe EHVQavITVn EHVvVRIsIS EHVvVRITIt EHVQaRlTlS EHVQVqlslS EHVaVtVsIt EHV------101 MsVHtWGEnp MTtHtWGEDA MTVHCWGEkA MTIHCWGErA MTtHsWdEDp MstHYWdEDp sTVrCWGErA -----W-E--

rPC1 rPC2 rPACE4 rPC5 rFurin rPC4 rPC7 Consensus

rPC1 rPC2 rPACE4 rPC5 rFurin rPC4 rPC7 Consensus

Fig. 3.3. Alignment of the P-domain of rat convertases.

eCIIkdnnfE hCVggsvq.n mCVatadK.r vCVestdr.q KCIIeIla.E KCtIrVvh.t sYVspmlK.E ----------

1 RtWrnVPekk kdWkTVPerf RkWTaVPsQh ekWTTVPqQh qnWTTVapQr RvWlptkpQk kiWTsVPyla -W--------

rPC1 rPC2 rPACE4 rPC5 rFurin rPC4 rPC7 Consensus

msgrMq...N fvgsap...q ipsQvRnpek tpsQLRnfkt ts....eanN kg....yyyN vgd...eplq ----------

LTSaaGTstv MTSPmGTkSi LiSPsGTkSq LTSPsGTRSq LiSPmGTRSt LTSPmGTRSt LfcPsGmmSl -----G----

PrAlkangEV PekIPPtgkl PrsIPvvqvl iKtIrPnsaV PKdIgkrlEV PtpIlPrmlV nKAVPrsphs ----------

eGrivnWkLI kGlLKEWTLm qGkLKEWsLI pGkLKEWsLV yGtLtkFTLV tGtLyycTLl vGiLqqWqLt -G------L-

LLAeReR.Dt LLsrRPRdDd LLAkRl.LDF LLAnRl.FDh LLAaRP.hDY LvAiRP.LDi igApRs.MDs ----R---D-

iveipTrACE vlTLqTnACE RtTalTnACa RsiykasgCs RKT.VTaclg pKn.VTvcCD levLwnvsrt ----------

144 LhGT LhGT LYGT LYGT LYGT LYGT LYGs L-G-

100 SPnGFkNWDF SkvGFdkWpF SnEGFtNWEF SmEGFkNWEF SaDGFNdWaF SgqGYNNWiF dPnGFNdWtF ---G---W-F

50 gqEN.aInsL gkEN.FVrYL DhsdqrVvYL DnpNhhVnYL Epnh..IsrL gsrrrLIrsL DlEmsglktL ---------L

58 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

The Mammalian Precursor Convertases

59

granules, it does not undergo C-terminal truncation into its 66 kDa form and accordingly exits the cell via the constitutive secretory pathway.61 Future work should define whether these results obtained for PC1 are applicable to other members of the PC family which undergo zymogen cleavage in the ER, such as furin, PACE4, PC4 and PC5, and whether the integrity of the RRGDL sequence is also critical for the formation of the C-terminally truncated, granule-associated, 65 kDa PC537 and/or 76 kDa furin.62 In addition, it will be informative to define whether the variant RRGSL motif found in PC7 also plays a similar role for this convertase.

Ontogeny, Tissue Expression and Subcellular Localization Based on their tissue distribution and intracellular localization, the mammalian subtilisin/kexin-like serine proteinases could be subdivided into four classes4 where: 1. furin and the recently discovered PC7 process precursors which reach the cell surface via the constitutive secretory pathway; 2. PC1, PC2 and the isoform PC5-A process precursors whose products are stored in secretory granules; 3. PACE4 and PC5, which are expressed in both endocrine and nonendocrine cells, conceivably process precursors in both the constitutive and regulated secretory pathways; and 4. PC4 is predominantly synthesized in testicular germ cells. The class I convertases furin63,64 and PC736 are localized within the TGN and could cycle to the cell surface and enter endosomal compartments.63 Class II convertases traverse the TGN but are ultimately localized within mature secretory granules.37,65 PC5 could be found in the TGN and/or in granules.37 However, its isoform PC5-B mostly resides in the TGN and cycles to cell surface processing precursors within the constitutive secretory pathway.37 The intracellular localizations of PC4 and PACE4 are not yet known with certainty. Defining the cellular colocalization of each PC with its cognate substrate(s) is necessary in order to ascribe a role for these enzymes in particular precursor processing events.65-71 In addition, dramatic developmental changes in the tissue expression of the PCs have been reported.4,71-74 In the adult, numerous publications dealt with the cell and tissue distribution of the PCs.75-82 They demonstrate a unique distribution of each PC,4,16,17,32 for example in the central nervous system (CNS),70,75-77 pituitary,66,71,77 peripheral nervous system (PNS),78 and some peripheral organs including the heart,79 as well as the thyroid,80 adrenals,29,81 gut and gonads.82 The study of the coregulation of PCs and their cognate substrate(s)17,66,70,71,83-87 also provided hints on their physiological role(s) in vivo. The expression of PCs has been observed in the development of the cerebral cortex, neuromuscular junctions, pituitary gland, pancreatic islets, as well as in the development of cardiovasculature, ossification and chondrification centers. The early expression of PCs suggests their participation in the plasticity of tissue development. However, little is known about the role of specific PCs during embryonic development. For example, it is likely that the growth, differentiation and patterning of embryonic tissue which depends on secretion of growth factors will require proteolytic maturation. Some already published data on the expression of PCs in fetal mice and rats suggest that they are involved in processing of many morphogenetic factors.4,35,73,74,82,87 It is, however, not known which critical steps of organogenesis depend upon PCs, whether they be cell differentiation, cell proliferation or both. Furin is already expressed in both endoderm and mesoderm in the primitive streak stage on embryonic day 7 (e7), while in embryonic ectoderm, from which the nervous system is derived, its mRNA transcripts are undetectable. The overall expression pattern outside the presumptive nervous system is maintained until e10. Higher furin expression is observed in the cardiovasculature and liver primordia, while cephalic regions only express

60

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

low to medium levels of transcripts.73 In the rat, furin is transiently expressed in Rathke’s pouch region from e13 to e18, suggesting a role in development of the future hypophysis.73 The nervous system remains negative for furin until e18 in the rat73 or e16 in the mouse.82 Maximal expression of furin appears postnatally, but is much lower in comparison to that found in some peripheral tissues (Fig. 3.4), including the lacrimal gland, Bowman’s glands in olfactory turbinate tissues, the liver and Bruner’s glands in the gut.82 Finally, appreciable hybridization levels can be seen in chondrification centers, including chondrocytes and their immediate precursors. Based on comparative expression studies, a role of furin, PACE4, PC5 and PC7 in processing of bone morphogenetic proteins has been suggested.35,74,87 In Figure 3.4, we present some original data to compare the ontogeny of furin and PC7 in whole mouse embryos, and in Figure 3.5 we also present evidence that their tissue mRNAs are sometimes coordinately regulated during development. In situ hybridization ontogeny studies using whole body sections provided a clear demonstration for the presence of furin and PC7 transcripts in each and every tissue. These include skin, muscles, bones and tooth, central and peripheral nervous system, endocrine and exocrine tissues, gut and reproductive organs (Fig. 3.4). However, they exhibit a mosaic expression revealing unique and developmentally regulated local concentrations for each convertase. A representative example is the cardiovascular apparatus in mouse embryo on day 10 (e10) in which a relatively high furin hybridization signal is detected (H in Fig. 3.4a), and the signal remains elevated on e18 (Fig. 3.4b), and then declines on postnatal day 16 (p16; Fig. 3.4c). In comparison, the cardiovascular levels of PC7 mRNA are also detectable on e10 and e18, and postnatally they do not decline as dramatically as for furin. On the other hand, on e18 we note the high expression levels overall of both furin (Fig. 3.4b) and PC7 (Fig. 3.4e), which postnatally (p16) exhibit a dramatic decrease (Fig 3.4c and f). These data suggest that both convertases are functionally implicated in the development of striatal muscle (B) within a narrow developmental window, occurring late in uterine life. Interestingly, this period is critical for the formation of neuromuscular junctions, and hence may require the activity of furin and/ or PC7 either directly or as part of an enzymatic cascade (see ADAM family below). Thus, a picture of furin and PC7 distribution cannot be sealed in a rigid frame of ubiquity, but has to be envisaged with notions that admit plasticity, heterogeneity and developmental regulation of expression patterns. Aside from areas which exhibit a transient increase in PCs’ mRNA concentration, one can also observe that in some tissues (e.g., in the stomach’s pyloric glands) both furin and PC7 transcripts show elevated levels on p16 (Fig. 3.4c and f). This result indicates that, in the adult, PC7 and furin may also be relevant to normal tissue physiology. In order to further probe the developmental changes that occur from birth up to adulthood, in Figure 3.5, Northern blots compare the relative levels of PC7 and furin in selected mouse tissues, revealing cell-specific trends in mRNA expression levels. Thus, in the thymus, brain and male submaxillary gland, mRNA levels of PC7 and/or furin decrease from p1 to p42 (young adults), suggesting that they are developmentally downregulated in these tissues. However, the levels of these PCs do not appreciably change in liver, duodenum and in the cardiac section of the stomach especially for PC7. In contrast, within this postnatal period, dramatic increases in PC7 and furin mRNA levels are observed within the glandular portion of the stomach (pyloric region) and in lacrimal glands. It is interesting that the observed dramatic rise in PC mRNAs occurs in tissues potentially exposed to external pathogens and which specialize in neutralizing the toxic effects of the hostile environment. Thus, the predicted involvement of PCs in host defense mechanisms deserve further studies. More confined expression patterns have been found for PC1 and PC2 mRNAs in embryos, indicating specialization within limited structures. They are already evident by midgestation (e10 and e11) in pancreatic primordia.72 Expression in pituitary primordia and in

The Mammalian Precursor Convertases

61

Fig. 3.4. In situ hybridization histochemistry (ISH) showing furin (a-c) and PC7 (d-f) mRNA expression sites at the anatomical level in mouse embryos on day 10 (e10) and 18 (e18) and on postnatal day 16 (p16), using antisense (as) (a-f) and, to exemplify the extent of nonspecific hybridization, control sense (ss) riboprobes (g-i). A hybridization signal was detected in all tissues studied, exhibiting some heterogeneity in terms of its local concentration. Whereas most tissues were labeled with furin and PC7 on e10, high signal was observed in embryonic cardiovasculature with furin only (H in a). On e18 (b) furin mRNA is detectable in all tissues, with a low signal in brain and spinal cord (Br) and high level hybridization in visceral tissues, striated muscles (Mu) and mandibular bone rudiments (M). The PC7 hybridization pattern on e18 (e) resembles that of furin, with the exception of thymus (Th) which was strongly labeled. ISH on p16 reveals PCspecific patterns of furin (c) and PC7 (f) mRNA distribution. High concentrations of furin mRNA were found in the olfactory neuroepithelium within olfactory turbinates (OT), submaxillary gland (SM), liver (L) stomach pyloric region (Stp), and kidney (k), but not cardiac region (Stc) nor kidney (K). PC7 mRNA was in general less abundant on p16 and, with the exception of the thymus (Th), its distribution resembled that of furin. Magnification x 3.5; bar in (e) = 1cm.

some neuronal structures including the cortical plate has been observed 1 or 2 days later. During the later periods of embryonic development (e16-e19), the expression of PC1 and PC2 reaches appreciable levels both in intensity and extent in the future brain, spinal cord and cranial ganglia, including the trigeminal ganglion (TriG) and spinal ganglia.78 PC1 expression seems to increase maximally by adulthood, while higher levels of PC2 expression are found postnatally in the brain78 and pituitary gland, showing transient presence in corticotrophs.71 Interestingly, PC2 expression coincides with short-time span production of des-acetyl-α-MSH in the same corticotrophs, suggesting that at a high level of this particular convertase, ACTH1-39 processing into ACTH1-17 and CLIP takes place.71 Thus, analysis of temporal expression patterns provides insights into physiologically-relevant functions of PCs. Transient production of des-acetyl-α-MSH in neonatal corticotrophs is an example.

62

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Fig. 3.5. Northern blot analysis showing the levels of (A) furin and (B) PC7 mRNA during postnatal developmental stages in mice on postnatal days 1, 16 and 42. The selected tissues are: thymus, brain, male submaxillary gland (S/MAX), liver, duodenum (DUOD), stomach cardiac (ST C) and stomach pyloric (ST P) regions, as well as lachrymal gland (LACR GL). 5 µg of total RNA were loaded in each lane. The X-ray film was exposed for 3 days. Northern blots were obtained with cRNA probes, as described.32,70,76 The observed sizes of the mRNAs of furin and PC7 are 4.4 and 3.9 kb, respectively. The migration position of the 28S ribosomal RNA is indicated.

PC5 expression has been noted in mouse fetal and postnatal ontogeny. A strong PC5 hybridization signal is detected in developing intestine tissue. Only a few telencephalic and mesencephalic centers in the spinal cord synthesize PC5. Its expression appears to be associated with connective tissue around the cardiovascular apparatus and ossification centers35,74,82 and with the exception of ribs, in regions of developing bone.87 These correlative data suggest an important role of PC5 in processing of factors involved in bone morphogenesis, such as the TGFβ-like bone morphogenic proteins BMP-2 and BMP-435,87 and the lefty and nodal proteins which are involved in establishing left-right asymmetry in the mouse.88 In a very recent study, PC5 was found to be expressed both during implantation and embryogenesis.87 Thus, strong PC5 mRNA expression was detected in maternal decidua during implantation, overlapping the region where TGFβ family members and the inhibitor TIMP-3 are expressed.87 This is the first example of the possible role of a PC in implantation and hence, a participation in the uterine-embryo dialogue that requires endocrine-mediated interactions between the embryo and the uterus for implantation to succeed. In summary, PCs demonstrate a remarkable temporal and spatial specificity of expression patterns. Based on this, it seems that they are available in various proportions and combinations in different loci. This raises questions about their redundancy vs. their specialization. Since the PC family actually counts 7 paralogs (Fig. 3.1) with some exhibiting additional isoforms produced by alternative splicing (PACE4 and PC5), the distribution picture remains open to future exploration. Essential steps for understanding PC functions in embryogenesis would involve their modulation in living embryos, with subsequent observation of changes in cell differentiation and organogenesis.

Structure, Loci, and Evolution of PC Genes The intron-exon organization of the genes for furin, PC1, PC2 and PC4 has been determined.89-92 It shows a remarkable conservation of exon order and size in the catalytic and

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63

P domains, an additional indication of a common origin of these genes. The Asp, His and Ser of the catalytic triad are each contained within a distinct exon, in line with the hypothesis that exons define functional domains. The Asn of the oxyanion hole is also contained within an exon. In the human PC2 gene, however, the homologous exon is interrupted; and the Asp of the oxyanion hole is located in the 3' portion of the split exon.90 The number and the size of exons flanking the conserved domains are more variable. They probably carry domains that determine properties unique to each enzyme, including its subcellular location, its cleavage site preference and its half life.93 For furin, PC1 and PC4, the use of alternate promoters, splicing sites or polyadenylation signals during transcription generates optional exons found only in some of their mRNA isoforms.91,92,94 The differences in size among PC genes largely result from variation of intron lengths. In this regard, the PC4 gene is noticeable by its short size (about 7 kb)92 and the PC2 gene by its large size (about 130 kb).90 The conservation of the catalytic and P domains and the variability around these domains suggests that PC genes evolved from a common ancestral gene through various mechanisms including duplications, translocations, insertions or deletions. Except for furin and PACE4, which are closely linked, all the other PCs gene are dispersed on various chromosomes (Table 3.7) (reviewed in ref. 95). The synteny between mouse and human for all the chromosomal regions carrying PC loci suggests that their multiplication and divergence occurred before the branching apart of human and murine evolutionary lines about 80 million years ago.

Antisense Transgene Inhibition Cellular Functions of PCs The multiplicity of PC genes suggests both redundancy and differentiation of functions among their products. Redundancy serves the survival of a biological system while differentiation affords it complexity. The challenge in such a case is in the delimiting of the degree of overlap and distinctiveness. Co-expression of substrates and convertases in transfected cells provided invaluable insights into the substrate and cleavage site preferences of many convertases. Some of these relationships have been confirmed in cells expressing transgene-directed antisense RNA that blocks endogenous expression of a convertase. Using this approach, Bloomquist et al84 showed that PC1 deficiency induced by antisense RNA in AtT20 cells caused POMC to accumulate and its conversion to ACTH to diminish, supporting a critical role of PC1 in the production of this corticotrophic hormone.96,97 Similarly, transgene-directed antisense RNA has been used to demonstrate the converting action on several other precursors in various cell types: furin on proparathyroid hormone-related peptide (proPTH-RP) in monkey kidney COS-7 cells;98 PC2 on POMC in rat somatomammotrope GH3;99 PC1 and PC2 on proglucagon in mouse glucagonoma aTC1-6 cells;100,101 PC2 on proneuropeptide Y (proNPY) in cultured sympathetic neurons;102 PC2 on proneurotensin (proNT) in rat medullo-thyroid carcinoma rMTC 6-23 cells;103 and PC1 on procholecystokinin (proCCK) in rat insulinoma Rin5F cells.104 In human colon carcinoma LoVo cells, spontaneous mutations have affected both alleles of the furin gene, making them deficient in furin activity.105,106 These cells are unable to process receptors for insulin-like growth factor (IGF-R) and hepatocyte growth factor (HGF-R),105,106 while they still can convert HIV gp160,107 probably through the action of coresident PACE4 and PC7.16,32 A dependence of IGF-R processing on furin may explain the anti-proliferative effect of antisense transgene or oligonucleotide on mouse gastric mucus GDM6 cells108 and on the well differentiated pancreatic beta cell line MIN6.109

20p (c)

15q (c)

15q (c, s)

11q (c, s)

PCSK2

PCSK3 PCSK4

PCSK5 PCSK6

PCSK7

PC2

Furin

PC5

PACE4

PC7

Pcsk7

Pcsk5 Pcsk6

Pcsk3 Pcsk4

Pcsk2

Pcsk1

Locus Symbol

9 (l)

7 (l)

19 (l)

10 (l)

7 (c, l)

2 (c, l)

13 (c, l)

Chr (c, s, l)

Mouse (m)

nd

nd

nd

7 (m)

12 (h)

> 130 (h)

35 (h), 41 (m)

Size (kb)

nd

nd

nd

15 (m)

16 (h)

12 (h)

14 (h) 15 (m)

No of Exons

nd

nd

nd

Reduced male fertility, preimplantation embryonic lethality (m)

Embryonic dysmorphisms and lethality (m)

Slow growth rate, hyperplasia of pancreatic alpha and delta cells (m)

Obesity, hypogonadotropic hypogonadism, hypercortisolism (h)

Gene Structure (Species) Morbid Phenotype (Species)

PC7. References on chromosomal localizations were reviewed by Mbikay et al (95). Localization was either by cytogenetic (c) methods (including in situ hybridization), by analysis of somatic (s) cell hybrids or by linkage (l) analysis in human (h) or mouse (m).

aReferences on PC gene structure and on morbid phenotypes of PC mutations are presented in the text. This has not been determined (nd) for PC5, PACE4 and

11q (c, s)

19 (s)

5q (c)

PCSK1

PC1

PC4

Chr (c, s, l)

Human (h)

Chromosomal Localization

Locus Symbol

PC

Table 3.7. PC Genes: chromososomal localization, structure, and phenotypic consequences of their inactivationa

64 Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

The Mammalian Precursor Convertases

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Implication of PCs in Disease States: The Model of Atherosclerosis The endovascular cell proliferation causing atherosclerosis is due in part to the activation of growth factors. Among them, many are produced as inactive precursors which are cleavable by one or more PCs (Table 3.1), e.g., platelet-derived-growth factors (PDGFs), epidermal-growth-factor (EGF), insulin-like growth factors (IGFs) and their receptors and transforming growth factors (TGFs). These molecules require activation by limited proteolysis at the motif (K/R)-Xn-R (where n = 0, 2, 4, 6). Based on the fact that high levels of PC5 transcripts were detected in cells lining rat coronary vessels,79 we explored the possibility that PC5 may play an important role in human arterial restenosis.110 Thus, by in situ hybridization histochemistry the PC expression in normal and atherosclerotic human coronaries revealed that occluded vessels exhibit a marked increase in the level of PC5 mRNA within smooth muscle cells, whereas coronaries without occlusions were PC5 negative. Accordingly, incubation of rabbit smooth muscle cells with a 17-mer PC5-specific antisense oligonucleotide caused a dose-dependent inhibition of cellular proliferation with a maximal effect of 81.6% ± 1.6% at 10 µM, a dose causing a significant reduction in the protein level of PC5.110 The results of this in vitro assay were further extended using an in vivo model of induced injury of rabbit carotid arteries. In this model, the selected PC5-derived 17-mer antisense phosphorothioate oligonucleotide caused a 50% inhibition of neointimal hyperplasia when compared to sense or random oligonucleotides.110 These data suggest that PC5 (and possibly other PCs) could play an important role in the development of arterial restenosis following injury to blood vessels.

Heritable Deficiency of PC in Human and Mouse Oftentimes in contemporary biology, efforts to understand the physiological function of a specific molecule gains significant momentum with the discovery of spontaneous mutant organisms or the production of induced ones. When a female patient with history of massive childhood obesity, hypogonadotropic hypogonadism and secondary hypocortisolism was also found to have large amounts of circulating proinsulin and des-64,65 proinsulin, a possible defect in PC1 was immediately suspected.111 This presumption has been recently confirmed: The patient is a compound PC1 heterozygote, carrying a missense mutation on one allele and a splicing mutation on the other allele.112 The product of the first allele carries a Gly→Arg483 mutation which leads to proPC1 retention in the ER. The second mutation on the second allele affects the donor site of intron 5 and causes skipping of exon 5 during transcription, resulting in the loss of 26 amino acids, a frameshift and creation of a premature stop codon within the catalytic domain, thus leading to an inactive PC1.112 The clinical picture of this patient clearly implicates PC1 in the intricate network of signaling pathways that control body mass and gonadal development, most likely through its processing/activating action on prohormones (proinsulin and POMC) and proneuropeptides. With a mouse model of PC1 deficiency, it should be possible to determine which proneuropetides fail to be appropriately processed and to assess their implication in the pathology by replacement therapy. The PC2 locus was recently inactivated in mouse by homologous recombination.113 Besides a small decrease in growth, the mouse is otherwise normal. However, pancreatic islets in these mice are deficient in their processing of precursors to glucagon, insulin and somatostatin. This deficiency is associated with a secondary hyperplasia of α and δ cells. The mild phenotype resulting from PC2 inactivation is so in contrast with its relative abundance in most neuronal cells, and some endocrine cells, that one is attempted to ascribe to this enzyme a backup role for PC1, its coresident homolog in many of these cells. These results imply that a PC1-null mouse may present a viable, albeit more morbid, phenotype and that PC1/PC2 double-nulls may not be viable.

66

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

Targeted inactivation of the PC4 locus in mouse results in a significant decrease in male fertility, consistent with its restricted expression in spermatogenic cells.114 Spermatogenesis is apparently normal in this mouse. In vitro, its spermatozoa are less efficient at fertilizing ova, and zygotes derived from them fail to develop to the blastocyst stage, suggesting that PC4 activates proproteins that are necessary for fertilization as well as early embryonic development. Whether the PC4 gene is expressed at these early stages remains to be determined. Furin, whose transcripts become detectable on embryonic day e7, is apparently expressed very early during development.73,74 It certainly plays a critical role in embryogenesis since its targeted inactivation leads to dysmorphic embryos that die on e11.5.115 The observed dysmorphisms lend support to the experimental data derived from cells transfected with inducible furin sense or antisense transgenes, which show that furin level influences the equilibrium between cell proliferation and cell differentiation.108,109 In conclusion, the availability of these PC mutant mice enriches our understanding of their biological functions and provides useful models of human pathologies, ultimately leading to the identification of their physiological substrates.

Inhibitors of PCs In view of the potential clinical and pharmacological role of the convertases,93 it was of interest to produce specific PC inhibitors. The proposed strategies involved the development of either peptide-based PC inhibitors,116-119 or protein-based inhibitors.120,121 So far, the in vitro peptide-based approach has not succeeded in effectively inhibiting the PCs intracellularly, and more work in this direction is needed in order to improve the cellular permeability of the designed inhibitors. In humans, the 394 amino acid α1-antitrypsin (α1-AT) is the physiological inhibitor of neutrophil elastase.122 A naturally occurring mutation, known as α1-AT-Pittsburgh (α1-PIT), at the α1-AT reactive site AIPM358 into AIPR358, changed the specificity of this serpin from an inhibitor of elastase into an inhibitor of thrombin.123 Consistent with the critical importance of Arg at the P1 and P4 positions of substrates recognized by furin,124,125 a second mutation in α1-PIT giving the sequence RIPR358 was engineered by Anderson et al, resulting in a potent inhibitor of this convertase.120 This new variant, called α1-AT-Portland (α1-PDX), was shown to inhibit 50% of the furin-catalyzed in vitro cleavage of a small fluorogenic substrate at 0.5 nM concentrations.120 Furthermore, Decroly et al demonstrated that, in vitro, α1-PDX is an inhibitor of all tested PCs.68 However, Vollenweider et al126 demonstrated that in AtT20 cells α1-PDX only partially inhibits the endogenous processing of gp160 or exogenous processing by furin, PACE4 and PC5-B. This observation was recently rationalized by the fact that α1-PDX acts primarily within the constitutive secretory pathway.127 Biosynthetic and immunocytochemical analyses of AtT20 cells stably transfected with α1-PDX cells demonstrated that this 64 kDa serpin is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as an inactive 56 kDa product resulting from cleavage of the active 64 kDa form at the engineered RIPR358 site.127 Furthermore, expression of α1-PDX resulted in modified contents of mature secretory granules with increased levels of partially processed products, suggesting a delayed processing. Accordingly it became apparent that α1-PDX may not inhibit the processing of all precursors to a similar extent and that processing inhibition occurs primarily within the constitutive secretory pathway. Therefore, α1-PDX is a very useful lead protein to inhibit processing of precursors including endogenous growth factors and imported viral surface glycoproteins in constitutive cells, and may exhibit a limited toxicity to cells in vivo. Finally, further variations in the structure of this serpin may lead to a more specific inhibitor which may better discriminate between the convertases.

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Swapping of the pro-domains of PCs demonstrated that the prosegment of PC1 can replace that of PACE4128 and that the furin prosegment can replace that of either PC1 or to a lesser extent that of PC2.128 However, the prodomains of PC1 and PC2 are not interchangeable and the prodomain of PC2 cannot replace that of furin.129 These results further emphasize the uniqueness of PC2, which is the only convertase undergoing pro-segment removal late along the secretory pathway (TGN/immature granules), which has an Asp instead of the usual Asn at the site of the oxyanion hole, and which requires the participation of a specific binding protein 7B2 for its efficient zymogen activation. Nevertheless, these domain-swap data suggested that some of the pro-segments are interchangeable and hence could act as inhibitors of more than one PC. Accordingly, in an effort to design specific protein-based inhibitors which would selectively inhibit the various members of the PC family, the potency and selectivity of their pro-segments were tested in vitro. Thus, a recent report demonstrated that the prosegment of furin exhibits in vitro an inhibitory activity on furin with a K0.5 of 14 nM when used as a fusion protein to glutathione S-transferase.130 We have also recently shown that the pro-segment of PC7 itself when purified from bacterial cultures is a very potent slow binding inhibitor of PC7 in vitro, exhibiting a marked preferential inhibition of PC7 over furin (M. Zhong, J. S. Munzer, N.G. Seidah, unpublished data). Additionally, Lazure et al demonstrated that the pro-segment of PC1 is also a very potent inhibitor of this enzyme in vitro131 and Basak et al132 showed that synthetic peptides derived from the prosegments of proprotein convertase PC1 and furin are potent inhibitors of both proteinases. A recently identified peptide-based inhibitor which shows promise in terms of selectivity and potency is the C-terminal 31 amino acid of the PC2-specific binding protein 7B2,51,52,133,134 called CT-peptide.132 This peptide is highly selective for PC2 and requires the presence of the Lys-Lys bond in the CT-peptide of 7B2 for maximal activity.135 Another very recently identified inhibitory polypeptide is the C-terminal segment of PC1, which is a potent inhibitor of this enzyme, dampening its activity on certain substrates, e.g., proRenin, until the enzyme/substrate complex reaches immature secretory granules.136 In conclusion, future efforts to understand the selectivity embedded within the prosegment, C-terminal sequence of certain PCs and of 7B2's CT-peptide may shed some light on the structures needed to achieve both potent and selective PC inhibition.

Enzymatic Cascades: ADAM Family and PCs In studies involving the definition of the cleavage specificity of the PCs, human aspartyl protease proRenin, responsible for the processing of angiotensinogen into angiotensin I, was shown to be processed quite efficiently by either PC141 or PC5.137 This was the first example of a cascade phenomenon where a PC would activate an enzyme which in turn would process another enzyme precursor. The second example came from the work on the human metalloproteinase Stromelysin-3 (Table 3.1), which is effectively processed by either furin or PACE4.138,139 In a similar vein, in vitro furin can activate the type I membraneassociated matrix metalloproteinase MT1-MMP at the Arg-Arg-Lys-Arg111 ↓ Tyr112-Ala site,140 though the authors report that another enzyme found in CHO-cells can process MT1-MMP intracellularly at the alternative Arg-Arg-Lys-Arg-Tyr112 ↓ Ala113-Ile site,140 a motif similar to that recognized in SREBPs (Table 3.2). Recently, a new family of type I membrane-bound metalloendopeptidases was identified. The members of this so called “ADAM” family contain both a disintegrin and metalloprotease domain. This is an emerging gene family, which in the mouse genome consists of at least 20 members (for review see ref. 141). As prototype paralogs of this family we can mention the fertilins α (ADAM-1) and β (ADAM-2), which are proteins at the surface of sperm implicated in sperm-egg binding during fertilization via an interaction with an

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

68

integrin α6β1 receptor.142 Our results in PC4-null mice suggested that the processing of fertilins, and/or Cyritestin (ADAM-3) could be affected in these mice (ref. 88 and M. Mbikay, unpublished results) and thus provide other examples of enzymatic cascades, whereby a PC would activate an ADAM protease, which in turn would cleave other substrates. Two other examples emerged recently suggesting the generality of this mechanism. One of them involves tumor necrosis factor-alpha-converting enzyme (TACE), which processes membranebound proTNFα to TNFα at the cell surface during an inflammatory response.143,144 In this example, proTACE is first processed intracellularly into active TACE at the type I precursor sequence Arg-Val-Lys-Arg214 ↓ Arg-Ala143 (Table 3.1). The other example involves the likely furin activation of the novel ADAM protease KUZ (Table 3.1), needed for the downstream processing of the Notch protein.145 The latter encodes an ≈300 kDa transmembrane protein146 that acts as a receptor in a cell-cell signaling mechanism controlling cell fate decisions throughout development.146,147 Finally, since proteolytic cleavage at pairs of basic residues found at interdomain boundaries of ADAM proteinases regulates some of their functions, the PCs are likely to exert a key regulatory role by processing various ADAM precursors.

Conclusions The recent identification of a family of serine proteinases of the subtilisin/kexin-type and proof of their function(s) as intracellular processing enzymes recognizing the general motif Arg-(X)n-Arg↓ where n = 0, 2, 4 or 6, resolved a longstanding quest for some of the precursor convertases. Other processing sites, including those occurring at single or pairs of hydrophobic and/or small amino acids are yet to be identified. Furthermore, the generation of PC1-, PC2-, PC4- and furin-null animals suggested that while a limited redundancy in the in vivo activities of these enzymes is possible, convertases expressed early during development (e.g., furin) are indispensable and their silencing inevitably leads to a lethal phenotype. Structure-function and cellular localization studies performed on the PCs exposed the functional complexity of their various domains and the likely cooperativity between some of them. What is emerging are complex, well orchestrated, genetic events, mRNA regulation and protein-protein interactions that regulate the temporal expression, tissue and intracellular localization, and the fine substrate specificity of processing events. Additionally, the involvement of new convertases, distinct from the PCs (see chapters 2, 5, and 7), further emphasize the complexity of regulatory steps needed for high fidelity and efficient precursor processing. Medical applications of this new, but still incomplete and fragmented, knowledge are now beginning to take shape, and it is hoped that in the future these will lead to novel rational therapeutical approaches in a number of pathologies, including cancer, neurological disorders, proliferative diseases and opportunistic pathogenic infections.

Acknowledgments This work was supported by grants from Medical Research Council of Canada (GR 11474), NeuroScience Network and Protein Engineering Network of Centres of Excellence (PENCE). We thank Jon Scott Munzer for Figure 3.1.

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41. Benjannet S, Reudelhuber T, Mercure C, Rondeau N, Chrétien M, Seidah NG. Pro-protein conversion is determined by a multiplicity of factors including convertase processing, substrate specificity and intracellular environment. J Biol Chem 1992; 267:11417-11423. 42. Benjannet S, Rondeau N, Paquet L, Boudreault A, Lazure C, Chrétien, Seidah NG. Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: Glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2. Biochem J 1993; 294:735-743. 43. Lindberg I. Evidence for cleavage of the PC1/PC3 pro-segment in the endoplasmic reticulum. Mol Cell Neurosci 1994; 5:263-268. 44. Milgram SL, Mains RE. Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins. J Cell Sci 1994; 107:737-745. 45. Rehemtulla A, Barr PJ, Rhodes CJ, Kaufman RJ. PACE4 is a member of the mammalian family that has overlapping but not identical substrate specificity to PACE. Biochemistry 1993; 32:11586-11590. 46. Zhong M, Benjannet S, Lazure C, Munzer S, Seidah NG. Functional analysis of human PACE4-A and PACE4-C isoforms: Identification of a new PACE4-CS isoform. FEBS Lett 1996; 396:31-36. 47. Mains RE, Berard CA, Denault JB, Zhou A, Johnson RC, Leduc R. PACE4-a subtilisin-like endoprotease with unique properties. Biochem J 1997; 321:587-593. 48. Hsi KL, Seidah NG, De Serres G, Chrétien M. Isolation and NH2-terminal sequence of a novel porcine anterior pituitary polypeptide: Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60. FEBS Lett 1982; 147:261-266. 49. Seidah NG, Hsi KL, De Serres G, Rochemont J, Hamelin J, Antakly T, Cantin M, Chrétien M. Isolation and NH2-terminal sequence of a highly conserved human and porcine pituitary protein belonging to a new superfamily: Immunocytochemical localization in pars distalis and pars nervosa of the pituitary and in the supraoptic nucleus of the hypothalamus. Arch Biochem Biophys 1983; 225:525-534. 50. Martens GJ, Braks JA, Eib DW, Zhou Y, Lindberg I. The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2. Proc Nat Acad Sci USA 1994; 91:5784-5787. 51. Braks JAM, Martens GJM. 7B2 is a neuroendocrine chaperone that transiently interacts with prohormone convertase PC2 in the secretory pathway. Cell 1994; 78:263-273. 52. Benjannet S, Savaria D, Chrétien M, Seidah NG. 7B2 is a specific intracellular binding protein of the prohormone convertase PC2. J Neurochem 1995; 64:2303-2311. 53. Benjannet S, Lusson J, Hamelin J, Savaria D, Chrétien M, Seidah NG. Structure-function studies on the biosynthesis and bioactivity of the precursor convertase PC2 and the formation of the PC2/7B2 complex. FEBS Lett 1995; 362:151-155. 54. Seizen RJ, Leunissen JAM. Subtilases: The superfamily of subtilisin-like serine proteases. Prot Sci 1997; 6:501-523. 55. Gluschankof P, Fuller R. A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease. EMBO J 1994; 13:2280-2288. 56. Taylor NA, Shennan KIJ, Cutler DF, Docherty K. Mutations within the propeptide, the primary cleavage site or the catalytic site, or deletion of C-terminal sequences, prevents secretion of proPC2 from transfected COS-7 cells. Biochem J 1997; 321:367-373. 57. Hatsuzawa K, Murakami K, Nakayama K. Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. J Biochem (Tokyo) 1992; 111:296-301. 58. Creemers JWM, Siezen RJ, Roebroek AJM, Ayoubi TAY, Huylebroeck D, van de Ven WJM. Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. J Biol Chem 1993; 268:21826-21834. 59. Creemers JWM, Usac EF, Bright NA, van de Loo JW, Jansen E, van de Ven, WJM, Hutton JC. Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. J Biol Chem 1996; 271:25284-25291.

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60. Ruoslahti E. RGD and other recognition sequences for integrins [review]. Annu Rev Cell Develop Biol 1996; 12:697-715. 61. Lusson J, Benjannet S, Hamelin J, Savaria D, Chrétien M, Seidah NG. The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing, and for its cellular trafficking. Biochem J 1997; 326:737-744. 62. Hill RM, Ledgerwood EC, Brennan SO, Pu LP, Loh YP, Christie DL, Birch NP. Comparison of the molecular forms of the kex2/subtilisin-like serine proteases SPC2, SPC3, and furin in neuroendocrine secretory vesicles reveals differences in carboxyl-terminus truncation and membrane association. J Neurochem 1995; 65:2318-2326. 63. Sariola M, Saraste J, Kuismanen E. Communication of post-golgi elements with early endocytic pathway: Regulation of endoproteolytic cleavage of semliki forest virus p62 precursor. J Cell Sci 1995; 108:2465-2475. 64. Shapiro J, Sciaky N, Lee J, Bosshart H, Angeletti RH, Bonifacino JS. Localization of endogenous furin in cultured cell lines. J Histochem Cytochem 1997; 45:3-12. 65. Malide D, Seidah NG, Chrétien M, Bendayan M. Electron microscopy immunocytochemical evidence for the involvement of the convertases PC1 and PC2 in the processing of proinsulin in pancreatic β-cells. J Histochem Cytochem 1995; 43:11-19. 66. Day R, Schäfer MKH, Watson SJ, Chrétien M, Seidah NG. Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary. Mol Endocrinol 1992; 6:485-497. 67. Wetsel WC, Liposits Z, Seidah NG, Collins S. Expression of candidate pro-GnRH processing enzymes in rat hypothalamus and an immortalized hypothalamic neuronal cell line. Neuroendocrinology 1995; 62:166-177. 68. Decroly E, Wouters S, Di Bello C, Lazure C, Ruysschaert J-M, Seidah NG. Identification of the paired basic convertases implicated in HIV gp160 processing based on in vitro assays and expression in CD4+ cell lines. J Biol Chem 1996; 271:30442-30450. 69. Schaner P, Todd RG, Seidah NG, Nillni E. Processing of prothyrotropin-releasing hormone by the family of prohormone convertases. J Biol Chem 1997; 272:19958-19968. 70. Dong W, Seidel B, Marcinkiewicz M, Chrétien M, Seidah NG, Day R. Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: Selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids. J Neurosci 1997; 17:563-575. 71. Marcinkiewicz M, Day R, Seidah NG, Chrétien M. Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin. Proc Natl Acad Sci USA 1993;. 90:4922-4926. 72. Marcinkiewicz M, Ramla D, Seidah NG, Chrétien M. Developmental expression of the prohormone convertases PC1 and PC2 in mouse pancreatic islets. Endocrinology 1994; 135:1651-1660. 73. Zheng M, Streck RD, Scott REM, Seidah NG, Pintar JE. The developmental expression in rat of proteases furin, PC1, PC2, and carboxypeptidase E: Implications for early maturation of proteolytic processing capacity. J Neurosci 1994; 14:4656-4673. 74. Zheng M, Seidah NG, Pintar JE. The developmental expression in the rat CNS and peripheral tissues of proteases PC5 and PACE4 mRNAs: Comparison with other proprotein processing enzymes. Dev Biol 1997; 181:268-283. 75. Schäfer MKH, Day R, Cullinan WE, Chrétien M, Seidah NG, Watson SJ. Gene expression of prohormone and proprotein convertases in the rat CNS: A comparative in situ hybridization analysis. J Neurosci 1993; 13:1258-1279. 76. Day R, Schäfer, MKH, Cullinan WE, Watson SJ, Chrétien M, Seidah NG. Region specific expression of furin mRNA in the rat brain. Neurosci Lett 1993; 149:27-30. 77. Dong W, Marcinkiewicz M, Vieau D, Chrétien M, Seidah NG, Day R. Distinct mRNA expression of the highly homologous convertases PC5 and PACE4 in the rat brain and pituitary. J Neurosci 1995; 15:1778-1796. 78. Marcinkiewicz M, Seidah NG, Chrétien M. Les convertases des prohormones et le système nerveux. Médecine/Sciences 1993; 9:553-561.

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79. Beaubien G, Schäfer MKH, Weihe E, Dong W, Chrétien M, Seidah NG, Day R. The distinct gene expression of the pro-hormone convertases in the rat heart suggests potential substrates. Cell Tissue 1995; Res 279:539-549. 80. Hendy GN, Bennett HPJ, Gibbs BF, Lazure C, Day R, Seidah NG. Proparathyroid hormone is preferentially cleaved to parathyroid hormone by the prohormone convertase furin: A mass spectrometric study. J Biol Chem 1995; 270:9517-9525. 81. Seidah NG, Day R, Chrétien M. The family of pro-hormone and pro-protein convertases. Biochem Soc Trans 1993; 21:685-691. 82. Marcinkiewicz M, Seidah NG, Chrétien M. Implications of the subtilisin/kexin-like precursor convertases in the development and function of nervous tissues. Acta Neurobiol Exp 1996; 56:287-298. 83. Alarcon C, Lincoln B, Rhodes CJ. The biosynthesis of the subtilisin-related proprotein convertase PC3, but not that of the PC2 convertase, is regulated by glucose in parallel to proinsulin biosynthesis in rat pancreatic islets. J Biol Chem 1993; 268:4276-4280. 84. Bloomquist BT, Eipper BA, Mains RE. Prohormone-converting enzymes—Regulation and evaluation of function using antisense RNA. Mol Endocrinol 1991; 5:2014-2024. 85. Johnson RC, Darlington DN, Hand TA, Bloomquist BT, Mains RE. PACE4: A subtilisinlike endoprotease prevalent in the anterior pituitary and regulated by thyroid status. Endocrinology 1994; 135:1178-1185. 86. Oyarce AM, Hand TA, Mains RE, Eipper BA. Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary. J Neurochem 1996; 67:229-241. 87. Rancourt SL, Rancourt DE. Murine subtilisin-like proteinase SPC6 is expressed during embryonic implantation, somitogenesis and skeletal formation. Dev Genet 1997; 21:75-81. 88. Meno C, Saijoh Y, Hideta F, Ikeda M, Yokoyama T, Yokoyama M, Toyoda Y, Hamada H. Left-right aymmetric expression of the TGF-β-family member lefty in mouse embryos. Nature 1996; 381:151-155. 89. Barr PJ, Mason OB, Landsberg KE, Wong PA, Kiefer MC, Brake AJ. cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues. DNA Cell Biol 1991; 10:319-328. 90. Ohagi S, LaMendola J, LeBeau MM, Espinosa R, Takeda J, Smeekens SP, Chan SJ, Steiner DF. Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues. Proc Natl Acad Sci USA 1992; 89:977-4981. 91. Ftouhi N, Day R, Mbikay M, Chrétien M, Seidah NG. Gene organization of the mouse pro-hromone and pro-protein convertase PC1. DNA Cell Biol 1994; 13:395-407. 92. Mbikay M, Raffin-Sanson ML, Tadros H, Sirois F, Seidah NG, Chrétien M. Structure of the gene for the testis-specific proprotein convertase 4 and of its alternate messenger RNA isoforms. Genomics 1994; 20:231-237. 93. Chrétien M, Mbikay M, Gaspar L, Seidah NG. Proprotein convertases and the pathophysiology of human diseases: prospective considerations. Proc Assoc Am Physicians 1995; 107:47-66. 94. Ayoubi TAY, Creemers JWM, Roebroek AJM, Vandeven WJM. Expression of the dibasic proprotein processing enzyme furin is directed by multiple promoters. J Biol Chem 1994; 269:9298-9303. 95. Mbikay M, Seidah NG, Chrétien M, Simpson EM. Chromosomal assignment of the genes for proprotein convertases PC4, PC5 and PACE4 in mouse and human. Genomics 1995; 26:123-129. 96. Benjannet S, Rondeau N, Day R, Chrétien M, Seidah NG. PC1 and PC2 are pro-protein convertases capable of cleaving POMC at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-3568. 97. Thomas L, Leduc R, Thorne BA, Smeekens SP, Steiner DF, Thomas G. Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 1991; 88:5297-5301. 98. Liu B, Goltzman D, Rabbani SA. Processing of pro-PTHRP by the prohormone convertase, furin: Effect on biological activity. Am J Physiol 1995; 268:E832-E838.

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99. Friedman TC, Cool DR, Jayasvasti V, Louie D, Loh YP. Processing of pro-opiomelanocortin in GH3 cells: Inhibition by prohormone convertase 2 (PC2) antisense mRNA. Mol Cell Endocrinol 1996; 116:89-96. 100. Rouillé Y, Westermark G, Martin SK, Steiner DF. Proglucagon is processed to glucagon by prohormone convertase PC2 in alpha TC1-6 Cells. Proc Natl Acad Sci USA 1994; 91:3242-3246. 101. Rothenberg ME, Eilertson CD, Klein K, Mackin RB, Noe BD. Evidence for redundancy in propeptide/prohormone convertase activities in processing proglucagon: An antisense study. Mol Endocrinol 1996; 10:331-341. 102. Paquet L, Massie B, Mains RE. Proneuropeptide Y processing in large dense-core vesicles: Manipulation of prohormone convertase expression in sympathetic neurons using adenoviruses. J Neurosci 1996; 16:964-973. 103. Rovère C, Barbero P, Kitabgi P. Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin. J Biol Chem 1996; 271:11368-11375. 104. Yoon JY, Beinfeld MC. Prohormone convertase 1 is necessary for the formation of cholecystokinin 8 in Rin5f and STC-1 cells. J Biol Chem 1997; 272:9450-9456. 105. Takahashi S, Kasai K, Hatsuzawa K, Kitamura N, Misumi Y, Ikehara Y, Murakami K, Nakayama K. A mutation of furin causes the lack of precursor-processing activity in human colon carcinoma LoVo cells. Biochem Biophys Res Commun 1993; 195:1019-1026. 106. Takahashi S, Nakagawa T, Kasai K, Banno T, Duguay SJ, Van de Ven WJM, Murakami K, Nakayama K. A second mutant allele of furin in the processing-incompetent cell line, lovo. evidence for involvement of the homo b domain in autocatalytic activation. J Biol Chem 1995; 270:26565-26569. 107. Ohnishi Y, Shioda T, Nakayama K, Iwata S, Gotoh B, Hamaguchi M, Nagai Y. A furindefective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1. J Virol 1994; 68:4075-4079. 108. Konda Y, Yokota H, Kayo T, Horiuchi T, Sugiyama N, Tanaka S, Takata K, Takeuchi T. Proprotein-processing endoprotease furin controls the growth and differentiation of gastric surface mucous cells. J Clin Invest 1997; 99:1842-1851. 109. Kayo T, Sawada Y, Suzuki Y, Suda M, Tanaka S, Konda Y, Miyazaki J, Takeuchi T. Proprotein-processing endoprotease furin decreases regulated secretory pathway-specific proteins in the pancreatic beta cell line MIN6. J Biol Chem 1996; 271:10731-10737. 110. Chrétien M, Fleser A, Day R, Martel R, Leclerc G, Seidah NG. Role of the pro-protein convertases (PCs) in arterial restenosis: a novel therapeutical approach. J Invest Med 1997; 45(3):197A-293A. 111. O’Rahilly S, Gray H, Humphreys PJ, Krook A, Polonsky KS, White A, Gibson S, Taylor K, Carr C. Brief report: Impaired processing of prohormones associated with abnormalities of glucose homeostasis and adrenal function. New Engl J Med 1995; 333:1386-1390. 112. Jackson RS, Creemers JWM, Ohagi S, Raffin-Sanson M-L, Sanders L, Montague CT, Hutton JC, O’Rahilly S. Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nat Genet 1997; 16:303-306. 113. Furuta M, Yano H, Zhou A, Rouillé Y, Holst JJ, Carroll, Ravazzola M, Orci L, Furuta H, Steiner DF. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 1997; 94:6646-6651. 114. Mbikay M, Tadros H, Ishida N, Lerner CP, de Lamirande E, Chen A, El-Alfy M, Clermont Y, Seidah NG, Chrétien M, Gagnon, Simpson EM. Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 1997; 94:6842-6846. 115. Roebroek AJM, Pauli I, van Leuven F, Van de Ven WJM. Targeted inactivation of the FUR gene in mice. Keystone Symposium: Molecular and Cellular Biology. Processing of Peptide Hormones, Neurotransmitters, Growth Factors and Viral Proteins. Taos, New Mexico. March 3-9, 1997; Abstract #218. 116. Jean F, Boudreault A, Basak A, Seidah NG, Lazure C. Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor. J Biol Chem 1995; 270:19225-19231.

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117. Decroly E, Vandenbranden M, Cogniaux J, Ruysschaert JM, Howard SC, Jacob G, Marshall G, Kompelli A, Basak A, Jean F, Lazure C, Benjannet S, Chrétien M, Day R, Seidah NG. The convertases furin and PC1 can both cleave the human immunodeficiency virus (HIV)-1 envelope glycoprotein gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). J Biol Chem 1994; 14:12240-12247. 118. Hallenberger S, Bosch V, Angliker H, Shaw E, Klenk H-D, Garten W. Inhibition of furinmediated cleavage activation of HIV-1 glycoprotein gp160. Nature 1992; 360:358-361. 119. Jean F, Basak A, DiMaio J, Seidah NG, Lazure C. An internally quenched fluorogenic substrate of prohormone convertase 1 and furin leads to a potent prohormone convertase inhibitor. Biochem J 1995; 307:689-695. 120. Anderson ED, Thomas L, Hayflick JS, Thomas G. Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant. J Biol Chem 1993; 268:24887-24891. 121. Lu WY, Zhang WL, Molloy SS, Thomas G, Ryan K, Chiang YW, Anderson S, Laskowski M. Arg(15)-Lys(17)-Arg(18) turkey ovomucoid 3rd domain inhibits human furin. J Biol Chem 1993; 268:14583-14585. 122. Kurachi K, Chandra T, Friezner Degen SJ, White TT, Marchioro TL, Woo SLC, Davie EW. Cloning and sequence of cDNA coding for alpha 1-antitrypsin. Proc Natl Acad Sci USA 1981; 78:6826-6830. 123. Owen MC, Brennan SO, Lewis JH, Carrell RW. Mutation of antitrypsin to antithrombin alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder. N Engl J Med 1983; 309:694-698. 124. Hatsuzawa K, Murakami K, Nakayama K. Molecular and enzymatic properties of furin, a Kex2-cleavage at Arg-X-Lys/Arg-Arg sites. J Biochem Tokyo 1992; 111:296-301. 125. Bresnahan PA, Leduc R, Thomas L, Thorner J, Gibson HL, Brake AJ, Barr PJ, Thomas G. Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-b-NGF in vivo. J Cell Biol 1990; 111:2851-2859. 126. Vollenweider F, Benjannet S, Decroly E, Savaria D, Lazure C, Thomas G, Chrétien M, Seidah NG. Comparative cellular processing of the human immunodeficiency virus (HIV1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases. Biochem J 1996; 314:521-532. 127. Benjannet S, Savaria D, Laslop A, Chrétien M, Marcinkiewicz M, Seidah NG. α1-antitrypsin-Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. J Biol Chem 1997; 272:26210-26218. 128. Zhou A, Paquet L, Mains RE. Structural elements that direct specific processing of different mammalian subtilisin-like prohormone convertases. J Biol Chem 1995; 270:21509-21516. 129. Rehemtulla A, Dorner AJ, Kaufman RJ. Regulation of PACE propeptide-processing activity—requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation. Proc Natl Acad Sci USA 1992; 89:8235-8239. 130. Anderson ED, Vanslyke JK, Thulin CD, Jean F, Thomas G. Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage. EMBO J 1997; 16:1508-1518. 131. Lazure C, Boudreault A, Gauthier D, Seidah NG, Chrétien M, Basak A. The use of recombinant baculovirus expressed mPC1 and mPC2 and synthetic peptides to probe the role of their respective propeptide. Keystone Symposia: Molecular & Cellular Biology. Processing of Peptide Hormones, Neurotransmitters, Growth Factors and Viral Proteins. Taos, New Mexico. March 3-9, 1997; Abstract #111. 132. Basak A, Gauthier D, Seidah NG, Lazure C. Synthetic peptides derived from the prosegments of proprotein convertase PC1 and furin are potent inhibitors of both proteases. Fifteenth American Peptide Symposium, Nashville, TN. June 14-19, 1997. 133. Martens GJM. Cloning and sequence analysis of human pituitary cDNA encoding a novel polypeptide 7B2. FEBS Lett 1988; 234:160-164. 134. Mbikay M, Grant SGN, Sirois F, Tadros H, Skowronski J, Lazure C, Seidah NG, Hanahan D, Chrétien M. cDNA sequence of neuroendocrine protein 7B2 is expressed in beta cell tumors of transgenic mice. Int J Pept Protein Res 1989; 33:39-45.

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135. Zhu X, Rouillé Y, Lamango NS, Steiner DF, Lindberg I. Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: A potential mechanism for its inactivation. Proc Natl Acad Sci USA 1996; 93:4919-4924. 136. Jutras I, Seidah NG, Reudelhuber TL, Brechler V. Two activation states of the prohormone convertase PC1 in the secretory pathway. J Biol Chem 1997; 272:15184-15188. 137. Mercure C, Jutras I, Day R, Seidah NG, Reudelhuber TL. Prohormone convertase PC5 is a candidate processing enzyme for prorenin in the human adrenal cortex. Hypertension 1996; 28:840-846. 138. Santavicca M, Noel A, Stoll I, Angliker H, Stoll I, Segain J-P, Anglard P, Chrétien M, Seidah NG, Basset P. Characterization of structural determinants and molecular mechanisms involved in pro-stromelysin-3 activation by 4-aminophenylmercuric acetate and furintype convertases. Biochem J 1996; 315:953-958. 139. Pei DQ, Weiss SJ. Furin-dependent intracellular activcation of the human Stromelysin-3 zymogen. Nature 1995; 375:244-247. 140. Sato H, Kinoshita T, Takino T, Nakayama K, Seiki M. Activation of a recombinant membrane type 1-matrix metalloproteinase (MT1-MMP) by furin and its interaction with tissue inhibitor of metalloproteinase (TIMP) 2. FEBS Lett 1996; 393:101-104. 141. Wolfsberg TG, Primakoff P, Myles DG, White JM. ADAM, a novel family of membrane proteins containing a disintegrin and metalloprotease domain: Multipotential functions in cell-cell and cell-matrix interactions. J Cell Biol 1995; 131:275-278. 142. Blobel CP, Wolfberg TG, Turck CW, Myles DG, Primakoff P, White JM. A potential fusion peptide and an integrin ligand domain in a protein active sperm-egg fusion. Nature 1992; 356:248-252. 143. Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson M F, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP. A metalloproteinase disintegrin that releases tumor necrosis factor-α from cells. Nature 1997; 385:729-733. 144. Moss ML, Jin SLC, Milla ME, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Warner J, Willard D, Becherer JD. Cloning of a disintegrin metalloproteinase that processes precursor tumor-necrosis factor-α. Nature 1997; 385:733-736. 145. Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah HG, Israel A. The Notch1 receptor is constitutively cleaved by a furin-like convertase. Proc Natl Acad Sci, USA 1998; 95:8108-8112. 146. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during drosophila and vertebrate neurogenesis. Cell 1997; 90:271-280. 147. Blaumueller CM, Qi H, Zagouras P, Artavanis-Tsakonas S. Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane. Cell 1997; 90:278-291.

CHAPTER 4

The Neuroendocrine Prohormone Convertases PC1, PC2 and PC5 Margery C. Beinfeld

Introduction

T

he cloning of the cDNAs of most of the major peptide prohormones revealed that the active moieties are almost always flanked by single or paired basic residues. The recognition that the processing of these prohormones probably takes place in acidifying secretory granules in the presence of significant calcium concentrations has defined the location and some of the biochemical requirements of potential prohormone processing enzymes. They should be trypsin-like enzymes which have the ability to be sorted into regulated secretory vesicles, which are activated by calcium and which operate at moderately acidic pH (5.5-6.5). The discovery of the subtilisin family of enzymes, with the correct anatomical and subcellular distribution and precisely these biochemical properties, has provided a number of likely candidates for the endoproteolytic cleavage of a number of proproteins, prohormones and propeptides in both the regulated and constitutive secretory pathway. This chapter focuses on the three members of this family (PC1, PC2 and PC5) whose distribution in neuroendocrine tissues and endocrine tumor cells and their ability to process a wide range of precursors makes them most likely to participate in the processing of a number of prohormones and proneuropeptides in the regulated secretory pathway.

The Discovery of the Subtilisin Family of Prohormone Convertases Discovery of the prohormone convertase (PC) family of enzymes which now numbers 7 members represented a major advance in the prohormone processing field.1,2 These mammalian enzymes include PC1 (also known as PC3),3,4 PC2,5 PC4,6 PACE 4,7 PC5 (also known as PC6),8,9 PC7/8,10 and furin (also known as SPC1 or PACE, paired basic amino acid cleaving enzyme).11,12 This confusing nomenclature has arisen from the near simultaneous discovery of the same enzymes by different groups. PC5 also has multiple splice variants (PC5B), further complicating the nomenclature. The relationship between these enzymes is depicted schematically in Figure 4.1. Discovery of the Kex2 yeast enzyme which processes the alpha mating factor and killer toxin precursor inspired the search for similar enzymes which could process mammalian precursors. PC25 was cloned by PCR based on its similarity to the catalytic domain of kex2. A database search with the PC2 sequence uncovered the partial sequence of a related human protein fur, found in the c-fes/fps protooncogene. Cloning of this protein, which was later called furin, provided another member of the family.11,12 The other family members PC1, PC4, PC5, PC7/8 and PACE4 were subsequently cloned based on their highly homologous Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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Fig. 4.1. Schematic representation of the structure of the subtilisin family of prohormone convertases. The catalytic domain of subtilisin, containing active side D (asp), H (His), N (Asn) and S (Ser) residues is strongly conserved in all family members. P is the P domain, AH is the amphipathic alpha helical domain, TM is the transmembrane spanning domain. PC5 and PC5B are alternately spliced proteins produced from the same gene. The arrow shows where the protein sequences diverge.

catalytic domains. PC5 and PC5B are alternate splice variants of the same gene, as shown in Figure 4.1. PC5B is much larger, has a transmembrane spanning domain and cycles to the cell surface. PC1, PC2, PC4 and PC5 are sorted and activated in the regulated secretory pathway and are candidates for processing prohormones whose secretion is regulated. PC4 is found only in testis and may be involved in prohormone processing in this tissue. The others like furin, PACE4, PC5B and PC7/8 have a transmembrane spanning domain, cycle to the cell surface and are involved in the processing of proteins and precursors in the constitutive pathway.13 These calcium-activated serine proteases which work at mildly acidic pH all have a signal sequence, an amino terminal propeptide of about 80-90 amino acids which is removed at a cleavage site with the motif RXK/RR. They all have a highly conserved catalytic domain with active site aspartic, histidine and serine residues which is very similar to sub-

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tilisin from Bacillus subtilis. They also have a conserved P or Homo B domain of about 150 residues with an integrin recognition motif RDG.

Distribution of PC1, PC2 and PC5 PC1 and PC2 mRNA are widely distributed in neural and endocrine tissues (as well as tumor cells derived from these tissues) including pituitary, adrenal, pancreas, islet cells, brain and intestine.3,4 PC2 is more abundant than PC1 in most tissues. AtT20 cells are the most abundant source of PC1 mRNA. PC1 and PC2 immunoreactive cells have a distinct distribution in the gastrointestinal tract.14 PC1 is widely distributed throughout the gut and is colocalized with a number of G.I. peptides. PC2 is more abundant in the gastric pylorus and proximal duodenum where it is colocalized with gastrin, CCK and somatostatin. In the stomach, the PCs are more abundant in the mucosa than in the muscular layer. Both PC1 and PC2 are present in the pancreas, but PC2 is much more abundant in pancreatic β cells where it is colocalized with glucagon, somatostatin and pancreatic polypeptide, while PC1 is colocalized with insulin in islet cells.15 PC1 and PC2 are both present in superior cervical ganglia.16 PC1 is present in the ovary. PC1 and PC2 have been visualized in bovine chromaffin granules and their release is stimulated by carbamoylcholine chloride. 17 PC2 has been colocalized with proenkephalin in human adrenal medulla and in many human pheochromocytomas.18 PC1 and PC2 have been found in rat neutrophils and macrophages.19 PC1, PC2 have been visualized by in situ hybridization in the intracardiac para-aortic ganglia of the rat heart.20 PC5 is expressed in brain while both PC5 and PC5B are expressed in intestine. The alternate splice variant PC5B is much larger, has a transmembrane spanning domain, cycles to the cell surface like furin,21 and is probably involved in protein processing in the constitutive pathway.22,23 PC5 is fairly widely distributed, but it is much more abundant in intestine than either PC1 or PC2, as well as being more abundant in brain than PC1. PC5 has also been localized in glucagon-containing dense core secretory granules in the pancreas.23 PC5 has been found in the endothelial cells lining the coronary vessels and the valve leaflets of the rat heart.20 PC5 is known to be expressed in PC12, NB-1 neuroblastoma cells and adrenocortical Y-1 cells, but it is not expressed in RIN5F or AtT20 cells.22

Biosynthesis and Activation of PC1, PC2 and PC5 Detailed examination of the life cycle, intracellular localization, catalytic activity and tissue distribution of these PCs has yielded a gold mine of information. During their biosynthesis, they all undergo autoactivation with the removal of the propeptide. ProPC1 (88 kDa) is converted into 83 kDa, and an 84 kDa sulfated and glycosylated form is secreted.24 The carboxyl terminal of PC1 is not required for maturation although the active site mutant (Ser to Ala mutation) is not able to mature itself.25 PC1 activation is thought to occur in the endoplasmic reticulum with a pH of 7-8 with no added calcium, while PC2 is activated much more slowly than PC1. It is thought to occur in the trans Golgi network with a pH of about 5.5 to 6 and a millimolar calcium concentration.26 This difference in the time course of activation may explain why PC1 frequently cleaves prohormones before PC2. PC1 and PC2 are both glycosylated, and inhibition of PC2 glycosylation by tunicamycin causes intracellular degradation of PC2.24 PC2 is converted from 76 to 64/66 kDa with a half time of about 140 min. Its release is stimulated by glucose in islet cells. PC1 and insulin biosynthesis are also stimulated at the translational level by glucose.27 Production of active PC2 requires an additional protein called 7B2.28,29 It serves as a chaperone, helping PC2 to reach its correct cellular destination. The amino terminal 21 kDa

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protein is responsible for this chaperone action and enhances transport of PC2, while the 31 amino acid peptide is an inhibitor of PC2 activity which is active at a nanomolar concentration. Activation of PC2 in the trans Golgi network is accompanied by the cleavage of this inhibitory domain of 7B2 by PC2.30 The PC enzymes have been reported to be sulfated in vivo but whether this sulfate is on tyrosine or sugar resides is not clear. Much less is known about PC5. When it is expressed in AtT20 cells, it is processed to 117 kDa and 65 kDa forms which are secreted into the media, and their secretion is stimulated by 8Br-cAMP.23 The major form found in rat brain tissue extracts is the 65 kDa form.31 PC5B is expressed as a 210 KDa protein intracellularly and a shorter form of 170 kDa is secreted, although its secretion is not stimulated by 8Br-cAMP, suggesting that it is secreted constitutively.

Regulation of PC Expression Overexpression of CCK mRNA in AtT20 cells does not alter PC1 expression in these cells,32 although pharmacological elevation of cAMP levels and treatment with phorbol esters increased CCK, PC1 and PC2 mRNA levels in rat thyroid medullary carcinoma WE cells and in human neuroblastoma cells, suggesting that the expression of both genes is probably regulated by protein kinase A and C.33 The PC1 gene has multiple transcriptional start sites and its 5' promoter confers both basal and hormone-regulated activity.34 The PC1 and PC2 promoters contain AP-1, SP-1 and cAMP response elements. In rat pituitary, the expression of PC1, PC2, POMC, the amidating enzyme and carboxypeptidase H are all decreased by dopamine agonists.35,36 Birch et al37 have shown that both PC1 and PC2 are present in the supraoptic and paraventricular nuclei of the hypothalamus (PVN), and that expression of both of them is increased by chronic osmotic stimulus. CCK synthesis in this region is increased by the same stimulus.38 PC1 expression in PVN is regulated by glucocorticoids39 and PC1 expression is transiently increased in pilocarpine-induced and kindled seizures.40 In AtT20 cells ACTH secretion is also regulated by ICER (inducible cAMP early repressor), which appears to work through transcriptional control of the PC1 gene rather than expression of the POMC gene.41

Experimental Systems Used to Study Processing Much of the experimental work on the temporal order of cleavages and elucidation of the enzymes responsible for these cleavages initially relied on COS-7 or other fibroblastic cells or neural and endocrine tumor cells in culture. More recently the production of recombinant prohormones and recombinant processing enzymes has allowed processing reactions to be studied in vitro under controlled conditions. The production of processing enzyme mutant mice has added yet another important model. None of these techniques are ideal and utilization of several of them may be required to completely understand the processing of specific prohormones. A wide variety of neural and endocrine tumor cells have been used: neuroblastomas, pituitary (AtT20 and GH3), pancreatic (RIN5F), intestinal (STC-1), adrenal (PC12), and thyroid (WE) cells which normally express peptide mRNAs and some of the processing enzymes and secrete processed peptides. Secretion of these processed peptides is stimulated in response to secretagogues like potassium or cAMP.32,42-44 If these cells do not express the peptide mRNA of interest, it can be stably transfected. Specific processing enzymes can also be coexpressed in these cells with the prohormone with standard techniques, or by infection with vaccinia45 or adenovirus46 vectors. As a first approximation, these endocrine cells appear to be a good model of peptide processing. Some of these endocrine cells normally

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express several PC enzymes, so knowing which of them is responsible for specific cleavages is sometimes difficult. COS-7, L cells and other fibroblastic cells have been used to transiently express peptide prohormones (like proCCK) with individual processing enzymes. When expressed in fibroblastic cells in the absence of specific processing enzymes, the intact prohormones (like proCCK) are generally secreted into the media. When PC1 is transiently coexpressed with proCCK in these cells, the CCK prohormone is processed at appropriate sites and these products are secreted into the media. This makes another system to study the ability of individual processing enzymes to cleave specific prohormones. Many of these cells may also express furin and other related enzymes, which may complicate the analysis because they may also have some ability to cleave expressed prohormones. PC enzyme mRNAs have also been successfully expressed in Xenopus oocytes,26 where they are activated as in mammalian cells. The production of recombinant prohormones and processing enzymes in quantity in bacteria,47 mammalian cells29,48 and insect cells with baculovirus vectors49 has permitted several studies of prohormone processing in vitro.29,48,50-53 These experiments, which take place under more defined conditions, have allowed a more precise determination of the properties of these enzymes with their physiological substrates as well as with synthetic fluorogenic and chromogenic substrates. The Km for most of these enzymes with synthetic substrates is in the 100-200 micromolar range.29,48,52 This is not surprising, as prohormones are known to be considerably concentrated in the secretory granules and this may not be far from their physiological concentration. These studies have provided many new insights, but in some cases there have been discrepancies between cleavages observed in transfection experiments in endocrine cells and in vitro incubations. These differences can perhaps be attributed to the difficulty in reproducing the milieu of the condensing secretory vesicles in vitro.

Enzymatic Activity of PC1, PC2, and PC5 These enzymes cleave mainly at dibasic pairs such as Lys-Arg, Arg-Lys, and Arg-Arg, while some monobasic sites (mainly Arg) are also cleaved.52,54 PC1 and PC2 are widely distributed in neural and endocrine tissues and cell lines and have been shown to be good candidates for the processing of POMC (proopiomelanocortin),45,55,56 insulin,57 glucagon,58 CCK,52,59,60 gastrin,61 dynorphin,54 enkephalin,62 TRH,63 neurotensin,64 somatostatin,65,66 NPY,16 chromogranin A,67 secretogranin II,68 and procorticotropin releasing hormone.69 The newly discovered PC5 is a good candidate for prohormone processing in the brain and intestine.8,9,22 Little is known about the activity of PC5. When studied with renin substrates, PC5 appears to resemble PC1 in terms of its catalytic activity, but with a stricter requirement for paired basic over single basic sites.

Antisense PC1 and PC2 Strategies to Study Proneuropeptide Processing In order to address which of these enzymes is responsible for proneuropeptide cleavage in specific endocrine cells, an antisense strategy has been used because there are no specific, nontoxic inhibitors of these enzymes. This strategy, in which partial PC1 or PC2 cDNAs consisting of the proregions are expressed stably in endocrine cells in the antisense orientation, has provided evidence that PC1 and/or PC2 is involved in POMC, 70 proCCK,59,60,71 proenkephalin,72 neurotensin64 and glucagon processing.58 This is a technique limited to cells that can be transfected or infected. It is a successful strategy in general, but complete inhibition of PC expression with antisense methods has been difficult to achieve.

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Possible toxic effects of overexpression of antisense mRNAs on the expression of peptide or other processing enzyme mRNAs may also limit this technique. Although complete inhibition of PC expression was not always achieved with this technique, there was a large enough decrease in enzyme protein expression to see alteration of prohormone processing. In the case of CCK, inhibition of PC1 in STC-1 and RIN 5F cells caused a selective depletion of CCK-8 while sparing CCK-22, while inhibition of PC2 depleted CCK-22, sparing CCK-8. Antisense experiments have more recently used an inducible promoter system73 and adenovirus vectors to express the PC antisense mRNA.16

Endoproteases in CCK Processing, a Case in Point The question of which of these many enzymes are responsible for individual proCCK cleavages in tissues is difficult to answer. Although detailed studies of the colocalization of CCK with these enzymes have not been performed, PC1, PC2, and PC5 are found in CCKergic cell types and neuronal populations in both the brain and intestine. PC1, PC2 and PC5 are colocalized with oxytocin in the supraoptic and paraventricular nuclei of the hypothalamus. The oxytocin cells are known to also contain CCK,74 and thus these enzymes have a distribution which is consistent with a role in CCK processing.39,75 The colocalization of CCK with these three enzymes appears to hold throughout the rat CNS. AtT20 cells which express PC1 but neither PC2 nor PC5 when transfected with the CCK cDNA can generate CCK-8 in the absence of PC2 and PC5. PC1 by itself has the ability to cleave proCCK at the Arg-Asp and Arg Arg-Ser bonds flanking CCK-8 to generate CCK-8 Gly Arg Arg in L cells. This peptide is thought to be the immediate precursor of CCK-8 amide and accumulates in carboxypeptidase E deficient fat/fat mice.76 COS cells or L cells transfected with PC1 and proCCK can also cleave proCCK to liberate the propeptide (between the signal peptide cleavage site and the amino terminal of CCK-58), a peptide that is found in rat brain77 and endocrine cells.78 Antisense studies also support a role for PC1 and PC2 in proCCK processing. Inhibition of PC1 protein expression by expression of antisense PC1 mRNA in STC-1 and RIN 5F cells caused a selective depletion of CCK-8 while sparing CCK-22. Inhibition of PC2 protein expression by a similar strategy depleted CCK-22, sparing CCK-8. These results further support a role for PC1 in production of CCK-8 and suggest that PC2 activity may be responsible for the production of larger forms of amidated CCK (such as CCK-58, -33, and -22) found in gastrointestinal tissues. Recent work in progress has shown that other cell lines lacking PC1 (which have PC5 with or without PC2) can also process proCCK to CCK-8. This suggests that either PC5 can substitute for PC1 or that there is redundancy so that all three enzymes can produce CCK-8 Gly Arg Arg by themselves.

Processing Enzyme Knockouts and Mutations The most promising approach to understanding the physiological relevance of these enzymes is the production of mice in which these enzymes are specifically deleted. PC279 and furin knockout mice have been produced and efforts are underway to make PC1 knockout mice. PC2 mice are viable and fertile and have defective processing of insulin, somatostatin, glucagon, dynorphin, and enkephalin. Furin knockout mice are embryonic lethals. PC4 knockout mice are viable but the homozygous male mice have reduced fertility, and eggs fertilized in vitro by homozygous male sperm had reduced viability.80 A human patient with defective PC1 enzyme has been described.81 The affected individual was heteroallelic, one allele produced an abnormal Gly to Arg483 substitution while the other allele had a mutation producing a stop codon. The Gly to Arg substituted protein when expressed in CHO cells was retained in the ER, so no active PC1 enzyme was pro-

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duced. The affected individual had severe early-onset obesity, impaired glucose tolerance, secreted excess proinsulin and pro opiomelanocortin. She displayed hypogonadotrophic hypogonadism (but was not sterile) and hypocortisolism. All of her three children inherited one of the Gly to Arg substituted alleles but were clinically unaffected. The ability to use human monocyte-derived macrophage RNA for diagnostic analysis should greatly simplify genetic screening for other human patients with prohormone convertase deficiencies.82

Future Challenges Knowledge of the actual enzymes responsible for prohormone cleavages in specific tissues lags behind progress in other areas of the field due to the technical difficulty of working with intact tissues and whole animals. It is clear that the subtilisin-like PC enzymes are major players, but that there may be additional enzymes which are not in this family which are also required for specific cleavages. Progress in our ability to develop specific, bioavailable inhibitors for these enzymes as well as to develop conditional, tissue-specific knockouts will allow us to more directly address the question of which enzymes are physiologically relevant. Detailed analysis of the recently produced knockout mice should provide much insight into the physiological role of these enzymes. Detailed investigation of the regulation of these processing enzymes and the complex process of their biosynthetic activation is likely to provide many answers. The possibility that tissue-specific differences in their activation and the presence of tissue specific inhibitory substances which play a role cannot be excluded. The most interesting questions are still unanswered, although work is in progress to address them. What structural features of the prohormone determine where it will be processed and how it will be recognized as secretory material by the sorting machinery? How is tissue-specific processing determined and regulated? What is the physiological and clinical significance of this regulation? In summary, the last five years have seen enormous progress but many of the most interesting questions remain to be answered.

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28. Hsi KL, Seidah NG, DeSerres G et al. Isolation and NH2-terminal sequence of a novel porcine anterior pituitary polypeptide. Homology to proinsulin, secretin and Rous sarcoma virus transforming protein TVFV60. FEBS Lett 1982; 147:261-6. 29. Lamango NS, Zhu Z, Lindberg I. Purification and enzymatic characterization of recombinant prohormone convertase 2: Stabilization of activity by 21 kDa 7B2. Arch Biochem Biophys 1996; 330:238-50. 30. Martens GJM, Braks JAM, Eib DW et al. The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2. Proc Natl Acad Sci USA 1994; 91:5784-5. 31. Dong W, Marcinkiewicz M, Vieau D et al. Distinct mRNA expression of the highly homologous convertases PC5 and PACE4 in the rat brain and pituitary. J Neurosci 1995; 15:1778-96. 32. Yoon JY, Beinfeld MC. A mouse intestinal tumor cell line, STC-1, expresses CCK, PC1, and PC2 mRNA, processes pro CCK to CCK-8, and displays cAMP regulated release. Endocrin 1994; 2:973-7. 33. Mania-Farnell BL, Botros I, Day R et al. Differential modulation of prohormone convertase mRNA by second messenger activators in two cholecystokinin-producing cell lines. Peptides 1996; 17:47-54. 34. Jansen E, Torik AY, Meulemans SMP et al. Neuroendocrine-specific expression of the human prohormone convertase 1 gene. J Biol Chem 1995; 270:15391-7. 35. Eipper BA, Bloomquist BT, Husten EJ et al. Peptidylglycine α-amidating monooxygenase and other processing enzymes in the neurointermediate pituitary. Ann N Y Acad Sci 1993; 147-60. 36. Oyarce AM, Hand TA, Mains RE et al. Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary. J Neurochem 1996; 67:229-41. 37. Birch NP, Hakes DJ, Dixon JE et al. Distribution and regulation of the candidate prohormone processing enzymes SPC2 and SPC3 in adult rat brain. Neuropeptides 1994; 27:307-22. 38. Beinfeld MC, Meyer DK, Brownstein MJ. Cholecystokinin octapeptide in the rat hypothalamo-neuro-hypophysial system. Nature 1980; 288:376-8. 39. Dong W, Seidel B, Marcinkiewicz M et al. Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: Selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids. Journal of Neuroscience 1997; 17:563-75. 40. Marcinkiewicz M, Nagao T, Day R et al. Pilocarpine-induced seizures are accompanied by a transient elevation in the messenger RNA expression of the prohormone convertase PC1 in rat hippocampus: Comparison with nerve growth factor and brain-derived neurotrophic factor expression. Neurosci 1996; 76:425-39. 41. Lamas M, Molina C, Foulkes NS et al. Ectopic ICER expression in pituitary corticotroph AtT20 cells: Effects of morphology, cell cycle, and hormonal production. Mol Endo 1997; 11:1425-34. 42. Buonassisi V, Sato G, Cohen AI. Hormone-producing cultures of adrenal and pituitary tumor origin. Proc Natl Acad Sci USA 1962; 48:1184-90. 43. Beinfeld MC. CCK mRNA expression, pro-CCK processing, and regulated secretion of immunoreactive CCK peptides by rat insulinoma (RIN 5F) and mouse pituitary tumor (AtT-20) cells in culture. Neuropeptides 1992; 22:213-7. 44. Gazdar AF, Chick WL, Oie HK et al. Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci USA 1980; 77:3519-23. 45. Thomas L, Leduc R, Thorne BA et al. Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendogrine processing enzymes. Proc Natl Acad Sci USA 1991; 88:5297-301. 46. Irminger J-C, Meyer K, Halban P. Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus. Biochem J 1996; 320:11-5.

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47. Hook VYH, Schiller MR, Azaryan AV. The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70kDa aspartic proteinase show preference among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. Arch Biochem Biophys 1996; 328:107-14. 48. Zhou Y, Lindberg I. Purification and characterization of the prohormone convertase PC1(PC3). J Biol Chem 1993; 268:5615-23. 49. Wang W, Yum L, Beinfeld MC. Expression and purification of rat pro CCK in bacteria and from media from insect cells infected with recombinant baculovirus. Peptides 1997; 18:1295-1299. 50. Hook VYH, Hegerle D, Affolter HU. Cleavage of recominant enkephalin precursor by endoproteolytic activity in bovine chromaffin granules. Biochem Biophys Res Commun 1990; 167:722-30. 51. Andreasson KI, Tam WWH, Feurst TO et al. Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by a POMC-converting enzyme. FEBS Lett 1989; 248:43-7. 52. Wang W, Beinfeld MC. Cleavage of CCK-33 by recombinant PC 2 in vitro. Biochem Biophys Res Commun 1997; 231:149-52. 53. Rufaut NW, Brennan SO, Hakes DJ et al. Purification and characterization of the candidate prohormone processing enzyme SPC3 produced in a mouse L cell line. J Biol Chem 1993; 268:20291-8. 54. Dupuy A, Lindberg I, Zhou Y et al. Processing of prodynorphin by the prohormone convertase PC1 results in high molecular weight intermediate forms. FEBS Lett 1994; 337:60-5. 55. Benjannet S, Rondeau N, Day R et al. PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-8. 56. Zhou A, Bloomquist BT, Mains RE. The prohormone convertases PC1 and PC2 mediate distinct endoproteolytic cleavages in strict temporal order during proopiomelanocortin biosynthetic processing. J Biol Chem 1993; 268:1763-9. 57. Smeekens SP, Montag AG, Thomas G et al. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC3, and PC3. Proc Natl Acad Sci USA 1992; 89:8822-6. 58. Rouille Y, Westermark G, Martin SK et al. Proglucagon is processed to glucagon by prohormone convertase PC 2 in α TC1-6 cells. Proc Natl Acad Sci USA 1994; 91:3242-6. 59. Yoon JY, Beinfeld MC. Prohormone convertase 1 (PC 1) is necessary for the formation of CCK-8 in Rin5F and STC-1 cells. J Biol Chem 1997; 272:9450-6. 60. Yoon JY, Beinfeld MC. Prohormone convertase 2 (PC2) in necessary for the formation of CCK-22 but not CCK-8 in RIN5F and STC-1 cells. Endo 1997; 138:3620-3. 61. Dickinson CJ, Sawada M, Guo YJ et al. Specficity of prohormone convertase endoproteolysis of progastrin in AtT-20 cells. J Clin Invest 1995; 96:1425-31. 62. Breslin MB, Lindberg I, Benjannet S et al. Differential processing of proenkephalin by prohormone convertase I (3) and 2 and furin. J Biol Chem 1993; 268:27084-93. 63. Nillni EA, Friedman TC, Todd RB et al. Pro-thyrotropin-releasing hormone processing by recombinant PC1. J Neurochem 1995; 65:2462-72. 64. Rovere C, Barbero P, Kitabgi P. Evidence that PC2 is the endogenous pro-neurotensin convertase in rMTC 6-23 cells and that PC1- and PC2-transfected PC12 cells differentially process pro-neurotensin. J Biol Chem 1996; 271:11368-75. 65. Galanopoulou AS, Kent G, Rabbani SN et al. Heterologous processing of prosomatostatin in constitutive and regulated secretory pathways. Putative role of the endoproteases furin, PC 1 and PC 2. J Biol Chem 1993; 268:6041-9. 66. Galanopoulou AS, Seidah NG, Patel YC. Direct role of furin in mammalian prosomatostain processing. Biochem J 1995; 309:33-40. 67. Arden SD, Rutherford NG, Guest PC et al. The post-translational processing of chromogranin A in the pancreatic islet: Involvement of the eukaryote subtilisin PC2. Biochem J 1994; 298:521-8.

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68. Hoflehner J, Eder U, Laslop A et al. Processing of secretogranin II by prohormone convertases: Importance of PC1 in generation of secretoneurin. FEBS Lett 1995; 360:294-8. 69. Perone MJ, Ahmed I, Linton EA et al. Procorticotrophin releasing hormone is endoproteolytically processed by the prohormone convertase PC2 but not PC1 with stably transfected CHO-K1 cells. Biochemical Society Transactions 1996; 24:497S 70. Bloomquist BT, Eipper BA, Mains RE. Pro-hormone converting enzymes: Regulation and evaluation of function using antisense RNA. Mol Endo 1991; 5:2014-24. 71. Yoon JY, Beinfeld MC. Expression of antisense PC1 in stably transfected RIN5F cells significantly reduces CCK-8 biosynthesis. Reg Peptides 1995; 59:221-7. 72. Johanning K, Mathis JP, Lindberg I. Role of PC2 in proenkephalin processing: Antisense and overexpression studies. J Neurochem 1996; 66:898-907. 73. Eskeland NL, Zhou A, Dinh TQ et al. Chromoganin A processing and secretion: Specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway. J Clin Invest 1996; 98:148-56. 74. Vanderhaeghen JJ, Lotstra F, Vandesande F et al. Co-existence of cholecystokinin and oxytocin-neurophysin in some magnocellular hypothalamophypophyseal neurons. Cell Tis Res 1981; 221:227-31. 75. Shafer MKH, Day R, Cullinan WE et al. Gene expression of prohormone and proprotein convertases in the rat CNS: A comparative in situ hybridization analysis. J Neurosci 1993; 13:1258-79. 76. Cain BM, Wang W, Beinfeld MC. Cholecystokinin (CCK) levels are severely decreased in the forebrains of Cpefat/Cpefat mice: A regional difference in the involvement of carboxypeptidase E (CPE) in pro CCK processing. Endo 1997; 138:4034-7. 77. Beinfeld MC. Cholecystokinin (CCK) gene-related peptides: Distribution and characterization of immunoreactive pre-pro CCK, pro-CCK and an amino terminal pro-CCK fragment in rat brain. BrainRes 1985; 344:351-5. 78. Cao GH, Beinfeld MC. Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells. Peptides 1992; 13:1087-90. 79. Furuta M, Yano H, Zhou A et al. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc Natl Acad Sci USA 1997; 94:6646-51. 80. Mbikay M, Tadros H, Ishida N et al. Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc Natl Acad Sci USA 1997; 94:6842-6. 81. Jackson RS, Creemers JWM, Ohagi S et al. Obesity and impared prohormone processing associated with mutations in the human prohormone convertase 1 gene. Nature Genetics 1997; 16:303-6. 82. LaMendola J, Martin SK, Steiner DF. Expression of PC3, carboxypeptidase E and enkephalin in human monocyte-derived macrophages as a tool for genetic studies. FEBS Lett 1997; 404:19-22.

CHAPTER 5

‘Prohormone Thiol Protease’ (PTP), a Novel Cysteine Protease for Proenkephalin and Prohormone Processing Vivian Y.H. Hook, Yuan-Hsu Kang, Martin Schiller, Nikolaos Tezapsidis, Jane M. Johnston and Ada Azaryan

Introduction

P

roduction of peptide hormones and neurotransmitters requires several steps which involves transcription of the pro-hormone gene, translation of the corresponding mRNA, packaging of the prohormone into secretory vesicles, processing by proteolytic mechanisms, storage of mature neuropeptides in secretory vesicles, and regulated secretion of bioactive peptides. Among these steps, posttranslational processing is required for converting the inactive protein precursor into biologically active neuropeptides. Clearly, limited proteolysis is critical for generating neuropeptides.1-3 Endoproteases and exoproteases are required for prohormone processing, which occurs in the regulated secretory pathway of neuroendocrine cells. These potent neuropeptides are stored and secreted from secretory vesicles. The released peptide hormones and neurotransmitters mediate cell-cell communication in neuroendocrine systems.

Features of Prohormone Processing Several proteolytic steps are involved in prohormone processing (Fig. 5.1). The preprohormone is first synthesized from its mRNA at the rough endoplasmic reticulum (RER). The signal peptide of the preprohormone is then removed at the RER by signal peptidase. The resultant prohormone is routed through the Golgi apparatus and packaged into newly formed secretory vesicles, the primary site of prohormone processing.2 Endoproteolytic processing occurs at characteristic paired basic residues—Lys-Arg, ArgArg, Lys-Lys, or Arg-Lys—that flank the NH2- and COOH-termini of the active peptide within its precursor (Fig. 5.1). In addition, processing occasionally occurs at the monobasic arginine residues of some prohormones.4 Endoproteolytic processing at the paired basic residues may occur at three possible sites: 1. at the COOH-terminal side of the dibasic residues; 2. between the dibasic residues; or 3. at the NH2-terminal side of the paired basic residues. Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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Fig. 5.1. Proteolysis in Prohormone Processing. The illustrated model neuropeptide precursor contains one copy of the active peptide. The NH2-terminal signal sequence is first removed by signal peptidase at the rough endoplasmic reticulum. Upon packaging of the prohormone into secretory vesicles, endoproteolytic processing occurs at paired basic residues—Lys-Arg (shown in this figure), Arg-Arg, Lys-Lys, or Arg-Lys—at one of three cleavage sites. Endoproteolytic cleavage at paired basic residues may occur at the positions indicated by #1, #2, or #3. Processing by carboxypeptidase and aminopeptidase exoproteolytic processing enzymes is then required to remove basic residues from COOH- and NH2-termini of peptide intermediates, respectively, to complete the series of proteolytic steps needed to produce active neuropeptides.

Resultant peptide intermediates then require removal of the basic residues at their COOH- and NH2-termini by carboxypeptidase and aminopeptidase enzymes,3,5 respectively, to complete the series of proteolytic steps required to generate active neuropeptides.

Evidence for Neurosecretory Vesicles as a Major Site of Prohormone Processing Two important experimental approaches have provided evidence that prohormone processing occurs primarily within secretory vesicles of neuroendocrine tissues. In one approach, the defined neuroanatomical features of the rat hypothalamo-neurohypophyseal system was advantageously utilized to demonstrate that provasopressin and prooxytocin processing occurs during axonal transport of secretory vesicles from neuronal cell bodies of the hypothalamus to nerve terminals of posterior pituitary.6 In a second approach, studies of proinsulin processing by immunoelectron microscopy of proinsulin and insulin, with pH-sensitive indicators, demonstrated that proinsulin processing occurs during maturation and acidification of secretory vesicles.7,8

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Fig. 5.2. The hypothalamo-neurohypophyseal system: Prohormone processing during axonal transport of secretory vesicles. Hypothalamic neuronal cell bodies located in the SON (supraoptic nucleus) and PVN (paraventricular nucleus) send axons through the ME (median eminence) and pituitary stalk, which terminate in the posterior pituitary. Secretory vesicles are axonally transported from cell bodies to nerve terminals.

The hypothalamo-neurohypophyseal system consists of neuronal cell bodies of the SON (supraoptic nucleus) and PVN (paraventricular nucleus) in the hypothalamus, which project axons through the median eminence to the posterior pituitary, known as the hypothalamo-neurohypophyseal system (HNS, Fig. 5.2). Microdissection of these regions allows analyses of radiolabeled 35S-cysteine forms of vasopressin and oxytocin in hypothalamic cell bodies, axons of the median eminence, and nerve terminals in the posterior pituitary. Analyses of 35S-(Cys)-provasopressin and 35S-(Cys)-prooxytocin processing in the hypothalamo-neurohypophyseal system indicated the presence of precursor forms of vasopressin and oxytocin in neuronal cell bodies of the hypothalamus, partially processed peptide forms in axons of the median eminence, and fully processed vasopressin and oxytocin peptides in nerve terminals of the posterior pituitary. These results demonstrate processing of vasopressin and oxytocin precursors during axonal transport of vesicles to nerve terminals. Elegant studies of proinsulin processing have been achieved by immunoelectron microscopic detection of proinsulin and insulin (with specific antibodies to proinsulin and insulin), combined with measurement of internal vesicle pH with the pH-sensitive reagent DAMP (3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine). These studies demonstrated that proinsulin processing occurs within the maturing secretory vesicle while it undergoes internal acidification.7,8 Significantly, proinsulin was present in immature secretory vesicles with an internal pH near 6.0. However, insulin was present in mature vesicles with a more acidic internal pH near 5.0. These results demonstrate that proinsulin processing takes place during vesicle maturation and acidification, suggesting that the processing enzymes within the vesicle become active as the internal vesicular pH becomes acidified to allow conversion of proinsulin to insulin.

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Chromaffin Granules as a Model Neurosecretory Vesicle System for Identification of Proenkephalin and Prohormone Processing Enzymes Evidence for prohormone processing occurring within secretory vesicles6-8 indicates that relevant processing enzymes should be present within isolated secretory vesicles. The bovine adrenal medulla provides an abundant source of homogeneous neurosecretory vesicles9,10 that contain high levels of the opioid peptide (Met)enkephalin (Tyr-Gly-GlyPhe-Met) (Fig. 5.3),11-13 as well as enkephalin-related peptides derived from proenkephalin.14,15 Proenkephalin-derived intermediates (10-25 kDa) and peptides are well-defined in chromaffin granules.12,13,16-18 The chromaffin granule, therefore, serves as an excellent model secretory vesicle system to investigate proenkephalin processing proteases. In vitro and cellular studies of relevant proenkephalin cleaving activity in chromaffin granules have resulted in identification and characterization of relevant proteases that are responsible for converting proenkephalin to enkephalin peptide products. In addition to (Met)enkephalin, chromaffin granules contain a number of neuropeptides including neuropeptide Y (NPY),19 somatostatin,20 vasoactive intestinal polypeptide (VIP),21 and galanin.22 These neuropeptides are also synthesized as precursors that require proteolytic processing at paired basic residues to generate the smaller active peptides. Knowledge gained from studies of proenkephalin processing enzymes can be extended in future investigations to determine the proteases that are involved in processing other proneuropeptides.

The Novel ‘Prohormone Thiol Protease’ (PTP): A Major Proenkephalin Processing Enzyme in Chromaffin Granules Relevant prohormone processing enzymes should meet the following criteria to be considered as serious candidate proteases for authentic prohormone processing.1-3 The processing enzyme should: 1. be colocalized in secretory vesicles with peptide products; 2. demonstrate activity at the acidic pH range of pH 5.5-6.0 that corresponds to the intravesicular pH in vivo; 3. convert precursor substrate to products that are present in vivo; 4. undergo full characterization for comparison to other processing enzymes and proteases; and 5. be coordinately regulated with cellular stimulation of neuropeptide levels. Finally, inhibition of the processing enzyme should reduce cellular levels of the neuropeptide. This chapter describes how the novel cysteine protease known as ‘prohormone thiol protease’ (PTP) meets these criteria, to indicate PTP as an important proenkephalin and prohormone processing enzyme.

Recombinant Enkephalin Precursor as Substrate for Processing Enzymes Identification and characterization of relevant proenkephalin and prohormone processing enzymes requires use of full-length precursor substrates, since the prohormone processing enzyme(s) may require the conformation of the precursor protein for proper recognition and specificity of processing. For this reason, recombinant 35S-(Met)enkephalin precursor was generated from the corresponding rat preproenkephalin cDNA14 by in vitro transcription and translation.23-25 The resultant 35S-(Met)-preproenkephalin (35S-(Met)-PPE) was utilized as a model substrate for detection of candidate proenkephalin processing proteases. 35S-(Met)-PPE was generated from the rat preproenkephalin (PPE) cDNA subcloned downstream of the SP6 promoter of the pSP65 transcription vector. Efficient in vitro transcription with SP6 RNA polymerase routinely generates approximately 20 µg RNA from

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93 Fig. 5.3. Immunoelectron microscopy of (Met)enkephalin in chromaffin granules. Sections of isolated chromaffin granules were incubated with anti-(Met)enkephalin (rabbit) serum in PBS (phosphate-buffered saline), and incubated with anti-rabbit IgG labeled with 5 nm gold. Electron dense 5 nM gold particles are visualized directly over the granules.

one µg plasmid DNA.24 Subsequent in vitro translation with wheat germ extract produces up to 20 million cpm 35S-(Met)enkephalin precursor from 4-5 µg RNA. Overall, one µg plasmid DNA can yield 1 x 108 cpm 35S-(Met)enkephalin precursor of high radiospecific activity.23,24 This approach of in vitro transcription and translation of cloned prohormone cDNAs allows efficient production of 35S-(Met)-precursors for studies of in vitro processing.

Purification and Biochemical Characterization of PTP Purification of 35S-(Met)enkephalin precursor cleaving activity from bovine adrenal medullary chromaffin granules results in the isolation of the novel ‘prohormone thiol protease’ (PTP) as the major proenkephalin processing activity.23,25 The majority, 80-90%, of enkephalin precursor cleaving activity from these granules is present in the soluble component of granules.22 Purification by Concanavalin A-Sepharose, Sephacryl S200 gel filtration, chromatofocusing, and thiopropyl-Sepharose results in purification of 33 kDa PTP. PTP is a glycoprotein with a pI of 6.0 and pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0.23 The thiol dependence of PTP is indicated by its stimulation by DTT (dithiothreitol), as well as by its inhibition by the cysteine protease reagents p-hydroxymercuribenzoate, mercuric chloride, cystatin C, E-64c, and E-64d. Importantly, peptide microsequencing of PTP indicates that it possesses a unique NH2-terminal primary sequence that bears no homology to any other known proteases.26 PTP has been characterized with recombinant proenkephalin (PE) in in vitro processing studies.27 In vitro concentrations of PE near estimated in vivo levels29 at 10–5 to 10–4 M were achieved by high level expression of PE in E. coli.27,28 PTP converted purified recombinant PE to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by SDS-PAGE gels and peptide microsequencing. Also, products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOHterminus of (Met)enkephalin-Arg6-Gly7-Leu8. Thus, PTP generates PE products in vitro (Fig. 5.4) that resemble those in adrenal medulla in vivo. These results indicate that PTP possesses appropriate cleavage sites for paired basic residues, and generates relevant PE-related peptide products. Kinetic studies with recombinant proenkephalin showed that PTP possesses a Km(app) of 18.6 µM PE and Vmax(app) of 1.98 mmol/hr-mg.27 PTP’s affinity for PE is compatible with the estimated concentration of PE within the secretory vesicle of approximately 10–4 M,29 indicating that PTP would be functional in vivo.

Cleavage Site Specificity at Dibasic and Monobasic Residue Sites PTP cleavage site specificity has been assessed by examining PTP cleavage of the enkephalin-containing peptides (Fig. 5.5) known as peptide F and BAM-22P that are generated in vivo from proenkephalin.23,30 PTP cleaves peptide F at Lys-Lys and Lys-Arg paired basic residue sites, with cleavage occurring between the two basic residues and at the

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Fig. 5.4. PTP in vitro processing of proenkephalin. PE products generated by PTP are illustrated below the schematic structure of PE. Bars with apparent molecular sizes indicate products that were identified by SDS-PAGE, peptide microsequencing, and reactivity to PE-18 monoclonal antibody.

Fig. 5.5. PTP cleavage of enkephalin-containing peptides. PTP cleavage sites within the enkephalin-containing neuropeptides known as peptide F and BAM-22P are shown by the arrows. The active opioid peptide (Met)enkephalin, YGGFM, is underlined. Amino acids representing paired basic or monobasic cleavage sites are shown as bold letters. Results indicate that PTP generates the final peptide (Met)enkephalin from the proenkephalin-derived intermediates known as peptide F and BAM-22P.

NH2-terminal side of the dibasic residues. PTP also cleaves the Arg-Arg site within BAM-22P at the NH2-terminal side of the dibasic residues. Interestingly, PTP cleaves at a monobasic arginine site within BAM-22P. Cleavage at this monobasic site in vivo is predicted as a necessary processing step in the formation of metorphamide, a bioactive opioid peptide.31 PTP thus possesses appropriate cleavage specificity for paired basic residues and monobasic arginine residues that are required for proenkephalin processing. Importantly, PTP processing at these basic residue sites generates the final product (Met)enkephalin.23,30 PTP’s cleavage sites at paired basic residues differs from the subtilisin-like PC1/3 and PC2 proteases (PC = prohormone convertase)32-38 and the pituitary aspartyl protease (known as ‘POMC converting enzyme,’ or PCE).39,40 PTP cleaves enkephalin-containing peptides at the NH2-terminal side of the dibasic residues or between the dibasic residues, as well as on the NH2-terminal side of monobasic arginine residues.23,30 In contrast, PC1/3, PC2, and PCE cleave at the COOH-terminal side of the dibasic residues, and between the dibasic residues.32-38

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Table 5.1. Comparison of PTP, PC1/3, and PC2 hydrolysis of peptide-MCA substrates, in the absence and presence of aminopeptidase M Peptide-MCA Substrate

-APM

+APM

-APM

+APM

-APM

+APM

PTP Activity (nmol AMC/h/mg)

PC1/3 Activity (nmol AMC/h/mg)

PC2 Activity (nmol AMC/h/mg)

Peptide-MCA Substrate

-APM

+APM

-APM

+APM

-APM

+APM

Dibasic substrates: Z-Arg-Arg-MCA Boc-Gln-Arg-Arg-MCA Boc-Gly-Arg-Arg-MCA Z-Arg-Val-Arg-Arg-MCA Boc-Gly-Lys-Arg-MCA Boc-Glu-Lys-Lys-MCA

16,000 15,000 7,000 19,000 21,000 7,000

49,000 118,000 42,000 50,000 73,000 26,000

ND 125 116 351 208 32

ND 192 118 345 300 51

ND 345 336 762 515 81

ND 364 360 747 552 104

Monobasic substrates: Z-Phe-Arg-MCA 1,176,000 1,915,000 Bz-Arg-MCA 7,000 24,000 Boc-Gln-Gly-Arg-MCA 6,000 84,000 Bz-Val-Leu-Lys-MCA 695,000 885,000 Ac-Lys-MCA 0 3,000

ND ND 2 ND ND

ND ND 29 ND ND

ND ND 6 ND ND

ND ND 36 ND ND

PTP, PC1/3, and PC2 were purified from bovine chromaffin granules, as previously described.23,38 Proteolytic activities with peptide-MCA substrates for each enzyme was determined under the established optimum buffer conditions for each enzyme. ND = not determined.

Further analysis of PTP cleavage sites utilized fluorogenic peptide-MCA substrates containing dibasic and monobasic residues.41,42 PTP cleavage of these peptide substrates indicates cleavage at the COOH-terminal side of dibasic and monobasic residues (Table 5.1). However, it is noted that if cleavage occurs at the NH2-terminal side of basic residues, resultant peptide-MCA products are not detected since only free AMC (not peptide-MCA products) is fluorometrically detected. Therefore, to assess cleavage between dibasic residues, or at their NH2-terminal side, aminopeptidase M (APM) was utilized after PTP hydrolysis to convert peptide-MCA products to free AMC that can be detected fluorometrically. Assays with APM showed several-fold higher activity compared to PTP alone, indicating that PTP preferentially cleaves at the NH2-terminal side of basic residues (Table 5.1). PTP’s preference for cleavage at NH2-terminal sides of basic residues compared to cleavage at the COOH-terminal sides, is illustrated in a bar graph plot (Fig. 5.6). In contrast, PC1/3 and PC2 cleave the dibasic peptide-MCA substrates primarily at the COOH-terminal side, since addition of APM makes little difference in their proteolytic activity (Table 5.1). In contrast to PTP, PC1/3 and PC2 possess much lower activity with monobasic peptide substrates. These results demonstrate PTP’s high specific activity for cleaving both dibasic and monobasic processing sites. A striking difference between PTP and the PC enzymes is the high PTP activity detected with these model substrates. PTP specific activity is 100-500 times greater (average range) than the specific activity measured for PC1/3 and PC2 (Table 5.1). The high activity of PTP, compared to PC1/3 and PC2, is also evident with several prohormone substrates, discussed in the next section.

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Proteolytic Activity (umol AMC/hr/mg)

a

Dibasic Peptides

Proteolytic Activity (umol AMC/hr/mg)

Peptide-MCA substrate

Monobasic Peptides NH2-Terminal Side COOH-Terminal Side

Peptide-MCA substrate Fig. 5.6. PTP cleavage of peptide-MCA substrates at NH2- and COOH-terminal sides of basic residues in peptide-MCA substrates. Cleavage at NH2-terminal (filled bars) and COOH-terminal (hatched bars) sides of basic residues are represented by PTP activity with aminopeptidase M (APM) minus activity without APM, and PTP activity without APM, respectively. (a) Dibasic peptides: Dibasic peptides numbered 1-6 are Z-Arg-ArgMCA, Boc-Gln-Arg-Arg-MCA, Boc-Gly-Arg-Arg-MCA, Z-Arg-Val-Arg-Arg-MCA, BocGly-Lys-Arg-MCA, and Boc-Glu-Lys-Lys-MCA, respectively (indicated in Table 5.1). (b) Monobasic peptides: Monobasic peptides numbered 1-5 are Z-Phe-Arg-MCA, Bz-ArgMCA, Boc-Gln-Gly-Arg-MCA, Bz-Val-Leu-Lys-MCA, and Ac-Lys-MCA, respectively (indicated in Table 5.1).

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Fig. 5.7. Recombinant prohormones generated with the pET3c expression vector. Proenkephalin, proneuropeptide Y, and proopiomelanocortin (POMC) structures are illustrated. Proenkephalin contains four copies of (Met)enkephalin (M), one copy of (Leu)enkephalin (L), and the enkephalin-related octapeptide (O) and heptapeptide (H). ProNPY contains NPY- and COOHterminal peptide (CTP) sequences. POMC contains ACTH and β-endorphin-related bioactive peptides.

Selectivity for Prohormone Substrates Although prohormones contain similar dibasic or monobasic processing sites, the unique primary structures of prohormones predict that each prohormone possesses a unique conformation that may be preferentially recognized by particular processing enzyme(s). To test the hypothesis that PTP and other processing enzymes may possess selectivity for different prohormone substrates, recombinant proenkephalin (PE), proneuropeptide Y (proNPY), and proopiomelanocortin (POMC) (Fig. 5.7) were expressed in E. coli, and the relative rates of in vitro processing of these precursors were measured (Fig. 5.8).27,28,43,44 PTP efficiently processed PE and proNPY at rates of 383 and 420 µmol/hr/mg enzyme, respectively; however, PTP was much less effective in cleaving POMC. Comparison of four candidate processing enzymes showed that PTP processed PE at rates that were 1,665 and 383,000 times greater than that by the 70 kDa aspartyl protease (also known as ‘POMCconverting enzyme’ or PCE) or PC (PC1/3 and PC2) enzymes, respectively. PTP also processed proNPY 35,000 and 84,000 times more efficiently than the 70 kDa aspartyl protease or the PC enzymes, respectively. In contrast, the 70 kDa aspartyl protease was most effective in POMC processing,43,45,46 and the PC enzymes were also very good;43 however, PTP showed little cleavage of POMC. Furthermore, PTP does not cleave proinsulin (Hook and Gorman, unpublished observations). These results indicate that PTP possesses a high degree of selectivity for particular prohormone substrates.

Immunohistochemistry and Immunoelectron Microscopy of PTP: Localization to Secretory Vesicles To develop antisera for immunolocalization studies of PTP, antisera were generated against a synthetic peptide that corresponds to the NH2-terminus of purified PTP that was determined by peptide microsequencing. The determined NH2-terminal peptide sequence of PTP is a novel sequence,26 as assessed by comparison with sequences in the protein database. The anti-PTP serum detects 33 kDa PTP26 in chromaffin granules, as well as a band of 55 kDa that may represent a zymogen form of PTP. The antibody immunoprecipitates

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Fig. 5.8. Comparison of PTP, 70 kDa apartyl protease, and PC enzymes in rates of processing recombinant proenkephalin, proNPY, and POMC. The relative rates of processing proenkephalin, proNPY, and POMC—panels (a), (b) and (c), respectively—by PTP, 70 kDa aspartyl protease (PCE, indicated as AP), PC1/3, and PC2 were determined by incubating recombinant prohormones with purified processing enzymes, and the rates of prohormone cleavages were quantitated by SDS-PAGE gels and densitometric scanning of prohormone bands. The specific activity of prohormone processing was calculated as µmol prohormone cleaved per hour per milligram enzyme protein (µmol/h/mg enzyme).

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Fig. 5.9. Chromaffin cell localization of PTP by immunoelectron microscopy. Chromaffin granules were prepared for immunoelectron microscopy, as described in Figure 5.3. Sections of isolated chromaffin granules were incubated with anti-PTP (rabbit) serum in PBS (phosphate-buffered saline), and incubated with anti-rabbit IgG labeled with 5 nm gold. Electron dense 5 nM gold particles are visualized directly over the granules. The average diameter of the chromaffin granule is approximately 0.1 to 0.2 µM.53

purified PTP activity, indicating that the determined NH2-terminal sequence corresponds to PTP. Immunofluorescence immunocytochemistry indicates punctate, perinuclear PTP staining of chromaffin cells in primary culture (Hook et al, manuscript in preparation). This discrete staining pattern is consistent with a secretory vesicle localization of PTP. The subcellular localization was further examined by immunoelectron microscopy of PTP in isolated chromaffin granules (Fig. 5.9). Immunogold labeling shows the presence of PTP within the chromaffin granules. These results provide definitive evidence for the secretory vesicle localization of PTP.

(Met)enkephalin and PTP are Coordinately Regulated by cAMP in Chromaffin Cells Indication of PTP as the primary proenkephalin processing activity from in vitro studies suggests that PTP may be involved in the regulation of (Met)enkephalin biosynthesis. Forskolin, a stimulator of adenylate cyclase, raises intracellular cAMP, and leads to a 2-fold elevation of (Met)enkephalin levels in chromaffin cells.26,47 During this stimulation, enkephalin precursor cleaving activity from forskolin-treated chromaffin cells was elevated 2-fold in chromaffin granules. Importantly, the elevated processing activity was immunoprecipitated by anti-PTP antibodies, and was inhibited by the cysteine protease inhibitor E64c that is a potent inhibitor of PTP. These results indicate cAMP stimulation of PTP and (Met)enkephalin in chromaffin cells. Moreover, a role for PTP in cellular PE processing is suggested by blockade of forskolin-stimulated (Met)enkephalin production by incubation of cells with Ep453,26 which is converted intracellularly to E-64c that potently inhibits PTP.41

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Table 5.2. Properties of ‘prohormone thiol protease’ (PTP) – Secretory vesicle localization – 33 kDa glycoprotein, with pI of 6.0. – Cysteine protease, as determined by protease inhibitors. – pH optimum of 5.5, consistent with intravesicular pH. – Cleaves dibasic and monobasic sites: Dibasic: Lys-Arg, Lys-Lys, Arg-Arg Monobasic: Arg – High specific activity compared to other processing enzymes. – Distinct selectivity for prohormone substrates.

These results demonstrate that cAMP-stimulation of PTP is involved in regulating cellular production of (Met)enkephalin.

Participation of PC1/3 and PC2 Subtilisin-Like Proteases, and 70 kDa Aspartyl Protease (PCE) in Proenkephalin Processing in Chromaffin Granules It is noted that while PTP represents the major proenkephalin processing enzyme in chromaffin granules, lesser activities were identified as the subtilisin-like PC1/3 and PC2 proteases,38 as well as the 70 kDa aspartyl protease45,46 (also known as ‘POMC converting enzyme’ or PCE). The presence of the subtilisin and aspartyl processing proteases in chromaffin granules indicates that PC1/3, PC2 and PCE may also be involved in certain steps of proenkephalin processing. Studies described in this chapter have compared proteases that generate the first cleavage(s) in proenkephalin and other prohormones. Based on differences in primary and therefore, tertiary, structures of prohormones and proteolytic products, processing of intermediates may involve similar or different proteases. It will be important in future studies to determine the order of proteases that are responsible for proteolytic steps of the prohormone processing pathway.

Conclusions Results of these studies indicate the ‘prohormone thiol protease’ (PTP) as a distinct protease among candidate prohormone processing enzymes (Table 5.2). Determination of PTP’s NH2-terminal peptide sequence indicates it as a unique cysteine protease. PTP is a 33 kDa glycoprotein localized to secretory vesicles. It displays a pH optimum of 5.5 that is consistent with the intravesicular pH.1-3 PTP preferentially cleaves dibasic residue sites between the two residues, and at the NH2-terminal side of the paired basic residues; PTP also cleaves at the NH2-terminal side of monobasic arginine residues. PTP possesses extremely high specific activity compared to PC1/3 and PC2 when assayed with dibasic and monobasic peptide-MCA substrates (Table 5.1). Significantly, PTP possesses a high degree of selectivity for proenkephalin and certain prohormone substrates. These properties suggest PTP as an important proenkephalin processing enzyme that possesses selectivity for particular prohormones. It is of interest to note that the primary proteolytic activities within secretory vesicles in vivo from different tissues parallel each protease’s preferences for prohormone substrates (Table 5.3). Proenkephalin processing in chromaffin granules is achieved primarily by PTP, and PTP shows high activity with PE as substrate. POMC processing in pituitary secretory vesicles is primarily accomplished by PCE,39,40 and PCE (which presumably is represented by the 70 kDa aspartyl protease) shows highest activity with POMC (compared to PE and

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Table 5.3. Primary processing proteases identified in isolated neuroendocrine secretory vesicles

Secretory Vesicles

Endogenous Prohormone

Primary Protease for Processing the Endogenous Prohormone

Bovine adrenal medulla chromaffin granules

Proenkephalin

‘Prohormone Thiol Protease’(PTP)*

Pituitary intermediate lobe secretory vesicles

POMC

‘POMC Converting Enzyme’ (PCE)+

Pancreatic insulin vesicles

Proinsulin

PC1/3 and PC2# (PC = prohormone convertase)

* PTP studies23,27,28,30,43,44;+ PCE studies39,40; # PC1/3 and PC2 studies1-3,48,49

proNPY) as substrate. Furthermore, pancreatic insulin secretory vesicles contain PC1/3 and PC2 as primary proinsulin processing proteases.48,49 These observations suggest that distinct, specific processing proteases may be involved in converting different prohormones into active neuropeptides. These studies of PTP, combined with other studies in the field of prohormone processing, indicate that at least three mechanistic groups of proteases are involved in prohormone processing: 1. the unique cysteine protease PTP; 2. the subtilisin-like PC1/3 and PC2 enzymes; and 3. the 70 kDa aspartyl protease known as ‘POMC converting enzyme’ (PCE). It will be important in future studies to determine the specific roles of multiple processing proteases in the conversion of different precursors into active peptide hormones and neurotransmitters.

Acknowledgments This work was supported by grants from the National Institute of Drug Abuse (NIH), National Institute of Neurological Disease and Stroke (NIH), and the National Science Foundation.

References 1. Docherty K, Steiner DF. Post-translational proteolysis in polypeptide hormone biosynthesis. Annu Rev Physiol 1982; 44:625-638 2. Gainer H, Russel JT, Loh YP. The enzymology and intracellular organization of peptide precursor processing: The secretory vesicle hypothesis. Neuroendocrinology 1985; 40:171-184 3. Hook VYH, Azaryan AV, Hwang SR, Tezapsidis N. Proteases and the emerging role of protease inhibitors in prohormone processing. FASEB J 1994; 8:1269-1278. 4. Devi L. Consensus sequences for processing of peptide precursors at monobasic sites. FEBS Lett 1991; 280:189-194. 5. Fricker LD. Peptide processing exopeptidases: Amino and carboxypeptidases involved with peptide biosynthesis. In: Fricker LD, ed. Peptide Biosynthesis and Processing. Boca Raton: CRC Press, 1991:199-229. 6. Gainer H, Sarne Y, Brownstein MG. Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides. J Cell Biol 1977; 73:366-381.

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7. Orci L, Ravazzola M, Storch MG, Anderson RGW, Vassalli JD, Perrelet A. Proteolytic maturation of insulin is a post-golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987; 49:865-868. 8. Orci L, Halban P, Perrelet A, Amherdt M, Ravazzola M, Anderson RGW. pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle. J Cell Biol 1994; 126:1149-1156. 9. Carmichael SW, Winkler H. The adrenal chromaffin cell. Sci Am 1985; 253:40-49. 10. Hook VYH, Eiden LE. Two peptidases that convert 125I-Lys-Arg-(Met)enkephalin and 125I(Met)enkephalin-Arg6, respectively, to 125I-(Met)enkephalin in bovine adrenal medullary chromaffin granules. FEBS Lett 1984; 171:212-218. 11. Schultzberg M, Hokfelt T, Lundberg JM, Terenius L, Elfvin LG, Elde R. Enkephalin-like immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medullary gland cells. Acta Physiol Scand 1978; 103:475-477. 12. Udenfriend S, Kilpatrick DL. Biochemistry of the enkephalins and enkephalin-containing peptides. Arch Biochem Biophys 1983; 221:309-323. 13. Liston DR, Vanderhaeghen JJ, Rossier J. Presence in brain of synenkephalin, proenkephalinimmunoreactive protein which does not contain enkephalin. Nature 1983; 302:62-65. 14. Yoshikawa K, Williams C, Sabol SL. Rat brain preproenkephalin mRNA, cDNA cloning, primary structure, and distribution in the central nervous system. J Biol Chem 1984; 259:14301-14308. 15. Gubler U, Seeburg P, Hoffman BJ, Gage LP, Udenfriend S. Molecular cloning establishes proenkephalin as precursor of enkephalin-containing peptides. Nature 1982; 295:206-208. 16. Spruce BA, Jackson S, Lowry PJ, Lane DP, Glover D. Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. J Biol Chem 1988; 263:19788-19795. 17. Birch NP, Christie DL. Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum. J Biol Chem 1986; 261:12213-12221. 18. Hook VYH, Liston D. Distribution of enkephalin containing peptide within bovine chromaffin granules. Neuropeptides 1987; 9:263-268. 19. Carmichael WS, Stoddard SL, O’Connor DT, Yaksh TL, Tyce GM. The secretion of catecholamines, chromogranin A and neuropeptide Y from the adrenal medulla of the cat via the adrenolumbar vein and thoracic duct: Different anatomic routes based on size. Neuroscience 1990; 34:433-440. 20. Lundberg JM, Hamberger B, Schultzberg M, Hokfelt T, Granberg PO, Efendie S, Terenius L, Goldstein M, Luft R. Enkephalin-and somatostatin-like immunoreactivities in human adrenal medulla and pheochromocytoma. Proc Natl Acad Sci USA 1979; 76:4079-4083. 21. Holzward MA. The distribution of vasoactive intestinal peptide in the rat adrenal cortex and medulla. J Auton Nerv Syst 1984; 11:269-283. 22. Rokaeus A, Brownstein MG. Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor. Proc Natl Acad Sci USA 1986; 83:6287-6291. 23. Krieger TK, Hook VYH. Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor. J Biol Chem 1991; 266:8376-8383. 24. Hook VYH, Schiller MR, Nguyen C, Yasothronsrikul S. Production of a radiolabeled neuropeptide precursors by in vitro transcription and translation. Peptide Res 1996; 9:183-187. 25. Hook VYH, Hegerle D, Affolter HU. Cleavage of recombinant enkephalin precursor by endoproteolytic activity in bovine adrenomedullary chromaffin granules. Biochem Biophys Res Commun 1990; 167:722-730. 26. Tezapsidis N, Noctor S, Kannan R, Krieger TK, Mende-Mueller L, Hook VYH. Stimulation of ‘prohormone thiol protease’ (PTP) and (Met)enkephalin for forskolin: Blockade of elevated (Met)enkephalin by cysteine protease inhibitor of PTP. J Biol Chem. 1995; 270:13285-13290. 27. Schiller MR, Mende-Mueller L, Miller KW, Hook VYH. ‘Prohormone thiol protease’ (PTP) processing of recombinant proenkephalin. Biochemistry 1995; 34:7988-7995.

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28. Hook VYH, Moran K, Kannan R, Kohn A, Lively MO, Azaryan A, Schiller M, Miller K. High-level expression of the prohormone proenkephalin, proneuropeptide Y, proopiomelanocortin, and protachykinin for in vitro prohormone processing. Prot Express Purif 1997; 10:80-88. 29. Ungar A, Phillips JH. Regulation of the adrenal medulla. Physiol Rev 1983l 63:787-843. 30. Kreiger TK, Mende-Mueller L, Hook VYH. Prohormone thiol protease and enkephalin precursor processing: Cleavage at dibasic and monobasic sites. J Neurochem 1992; 59:26-31. 31. Weber E, Esch FE, Bohlen P, Paterson S, Corbett AD, McKnight AT, Kosterlitz HW, Barchas JD, Evans CJ. Metorphamide: Isolation, structure, and biologic activity of an amidated opioid octapeptide from bovine brain. Proc Natl Acad Sci USA 1982; 80:7362-7366. 32. Benjannet D, Rondeau N, Day R, Chretien M, Seidah NG. PC1 and PC2 are proprotein convertases capable of cleaving propiomelanocortin at distinct pairs of basic residues. Proc Natl Acad Sci USA 1991; 88:3564-3568. 33. Thomas L, Leduc R, Thorne B, Smeekens SP, Steiner DF, Thomas G. KEX2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: Evidence for a common core of neuroendocrine processing enzymes. Proc Natl Acad Sci USA 1991; 88:5297-5301. 34. Sheenan KIJ, Smeekens SP, Steiner DF, Docherty R. Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes. FEBS Lett 1991; 284:277-280. 35. Zhou Y, Lindberg I. Purification and characterization of the prohormone convertase PC1 (PC3). J Biol Chem 1993; 268:5615-5623. 36. Friedman TC, Loh YP, Birch NP. In vitro processing of proopiomelanocortin by recombinant PC1 (PC3). Endocrinology 1994; 135:854-862. 37. Nillni EA, Friedman TC, Todd RB, Birch NP, Loh YP, Jackson IM. Pro-thyrotropin-releasing hormone processing by recombinant PC1. J Neurochem 1995; 65:2462-2472. 38. Azaryan AV, Krieger TK, Hook VYH. Characteristics of the candidate prohormone processing proteases, PC2 and PC1/3, from bovine adrenal medulla chromaffin granules. J Biol Chem 1995; 270:8201-8208. 39. Loh YP, Parish DC, Tuteja R. Purification and characterization of paired basic residuespecific pro-opiomelanocortin converting enzyme from bovine pituitary intermediate lobe secretory vesicles. J Biol Chem 1985; 260:7194-9205. 40. Loh YP, Beinfeld MC, Birch NP. Proteolytic processing of prohormone and pro-neuropeptides. In: Loh YP, ed. Mechanisms of Intracellular Trafficking and Processing of Proproteins. CRC Press 1993:179-224 41. Azaryan AV, Hook VYH. Unique cleavage site specificity of ‘prohormone thiol protease’ related to proenkephalin processing. FEBS Lett 1994; 341:197-202. 42. Azaryan AV, Hook VYH. Distinct properties of ‘prohormone thiol protease’ (PTP) compared to cathepsins B, L, and H: Evidence for PTP as a novel cysteine protease. Arch Biochem Biophys 1994; 314:170-177. 43. Hook VYH, Schiller MR, Azaryan AV. The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70 kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates. Arch Biochem Biophys 1996; 328:107-1147. 44. Schiller MR, Kohn AB, Mende-Mueller L, Miller K, Hook VYH. Expression of recombinant pro-neuropeptide Y, proopiomelanocortin, and proenkephalin: Relative processing by ‘prohormone thiol protease’ (PTP). FEBS Lett 1996; 382:6-10. 45. Azaryan AV, Schiller M, Mende-Mueller L, Hook VYH. Characteristics of the chromaffin granules aspartic proteinase involved in proenkephalin processing. Neurochem 1995; 65:1771-179. 46. Azaryan AV, Schiller MR, Hook VYH. Chromaffin granule aspartic proteinase processes recombinant proopiomelanocortin (POMC). Biochem Biophys Res Comm 1995; 215:937-944.

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47. Hook VYH, Eiden LE, Pruss RM. Selective regulation of carboxypeptidase peptide hormone processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells. J Biol Chem 1985; 260:5991-5997. 48. Bennet DL, Bailyes EM, Nielson E, Guest PC, Rutherford NG, Arden SD, Hutton JC. Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. J Biol Chem 1992; 267:15229-14236. 49. Bailyes EM, Shennan KI, Seal AJ, Smeekens SP, Steiner DF, Hutton JC, Docherty K. A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. Biochem J 1992; 285:391-394. 50. Studier FW, Rosenberg AH, Dunn JJ, Dubenforff JW. Use of the T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 1990; 185:60-89. 51. Kang, YJ, Carl M, Watson LP, Yaffe L. Immunoelectron microscopic identification of human NK cells by FITC-conjugated anti-Leu-11a and anti-Leu-7 antibodies. J Immunol Methods 1985; 84:177-196. 52. Kang YH, Dwivedi RS, Lee CH. Ultrastructural and immunocytochemical study of the uptake and distribution of bacterial lipopolysaccharide in human monocytes. J Leuko Biol 1990; 48:316-332. 53. Darchen F, Senyshyn J, Brondyk WH, Taatjes DJ, Holz RW, Henry JP, Denizot JP, Macara IG. The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells. J Cell Sci 1995; 108:1639-1649.

CHAPTER 6

Regulation of Prohormone Conversion by Coordinated Control of Processing Endopeptidase Biosynthesis with that of the Prohormone Substrate Terence P. Herbert, Cristina Alarcon, Robert H. Skelly, L. Cornelius Bollheimer, George T. Schuppin and Christopher J. Rhodes

I

t has been established for many years that maintenance of intracellular store levels for mature polypeptide hormones in neuroendocrine cells can be regulated at the level of both gene expression and mRNA translation. Recent evidence has unveiled a specific coordinated control of the appropriate prohormone convertase(s) in parallel to their particular prohormone substrate, both at the level of transcription and translation. In general, translational regulation applies to the short-term (<2 hours) stimulation of prohormone biosynthesis, but for the longer term situation (>4 hours) there is additional transcriptional regulation. The specific parallel regulation of prohormone biosynthesis with that of the appropriate prohormone processing endopeptidases implicates unique common control mechanisms. For gene transcription this coordinated control likely resides in common ciselements in these genes and trans-acting factors peculiar to a given neuroendocrine cell type. For specific translational regulation, it is unlikely that this coordinated control is via initiation or elongation factor activity regulation, since this would affect protein synthesis translation in general. Thus, the focus has been on potential common regulatory elements in the untranslated regions of prohormone and proprotein convertase mRNAs.

Introduction Polypeptide hormones are often synthesized as larger inactive precursor prohormones which are post translationally processed into biologically active hormones by limited proteolysis.1,2 One feature of prohormone biosynthesis is the rapid changes in the demand for a particular hormone to exogenous stimuli (e.g., the increased demand for insulin in response to a rise in blood glucose concentration3,4). To ensure that optimal intracellular storage levels of a peptide hormone are maintained for regulated secretion, increases in demand for hormone secretion are often met by a corresponding increase in prohormone gene expression and/or biosynthesis. Such an increase in prohormone biosynthesis in turn leads to an increased demand in the specific proteolytic processing enzymes to assure efficient Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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processing of prohormone to biologically active hormone. A means by which this increased demand is met is the specific coordinate regulation of gene expression and/or biosynthesis of proteolytic processing enzymes with that of their prohormone substrate. This chapter focuses on the current knowledge for this latter aspect of the mechanism behind regulated polypeptide hormone production.

Coordinated Regulation of Prohormone and Processing Enzyme mRNA Levels There have been several reports of coordinate gene expression of a prohormone with its specific proprotein-processing endopeptidases. The most extensively studied of these is that of preproinsulin and proopiomelanocortin (POMC).

The Preproinsulin Gene Expression Example The pancreatic β cell is the site of production, storage and regulated secretion of insulin. Preproinsulin gene expression and proinsulin biosynthesis in β cells is regulated by many factors, including certain amino acids and some nutrients,4-10 but glucose is the most physiologically relevant.5,6,11 Proinsulin conversion to insulin is catalyzed by endopeptidic cleavage of human proinsulin at the carboxyl-terminal side of the dibasic amino acid sequences Arg31-Arg32 and Lys64-Arg65.1,12 PC1/PC3 cleaves at the Arg-Arg site, and PC2 has a strong preference for the Lys-Arg site.12-14 Once an endopeptidic cleavage has been made the newly exposed basic amino acids are removed by the exopeptidase carboxypeptidase-H (CP-H). Long term exposure of pancreatic β cells to glucose (>6 h) induces an increase in preproinsulin transcription,15-17 which in turn results in an increase in preproinsulin mRNA levels in the pancreatic beta cell.18,19 The specific levels of the proinsulin converting endopeptidases PC2 and PC1/3 have been shown to be coordinately regulated in parallel to preproinsulin mRNA levels in response to long term exposure to glucose (>12 h) in insulinproducing βTC3 cells.17 Interestingly, mRNA levels of the proinsulin processing exopeptidase CP-H did not appear to be affected by glucose in βTC3 cells. In an in vivo study, coordinate pancreatic islet expression of preproinsulin mRNA with that of its processing enzymes PC2, PC1/3 and CPH has been examined.20 Pancreatic islets were isolated from hyperglycemic rats infused with 50% (w/v) glucose over a 48 hour period. The hyperglycemia specifically increased levels of preproinsulin and PC1/3 mRNAs about 3-fold. However the expression of PC2 and CPH mRNA was unaffected by the treatment. The apparent discrepancy in the coordinate expression of PC2 with preproinsulin observed in βTC3 cells with that observed in pancreatic islets is likely explained by the heterogeneity of the islet cell population. The islet is a heterogeneous population of cells made up of α, β, δ and PP cells.21 PC2 is more abundantly expressed in α, PP and δ cells,22 and although glucose induces a specific induction of PC2 expression in β cells, it is masked by the expression of PC2 in islet non-β cells which are not responsive to glucose. The specific glucose-induced coordinate increase in mRNA levels for preproinsulin, PC1/3 and PC2 in the pancreatic β cell is likely a reflection of two factors: 1. the rate of gene transcription; and/or 2. the rate of mRNA decay. Nuclear run-on experiments were used to measure the rate of transcription of PC2, PC3 and preproinsulin genes in βTC3 cells in response to glucose.17 The transcription rate of preproinsulin, PC2 and PC1/3 genes increased approximately 2-fold in response to a prior 1 h exposure to glucose,17 implicating glucose regulation mediated at the transcriptional level. No glucose regulation of CP-H transcription in β cells has been observed. It should be noted, however, that although glucose can induce preproinsulin, PC2 and PC1/3 gene transcription within 1 hour,16,17,23 an increase in mRNA levels in the β cell cytoplasm is

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not reflected until >6 hours glucose exposure.15-17,24,25 This is due to pre-mRNA processing intron excision occurring in the nucleus prior to mRNA export, which does not appear to be regulated in pancreatic β cells.19,26 The human preproinsulin gene contains 3 exons and 2 intervening sequences,19,26 the mouse PC1/3 gene 15 exons/14 introns,27,28 the human PC2 gene 12 exons/11 introns,29 and rat CP-H 9 exons/8 introns.30 Cytosolic levels of mRNAs can also be altered by changes in their half-life. In pancreatic β cells, the half-life of preproinsulin mRNA is unusually long (>48 h),17,31 and has been shown to be slightly increased by very long term glucose exposure (>24 h).31 In contrast to preproinsulin mRNA, half-lives of PC2, PC1/3 and CP-H mRNAs in β cells (between 4-6 hours) are more characteristic of mRNAs in general, with no apparent glucose regulation of their stability.17 Thus, coordinate regulation of preproinsulin, PC1/3 and PC2 mRNA levels is primarily mediated at the gene transcription level.17 Nonetheless, it should be noted that glucose-induced preproinsulin gene transcription (and that of PC1/3 and PC217), minimally requires more than 6 hours exposure to elevated glucose levels.18,19 However, under normal circumstances in vivo, circulating glucose concentrations are tightly controlled by insulin secretion.12,32 Thus, glucose regulated preproinsulin, PC1/3 and PC2 gene transcription is really only applicable to unusual circumstances of prolonged hyperglycemia,33,34 or refeeding after starvation35 and/or hypoglycemia.23,36 The preproinsulin, PC1/3 and PC2 gene promoter sequences (see below) all contain a cAMP response element (CRE). However, elevation of cAMP in pancreatic β cells does not appear to induce specific transcription of preproinsulin, PC1/3 and PC2 genes, in spite of a marked cAMP induction of c-jun and c-fos genes in the same β cells and of phosphorylation activation of the CRE-binding protein transcription factor.16,17,25

The Proopiomelanocortin (POMC) Gene Expression Example The prohormone POMC is expressed mostly in the neuro intermediate lobe, as well as in ~5% of anterior pituitary cells.37 In the anterior lobe of the pituitary POMC is primarily processed into adrenocorticotropin (ACTH) and β-lipotrophin (LPH). In the neuro intermediate lobe of the pituitary POMC is processed more extensively to β-endorphin, corticotrophin-like intermediate lobe peptide (CLIP), melanocyte stimulating hormone (MSH), and LPH.37 The prohormone processing endopeptidases believed to be primarily responsible for the processing of POMC are PC2 and PC1/3.38-40 Coexpression of PC1/3 and POMC results only in the production of ACTH and β-LPH, whereas coexpression of both PC1/3 and PC2 results in the production of ACTH, LPH and β-endorphin in cells which contain only a constitutive pathway; in cells containing a regulated secretory pathway ACTH is further cleaved to MSH and CLIP.38,39 Complementary to these observations, levels of PC2 mRNA are higher than PC1/3 in the intermediate lobe of the pituitary, but, conversely, in the anterior pituitary lobe PC1/3 mRNA levels are higher than PC2 mRNA levels.41 In the intermediate lobe, POMC gene transcription can be regulated by the neurotransmitter dopamine via the D2 dopamine receptor.42 Bromocryptine, a dopamine agonist, decreases POMC mRNA, whereas haloperidol, a dopamine receptor agonist, increases POMC mRNA.42 It has been found that PC2 and PC1/3 mRNA levels coordinately increase in parallel to POMC mRNA in response to haloperidol.41,43,44 Concordantly, in response to bromocryptine, PC2 and PC1/3 mRNAs fell in parallel to that of POMC.41,43,44 Further, in intermediate pituitary cells, mRNA levels for CP-H and peptidylglycine alpha-amidating monooxygenase (PAM) also decrease in parallel to POMC after bromocryptine treatment and increase after haloperidol treatment.44 In pituitary cells coordinated regulation of CP-H mRNA levels, in parallel to the POMC mRNA levels, contrasts with that seen in pancreatic β cells where CP-H mRNA levels remain constant when preproinsulin mRNA levels increase.20 In contrast to intermediate pituitary cells, haloperitol treatment of anterior pituitary cells

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markedly increases PC2 mRNA levels, whereas PC1/3 mRNA levels do not change.41 Thus, regulation of PC1/3 gene expression differs in anterior versus intermediate pituitary cells. In pituitary cells, the dopamine D2 receptor signal transduction pathway involves heterotrimeric G protein coupled interaction with adenylate cyclase, which in turn alters intracellular cAMP levels in response to bromocriptine or haloperidol.42 This could subsequently result in phosphorylation activation of the CRE-binding protein and increased gene transcription of responsive genes that contain a cAMP-response element (CRE),45 including POMC, PC1/3 and PC2,27-29,42,46 and/or induction of transcription factors such as c-Fos and c-Jun17,45 which in turn induce transcription of other genes via binding activation to an AP-1 site in the promoter.47 It has been shown that under certain circumstances (e.g., regulation by CRH) that induction of POMC gene transcription is dependent on c-Fos/c-Jun expression.48 A similar c-Jun/c-Fos requirement might also be necessary for PC1/3 and PC2 gene expression in intermediate pituitary cells. It should be noted, however, that in intermediate lobe pituitary cells, unlike pancreatic β cells,17 elevated cAMP can induce POMC, PC1/3 and PC2 gene transcription, likely via CRE.41,43,44,49,50 However, in anterior pituitary cells haloperitol does not induce PC1/3 mRNA levels in spite of increased POMC and PC2 gene expression, and therefore different transcriptional regulation applies in different pituitary cell types. Nonetheless, the pattern in pituitary cells for the majority of the time is that effects on POMC expression are generally accompanied by a parallel increase/decrease in PC1/3 and PC2 gene expression. Other circumstances which have previously been shown to affect POMC gene expression, such as hypothyriodism or dexamethazone treatment, also affect PC1/3 and PC2 gene expression.41 Thus, in pituitary cells, dopamine, thyroid hormones and corticosteroids are implicated in regulation of PC1/3 and PC2 gene expression in parallel to that of POMC.

Processing Enzyme and Prohormone Substrate Promoter Regions Regulatory elements within the 5'-promoter regions of both the prohormone and its conversion enzyme genes may be shared, which in turn confers the ability for the transcription of these genes to be coordinately regulated in response to an appropriate stimulus in a given cell type. As outlined in Table 6.1, some common regulatory elements are found in both prohormone substrates (proinsulin and POMC are used as examples in this instance) and the relevant processing enzyme gene promoter sequences. However, a good deal of care should be taken in interpreting the significance of coincidental expression of transcription factor coding elements, and the interpretations must be experimentally tested before making any formative conclusions. Some transcription factors, such as SP-1, are ubiquitous19 and unlikely to be involved in specific regulation of gene expression. Certain other transcription factors, such as Pan-1 (a helix-loop-helix binding protein), form heterodimers with a variety of other transcription factors in a cell-specific manner, that in turn affects its DNA-binding and transcription of a given gene.19 In the example of the preproinsulin gene promoter in pancreatic β cells, Pan-1 is able to form heterodimers with the transcription factors Meso-1,51 Mash-1,52 NeuroD/Beta253 and also inhibitory transcription factors Id1 and Id3.19 However, it is not clear under what circumstances a particular Pan-1-containing transcription factor heterodimer complex will bind the preproinsulin gene promoter region and what will then be the consequences of that DNA binding for regulation of preproinsulin gene transcription. Furthermore, some transcription factors are apparently more critical than others. The homeodomain transcription factor pdx-1 (also known as IPF-1, STF-1, IUF-1, Idx-1) appears to be very important for not only preproinsulin gene transcription but also endocrine pancreatic development.54-57 Homozygous pdx-1 knockout transgenic mice do not develop a pancreas and die soon after birth.58 pdx-1 is also

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Table 6.1. Some consensus regulatory elements present in known promoter regions of processing enzyme and prohormone genes Gene

Consensus Regulatory Elements Present

Ref.

Human Preproinsulin Human POMC Murine PC1/3 Human PC2 Rat CPH Human 7B2

SP-1, CRE, Pan-1, NRE, TATA Box, CCAAT Box, GAGA Box SP-1, CRE, Pan-1, AP-1, TRS, TATA Box, GAGA Box, SP-1, CRE, Pan-1, AP-1, ICS, GHF-1, GAGA Box SP-1, CRE, AP-2, TRS, Pan-1 SP-1, AP-2, NF-1, Pan-1 CRE, AP-1, Pan-1, TRS, HSE, Pit-1/GHF-1

19 46 27 29 30 93

The 5'-promoter primary sequences for human preproinsulin, human POMC, murine PC1/3, human PC2, rat CP-H and human 7B2 (a specific chaperone for PC294) have been cloned and contain some putative regulatory elements. Abbreviations are: SP-1, consensus binding site for the ubiquitous transcription factor SP-1; CRE, cAMP response element; Pan-1, consensus binding site for the helixloop-helix transcription factor Pan-1; NRE, negative regulatory element; AP-1, consensus binding site for the AP-1 transcription factor complex containing c-fos; AP-2, consensus binding site for protein kinase-C sensitive transfactor complexes; TRS, thermal stress response consensus sequence; ICS, interferon consensus sequence; HSE, heat shock consensus sequence; Pit-1/GHF-1, consensus binding site for POU proteins; NF-1, consensus binding site for the transcription factor NF-1.

important for somatostatin gene transcription,56 indicative that certain transcription factors have multiple functions dependent on the cell type. To date the 5'-promoter region of the preproinsulin gene has been shown to bind at least twelve different transcription factors to different elements in that region.18,19 This, in turn, suggests that the combination of transcription factors required to drive expression of a prohormone gene are likely to be cell specific. Nonetheless, it remains to be shown under what regulatory circumstances certain factors associate to the preproinsulin gene promoter, and what combination of transcription factors is required to drive preproinsulin gene transcription in pancreatic β cells. In general, similar circumstances will also be appropriate for the regulated gene expression of other prohormones and processing enzymes in other endocrine cell types. However, although some transcription factors might be common, the final composition of factors required will ultimately be specific for a given prohormone and peculiar to the cell where that the prohormone is expressed. It should also be noted that presence of a primary sequence encoding for a transcription factor binding in a promoter region of a gene does not necessarily mean that it is functional, and could be dependent on whether that element is functional in a particular cell type. For example, a CRE is present in the preproinsulin, PC1/3 and PC2 gene promoter sequences, but cAMP does not appear to regulate expression of these genes in pancreatic β cells.17 In contrast to pancreatic β cells, in intermediate pituitary cells cAMP does appear to regulate gene expression of POMC, PC1/3 and PC2;41 in anterior pituitary cells only PC2 mRNA, and not that of PC1/3 is increased by cAMP mediated pathways.41 Therefore, as well as the combination of transcription factors required to drive expression of a given prohormone/processing enzyme being specific to an endocrine cell-type, another important consideration is that transcription factors are responsive to an appropriate signal transduction pathway in that cell.

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Coordinated Translational Regulation of Specific Prohormone and Processing Enzyme Biosynthesis Intracellular content of polypeptide hormones in endocrine cells in dense core secretory granules is continually maintained at optimal levels so that a hormone is readily available for rapid regulated release in response to an extracellular stimulus. The secreted hormone lost from the cell in response to the stimulus is rapidly replaced by a corresponding specific stimulation of prohormone biosynthesis. It is becoming apparent that this specific regulation of prohormone biosynthesis is mediated at the level of translation. Such an increase in the biosynthesis of a prohormone places an obvious increased demand on the proteolytic processing of that precursor; however this is provided for by a coordinated regulation of the appropriate proprotein convertase biosynthesis in parallel to the prohormone substrate. This is especially appropriate to the regulation of proinsulin biosynthesis in pancreatic β cells, which will be more extensively described here. It should be noted that the general mechanism for proinsulin biosynthesis translation could well apply to prohormones in other endocrine cell types; for example, there is recent evidence that the biosynthesis of POMC is also translationally regulated.44

Translational Regulation of Proinsulin Biosynthesis Proinsulin biosynthesis is specifically regulated in a pancreatic β cell by a variety of nutrients, hormones, pharmaceutical and physiological factors; however the most physiologically relevant of these is glucose.59 Short term (<2 h) exposure to glucose results in a 10- to 20-fold specific increase in proinsulin biosynthesis60-62 without any change in total preproinsulin mRNA levels in β cells59,62 (Fig. 6.1). It has also been shown that blocking transcription by the addition of actinomycin D (an inhibitor of mRNA synthesis) has no affect on short term glucose stimulated proinsulin biosynthesis.60,61,63 Therefore, for the short term (<2 h), glucose stimulated proinsulin biosynthesis is entirely regulated at the translational level. It is important to note that this is a specific induction, by increasing glucose concentrations in pancreatic β cells, of proinsulin biosynthesis translation above that of the vast majority of proteins synthesized in the β cell. Two-dimensional electrophoresis experiments have determined that the biosynthesis of between 50-100 β cell proteins is translationally regulated by glucose.64 When taking into consideration posttranslational modifications this number would be closer to 50 than 100. Thus, since it is likely that several thousand proteins are synthesized in a β cell, there is an exclusive small subset of β cell proteins that are translationally regulated by glucose. Interestingly, the majority of this β cell protein subset appear to be located in insulin secretory granules.64 Coordinate regulation of proinsulin biosynthesis with that of its prohormone processing endopeptidases PC2 and PC1/3 has been investigated.61,65,66 In isolated rat pancreatic islets, short term glucose stimulation specifically induced PC1/3 biosynthesis in parallel to that of proinsulin.61 These biosynthetic effects were independent of any changes in total mRNA levels in β cells59,65 (Fig. 6.1), and unaffected by the presence of actinomycin D, indicating that short term glucose stimulated biosynthesis of these proteins occurs posttranscriptionally.60,61,65 This evidence suggests that the mechanism by which proinsulin biosynthesis is regulated by glucose is at the level of translation, and that this mechanism may be shared by PC3. In contrast, in the same islet cells, it was initially apparent that biosynthesis of PC2 was unaffected by glucose. However, this was subsequently found to be due to the heterogeneity of cell types found in pancreatic islets.22 PC2 is more highly expressed in non-β cells of an islet than in β cells. This, in turn, masks increases in PC2 biosynthesis translation in response to glucose. This possibility was addressed by investigating the biosynthesis of PC2, PC1/3 and proinsulin in islets isolated from an obese strain of mouse (ob/ob) which has elevated populations of β cells within the islets.66 Both PC2 and PC1/3

Regulation of Prohormone Conversion by Control of Endopeptidase Biosynthesis

Fig. 6.1. Glucose-Regulation of Proinsulin Biosynthesis Translation in Pancreatic β cells. Biosynthesis and mRNA levels of proinsulin, PC1/3, PC2 and CP-H in isolated rat pancreatic islets (panel A) or cells from the glucose responsive pancreatic β cell line MIN6 (panel B) were examined as previously described.20,65 Total protein synthesis in β cells was determined by trichloroacetic acid precipitation.20,65

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biosyntheses were found to respond to glucose in ob/ob mouse islets but not in normal mouse islets. Furthermore, short term translational glucose stimulation of both PC2 and PC1/3 biosynthesis in the glucose responsive pancreatic β cell line MIN6 has also been reported65 (Fig. 6.1B). Thus, in pancreatic β cells short term (<2 h) glucose stimulated biosynthesis of the proinsulin processing endopeptidases PC2 and PC1/3 is coordinately regulated with that of their prohormone substrate at the translational level. In contrast, the third enzyme involved in proinsulin conversion, the exopeptidase CP-H, does not appear to be translationally regulated in β cells.60,61,65 However, this might not render too much of an adverse effect on proinsulin conversion in β cells, since CP-H is quite abundant relative to PC1/3 and PC2.60,67 The coordinate regulation of PC2, PC1/3 and proinsulin biosynthesis translation in response to glucose is likely regulated through a similar mechanism. In studies to date the regulation of proinsulin biosynthesis translation cannot be uncoupled from that of PC1/3 and PC2.60,61,65,66 In rat pancreatic islets mannoheptulose, an inhibitor of glucose phosphorylation and hence of glycolysis, inhibits glucose stimulated proinsulin, PC1/3 and PC2 biosynthesis translation,61,65 indicating that the stimulus-coupling mechanism shares a common activation pathway through glucose metabolism.4 Similarly, glucose-induced proinsulin, PC1/3 and PC2 biosynthesis translation can be potentiated by activation of protein kinase-A, but not by activation of Ca2+/calmodulin kinases or protein kinase-C.65 In general, nutrient secretagogues that trigger insulin exocytosis also stimulate proinsulin biosynthesis translation to maintain the balance in insulin content in the β cell.4,59 However, other than a requirement for nutrient metabolism, the downstream stimulus-coupling pathways between hormone secretion verses synthesis in the β cell are quite different.59 For example, the signaling pathway for insulin secretion is Ca2+-dependent,6 whereas that for proinsulin biosynthesis translation is Ca2+-independent.61,65 The molecular mechanism by which glucose induces proinsulin PC1/3 and PC2 biosynthesis translation is poorly understood. For secretory pathway proteins such as proinsulin, PC1/3 and PC2, initiation of translation occurs within the cytosol, followed by elongation.68,69 The emerging nascent signal peptide interacts with the 54 kDa subunit of SRP, an 11S ribonucleoprotein complex.70,71 The binding of signal recognition particle (SRP) to the nascent polypeptide chain blocks further elongation. SRP then mediates the transfer of the mRNA/ribosome/peptide complex to the rough endoplasmic reticulum (RER) where SRP interacts with the RER membrane SRP receptor, causing dissociation of SRP from the ribosome, triggering elongation to proceed.72 Newly synthesized preproprotein is translocated into the intralumenal space of the RER, and an RER associated signal peptidase removes the signal peptide, yielding proinsulin.73 Translational regulation of proinsulin biosynthesis is thought to be mediated at the levels of initiation, elongation, and SRP Barrett Barrett interaction with its receptor;74 however, the primary point of regulation is considered to be at the initiation phase of translation.59 Upon short term (<2 h) glucose stimulation, although there is no change in total preproinsulin mRNA in β cells, there is a redistribution of preproinsulin mRNA from a cytosolic pool to mono/polyribosome-associated mRNA on the RER,62,74,75 the major site of proinsulin synthesis in the β cell. The redistribution in preproinsulin mRNA may also be the result of an increase in the rate of SRP-mediated transfer of the SRP/signal peptide/mRNA to the RER.74 However, a number of cytosolic proteins (proteins that do not interact with SRP) are also specifically regulated by glucose in pancreatic β cells and a number of secretory proteins (proteins that do interact with SRP) that are not specifically regulated by glucose, e.g., CP-H.60,61 Therefore, SRP mediated arrest is not necessarily an essential element required for glucose stimulated proinsulin biosynthesis. There is also a glucose specific stimulation of proinsulin biosynthesis through an

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increase in the rate of elongation. This stimulation occurs at up to 5.6 mM glucose; however, the maximal rate of glucose stimulated proinsulin translational regulation occurs between 10-12 mM glucose and therefore the increase in the elongation rate does not explain the increase in proinsulin biosynthesis at physiologically relevant glucose concentrations.74 The redistribution of preproinsulin mRNA is likely to reflect an increase in the rate of initiation of preproinsulin mRNA, either by the sequestration of mRNA from a yet uninitiated cytosolic pool or/and an increase in the number of initiations per mRNA. The regulation of general protein synthesis translation in eukaryotic cells is mostly mediated at the level of translation initiation.68,76-78 In particular, the phosphorylation state of protein translation factors has been implicated as a key mechanism of translational control of protein synthesis.78 A large number of initiation factors, some elongation factors and ribosomal proteins are known to be phosphoproteins.68,76-78 The phosphorylation state of initiation factors and their effect on protein synthesis has been extensively studied. It is possible that such phosphorylation might play a role in glucose-induced translational regulation of proinsulin biosynthesis in the pancreatic β cell. We and others have investigated the effect of glucose on initiation factor phosphorylation in β cells in parallel to glucoseinduced proinsulin biosynthesis translation (see Table 6.2 for summary). The eukaryotic initiation factor 2 (eIF2α) plays an important role in the regulation of translation. The eIF2α forms a complex with fMet-tRNA and then escorts fMet-tRNA onto the 40S subunit, generating the 43S initiation complex. The 43S complex binds to the 5'-untranslated region of the mRNA and scans to the first start AUG codon on the mRNA. Upon recognition of the AUG codon, initiation of protein synthesis occurs.78 When eIF2α is phosphorylated, its activity is inhibited. Phosphorylation of eIF2α occurs in β cells but it does not appear to be regulated by glucose79 (Alarcon and Rhodes, unpublished data) (Table 6.2). Further, eIF2α requires GTP for its activity, which increases its affinity for the 40S ribosomal subunit. GTP hydrolysis to GDP must occur to allow detachment of eIF2α from the 40S ribosomal subunit, and hence recycling of eIF2α for another round of initiation. This reaction is catalyzed by a guanine exchange factor named eIF2B. Phosphorylation of eIF2α inhibits the ability of eIF2B to catalyze the reaction eIF2-GDP to eIF2-GTP. As both eIF2B and eIF2 are limited in quantity, recycling of eIF2-GDP to GTP can abolish protein synthesis. Investigation into the phosphorylation state of eIF2α and the activity of eIF2B in the presence and absence of glucose in pancreatic rat islets showed that the activity of eIF2B was slightly stimulated by glucose, whereas the phosphorylation state of eIF2α was unaltered, indicating that the rise in activity was not due to increased eIF2α phosphorylation.79 The eIF2B can itself be phosphorylated in vitro, which in turn modulates its exchange activity.76 A correlation between eIF2B activity and total protein synthesis has been observed in β cells; however this did not correlate with the specific glucose regulation of proinsulin biosynthesis translation76 (Table 6.2). Phosphorylation of eIF4E results in binding to the 7-methyl guanosine 5'-cap of mRNAs and is essential for protein synthesis initiation. The availability of eIF4E is itself regulated by another phosphoprotein, eIF4bp or PHAS-1.80,81 When phosphorylated, eIF4bp binds to and sequestrates eIF4E, thereby reducing the pool of eIF4E available for initiation. The phosphorylation state of eIF4E and eIF4bp in response to glucose was investigated in pancreatic islets. However, although both were phosphorylated, neither was found to be phosphorylated in response to glucose, and is thus not involved in regulating proinsulin biosynthesis translation79 (Alarcon and Rhodes, unpublished data) (Table 6.2). Similarly, we have also investigated the phosphorylation of initiation factors eIF3 p120, and eIF4F p220 in β cells, and found that both were constitutively phosphorylated but not in response to glucose (Table 6.2) (Alarcon, Etchison and Rhodes, unpublished data).

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Table 6.2. Examination of putative glucose-regulation of eukaryotic initiation factor (eIF) phosphorylation and ribosomal protein kinase phosphorylation activation in pancreatic β cells Translation Initiation Factor eIF2α eIF4E eIF3 p120 eIF4F p220 PHAS-1 (eIF4bp) eIF2B Ribosomal Protein Kinase p70S6K MAP-Kinase (erk-1 and erk-2)

Phosphorylation

Glucose Effect

Yes Yes Yes Yes Yes Yes

None None None None None Yes *

Phosphorylation

Glucose Effect

Yes Yes

None Yes **

Eukaryotic protein synthesis translation can be regulated by the phosphorylation state of initiation factors (eIFs) and/or phosphorylation activation of ribosomal protein kinase activities. These have been investigated in pancreatic β cells to see if they are regulated by glucose, which in turn might correlate with glucose-induced proinsulin, PC1/3 and PC2 biosynthesis translation. * Only correlates with total protein synthesis, not specific phosphorylation of proinsulin biosynthesis translation or eIF2α phosphorylation state.79 ** Correlates with translocation to the nucleus in β cells, not specific phosphorylation of proinsulin biosynthesis translation.

Phosphorylation of certain ribosomal proteins (e.g., S6) has been reported to affect the rate of protein synthesis translation. One protein kinase, p70S6K kinase is capable of phosphorylating S6, and is itself activated by a phosphorylation event which in turn has been correlated with the increase in protein synthesis translation.77,82 We have investigated the phosphorylation of p70S6K in pancreatic islets, but have found no effect on p70S6K phosphorylation by glucose (Table 6.2) (Herbert and Rhodes, unpublished data). Furthermore, a specific inhibitor of p70S6K activity, rapamycin,83,84 has no effect on glucose-induced proinsulin translation (Herbert and Rhodes, unpublished data). Another protein kinase, p90RSK, can also phosphorylate ribosomal proteins.85 As for p70S6K, p90RSK is also activated by serine phosphorylation.85 Phosphorylation of p90RSK can be catalyzed by certain MAP-kinase isoforms which themselves are also phosphorylation activated.85,86 We have investigated glucose-induced phosphorylation of MAPK isoforms erk-1 and -2 in isolated pancreatic islets and MIN6 cells, and found an increase phosphorylation state (Herbert and Rhodes, unpublished data).87 However, the glucose-induced MAPK phosphorylation we have observed only correlated with MAPK-translocation to the nucleus, rather than any specific increase in proinsulin biosynthesis. To date, there is very little evidence for glucose affecting the phosphorylation state of eukaryotic initiation factors or activation of ribosomal protein kinases in the pancreatic β cell, that in turn would specifically regulate proinsulin biosynthesis above that of total protein synthesis. However, one should consider that phosphorylation-induced change in initiation factor or ribosomal protein kinase activity will tend to influence the rate of general total protein synthesis, not the specific glucose regulation of proinsulin (and PC1/3 and

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Fig. 6.2. Conserved Secondary Structure in the 5'-Untranslated Regions of Preproinsulin, PC1/3 and PC2 mRNAs. Secondary structural analysis of the 5'-untranslated regions of mammalian preproinsulin, murine PC1/3, human PC2 and rat CPH mRNA was analyzed by RNAfold in GCG. A ‘stem-loop’ secondary structure close to the AUG start codon is predicted in glucose regulated mRNAs.

PC2) biosynthesis translation in the β cell. Therefore, there must be something specific to the proteins which are glucose-regulated at the translational level in β cells, perhaps in the untranslated regions (UTR) of their mRNAs.88 In this light, it is interesting to note that a ‘stem-loop’ secondary structure in the 5'-UTR of mammalian preproinsulin mRNA, and that of PC1/3 and PC2, is conserved across species (Fig. 6.2). The 5'-UTR mRNAs of nonglucose-regulated proteins such as CP-H do not predict such a ‘stem-loop’ structure (Fig. 6.2). Such a 5'-UTR mRNA ‘stem-loop’ structure has been defined to be key to iron regulation of ferritin biosynthesis translation.89 Iron metabolism is regulated by the expression of ferritin and the transferrin receptor by the binding of a repressor protein IRP-BP to

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the 5'-UTR of the ferritin mRNA, inhibiting translation, and to the 3' end of the transferrin receptor.89-92 It is possible that proinsulin biosynthesis and its proteolytic processing enzymes may be regulated by an analogous mechanism. The 5'-UTR and 3'-UTR of preproinsulin, PC1/3 and PC2 mRNAs are currently being examined to see if they contain any such glucose-regulatory translational control elements.

Acknowledgments We thank Dr. Iris Lindberg of Louisiana State University of Medicine, New Orleans, LA for supplying anti-PC3 antibodies; Lynn M. O’Brien (from Dr. Edgar Henshaw’s laboratory), University of Rochester, Rochester, NY, for eIF-2α antibody; and Dr. Dianne Etchison, University of Kansas, Kansas City, KA for eIF3 and eI4F p220 antisera. We are also grateful to Dr. Robert E. Rhoads of the Louisiana State University, Shreveport, LA and Prof. Michael S. Clemens of St. George Hospital Medical School, London, UK, for their valuable advice. The work from our laboratory has been supported by grants from the National Institutes of Health (DK47919 and DK50610), the Juvenile Diabetes Foundation International, and Pfizer Inc.

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57. Miller CP, McGehee RE, Habener JF. IDX-1: A new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene. EMBO J 1994; 13:1145-1156. 58. Jonsson J, Carlsson T, Edlund T, Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 1994; 371:606-609. 59. Rhodes CJ. Processing of the insulin molecule. In: Diabetes Mellitus. A Fundemental and Clinical Text. LeRoith D, Taylor SI, Olefsky JM, eds. Philadelphia, PA: Lippincott-Raven Publishers, 1996:27-41. 60. Guest PG, Rhodes CJ, Hutton JC. Regulation of the biosynthesis of insulin secretory granule proteins: Co-ordinate translational control is exerted on some but not all granule matrix constituents. Biochem J 1989; 257:431-437. 61. Alarcón C, Lincoln B, Rhodes CJ. The biosynthesis of the subtilisin-related proprotein covertase PC3, but not that of the PC2 convertase, is regulated by glucose in parallel to proinsulin biosynthesis in rat pancreatic islets. J Biol Chem 1993; 268:4276-4280. 62. Itoh N, Okamoto H. Translational control of proinsulin synthesis by glucose. Nature 1980; 283:100-102. 63. Permutt MA, Kipnis DM. 1. Insulin biosynthesis: On the mechanism of glucose stimulation. J Biol Chem 1972; 247:1194-1199. 64. Guest PC, Bailyes EM, Rutherford NG, Hutton JC. Insulin secretory granule biogenesis; Co-ordinate regulation of the biosynthesis of the majority of constituent proteins. Biochem J 1991; 274:73-78. 65. Skelly RH, Schuppin GT, Ishihara H, Oka Y, Rhodes CJ. Glucose-regulated translational control of proinsulin biosynthesis with that of the proinsulin endopeptidases PC2 and PC3 in the insulin-producing MIN6 cell line. Diabetes 1996; 45:37-43. 66. Martin SK, Carroll R, Benig M, Steiner DF. Regulation by glucose of the biosynthesis of PC2, PC3 and proinsulin in (ob/ob) mouse islets of Langerhans. FEBS Lett 1994; 356:279-282. 67. Guest PG, Pipeleers D, Rossier J, Rhodes CJ, Hutton JC. Co-secretion of carboxypeptidase-H and insulin from isolated rat islets of Langerhans. Biochem J 1989; 264:503-508. 68. Merrick WC. Mechanism and regulation of eukaryotic protein synthesis. Mic Revs 1992; 56:291-315. 69. Clemens MJ. Regulatory mechanisms in translational control. Curr Op Cell Biol 1989; 1:1160-1167. 70. Okun MM, Warren TG, Shields D. In vitro biosynthesis of multiple preproglucagons results from acetylation of the primary translation products. J Mol Endocrinol 1989; 2:137-144. 71. Wolin SL, Walter P. Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes. J Cell Biol 1993; 121:1211-1219. 72. Siegel V, Walter P. Functional dissection of the signal recognition particle. Trends Biochem Sci 1988; 13:314-317. 73. Lively MO. Signal peptidases in protein biosynthesis and intracellular transport. Curr Op Cell Biol 1989; 1:1188-1194. 74. Welsh M, Scherberg N, Gilmore R, Steiner DF. Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose. Biochem J 1986; 235:459-467. 75. Welsh N, Welsh M, Steiner DF, Hellerstrom C. Mechanisms of leucine- and theophyllinestimulated insulin biosynthesis in isolated rat pancreatic islets. Biochem J 1987; 246:245-248. 76. Pain VM. Inititation of protein synthesis in eukaryotic cells. Eur J Biochem 1996; 236:747-771. 77. Redpath NT, Proud CG. Molecular mechanisms in the control of translation by hormones and growth factors. Biochim Biophys Acta 1994; 1220:147-162. 78. Hershey JWB. Translational control in mammalian cells. Ann Rev Biochem 1991; 60:717-755. 79. Gilligan M, Welsh GI, Flynn A, Bujalska I, Diggle TA, Denton RM, Proud CG, Docherty K. Glucose stimulates the activity of the guanine nucleotide-exchange factor eIF2B in isolated rat islets of Langerhans. J Biol Chem 1995; 271:2121-2125.

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80. Lin TA, Kong X, Haystead TA, Pause A, Belsham G, Sonenberg N, Lawrence JC. PHAS-1 as a link between mitogen activated protein kinas and translation initiation. Science 1994; 266:653-656. 81. Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC, Sonenberg N. Insulindependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature 1994; 371:762-767. 82. Sturgill TW, Wu J. Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6. Biochim Biophys Acta 1991; 1092:350-357. 83. Terada N, Patel HR, Takase K, Kohno K, Nairn AC. Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc Natl Acad Sci USA 1994; 91:11477-11481. 84. Jeffires HBJ, Reinhard C, Kozma SC, Thomas D. Rapamycin selectively prepresses translation of the “polypyrimidine tract” mRNA family. Proc Natl Acad Sci USA 1994; 91:4441-4445. 85. Blenis J, Chung J, Erikson E, Alcorta DA, Erikson RL. Distinct mechanisms for the activation of the RSK kinases/MAP2 kinase/pp90rsk and pp70-S6 kinase signalling systems are indicated by inhibition of protein synthesis. Cell Growth Differ 1991; 2:279-285. 86. Cobb MH, Goldsmith EJ. How MAP kinases are regulated. J Biol Chem 1995; 270: 14843-14846. 87. Frodin M, Sekine N, Roche E, Filloux C, Prentki M, Wollheim CB, Van Obberghen E. Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1. J Biol Chem 1995; 270:7882-7889. 88. Knight SW, Docherty K. RNA-protein interactions in the 5'-untranslated region of preproinsulin mRNA. J Mol Endocrinol 1992; 8:225-234. 89. Klausner RD, Rouault TA, Harford JB. Regulating the fate of mRNA: The control of cellular iron metabolism. Cell 1993; 72:19-28. 90. Müllner EW, Neupert B, Kühn LC. A specific mRNA binding factor regulates the iron dependent stability of cytoplasmic trasferrin receptor mRNA. Cell 1989; 58:373-382. 91. Haile DJ, Hentze MW, Rouault TA, Harford JB, Klausner RD. Regulation of interaction of the iron-responsive element binding protein with iron-respnsive RNA elements. Mol Cell Biol 1989; 9:5055-5061. 92. Koeller DM, Casey JL, Hentze MW, Gerhardt EM, Chan L-NL, Klausner RD, Harford JB. A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA. Proc Natl Acad Sci USA 1989; 86:3574-3578. 93. Braks JAM, Broers CAM, Danger J-M, Martens GJM. Structural organization of the gene encoding the neurendocrine chaperone 7B2. Eur J Biochem 1996; 236:60-67.

CHAPTER 7

Carboxypeptidase and Aminopeptidase Proteases in Proneuropeptide Processing Vivian Y.H. Hook and Sukkid Yasothornsrikul

Introduction

A

series of proteolytic processing steps is required for converting neuropeptide precursors into potent peptide hormones and neurotransmitters.1-3 Illustration of precursor structures for several neuropeptides (Fig. 7.1)—i.e., proinsulin, proopiomelanocortin (POMC), proenkephalin, provasopressin, and others—indicates that active peptide sequences are typically flanked by paired basic residues, and sometimes by single arginine residues (such as for provasopressin). Subsequent to initial processing at these paired basic residues by endoproteolytic processing enzymes—including the subtilisin-like PC1/3 and PC2 enzymes (PC = prohormone convertase),4-6 the novel cysteine protease ‘prohormone thiol protease’ (PTP),3,7 and a 70 kDa aspartyl protease known as ‘POMC converting enzyme’ (PCE)8,9— removal of basic residues from COOH- and NH2-termini of peptide intermediates by carboxypeptidase3,10,11 and aminopeptidase3,11 proteases, respectively, is required to generate active neuropeptides. The carboxypeptidase E/H selectively removes lysine or arginine basic residues from the COOH-terminus of peptide intermediates (Fig. 7.2). This enzyme has been known as carboxypeptidase E or H (EC 3.4.17.10, also referred to as ‘enkephalin convertase’),12 and is, therefore, referred to as carboxypeptidase E/H. In addition to COOH-terminal processing by CPE/H activity, the NH2-terminus of peptide intermediates requires exoproteolytic processing by specific aminopeptidase(s) that remove NH2-terminal lysine or arginine residues (Fig. 7.2) The carboxypeptidase E/H was the first processing enzyme to be identified through biochemical, molecular, and cell biological approaches. More recently, molecular genetic studies of a mouse model of obesity13 indicate the important role of carboxypeptidase E/H in neuroendocrine functions. It is interesting that little is currently known about the aminopeptidase processing activity, which shares equal importance with the carboxypeptidase E/H as an exopeptidase processing enzyme. Thus, this chapter describes the novel carboxypeptidase E/H and aminopeptidase processing activities that participate in prohormone processing.

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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Fig. 7.1. Preprohormone structures. Precursor structures for several peptide hormones and neurotransmitters are illustrated. All preprohormones contain a NH2-terminal signal sequence. Active neuropeptide domains within the precursors are indicated as shaded areas. Abbreviations: GLP, glucagon-like peptide; MSH, melanocyte-stimulating hormone; JP, joining peptide; ACTH, adrenocorticotropic hormone; M (Met)enkephalin; O, (Met)enkephalin-Arg6-Gly7-Leu8; L, (Leu)enkephalin; H, (Met)enkephalin-Arg6-Phe7; AVP, arginine-vasopressin. K and R are single letter codes for lysine and arginine, respectively.

Neuroendocrine-Specific Carboxypeptidase E/H Biochemical Characterization Cellular site of prohormone processing Identification of relevant carboxypeptidase processing activity for removal of COOHterminal arginine and lysine residues first requires consideration of the appropriate subcellular compartment where this exoproteolytic step occurs. Several studies utilizing a variety of cell biological and biochemical approaches demonstrate that the secretory vesicle is a major site of prohormone processing.1-3 Studies of prooxytocin and provasopressin processing in the hypothalamo-neurohypophyseal system indicate proteolytic processing within secretory vesicles during their maturation and transport from neuronal cell bodies of the hypothalamus to nerve terminals in the posterior pituitary.14 Furthermore, analysis of proinsulin processing indicates that it is processed within secretory vesicles during vesicular maturation.15,16 Many neuropeptide precursors and their processed bioactive peptide products are located together within secretory vesicles. For example, the prohormones proenkephalin, POMC (proopiomelanocortin), proinsulin, and other precursors are colocalized with their peptide products within neurosecretory vesicles. These studies indicate that many proteases responsible for processing should reside together with substrate and peptide product in secretory vesicles.

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Fig. 7.2. Carboxypeptidase and aminopeptidase steps in neuropeptide precursor processing. Carboxypeptidase E/H specifically removes basic residues, lysine or arginine, from the COOH-terminus of peptide hormone (H) intermediates. Aminopeptidase(s) remove basic residues from the NH2-terminus of peptide intermediates. The combined actions of endoproteolytic processing, and processing by exoproteases, result in cellular production of bioactive peptide hormones and neurotransmitters.

Identification of carboxypeptidase E/H The search for the prohormone processing carboxypeptidase B-like enzyme utilized isolated secretory vesicles from bovine adrenal medulla that contains multiple neuropeptides including enkephalins,17,18 neuropeptide Y,19 galanin,20 and others.21,22 These vesicles contain carboxypeptidase B-like activity that converts (Met)enkephalin-Arg 6 to (Met)enkephalin.23 This carboxypeptidase activity is prominently detected with the model fluorescent peptide substrate dansyl-Phe-Leu-Arg24 or 3H-benzoyl-Phe-Ala-Arg.25 The chromaffin granule carboxypeptidase that removes arginine and lysine residues is optimally active at the acidic pH of 5.5-6.0 (Table 7.1), which is consistent with the intragranular pH;23-26 no activity is detected at neutral pH 7.4. The carboxypeptidase B-like activity, currently known as carboxypeptidase E or carboxypeptidase H (therefore indicated as CPE/H, and previously known as ‘enkephalin convertase’), is stimulated several-fold by Co2+, inhibited

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Table 7.1. Biochemical characteristics of carboxypeptidase E/H Property

Characteristic

pH optimum

pH 5.5-6.0

Molecular weight

50 kDa for soluble CPE/H 52-53 kDa for membrane CPE/

H Inhibition by metalloprotease inhibitors

Inhibited by EDTA and 1,10-phenanthroline

Inhibition by active site-directed inhibitors and Amino acid specificity arginine

Inhibited by GEMSA, APMSA, MGTA Removes COOH-terminal and lysine residues from

peptides Abbreviations: EDTA, ethylenediaminetetraacetic acid; GEMSA, guanidino-ethylmercaptosuccinic acid; APMSA, aminomercaptosuccinic acid; MGTA, 2-mercaptomethyl-3guanidinoethylthiopropanoic acid.

by metal ion chelators, and is potently inhibited by the active-site directed inhibitors GEMSA (guanidinoethylmercaptosuccinic acid) and GPSA (guanidinopropylsuccinic acid) in the nanomolar range.24-27 The unique properties of activation by Co2+, and inhibition by GEMSA or GPSA, distinguish the prohormone processing CPE/H from carboxypeptidase B-like activity in lysosomes.24 Pituitary secretory vesicles from anterior, intermediate, and neural lobes possess CPE/H activity for processing POMC- and provasopressin-derived peptide intermediates.28 Appropriate carboxypeptidase activity in secretory vesicles from anterior and intermediate pituitary cleaves COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) which represents a peptide product derived from POMC. CPE/H activity is also present in secretory vesicles of pituitary neural lobe (posterior pituitary), removing -Lys-Arg residues from (Arg8)vasopressin-Gly-Lys-Arg, a processing intermediate derived from provasopressin. The pituitary CPE/H is optimally active at the acidic pH of 5.5. The activity is inhibited by the metallocarboxypeptidase inhibitors GPSA and APMSA (aminomercaptosuccinic acid), EDTA, 1,10-phenanthroline, and stimulated by Co2+. Similar activities that represent carboxypeptidase E/H are present within insulin secretory vesicles,29,30 as well as vesicles of the mouse pituitary AtT20 cell line.31 These results indicate that the CPE/H characterized within secretory vesicles from pituitary, adrenal medulla, and many other neuroendocrine tissues is a metallo-exopeptidase that is active in the acidic pH range of 5.5-6.0. Purification and characterization Purification of CPE/H from the soluble fractions of bovine adrenal, brain, and pituitary utilized affinity chromatography on concanavalin A Sepharose, ion exchange on DEAE

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cellulose, gel filtration on Sephadex G-100, and a p-aminobenzoyl-Arg Sepharose affinity chromatography.24 The CPE/H purified from these different tissues was identical in biochemical properties. The purified enzyme is inhibited by metal chelators, stimulated by Co2+, potently inhibited by GEMSA and related inhibitors, and is modulated by Ca2+.26,32,33 The purified soluble CPE/H from these different tissues has an apparent molecular weight of 50 kDa on SDS-PAGE. The purified membrane-bound carboxypeptidase E/H has a slightly larger molecular weight of 52-53 kDa compared to soluble CPE/H of 50 kDa. The membrane-bound CPE/H shows nearly identical biochemical properties as those of soluble CPE/H. Purification of soluble and membrane-bound carboxypeptidase E/H from several neuroendocrine tissues suggests that a single carboxypeptidase exists for processing prohormone-derived peptide intermediates into active neuropeptides. Moreover, the distinguishing biochemical characteristics of the carboxypeptidase E/H demonstrates the enzyme as a novel neuroendocrine processing protease.

Tissue and Subcellular Distribution Carboxypeptidase E/H activity and 3H-GEMSA binding Studies of the tissue distribution of CPE/H indicate that its expression is restricted to neuroendocrine tissues. Characteristic stimulation by Co2+ and inhibition by GEMSA has allowed specific detection of CPE/H activity, and has allowed detection of the enzyme protein by 3H-GEMSA binding.35 There is good agreement for tissue distribution based on measuring Co2+-stimulated activity, and based on detection of CPE/H by 3H-GEMSA binding.35-39 The highest levels of CPE/H are found in pituitary, brain, pancreas, and adrenal, with lower levels detected in intestine, heart, eye, and testis. Brain regions that are particularly enriched in CPE/H activity and 3H-GEMSA binding are the hippocampus, thalamus/ hypothalamus, striatum, and brainstem. The principle of specific 3H-GEMSA binding has also been utilized to detect CPE/H in brain and endocrine areas by 3H-GEMSA autoradiography.36-39 Immunohistochemistry Production of antisera against CPE/H for immunohistochemistry has allowed immunocytochemical localization of the enzyme to secretory vesicles of neuroendocrine cells.29,30,40 The anti-CPE/H serum does not crossreact with carboxypeptidases B, N, A, and Y, indicating that CPE/H differs from other carboxypeptidases. CPE/H cellular immunostaining is detected in adrenal medulla (not adrenal cortex), anterior and intermediate pituitary, posterior pituitary, nuclei of the hypothalamus, and in insulin-containing cells of the islets of Langerhans. CPE/H immunostaining shows a discrete, punctate staining pattern in the cytosol region, but not the nucleus, of neuroendocrine cells, such as in primary cultures of adrenal medullary chromaffin cells. This pattern of cellular immunoreactivity is consistent with the localization of CPE/H to secretory vesicles. Immunoelectron microscopy demonstrates the localization of CPE/H within secretory vesicles in neuroendocrine cells including pituitary40 and pancreas.29,30 More extensive immunocytochemical localization of CPE/H in the rat central nervous system (CNS) shows that the enzyme is generally localized to peptidergic neurons.41 Neuronal soma of the supraoptic and paraventricular nuclei of hypothalamus contain immunoreactive CPE/H. The enzyme is also found in axons derived from these nuclei, which terminate in the posterior pituitary. Immunoreactive CPE/H is present in several other CNS locations, including the stria terminalis and amygdala. CPE/H in the hippocampus is most abundant in the dentate gyrus and in the pyramidal cells of CA3. It is interesting that CPE/H

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immunoreactivity does not exactly correlate with known neuropeptide distributions in brain. Several possible explanations for future investigations are: 1. low levels of CPE/H may be adequate for complete processing; 2. other related carboxypeptidases may exist; and 3. new, undiscovered neuropeptides may be colocalized with CPE/H. The CPE/H enzyme has also been detected by immunohistochemistry in the striatum of adult cats and monkeys.42 Specific staining was found in the neuropil in medium and large neuronal cell bodies. The CPE/H immunoreactivity was found in zones that are rich in enkephalin and substance P. Enzyme immunoreactivity was, however, also present in areas that are low in these two neuropeptides. Thus, colocalization with enkephalin and substance P is consistent with the proposed role of CPE/H in the production of these neuropeptides. These results suggest a role for CPE/H in the metabolism of multiple neuropeptides in vivo. These immunocytochemical results, combined with distinct biochemical properties of the enzyme, indicate CPE/H as a novel carboxypeptidase that is specifically involved in processing neuroendocrine precursors into active peptide hormones and neurotransmitters. Carboxypeptidase E/H mRNA Parallel studies of CPE/H mRNA indicates that the expression of this enzyme is restricted to neuroendocrine tissues. The distribution of CPE/H mRNA parallels that of its immunocytochemical distribution and detection by 3H-GEMSA binding. CPE/H mRNA is detected by Northern blots and in situ hybridization in pituitary, adrenal, heart, cerebral cortex, and other neuroendocrine tissues.43,44 CPE/H mRNA levels are high in most peptidergic regions including the hypothalamus, hippocampus, cortex, substantia nigra, pons and medulla, and spinal cord. Tissue distribution studies of CPE/H activity, 3H-GEMSA binding, immunocytochemistry, and Northern blots combined with in situ hybridization indicate that the CPE/H is the only basic residue cleaving carboxypeptidase with selective localization in the central nervous system and endocrine tissues.

Molecular Characterization of Carboxypeptidase E/H CPE/H cDNA from multiple species Molecular cloning of bovine CPE/H cDNA utilized degenerate oligonucleotides complementary to determined partial amino acid sequences of the purified enzyme.45 A nearly full-length bovine cDNA was obtained which included the NH2-terminus of the active enzyme. Subsequently, CPE/H cDNAs from rat44,46 and human 47 were obtained. The preprocarboxypeptidase E/H contains an NH2-terminal signal sequence, a short prodomain, and a pentabasic RRRRR processing site between the prodomain and NH2-terminus of the mature enzyme. Structural features of the deduced primary sequence of rat CPE/H cDNA include residues predicted as Zn2+ binding sites (His72 and Glu75), substrate binding sites (Arg147, His225, and Gln284), and residues involved in enzymatic catalysis (Lys131, Tyr227, Tyr278, and Glu300). The cDNA sequence also predicts consensus Asn glycosylation sites. The bovine,45 rat,44,46 and human47 CPE/H cDNAs share high homology. The 476 amino acid open reading frames of bovine and rat sequences share 94% homology; human and rat sequences share 96% homology in primary sequences. The predicted molecular mass of 53 kDa is consistent with soluble and membrane forms of the enzyme as 50 and 52-53 kDa glycoproteins. Northern blots demonstrate rat and human CPE/H mRNAs of 2.2-2.4 kb and 2.4 kb, respectively.44,46,47 Bovine CPE/H shows three mRNA species of 3.3, 2.6, and 2.1 kb;45 varia-

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tions in 3'-untranslated regions are hypothesized for the different sizes of bovine CPE/H mRNAs.45 CPE/H mRNA expression is restricted to the endocrine and nervous systems, as described in the section on tissue distribution of CPE/H in this chapter. Carboxypeptidase E/H gene Genomic blots of rat and bovine DNAs indicate a single CPE/H gene.48 The rat CPE/H gene spans 50 kilobases consisting of 9 exons. Each exon contains protein-coding regions. Primer extension and nuclease protection analyses indicates that the 5'-end of the CPE/H mRNA is 105 nucleotides upstream from the ATG methionine translational initiation site. Upstream consensus sites for SP-1, NF-1, Pan-1, and AP-2 enhancer elements are present. Homologies in 5'-upstream sequences with several neuroendocrine-specific genes—neuropeptide Y, oxytocin, and others—suggest the possibility of similar cis and trans regulatory elements that control neuroendocrine-specific gene expression of carboxypeptidase E/H. Cis-acting elements that control CPE/H gene expression are indicated by deletion analyses of 5'-flanking regions of the gene with luciferase reporter constructs transiently expressed in pituitary AtT20 and GH4C1, kidney HEK293 cells, liver SK-HEP-1, and COS-1 cells.49 These results indicate that basal expression of the CPE/H gene from its major transcription initiation site, which does not contain an upstream TATA box, is under the control of an initiator-like element with an upstream GC box.

Regulation of Carboxypeptidase E/H Cellular regulation The production of peptide hormones and neurotransmitters is highly controlled in neuroendocrine cells. Therefore, regulation of CPE/H was examined in chromaffin cells during elevation of cellular (Met)enkephalin levels by reserpine and forskolin.50 During increased (Met)enkephalin production (2-fold increase in enkephalin levels over controls), CPE/H activity in chromaffin granules was stimulated 3-fold (assayed in the presence of Co2+). Kinetic studies showed that reserpine treatment lowered the Km for (Met)enkephalinArg6 substrate. Cellular levels of immunoreactive CPE/H were not altered, indicating that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form that displays a higher affinity for (Met)enkephalin-Arg6. Forskolin treatment of chromaffin cells (a stimulator of cellular cAMP), however, had no effect on the carboxypeptidase activity. These results suggest that the carboxypeptidase may be selectively regulated during elevated enkephalin or neuropeptide formation. Selective regulation of CPE/H is demonstrated under a variety of physiological conditions. CPE/H and vasopressin mRNA levels increase in magnocellular neurons of the hypothalamus during dehydration, induced by salt-loading of animals.51 The dopamine antagonist haloperidol increases CPE/H mRNA in rat intermediate pituitary but not in other tissues tested.52 Systemic thyroid hormone increases CPE/H gene expression, and this increase is associated with a preferential utilization of novel upstream transcriptional initiation sites.53 In insulin-producing pancreatic cells, insulin levels are highly regulated by circulating levels of glucose;54 however, glucose has no effect on carboxypeptidase E/H. Also, regulation of ACTH production in AtT20 cells by corticotropin-releasing factor (CRF) and dexamethasone does not appear to involve the carboxypeptidase enzyme.55 These studies48-53 and several other investigations56-59 indicate that CPE/H is selectively coregulated, with altered rates of neuropeptide biosynthesis under certain conditions.

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Product inhibition Within the secretory vesicle, active CPE/H leads to an accumulation of final peptide products at millimolar levels.60 The presence of high molecular weight peptide intermediates, such as the high molecular weight enkephalin-containing intermediates in chromaffin granules, however, suggests that when in vivo peptide levels reach a certain level (millimolar),60 mechanisms exist to limit processing to maintain neuropeptides within mature secretory vesicles at a particular concentration. These considerations predict that the CPE/H activity may be regulated by product inhibition.61 Indeed, CPE/H is inhibited by millimolar levels (2-10 mM) of the peptides (Met)- and (Leu)enkephalin, vasopressin, oxytocin, ACTH1-14, and other neuropeptides; the enzyme is also inhibited by similar concentrations of free arginine and lysine that are also products of carboxypeptidase activity.62 Thus, product and feedback inhibition of carboxypeptidase E/H by its peptide products may limit the maximum peptide concentration within the secretory vesicle. Activities of membrane-associated and soluble forms of carboxypeptidase E/H The observation of changes in CPE/H activity without a change in total number of enzyme molecules in reserpine-treated chromaffin cells50 suggests subpopulations of carboxypeptidase E/H with different specific activities. The finding that carboxypeptidase activity and immunoreactivity are differentially distributed in soluble and membrane components of chromaffin granules indicates pools of enzyme that display different degrees of activity.63 Most of the enzyme activity (80%) is present in the soluble component of chromaffin granules; however, the number of enzyme molecules (measured by radioimmunoassay) is distributed equally between soluble and membrane components of the granule. Thus, the soluble enzyme is 5-6 times more active than the membrane-bound form. Possible conversion of membrane CPE/H to the more active soluble form of CPE/H may be a potential mechanism for the regulation of CPE/H activity. Indeed, a precursor-product relationship for membrane-associated and soluble CPE/H has been demonstrated (described in cell biology section of this chapter, below). Secretion The mature secretory vesicle releases active neuropeptides upon specific, regulated stimuli. The presence of CPE/H in the soluble component of secretory vesicles indicates that the enzyme is cosecreted with its neuropeptide product. Co-secretion of CPE/H immunoreactivity and activity with (Met)enkephalin from chromaffin cells has been demonstrated.64 Activation of the nicotinic-receptor induces the release of (Met)enkephalin and CPE/H into the culture medium. The specific activity of the released CPE/H (10 pmol (Met)enkephalin formed per ng enzyme) was greater than CPE/H in the soluble component of chromaffin granules (5.5 pmol (Met)enkephalin formed per ng enzyme). Apparently, a pool of carboxypeptidase molecules with a high state of activation is present in functionally mature granules whose contents are released by nicotinic receptor stimulation of bovine chromaffin cells. Secretion of CPE/H from cultured rat hypothalamic neurons,65 and from mouse pituitary AtT20 cells has been demonstrated.66 Interestingly, astrocytes from rat brain synthesize and secrete CPE/H. Thus, secretion from neuroendocrine cells is characteristic of CPE/H.

Cell Biology Activation of CPE/H in neurons during axonal transport Molecular cloning of CPE/H indicates that the enzyme is initially synthesized as a proenzyme,44-47 which then requires maturation and activation to generate functional enzyme.

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Since the proteolytic processing of prohormones and neuropeptide precursors occurs during maturation of secretory vesicles during axonal transport, it is possible that proCPE/H, like prohormone precursors, is processed and activated during axonal transport. Studies of CPE/H in the rat hypothalamo-neurohypophyseal system support the hypothesis that proCPE/H is processed and activated during axonal transport from neuronal perikarya of hypothalamic SON (supraoptic nucleus) to nerve terminals of the posterior pituitary.67 ProCPE/H of 65 kDa is converted to 55 kDa CPE/H within axons of the median eminence, so that fully processed 55 kDa CPE/H is the primary form of CPE/H present in nerve terminals of the posterior pituitary. Moreover, the specific activity of CPE/H increases as it is axonally transported to nerve terminals. Evidence for axonal transport of CPE/H has also been demonstrated in rat sciatic nerves.68 Thus, proteolytic processing of proCPE/H and prohormones are both proteolytically processed within secretory vesicles during axonal transport. Demonstration that proCPE/H is enzymatically active was indicated by studies of proCPE/H isolated from bovine pituitary.69 ProCPE/H has Km and kcat kinetic values that are similar to CPE/H.69 Additional studies show that processing of proCPE/H occurs in a post-Golgi compartment, that is, in secretory vesicles.70 Carboxypeptidase E/H domains for membrane-association and routing in the regulated secretory pathway Within the secretory vesicle, bovine carboxypeptidase E/H is present as membraneassociated (52-53 kDa)34 and soluble (50 kDa)26,69 forms. Because there is one CPE/H gene, the biosynthesis of membrane-bound and soluble forms of the enzyme most likely results from posttranslational modifications. Direct assessment of a precursor-product relationship for membrane-bound and soluble forms of CPE/H was demonstrated by pulse-chase studies in pituitary AtT20 cells.71 In bovine pituitary secretory granules, membrane-bound CPE/H is converted to the slightly smaller soluble CPE/H by limited proteolysis. These results are consistent with a hypothesis for conversion of the membrane-bound CPE/H to soluble CPE/H of higher specific activity.63 The mechanism for CPE/H binding to the membrane involves the COOH-terminal region of the enzyme protein that can form an amphiphilic α-helix containing pairs of hydrophobic residues separated by hydrophilic residues.72 The COOH-terminal region of the membrane-bound CPE/H is responsible for its membrane-binding. This COOH-terminal peptide segment of CPE/H that binds membranes is absent from the soluble CPE/H. When the COOH-terminal 51 amino acids of the membrane-bound CPE/H is expressed as a fusion protein with albumin in AtT20 cells, the albumin/COOH-terminal CPE/H fragment becomes membrane-associated.73 These results suggest that the COOH-terminal region of CPE/H functions as a membrane anchor. In further experiments, expression in AtT20 cells of CPE/H constructs possessing deletions of 14 or 23 residues of the COOH-terminal region resulted in primarily soluble CPE/H, whereas nontransfected cells contain 50% soluble and 50% membrane-associated CPE/H.74 In addition, this soluble CPE/H without the COOH-terminal 14 or 23 residues was secreted. These results indicate that separate domains of the COOH-terminal region of CPE/H are responsible for membrane binding and sorting into the regulated secretory pathway.

Molecular Genetic Analysis of Mutant Carboxypeptidase E/H in fat/fat Obese Mice: Effects of Inactive CPE/H on Prohormone Processing The importance of CPE/H in the prohormone processing pathway is demonstrated in obese fat/fat mice.13 Among several genetic mouse models of obesity, the mouse autosomal

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recessive fat gene is distinguished from other characterized genetic mutations in obesity.13 The fat mutation elicits a slowly developing obesity compared to the db or ob mutations. These mice possess chronically high levels of plasma insulin. The fat mutation maps to mouse chromosome 8 in the vicinity of carboxypeptidase E/H. Further studies indicate a missense mutation in the Cpe gene, with mutation of Ser-202 to Pro, which is associated with the virtual absence of CPE/H activity and enzyme protein in islets and pituitary. Using the baculovirus system, expression of recombinant CPE/H with the Pro202 mutation results in inactive CPE/H.75 When expressed in mouse pituitary AtT20 cells, the mutant CPE/H is rapidly degraded.75 Other processing enzymes involved in proinsulin and prohormone processing, the prohormone convertases (PC1/3 and PC2) and the peptidylglycine α-amidating mono-oxygenase, were not altered in fat/fat mice. The loss of CPE/H activity is associated with an increase in proinsulin and partially processed proinsulin-derived intermediates in β cells and serum. Together, these results indicate that carboxypeptidase E/H is the fat locus, and that the mutant Ser-202 CPE/H is inactive. Since CPE/H is required for processing of multiple peptide precursors in neuroendocrine tissues, it would be expected that fat/fat mice would possess deficits in the production of neuropeptides that regulate feeding behavior. Indeed, the processing of proneurotensin (proNT) and promelanin-concentration hormone (proMCH), which inhibit food intake,76 was aberrant. Hypothalamic and brain extracts from fat/fat mice possessed reduced levels of (greater than 80% reduction) of neuromedin, yet high levels of incompletely processed NT-Lys-Arg and NN-Lys-Arg intermediate peptides are present in fat/fat mice compared to controls. However, MCH, which does not require CPE/H for processing since MCH is located at the COOH-terminus of proMCH, was elevated 2- to 3-fold. Interestingly, since PC1/3 and PC2 are not altered in fat/fat mice, the elevated production of MCH suggests the presence of other novel processing proteases for proMCH processing. The lack of active CPE/H in fat/fat mice results in higher levels of prohormones and partially processed intermediates. Fat/fat mice show almost 10-fold increases in proinsulin levels in pancreas.13 Levels of prodynorphin, the precursor of the opioid dynorphin peptide, and proteolytic intermediates are markedly increased in the brain.77 In pituitary, POMC and high molecular weight POMC-derived intermediates are increased several-fold; levels of 4.5 kDa ACTH are reduced by approximately 70%.78 These findings indicate that inactive, mutant CPE/H results in an accumulation of many prohormones and reduced levels of processed neuropeptides.

Mutant CPE/H in fat/fat Mice Leads to Discovery of Novel Carboxypeptidase D and Carboxypeptidase Z The observation that measurable levels of insulin,13 neurotensin,75 (Leu)enkephalin,76 and ACTH77 are detected in CPE/H-deficient fat/fat mice suggests that carboxypeptidases other than CPE/H are present to allow low levels of peptide hormone and neurotransmitter production. Apparently, these low levels of neuropeptides allow survival of fat/fat mice in the obese condition. Further study of carboxypeptidase E/H-like activity in different tissue regions of fat/fat mice showed that carboxypeptidase activity, assayed by following dansylPhe-Ala-Arg cleaving activity, was not uniformly lowered in all tissues from the fat/fat mice.77 In pituitary of fat/fat mice, CPE/H-like activity was reduced to 6-7% of controls, but the activities in brain, adrenal, and testis were 50-57% of controls. Yet, in all these tissues, the lack of CPE/H was confirmed by affinity purification and immunodetection of the enzyme. These results indicate that other carboxypeptidases are present to allow a low degree of complete prohormone processing to occur. Based on these observations, two novel carboxypeptidases—carboxypeptidase D and carboxypeptidase Z—have been identified.

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Carboxypeptidase D Carboxypeptidase D represents a second carboxypeptidase processing enzyme which has similar and distinct properties compared to CPE/H. CPD and CPE/H from bovine pituitary are separated and purified by sequential elution from a substrate affinity column, p-aminobenzoyl-Arg Sepharose, by pH 8 elution of CPE/H, followed by elution with arginine to obtain CPD.79 CPD, purified from bovine pituitary membranes, has an apparent molecular weight of 180 kDa, in contrast to 50-56 kDa for CPE/H. CPD is a metallocarboxypeptidase with pH optimum of 5.5-6.5.79 CPD and CPE/H show similar kinetics (Km and kcat) for a series of dansylated peptides containing COOH-terminal Arg. CPD and CPE/H are inhibited by metalloprotease inhibitors, although CPD is more potently inhibited by GEMSA and hippuryl-Arg (10- and 100-fold more inhibition, respectively). CPD exists as soluble and membrane-bound forms.80 Purification of soluble CPD from bovine pituitary results in two proteins of 170 and 135 kDa. The NH2-terminal peptide sequence of these two soluble forms are identical. The 180 kDa membrane CPD also has the identical NH2-terminal peptide sequence as the two soluble forms. The soluble and membrane forms of CPD possess similar pH optima, inhibitor profiles, and kinetics. Molecular cloning shows that the rat CPD cDNA81,82 encodes a protein with 75% homology in primary sequence to duck gp180, a hepatitis B virus particle binding protein. The CPD polypeptide possesses an N-terminal signal peptide, three metallocarboxypeptidaselike domains, a predicted transmembrane domain, and a 60 amino acid cytoplasmic tail (Fig. 7.3). The first two domains of CPD contain amino acids corresponding to active site, substrate-binding, and metal-binding residues; however, the third carboxypeptidase domain lacks several of these critical residues. The second carboxypeptidase domain of CPD is most closely related to CPE/H. Northern blots show rat CPD mRNA of 8 and 4 kDa in many tissue regions, with additional species ranging from 1.4 to 5 kb in some areas. CPD mRNA expression is widespread; it is expressed in hippocampus, spinal cord, atrium of heart, colon, testes, and ovaries. The broader tissue distribution of CPD mRNA, compared to CPE/H, suggests involvement of CPD in processing a wide variety of polypeptides.

Carboxypeptidase Z The carboxypeptidase (CPZ) cDNA was found through search of the expressed sequence tag database for clones with homology to human CPE/H.83 DNA sequencing of these homologous clones, combined with RT-PCR of human salivary gland mRNA revealed the CPZ cDNA. The CPZ cDNA of 2.1 kb encodes a 641 residue polypeptide that contains active site residues of metallocarboxypeptidases for Zn2+ binding (His69, Glu72, and His196, using the CPA numbering system), substrate binding (Arg145 and Tyr248), and catalytic activity (Glu270) which make up the carboxypeptidase domain (Fig. 7.3). CPZ contains a putative NH2-terminal 18 residue signal peptide, followed by a 167 residue NH2-terminal domain that precedes the carboxypeptidase domain. CPZ mRNAs of 2.1 and 2.6 kb are detected abundantly in human placenta, and are also present in liver, skeletal muscle, kidney, and pancreas. Expression of the 641 residue CPZ in baculovirus-infected Sf9 cells shows CPZ activity with the substrate dansyl-Phe-Ala-Arg, with optimum activity at neutral pH of 7.4.83 Low activity at the intragranular pH of 5.5 suggests that CPZ would not function within the acidic intravesicular environment of secretory vesicles. CPZ is inhibited by the metallocarboxypeptidase inhibitors MGTA and GEMSA. Interestingly, it is noted that identification of two new members of the metallopeptidase family are in progress. These results demonstrate CPZ as a novel member of the CPE/H subfamily of metallocarboxypeptidases.

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Fig. 7.3. Structural domain comparisons of carboxypeptidases E/H, D, and Z. The structural domains of CPE/H is compared to those of CPD and CPZ. Key residues for metallocarboxypeptidase function are indicated as His69, Glu72, Arg145, His196, Tyr248, and Glu270 with numbering according to the CPA numbering system. Metallocarboxypeptidase Zn2+ binding involves His69, Glu72, and His196. Substrate binding is hypothesized to involve Arg145 and Tyr248; catalytic activity involves Glu270.

Evidence for CPE/H as a Sorting Receptor for the Intracellular Routing of POMC and Possibly Other Prohormones to the Secretory Vesicle The membrane-associated CPE/H has recently been discovered as a sorting receptor for routing the POMC (proopiomelanocortin) prohormone into secretory vesicles.84 Specific sorting of prohormones is known to be a selective process requiring a sorting signal. The sorting signal motif for the prohormone POMC has been demonstrated as a conformational configuration consisting of the NH2-terminal POMC residues 8-20.84 This conformational motif binds specifically to membrane-associated CPE/H, which apparently acts to route the prohormone to the regulated secretory pathway. The role of CPE in regulated secretion of POMC products was demonstrated in CPE/Hdeficient Neuro-2A cells expressing antisense CPE/H, and in Cpefat mice that are genetically deficient in CPE/H.84 In POMC transfected Neuro-2A cells deficient in CPE/H, release of ACTH (release stimulated by KCl depolarization) is reduced by 70% compared to control cells expressing CPE/H. In mouse neurointermediate lobe, regulated ACTH release is normally inhibited by dopamine. However, in Cpefat mice that lack CPE/H, ACTH release from neurointermediate lobe is not regulated by dopamine. Furthermore, regulated ACTH release from anterior pituitary of Cpefat mice is absent, since ACTH release was not stimulated by CRH (corticotropin releasing hormone) or KCl depolarization; only nonregulated constitutive ACTH secretion was detected. Similarly, growth hormone release from anterior pituitary of Cpefat mice did not respond to KCl depolarization. These results demonstrate that: 1. the NH2-terminal conformational motif of POMC binds directly to membraneassociated CPE/H; and 2. in cells deficient in CPE/H, ACTH is absent from the regulated secretory pathway and ACTH is missorted to the constitutive pathway. These results suggest a role for CPE/H in sorting POMC and prohormones into the regulated secretory pathway.

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Aminopeptidase(s) for Prohormone Processing Endoproteolytic processing of prohormones and neuropeptide precursors results in intermediates with basic residue extensions at the NH2- and COOH-termini of peptide intermediates (Fig. 7.2). Therefore, aminopeptidase activity specific for the removal of NH2terminal basic residues is needed for complete prohormone processing. Less is known about the aminopeptidase processing enzyme(s), compared to the carboxypeptidase processing enzyme(s).

Arginine Aminopeptidase Activity in Pituitary Secretory Vesicles POMC-containing secretory vesicles from bovine intermediate and posterior pituitary possess aminopeptidase activity that converts Arg-(Met)enkephalin to (Met)enkephalin.85 Characterization indicates thiol metallo-aminopeptidase activity within pituitary secretory vesicles. The arginine aminopeptidase activity is maximal at pH 6.0, inhibited by EDTA, and stimulated by Co2+ and Zn2+. Their secretory vesicle localization is confirmed by cellular secretion of arginine aminopeptidase activity that converts Arg-(Met)enkephalin to (Met)enkephalin from cultured bovine intermediate lobe pituitary cells.86

Arginine and Lysine Aminopeptidase Activities in Adrenal Medullary Chromaffin Granules Arginine and lysine aminopeptidase activities are present in bovine adrenal medulla chromaffin granules (secretory vesicles) that contain several neuropeptides including (Met)enkephalin,17 neuropeptide Y,19 galanin,20 and others.21,22 Chromaffin granules contain activity for the conversion of Lys-Arg-(Met)enkephalin to Arg-(Met)enkephalin, and subsequently (Met)enkephalin;87 the lysine and arginine residues are sequentially removed to form (Met)enkephalin. The activity is optimal at pH 6.0 and is inhibited by metal ion chelators, as well as the thiol reagents CuCl2 and PCMPSA (p-chloromercuriphenylsulfonic acid), indicating the presence of thiol metallo-aminopeptidase activities in chromaffin granules. More detailed characterization of arginine and lysine aminopeptidase activities in chromaffin granules utilized Arg-MCA and Lys-MCA as substrates.88 These fluorescent substrates allow rapid and sensitive detection of aminopeptidase activities. Arginine and lysine aminopeptidase activities in chromaffin granules show similar properties. Arg-MCA and Lys-MCA cleaving activities are optimum at pH 6.7 and 7.0, respectively (Table 7.2), with much activity at the intragranular pH of approximately pH 5.8-6.0.89 These aminopeptidase activities are strongly stimulated by β-ME, indicating the importance of reduced sulfhydryl groups for activity. Inhibition by EDTA and 1,10-phenanthroline indicates metalloprotease activity; inhibition is also observed by the aminopeptidase inhibitors bestatin, amastatin, arphamenine A, and arphamenine B. Kinetic studies show that arginine and lysine aminopeptidases possess Km of 104 µM Arg-MCA and 160 µM Lys-MCA, respectively; these affinities are consistent with in vivo levels of enkephalin and other neuropeptides.61 The majority of Arg-MCA and Lys-MCA cleaving activities are soluble, rather than membranebound. These properties illustrate that thiol metallopeptidases possess arginine and lysine aminopeptidase activities in chromaffin granules. Differences in Arg-MCA and Lys-MCA cleaving activities are evident.88 The Arg-MCA cleaving activity is stimulated by physiological levels of NaCl (150 mM), but the Lys-MCA cleaving activity is not affected by NaCl. The Arg-MCA activity is stimulated by 70-80% over controls by Co2+, but the Lys-MCA activity is minimally affected by Co2+. It will be of interest in future studies to determine whether arginine and lysine aminopeptidase activities are represented by identical or different exopeptidases.

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Table 7.2. Biochemical characteristics of arginine and lysine aminopeptidase activities in chromaffin granules Property

Arg-MCA

Lys-MCA

pH 6.7 Yes Yes Yes Yes 104 µM

pH 7.0 Yes Yes No No 160 µM

pH optimum Inhibition by metalloprotease inhibitors Sensitive to cysteinyl reagents Stimulation by Co2+ Stimulation by NaCl Affinity for substrate, Km

Metallo-aminopeptidases that remove basic residues, known as aminopeptidase B enzymes, have been isolated from many tissue sources including muscle,90,91 brain,92,93 kidney,93 and testes.94 However, the neurosecretory vesicle arginine and lysine aminopeptidase activities identified in chromaffin granules88 are distinct from aminopeptidase B activities. Aminopeptidase B is activated by chloride ion, but the chromaffin granule arginine and lysine aminopeptidase activities are not affected by chloride ion. The chromaffin granule basic residue cleaving aminopeptidases appear to differ from porcine muscle aminopeptidase B that is inhibited by β-mercaptoethanol,91 and from rat testes aminopeptidase B that is stimulated by Zn2+.94 These results suggest that the neurosecretory arginine and lysine aminopeptidase activities in chromaffin granules may represent distinct metalloproteases compared to aminopeptidase B. Results from studies of basic residue-cleaving aminopeptidases in secretory vesicles from pituitary85,86 and adrenal medulla87,88 indicate that thiol metallopeptidases are involved in NH 2-terminal processing of prohormone-derived peptide intermediates. The colocalization of these aminopeptidases in neurosecretory vesicles with endoproteolytic prohormone processing enzymes and neuropeptides are compatible with secretory vesicle processing, storage, and secretion of active peptides.

Conclusions and Future Perspectives Carboxypeptidase E/H was the first prohormone processing protease to be identified and characterized. A wealth of knowledge demonstrates that the carboxypeptidase step in prohormone processing is achieved primarily by the neuroendocrine-specific CPE/H. The importance of this exopeptidase step in prohormone processing is recently demonstrated in genetically obese fat/fat mice that possess a mutation at Ser-202 that renders CPE/H inactive. With inactive CPE/H, the processing of proinsulin, POMC, prodynorphin, and other neuropeptide precursors is greatly retarded. However, fat/fat mice possess neuroendocrine systems that allow physiological functions and survival of the animal. Thus, other processing enzymes besides CPE/H are clearly involved in prohormone processing. The novel carboxypeptidase D metalloprotease may participate in prohormone processing. It is also likely in fat/fat mice, or other conditions, that endoproteases cleaving at the NH2-terminal side of paired basic residue processing sites would preclude a requirement for carboxypeptidase processing activity. These results imply that the endoproteolytic processing enzyme ‘prohormone thiol protease’ (PTP), which possesses the ability to cleave at the NH2-terminal side of dibasic residues, may be involved in maintaining neuroendocrine functions under conditions of aberrant CPE/H. It will be interesting in future studies to test this hypothesis.

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The actions of endoproteolytic prohormone processing proteases—‘prohormone thiol protease’ (PTP), subtilisin-like PC1/3 and PC2 (PC = prohormone convertase), and a 70 kDa aspartyl protease known as ‘POMC converting enzyme’ (PCE)—in cleaving polypeptide substrates between the dibasic residues, or at the NH2-terminal side of the pair in the case of PTP, clearly indicate that aminopeptidase processing activity is required for complete processing. Knowledge of arginine and lysine aminopeptidase(s) has lagged behind progress in the understanding of carboxypeptidase E/H. It will be of primary importance in future studies to identify the aminopeptidase(s) specific for removal of basic residues from NH2-termini of peptide intermediates. Coordinate regulation of aminopeptidase and carboxypeptidase processing enzymes, together with the endoproteolytic processing proteases, will be key toward understanding regulatory mechanisms of neuropeptide precursor processing for the production of active peptide hormones and neurotransmitters.

Acknowledgments This work was supported by grants from NIDA and NINDS of the National Institutes of Health, and from the National Science Foundation.

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57. Fricker LD, Rigual RJ, Diliberto EJ, Viveros OH. Reflex splanchnic nerve stimulation increases levels of carboxypeptidase E mRNA and enzymatic activity in the rat adrenal medulla. J Neurochem 1990; 55:461-467. 58. Das B, Sabban EL, Kilbourne EJ, Fricker LD. Regulation of carboxypeptidase E by membrane depolarization in PC12 pheochromocytoma cells: Comparison with mRNAs encoding other peptide- and catecholamine-biosynthetic enzymes. J Neurochem 1992; 59:2263-2270. 59. Klein RS, Fricker LD. Differential effects of a phorbol ester on carboxypeptidase E in cultured astrocytes and AtT20 cells, a neuroendocrine cell line. J Neurochem 1993; 60:1615-1625. 60. Ungar A, Phillips JH. Regulation of the adrenal medulla. Physiol Rev 1983; 63:787-843. 61. Hook VYH, LaGamma EF. Product inhibition of carboxypeptidase H. J Biol Chem 1987; 262:12583-12588. 62. Hook VYH. Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme. Lif Sci 1990; 47:1135-1139. 63. Hook VYH. Differential distribution of carboxypeptidase-processing enzyme activity and immunoreactivity in membrane and soluble components of chromaffin granules. J Neurochem 1985; 45:987-989. 64. Hook VYH, Eiden LE. (Met)enkephalin and carboxypeptidase processing enzyme are coreleased from chromaffin cells by cholinergic stimulation. Biochem Biophys Res Commun 1985; 128:563-570. 65. Vilijn MH, Das B, Kessler JA, Fricker LD. Cultured astrocytes and neurons synthesize and secrete carboxypeptidase E, a neuropeptide processing enzyme. J Neurochem 1989; 53:1487-1493. 66. Klein RS, Das B, Fricker LD. Secretion of carboxypeptidase E from cultured astrocytes and from AtT20 cells, a neuroendocrine cell line: implications for neuropeptide biosynthesis. J Neurochem 1992; 58:2011-2018. 67. Hook VYH, Affolter HU, Palkovits M. Carboxypeptidase H in the hypothalamo-neurohypophysial system: Evidence for processing and activation of a prohormone-processing enzyme during axonal transport. J Neurosci 1990; 10:3219-3226. 68. Yajima R, Chikuma T, Kato T. A rapid anterograde axonal transport of carboxypeptidase H in rat sciatic nerves. J Neurochem 1994; 63:997-1002. 69. Parkinson D. Two soluble forms of bovine carboxypeptidase H have different NH2-terminal sequences. J Biol Chem 1990; 265:17101-17105. 70. Song L, Fricker L. Processing of procarboxypeptidase E into carboxypeptidase E occurs in secretory vesicles. J Neurochem 1995; 65:444-453. 71. Fricker LD, Devi L. Posttranslational processing of carboxypeptidase E, a neuropeptideprocessing enzyme, in AtT20 cells and bovine pituitary secretory granules. J Neurochem 1993; 61:1404-1415. 72. Fricker LD, Das B, Angeletti RH. Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10). J Biol Chem 1990; 265:2476-2482. 73. Mitra A, Song L, Fricker LD. The C-terminal region of carboxypeptidase E is involved in membrane binding and intracellular routing in AtT20 cells. J Biol Chem 1994; 269:19876-19881. 74. Varlamov O, Fricker LD. The C-terminal region of carboxypeptidase E involved in membrane binding is distinct from the region involved with intracellular routing. J Biol Chem 1996; 271:6077-6083. 75. Varlamov O, Leiter EH, Fricker L. Induced and spontaneous mutations at Ser202 of carboxypeptidase E, effect on enzyme expression, activity, and intracellular routing. J Biol Chem 1996; 271:13981-13986. 76. Novere C, Viale A, Nahan JL, Kitabgi P. Impaired processing of brain proneurotensin and promelanin-concentrating hormone in obese fat/fat mice. Endocrinol 1996; 137:2954-2958. 77. Fricker LD, Berman YL, Leiter DH, Devi LA. Carboxypeptidase E activity is deficient in mice with the fat mutation, effect on peptide processing. J Biol Chem 1996; 271: 30619-30624.

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78. Shen FS, Loh YP. Intracellular misrouting of pituitary hormones and endocrinological abnormalities in the Cpefat mouse associated with a carboxypeptidase E mutation. Proc Natl Acad Sci USA 1997; 94:5314-5319. 79. Song L, Fricker LD. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J Biol Chem 1995; 270:25007-25013. 80. Song L, Fricker LD. Tissue distribution and characterization of soluble and membranebound forms of metallocarboxypeptidase D. J Biol Chem 1996; 271:28884-28889. 81. Xin S, Varlamov O, Day R, Dong W, Bridget MM, Leiter EH, Fricker LD. Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D. DNA and Cell Biol 1998; (in press). 82. Fricker LD. Metallocarboxypeptidase D. In: Barrett AJ, ed. Handbook of Proteolytic Enzymes. Academic Press, 1998; (in press). 83. Song L, Fricker LD. Cloning and expression of human carboxypeptidase Z, a novel metallocarboxypeptidase. J Biol Chem 1997; (in press). 84. Cool DR, Normant E, Shen FS, Chen HC, Pannell L, Zhang Y, Loh YP. Carboxypeptidase E is a regulated secretory pathway sorting receptor: Genetic obliteration leads to endocrine disorders in Cpefat mice. Cell 1997; 88:73-83. 85. Gainer HG, Russel JT, Loh YP. An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from β-lipotropin 60-65. FEBS Lett 1984; 175:135-139. 86. Castro MG, Birch NP, Loh YP. Regulated secretion of pro-opiomelanocortin converting enzyme and an aminopeptidase B-like enzyme from dispersed bovine intermediate lobe pituitary cells. J Neurochem 1989; 52:1619-1628. 87. Hook VYH, Eiden LE. Two peptidases that convert 125I-Lys-Arg-(Met)enkephalin and 125I(Met)enkephalin-Arg6, respectively, to 125I-(Met)enkephalin in bovine adrenal medullary chromaffin granules. FEBS Lett 1984; 172:212-218. 88. Yasothornsrikul S, Hook VYH. Arginine and lysine aminopeptidase activities in chromaffin granules of bovine adrenal medulla: Relevance to prohormone processing. J Neurochem 1998; 70:153-163. 89. Pollard HB, Shindo H, Creutz, CE, Pazoles CJ, Cohen JS. Internal pH and state of ATP in adrenergic chromaffin granules determined by 31P nuclear magnetic resonance spectroscopy. J Biol Chem 1978; 254:1170-1177. 90. Mantle D, Lauffart B, McDermott JR, Kidd AM, Pennington RJT. Purification and characterization of two Cl-activated aminopeptidases hydrolysing basic termini from human skeletal muscle. Eur J Biochem 1985; 147:307-312. 91. Flores M, Aristoy MC, Toldra F. HPLC purification and characterization of porcine muscle aminopeptidase B. Biochemie 1993; 75:861-7. 92. Gomez S, Gluschankof P, Lepage A, Cohen P. Relationhip between endo- and exopeptidases in a processing enzyme system: Activation of an endoprotease by the aminopeptidase B-like activity in somatostatin-28 convertase. Proc Natl Acad Sci USA 1988; 85:5468-5472. 93. Mantle D. Comparison of soluble aminopeptidases in human cerebral cortex, skeletal muscle and kidney tissues. Clinica Chimica Acta 1992; 207:107-118. 94. Cadel S, Pierotti AR, Foulon T, Creminon C, Barre N, Segretain D, Cohen P. Aminopeptidase-B in the rat testes: Isolation, functional properties and cellular localization in the seminiferous tubules. Mol Cell Endocrinol 1995; 110:149-160.

CHAPTER 8

The Neuroendocrine Polypeptide 7B2 as a Molecular Chaperone and Naturally Occurring Inhibitor of Prohormone Convertase PC2 A. Martin Van Horssen and Gerard J.M. Martens

Introduction

A

hallmark of neuroendocrine cells is their ability to synthesize, store and release biologically active peptides in a regulated manner. To fulfill this highly specialized task, these cells possess a unique, regulated secretory pathway which is characterized by specific storage organelles, the secretory granules. Besides peptide hormones and processing enzymes, these granules contain a group of acidic secretory proteins of unresolved function, named the granin (chromogranin/secretogranin) family.1 In addition to the three classical granins, chromogranin A (CgA, secretory protein I), chromogranin B (CgB, secretogranin I) and secretogranin II (SgII, chromogranin C), other neuroendocrine-specific proteins like the 1B1075 protein (secretogranin III, SgIII), the HISL-19 antigen (secretogranin IV, SgIV) and the 7B2 protein (secretogranin V, SgV) have been proposed to belong to this family. Apart from CgA and CgB, the granins are structurally unrelated, but share certain biochemical properties, as well as a broad neuroendocrine tissue distribution.1 Since the granins undergo proteolytic cleavage in secretory granules at pairs of basic amino acid residues before secretion, they have been postulated to be precursor proteins of peptide hormones and neuropeptides.2-7 However, the granins have also been suggested to act intracellularly as helper proteins in prohormone packaging and sorting and as modulators of processing enzymes in the secretory pathway.1,8-10 Consistent with the latter notion is the recent discovery that the granin family member 7B2 acts inside the cell. In this review, we will discuss various aspects of 7B2 and in particular pay attention to its intracellular role.

History of 7B2 The history of the 7B2 protein commences with its isolation and HPLC purification from porcine anterior pituitary by Seidah and colleagues, and the protein was therefore initially referred to as anterior pituitary pig (APPG). Amino-terminal amino acid sequence analysis resulted in the identification of the first 50 amino acid residues.11 From whole human pituitary glands, an ~21 kDa 7B2 protein was subsequently purified and the first 77 amino acid residues of the protein were determined.12 A preliminary immunocytochemical Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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study showed that 7B2 is present in the anterior and posterior lobe of the pituitary gland and in the supraoptic nucleus of the hypothalamus.12 Seidah and colleagues initially suggested that 7B2 could act as a growth factor, based on its homology with proinsulin and Rous sarcoma virus transforming protein.12

The 7B2 Gene and Its Regulation The structural organization of the 7B2 gene has been partially elucidated in human,13,14 rat (accession number M63965)15 and the amphibian Xenopus laevis (accession number X94303).14 The 7B2 gene carries six exons: Exon 1 represents the 5'-untranslated region (UTR), exon 2 corresponds to the remainder of the 5'-UTR and codes for the signal peptide, exons 3-5 encode most of the 7B2 protein and exon 6 codes for the remainder of the 7B2 protein and corresponds to the 3'-UTR. The 7B2 gene (locus SGNE-1 for secretory granule neuroendocrine protein-1) has been mapped to human chromosome region 15q13-1413,16 and mouse chromosome 2 E3-F3.13 The presumptive 7B2 gene promoter has been identified in rat15 and human.14 Although the 7B2 gene is selectively expressed in neurons and endocrine cells, the human 7B2 promoter displays characteristics of housekeeping genes and genes coding for heat-shock proteins, as well as proteins associated with cell growth and proliferation. The putative promoter lacks the TATA box and CAAT box elements common to most eukaryotic genes. The lack of a TATA box is a property of housekeeping genes and genes associated with cell growth and proliferation.17 Besides a cAMP responsive element and an AP-1 site, the putative promoter carries heat-shock element-like (HSE) sequences and a thermal response element (TRS, C4T).14 In addition to these elements, reminiscent of proteins with a broad tissue distribution, the 7B2 promoter carries motifs also found in promoters of genes coding for neuroendocrine-specific proteins such as human proenkephalin, bovine proopiomelanocortin (POMC) and human prohormone convertase (PC) 2.14,18-20 Like other TATA-less genes, the human 7B2 gene is transcribed from multiple transcription initiation sites.21 7B2 is expressed in parallel with POMC in both Xenopus intermediate pituitary22 and mouse anterior pituitary-derived AtT20 cells,23 suggesting a role for 7B2 in the biosynthesis and secretion of peptide hormones in neuroendocrine cells. In Xenopus intermediate pituitary, POMC and 7B2 mRNA levels are about 20-fold higher in the active peptide hormone-secreting cells of black-adapted animals versus the inactive cells of white-adapted animals.22,24 The transcription factors controlling 7B2 gene activity have not been identified. In human pituitary, two mRNAs for 7B2 have been identified, differing by the presence or absence of an alanine codon at amino acid position 100 (Fig. 8.1). This difference is probably due to alternative splicing as revealed by analysis of the acceptor splice site in the human 7B2 gene. Dimorphism at this position was also found in other human endocrine tissues, and in different species including mouse, rat, pig, cow and monkey.25 In Northern blot analysis, a major 7B2 gene transcript of ~1.35 kb and two minor transcripts of ~0.9 and ~1.2 kb were detected in human pituitary.26 The identity of the two smaller transcripts is unclear. The presence of two different 7B2 mRNAs in Xenopus intermediate pituitary is likely to result from a Xenopus genome duplication about 30 million years ago.27,28 The regulation of transcriptional activity of the 7B2 gene has been studied in a number of systems and at different levels. In a β cell-like insulinoma cell line, the transcription of the 7B2 gene is regulated by protein kinase A and C activators while in an α cell-like insulinoma cell line, 7B2 gene transcription seemed to be constitutively activated.21 Castration significantly reduced 7B2 mRNA levels in mouse pituitary.29 In human lung cancer cells, the 7B2 gene is differentially expressed.30 Since 7B2 protein levels have been found to be elevated in plasma and tissues of patients with endocrine tumors of various origin, 7B2 has been proposed to be a specific neuroendocrine tumor marker.31-36 In human cerebrospinal fluid and

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Fig. 8.1. Alignment of the amino acid sequences of the known 7B2 proteins. The single amino acid code is used. Gaps are introduced for optimal alignment. The putative signal peptides (SP) of the vertebrate sequences are overlined. The Lymnaea SP corresponds to Met-17 to Ala-1. Amino acid residues are boxed when they are identical among four or more of the aligned sequences. Potential cleavage sites for proprotein convertases are indicated in white letters on a black background. The 7B2 cDNA sequences are available (except for salmon) from the EMBL database under the following accession numbers: human (Y00757),26 porcine (M23654),43 rat (M63901),15 mouse (X15830),44 Xenopus (X15608/X14628)22 and Lymnaea (U72709).45

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in blood plasma, 7B2 levels have been found to diminish with age.37-39 In patients suffering from the Prader-Willi and the Angelman syndrome, deletions have been found close to the chromosomal subregion to which the human 7B2 gene is located.13,16,40,41 In the hypothalami of the majority of Prader-Willi patients, 7B2 expression has indeed been shown to be diminished.42

Evolutionary Aspects A differential screening approach to identify genes associated with the specialized secretory function of peptide-hormone producing cells led to the identification of a partial Xenopus and a full length human pituitary cDNA coding for 7B2.26 The 7B2 cDNAs from human, porcine,43 Xenopus laevis,22 mouse,44 rat and salmon15 and the mollusk Lymnaea stagnalis45 code for proteins of 207-273 amino acid residues with a calculated molecular mass of ~23-30 kDa (Fig. 8.1). The first 17-26 amino-terminal amino acid residues represent a signal peptide, allowing entrance into the endoplasmic reticulum (ER). Except for the Lymnaea homologue, the 7B2 proteins are highly conserved among the different species (Fig. 8.1). The overall amino acid sequence identity among mammalian, amphibian and fish 7B2 is 71-99% (the signal peptides not included). During evolution, the amino-terminal domain is conserved to a higher degree than the carboxy-terminal domain (with the amino-terminal 76 amino acids displaying 90-100% identity and the carboxy-terminal 106-113 amino acids 64-99% identity). The recent identification of a cDNA coding for the first invertebrate 7B2 protein from the brain of Lymnaea revealed that the overall amino acid sequence identity between invertebrate 7B2 and its vertebrate homologues is surprisingly low (29%). The sequence identity is confined to a proline-rich region in the middle of the protein and to a carboxy-terminal region carrying a pair of basic amino acids45 (Fig. 8.1). As discussed below, these regions are important for the functional role of 7B2. Except for salmon 7B2, three potential cleavage sites for proprotein convertases are conserved among all known 7B2 sequences (Fig. 8.1). Apart from these three sites, rat, mouse and Lymnaea 7B2 carry an additional pair of basic amino acid residues (Fig. 8.1). The 7B2 protein has some functional and structural characteristics in common with a number of protein families. Based on its biochemical properties and its neuroendocrinespecific tissue distribution, 7B2 has been proposed to belong to the granin family.1 The amino-terminal half of 7B2 shares a low degree of sequence similarity with a subclass of molecular chaperones, the chaperonins-60 and chaperonins-10 (including the bacterial chaperone GroEL and its cochaperone GroES, respectively).46 The carboxy-terminal half of 7B2 is distantly related to members of the potato inhibitor I family (including inhibitors of the bacterial subtilisin family of processing enzymes).47 However, 7B2 cannot be classified into one of these families and its evolutionary origin therefore remains obscure.

7B2 is a Neuroendocrine-Specific Polypeptide Immunocytochemical analyses have shown that 7B2 is broadly distributed in neurons and endocrine cells. Immunoreactivity to 7B2 has been reported in pituitaries of rat,34,48,49 mouse,34 human34 and Xenopus,50,51 as well as in the brain34,38,48,49,52,53 and spinal cord.49,54 The protein has also been detected in various endocrine organs including the adrenal medulla,48,55 pancreas,32,48,56 gut56,57 and thyroid gland.33 Within neuroendocrine cells, 7B2 is located in dense core secretory granules.53,58,59 Northern blot analysis showed that in mouse, 7B2 mRNA is most abundant in the pituitary gland and in various brain areas (in particular the hypothalamus). 7B2 mRNA has also been detected in several peripheral organs including the thyroid, stomach, kidney, submaxillary gland, thymus, lung, intestine, spleen, ovary and testis. The amount of 7B2 mRNA in these tissues is about 100-fold lower than in the

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pituitary.29 The 7B2 protein thus appears to be selectively present in peptide hormone-producing cells possessing a regulated secretory pathway.

Biochemical Characteristics of 7B2 Like many neuroendocrine secretory proteins, 7B2 is acidic. The calculated isoelectric point (pI) for the ~27 kDa intact form is ~6.3, and for the ~21 kDa processed form ~4.7. In line with this estimated pI, mammalian 21 kDa 7B2 indeed appeared to have a pI value of ~5, as determined by two-dimensional SDS-PAGE/isoelectric focusing.12,60 In SDS-PAGE gels, intact Xenopus 7B2 migrates as a 25 kDa protein61 and mouse 7B2 as a 29 kDa protein.62 The mobility of 7B2 is slower than predicted from its amino acid sequence (calculated molecular mass is 20.4 and 20.9 kDa for Xenopus and mouse, respectively). The highly acidic nature of 7B2 probably causes this anomalous behavior on SDS-PAGE. Similar discrepancies have been observed for other members of the granin family.63-65 The 7B2 protein shares various biochemical characteristics with the granins. First, the granins are hydrophilic with a high proportion of acidic amino acid residues. Second, they contain numerous pairs of basic amino acids that are potential sites for proteolytic processing enzymes (Fig. 8.1). Third, 7B2 and other members of the granin family bind calcium and aggregate under conditions representing those in the trans-Golgi network (TGN) (pH 6.4 and 3 mM Ca2+),1,66 and these characteristics are probably essential for the sorting of these proteins to the regulated pathway, away from those destined for constitutive secretion.

Posttranslational Modifications of 7B2 During transit through the regulated secretory pathway, 7B2 undergoes various posttranslational modifications including disulfide bond formation, phosphorylation, sulfation and proteolytic processing. No putative acceptor site for N-linked glycosylation (Asn-XSer/Thr)67 is present in any known 7B2 protein sequence (Fig. 8.1), and porcine and human 7B2 are indeed not glycosylated.12 The 7B2 protein carries two conserved cysteine residues (at positions 94 and 103 in the human protein) that may participate in the formation of a disulfide bridge. In newly synthesized 7B2 produced by Xenopus intermediate pituitaries, disruption of the disulfide bond by the reducing agent dithiothreitol (DTT) results in a slower mobility of the protein in nonreducing SDS-PAGE gels (A. M. Van Horssen, J. A. M. Braks and G. J. M. Martens; unpublished results), indicating that an intrachain disulfide bond is indeed present. The two highly conserved serine residues at positions 10 and 178 in human 7B2 are part of a casein kinase II consensus phosphorylation sequence (Ser/Thr-X-X-acidic residue).68 In bovine adrenal medulla chromaffin vesicles, Ser178 is indeed phosphorylated.69 Whether phosphorylation of 7B2 occurs before or after its cleavage has not been established, nor has the significance of this modification for the proper functioning of 7B2 and its fate in the secretory pathway. In Xenopus intermediate pituitary, no phosphorylation of intact or cleaved 7B2 has been detected (R. P. Kuiper, J. A. M. Braks and G. J. M. Martens; unpublished results). Except for the Xenopus and Lymnaea homologues, 7B2 contains a putative sulfation site on Tyr130. In anterior pituitary-derived AtT20 cells infected with recombinant vaccinia virus, tyrosine sulfation of mouse 7B2 precedes its proteolytic processing, indicating that the protein arrives intact in the TGN, the compartment where sulfation takes place.70,71 Xenopus 7B2 is not sulfated (R. P. Kuiper and G. J. M. Martens; unpublished results), indicating that tyrosine sulfation of 7B2 is not evolutionarily conserved. Tyrosine sulfation has been suggested to affect the biological activity and/or intracellular transport,70 but the significance of 7B2 sulfation is at present unclear. That sulfation of 7B2 is not evolutionarily conserved argues against an important role for this posttranslational modification.

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Finally, 7B2 undergoes proteolytic cleavage at pairs of basic amino acid residues. These pairs are located in the carboxy-terminal ends of the 7B2 proteins (with salmon containing two sites, human, porcine and Xenopus possessing three and mouse, rat and Lymnaea carrying four di- or oligobasic sites) (Fig. 8.1). Proteolytic processing of 7B2 occurs at different sites, depending on the species involved. In Xenopus intermediate pituitary, the intact 25 kDa form is cleaved at the Lys138-Lys139 site, yielding the secreted, 18 kDa form.61 The purification of a 23 kDa 7B2-derived peptide from porcine anterior pituitary indicates that the 29 kDa 7B2 precursor protein is cleaved at a site marked by five basic amino acid residues (Arg151-Arg-Lys-Arg-Arg155).72 Consistent with this finding is the isolation of the carboxyterminal peptide of 7B2, resulting from cleavage at the pentabasic site, from the culture media of AtT20 cells.62 The purification of the carboxy-terminal tridecapeptide (corresponding to Ser173-Glu185) from bovine adrenal medulla chromaffin vesicles indicates that Lys171-Lys172 is a site for proteolytic cleavage.69 In insulinoma and anterior pituitary cell lines transfected with 7B2, 3.5 kDa and 1.5 kDa carboxy-terminal peptides have been characterized, corresponding to the peptides which result from processing at the pentabasic site, and at the internal Lys171-Lys172 site, respectively.73 The proprotein convertase(s) responsible for 7B2 cleavage in vivo has not been identified. The broadly distributed proteinase furin is able to cleave 7B2 at the pentabasic site.71 In Xenopus intermediate pituitaries, 7B2 cleavage yielding the 18 kDa processing product cannot be the result of furin activity, since the site used in this animal (Lys138-Lys139) is a potential PC2 site, not susceptible to furin cleavage. In vitro, the prohormone convertase PC2 is able to cleave 7B2 at the Lys171-Lys172 site.73

Regulated Secretion of 7B2 Secretion of 7B2 has been studied in cultured rat pituitary cells, the rat pheochromocytoma cell line PC12, bovine chromaffin cells, isolated rat hypothalami, rat adrenals, the mouse anterior pituitary-derived cell line AtT20 and the rat pituitary cell line GH3.23,31,48,55,74-77 The ~21 kDa 7B2 cleavage product (but not the precursor form) is secreted via the regulated secretory pathway.23,48,61 From cultured bovine adrenal medulla chromaffin cells, nicotine-induced 7B2 is released in parallel with catecholamines.48 Secretion of 18 kDa 7B2 from the melanotrope cells of Xenopus intermediate pituitary is inhibited by the dopamine D2 receptor agonist apomorphine.61 In vivo, dopamine is one of the secretagogues of hypothalamic origin that inhibits the release of regulated secretory proteins from Xenopus melanotropes.78 Secretion of 7B2 from AtT20 cells is stimulated by cAMP.23

The Quest for the Role of 7B2 The physiological roles of 7B2 and of the other granins have been unclear for many years (the “oldest” granin, CgA, was isolated in 1967).79 Since the granins undergo proteolytic processing in secretory granules before their release, they have been suggested to be precursor proteins for biologically active peptides.1 This notion was supported by the finding that the CgA-derived peptides chromostatin and pancreastatin, as well as the SgII cleavage product secretoneurin, display some activity as autocrine and paracrine modulators of secretion from neuroendocrine cells.2-7 However, evidence has been provided that granins may function intracellularly and act as helper proteins in prohormone sorting and processing. For instance, CgA has been suggested to modulate prohormone processing in vitro by acting as a competitive substrate for endoproteases.8 Furthermore, CgB overexpression in AtT20 cells has been shown to promote sorting of POMC-derived cleavage products into immature secretory granules.10

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In the decade following the purification of 7B2 from porcine and human pituitary glands,11,12 detailed information has been collected on its intracellular location and on its distribution in normal and neoplastic tissues. Despite many years of investigation, the physiological role of 7B2 remained elusive. That 7B2 cannot be classified into any protein family hampered the hunt for its function. In parallel with the other granins, both an extracellular role for 7B2-derived cleavage products as peptide hormones, as well as an intracellular function as a helper protein in prohormone maturation, has been postulated.1,9,12,53,55,58

7B2 as a Precursor Protein for Bioactive Peptides Based on its homology with proinsulin and Rous sarcoma virus transforming protein, Seidah and colleagues initially suggested that 7B2 could act extracellularly as a growth factor.12 Consistent with a role outside the cell was the finding that a naturally occurring 7B2 cleavage product (corresponding to the carboxy-terminal 13 amino acids) induced depolarization of oxytocin- and vasopressin-producing neurons in the hypothalamic supraoptic nucleus of the rat.80 Recent studies, however, provided evidence that 7B2 has a major role inside the cell.

7B2 Associates with PC2 An important clue concerning a possible intracellular role for 7B2 came with the finding that, when incubated with newly synthesized proteins produced by Xenopus intermediate pituitary cells, recombinant 7B2 specifically associated with the prohormone convertase PC2.46 In vivo, in both Xenopus and mouse intermediate pituitary cells, the intact form of 7B2 transiently associates with PC2.46,81 The neuroendocrine tissue distribution of the 7B2 protein is similar, if not identical, to that of PC2. As shown for 7B2, the PC2 enzyme is broadly distributed in neuroendocrine cells and is located in secretory granules, where it cleaves prohormones at dibasic sites, liberating peptide hormones and neuropeptides from their biologically inactive, precursor proteins.82-84 In Xenopus intermediate pituitary, 7B2 and PC2 are coordinately expressed with POMC, suggesting a role for these proteins in the biosynthesis and secretion of peptide hormones in neuroendocrine cells.22 In the early compartments of the secretory pathway, the liaison between the two proteins commences with the binding of intact 7B2 to the proenzyme form of PC2. In the TGN/immature secretory granules, 7B2 is cleaved, the 7B2/proPC2 complex dissociates and proPC2 matures to its enzymatically active form.46 7B2 cleavage and dissociation of its aminoterminal fragment from proPC2 precedes, and is thus not intimately linked with, proPC2 maturation.81 The 7B2 protein specifically associates with PC2 and not with the related endoproteases PC1/PC3, PC5, furin and PACE4 (paired basic amino acid residue cleaving enzyme 4).46,85

7B2 as a Naturally Occurring Inhibitor of PC2 The association between 7B2 and PC2, and in particular, the effect of recombinant 7B2 on PC2 enzyme activity, has also been studied in the test tube. In two different in vitro enzyme assays, recombinant, intact, but not processed, 7B2 inhibits PC2 enzyme activity (producing half-maximal inhibition of PC2 at nanomolar concentrations) and prevents proPC2 cleavage. The activity of PC1/PC3 is not affected by either form of 7B2, indicating that 7B2 specifically inhibits PC2.47,86,87 The naturally occurring carboxy-terminal peptide of 7B2 (7B2-CT), encompassing the last 31 amino acid residues, is sufficient for this inhibition.86 A potential PC2 cleavage site (Lys171-Lys172) located within this region is essential, but not fully responsible, for the inhibitory potency of 7B2.86,87 Lymnaea 7B2 carries two regions in its carboxy-terminal region which share a substantial degree of sequence similarity with the inhibitory region of vertebrate 7B2-CT. Both domains in Lymnaea 7B2 are able

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to inhibit PC2 activity.45 The liaison between 7B2 and PC2, as well as the inhibitory action of 7B2-CT, is evolutionarily conserved from snails to mammals.45-47,81,88 This implies that the interaction between the two proteins has survived ~600 million years of evolution.

7B2 as a Neuroendocrine Chaperone of PC2 The amino-terminal half of 7B2 is distantly related to the so-called chaperonins, a subclass of molecular chaperones.46 In transfection studies with anterior pituitary-derived AtT20 and rat insulinoma Rin5f cell lines, 21 kDa 7B2 is sufficient for facilitating proPC2 transport and maturation.88 This is consistent with the finding that 21 kDa 7B2 is required for activation of proPC2 in transfected non-neuroendocrine CHO cells.88,89 Immunofluorescence studies showed that in transfected AtT20 cells, the efficient transport of proPC2 out of the ER required 7B2 overexpression (C. A. M. Broers, J. A. M. Braks, R. P. Kuiper, A. M. Van Horssen and G. J. M. Martens; unpublished results). In vitro, recombinant 21 kDa 7B2 possesses chaperone-like activity, since it was able to stimulate PC2-mediated prohormone cleavage.90 These observations suggest that 7B2 is required for the proper transport of proPC2 from the ER to the TGN/immature secretory granules, and for its maturation and activation, and support the notion that 7B2 is a neuroendocrine chaperone for PC2. Within 21 kDa 7B2, a number of regions have been implicated in the ability of 7B2 to act as a helper protein for PC2. A proline-rich region in the middle of 7B2 (Pro88-Pro95) has recently been reported to be essential for proPC2 binding and maturation. The distribution of these prolines is similar to that found in Src homology 3 domain (SH3) ligands. In addition, a probable α-helix (corresponding to residues 109 to 121), carboxy-terminal to the polyproline-stretch, also participates in the association of 7B2 with PC2.91 Moreover, a short segment of twelve amino acids (His120-Pro131) is crucial, and the aromatic residue Tyr130 within this segment is particularly important, for the ability of 7B2 to act as a helper protein for PC2 (A. M. Van Horssen and G. J. M. Martens; unpublished results). One 7B2-binding site within (pro)PC2 has been identified. Site-directed mutagenesis demonstrated that the oxyanion hole residue Asp309 in the catalytic domain of PC2 is required for its association with 7B2.92 That this Asp residue is unique to PC2 (in PC1/PC3, furin and PACE4, the corresponding amino acid is an Asn residue) and essential for 7B2 binding, suggests that Asp309 may account for the specific association of 7B2 with PC2. In general, molecular chaperones are common to all cell types and direct their activities towards a variety of proteins.93-95 In the secretory pathway, chaperones are mostly found in the ER where they are thought to assist in protein translocation, to participate in folding and quality control of newly synthesized proteins and to retain misfolded and unassembled proteins.96 The ER-resident chaperones include the binding protein BiP,97,98 the glucoseregulated protein Grp9499,100 and the related proteins calnexin101-103 and calreticulin.104,105 7B2 differs in various aspects from these ER-resident chaperones. First, 7B2 has a neuroendocrine-specific tissue distribution. Second, 7B2 apparently binds a single physiological target, PC2. Third, 7B2 appears to act in the ER, as well as in later compartments of the regulated secretory pathway. In addition to 7B2, similar so-called private chaperones have been shown to act in the secretory pathway and direct their activities to a single protein or protein family. For instance, the 47 kDa heat shock protein Hsp47 specifically associates with procollagen,106,107 the invariant chain Ii interacts with the major histocompatibility complex MHC class II108 and the receptor associated protein RAP binds to the lipoprotein receptor-related protein LRP, and to other members of the family of lipoprotein endocytotic receptors.109,110 Similar to what holds for the association of 7B2 with (pro)PC2, these private chaperones accompany their specific physiological targets during their intracellular transport and facilitate their in vivo fate.

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Model of the Interaction Between 7B2 and PC2 Based on the findings discussed above, the following model can be put forward for the interaction between 7B2 and PC2 during their transport through the secretory pathway (Fig. 8.2). Soon after synthesis, the association between 7B2 and proPC2 commences in the ER. Following transport of the 7B2/proPC2 complex through the Golgi apparatus, 7B2 is cleaved in the TGN/immature secretory granules. The cleavage destabilizes the complex and the amino-terminal 21 kDa fragment of 7B2 dissociates from proPC2, while 7B2-CT remains associated. After cleavage of associated 7B2-CT and subsequent dissociation, proPC2 matures into its enzymatically active form, which is thought to occur by autocatalytic cleavage.111 Mature PC2 is then able to liberate peptide hormones and neuropeptides from their precursors. The carboxy-terminal region of 7B2 is thought to block premature activation of proPC2 in the early compartments of the secretory pathway. In this way, the liaison between 7B2 and proPC2 provides a safeguard mechanism to prevent proPC2 maturation before it arrives in the organelles where it is supposed to be active. This association may account for the finding that of the two convertases mediating prohormone processing in neuroendocrine cells, PC2 is activated later in the secretory pathway than PC1/PC3.112-116 Consistent with such a role for 7B2-CT in the cell are the following observations. First, 7B2 cleavage and dissociation of its amino-terminal fragment from proPC2 clearly precedes, and is thus not intimately linked with, proPC2 maturation.81 Second, 7B2-CT is a potent inhibitor of PC2 activity in vitro.86 Third, in Xenopus intermediate pituitary, a peptide which comigrates with 7B2-CT in SDS-PAGE gels was found associated with proPC2 (R. P. Kuiper and G. J. M. Martens; unpublished results). Arguing against such an in vivo role is the finding that the kinetics of proPC2 cleavage and PC2 secretion are identical in AtT20 cells transfected with PC2 and either 27 kDa 7B2 or 21 kDa 7B2, even though the latter form lacks inhibitory 7B2-CT.88 Whether 7B2-CT indeed inhibits proPC2 maturation in the cell and delays activation until the proenzyme arrives in the proper compartment remains to be established. Dissociation of the 7B2/proPC2 complex and subsequent proPC2 maturation may be triggered by the proteolytic processing of intact 7B2 and of its inhibitory 7B2-CT. Two observations support the concept that proPC2 activation is indeed associated with these two cleavage steps. When processing of intact 7B2 to its 21 kDa form was blocked by mutation of the pentabasic processing site, the mutated protein was able to bind to proPC2, but remained associated and was not able to facilitate maturation of the proenzyme.88 The cleavage of 7B2-CT at the internal Lys171-Lys172 site, and the subsequent removal of the terminal lysines, greatly diminished its inhibitory potency against PC2.73 This cleavage may thus trigger dissociation of the 7B2-CT/proPC2 complex. As will be discussed below, the link between 7B2 processing and proPC2 maturation is reminiscent of the two cleavage steps required for the activation of furin. Among the endoproteases, PC2 is unique in that it seems to be the only processing enzyme that requires a helper protein for its proper transport and controlled activation. In general, the propeptides of zymogens are thought to act as intramolecular chaperones assisting in the folding of their enzymatically active forms.117,118 For instance, the propeptides of subtilisin and α-lytic protease, which are bacterial proteinases related to the PC family, are required for proper folding of the catalytic domains of these enzymes.118-121 After autocatalytic cleavage, the propeptides remain associated with the catalytic domains and act as potent autoinhibitors.122,123 The propeptide of subtilisin E is degraded, allowing the enzyme to cleave its substrates.120 Consistent with this concept is the finding that the propeptide of furin is essential for proper enzyme activation. Autoproteolytic cleavage of the propeptide is necessary, but not sufficient, for furin activation.124-126 The propeptide remains associated with the enzyme and functions as a potent inhibitor of furin activity. An internal cleavage

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Fig. 8.2. Model of the interaction between 7B2 and PC2 during their transit through the secretory pathway. Soon after synthesis, the liaison between 7B2 and proPC2 commences in the endoplasmic reticulum. After transport of the proPC2/7B2 complex through the Golgi stacks, 7B2 is cleaved (open arrow) in the trans-Golgi network/immature secretory granules, allowing dissociation of the amino-terminal 21 kDa fragment of 7B2 from proPC2, while the carboxy-terminal peptide (7B2-CT) remains associated. After cleavage of 7B2-CT and subsequent dissociation of the 7B2-CT/proPC2 complex, proPC2 matures into its enzymatically active form, which is thought to take place by autocatalytic cleavage. Mature PC2 is then able to liberate peptide hormones from their prohormones. The peptide hormones are stored in secretory granules and released from the cell upon stimulation by an extracellular signal (adapted from Braks132).

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within the propeptide (at a site carrying a P6 and P1 arginine) triggers dissociation of the furin/propeptide complex. Such a multi-step activation pathway has also been suggested for the other members of the PC family. However, some convertases may be activated by an alternative pathway. For instance, the PC4 and PC7 propeptides lack an internal cleavage site marked by basic amino acids.127 In addition, proPC2 requires 7B2 for its proper maturation and activation, suggesting that the propeptide of PC2 is not sufficient. Hence, the PC2 activation pathway differs from that of furin. From these observations, the hypothesis emerges that during evolution the propeptide of PC2 may not have been able to act as an intramolecular chaperone and a potent autoinhibitor. As a consequence, PC2 needed an additional chaperone-like protein for its proper transport and functioning, a role filled by the 7B2 protein. Before 7B2 started to fulfill this task, it might have been a precursor protein for biologically active peptides which was cleaved by PC2. During evolution, the 7B2 protein may have developed from a physiological substrate to a potent inhibitor of PC2. In general, proteinase inhibitors are thought to be optimal substrates for “their” proteinases.128 The question remains why inhibitors such as 7B2 are inhibitors and not just excellent substrates.

Implications and Future Prospects Now that the role of 7B2 appears to be established, the possibility arises that the other granins also exert a function inside the cell. Previous studies suggest that at least some granins (CgA and CgB) indeed act intracellularly as helper proteins in prohormone sorting and processing.8,10 In this connection it should be noted, however, that the concept of the granin family and which proteins belong to this family is still a matter of debate. Since the proposed granin family members share no structural relationship (except for CgA and CgB),1 the granins should perhaps be considered as a group, whose members have certain biochemical properties and a broad neuroendocrine tissue distribution in common, rather than as members of a protein family. Based on their characteristics, the neuroendocrine polypeptides VGF129 and NESP55 (neuroendocrine secretory protein of 55 kDa)130 may also belong to this group. Concerning the possibility that the granins act inside, rather than outside, the cell and may associate with other proteins in the secretory pathway, the use of the yeast two-hybrid system may be considered to characterize such granin-associated proteins. However, when fused with a DNA-binding domain, the granin family members SgII, SgIII and 7B2,131 as well as mature PC2, are themselves able to transcriptionally activate the reporter genes in this system (A. M. Van Horssen and G. J. M. Martens; unpublished results), probably due to the highly acidic nature of these regulated secretory proteins. Hence, the yeast two-hybrid system should be used with caution in the search for granin-associated proteins. To gain further insight into the association between 7B2 and PC2, a number of studies are worth future consideration. The generation of a 7B2 knockout mouse will contribute to a better understanding of the role of 7B2 in the living animal. A comparison of the phenotype of such a mouse with that of a PC2 knockout mouse would be of particular interest. Elucidation of the crystal structure of the 7B2 protein and the 7B2/(pro)PC2 complex in combination with mutational analysis of the two proteins will provide further insight into their liaison, and the essential domains and amino acid residues involved in this interaction. Moreover, such studies may reveal why 7B2 selectively binds to (pro)PC2, and not to the other, related proprotein convertases. The identification of 7B2 as a private chaperone for PC2 raises the question of whether other proprotein convertases also require selective helper proteins. So far, no such proteins have been identified. Furthermore, there is no evidence that 7B2-related proteins exist. Whether 7B2 actually prevents proPC2 aggregation and/or assists in the proper folding of proPC2 is not clear, and this point needs further attention.

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The possible involvement of 7B2 in neuroendocrine disorders such as the Prader-Willi and the Angelman syndrome is of interest. In general, 7B2 may represent a target for therapeutic regulation of PC2 enzyme activity in neuroendocrine disorders associated with defects in PC2-mediated processing of a variety of precursors for peptide hormones and neuropeptides, including insulin and opioids. Moreover, based on its role as a chaperone-like protein for PC2, 7B2 may be a useful tool for the efficient production of biologically active peptide hormones and neuropeptides in biotechnological systems. In conclusion, the identification of the 7B2 protein as a neuroendocrine chaperone and inhibitor of the prohormone convertase PC2 has led to a better understanding of the biosynthesis of neuropeptides in the brain and peptide hormones in a variety of endocrine glands.

Acknowledgments We are grateful to DW Eib, RP Kuiper and FJM Van Kuppeveld for critically reading the manuscript.

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79. Blaschko H, Comline RS, Schneider FH et al. Secretion of a chromograffin granule protein, chromogranin, from the adrenal gland by splanchnic nerve stimulation. Nature 1967; 215: 58-59. 80. Senatorov VV, Yang CR, Marcinkiewicz M et al. Depolarizing action of secretory granule protein 7B2 on rat supraoptic neurosecretory neurons. J Neuroendocrinol 1993; 5:533-536. 81. Braks JAM, Van Horssen AM, Martens GJM. Dissociation of the complex between the neuroendocrine chaperone 7B2 and prohormone convertase PC2 is not associated with proPC2 maturation. Eur J Biochem 1996; 238:505-510. 82. Barr PJ. Mammalian subtilisins: The long-sought dibasic processing endoproteases. Cell 1991; 66:1-3. 83. Seidah NG, Chrétien M. Proprotein and prohormone convertases of the subtilisin family. Recent developments and future perspectives. Trends Endocrinol Metabol 1992; 3:133-140. 84. Steiner DF, Smeekens SP, Ohagi S et al. The new enzymology of precursor processing endoproteases. J Biol Chem 1992; 267:23435-23438. 85. Benjannet S, Savaria D, Chrétien M et al. 7B2 is a specific intracellular binding protein of the prohormone convertase PC2. J Neurochem 1995; 64:2303-2311. 86. Lindberg I, Van den Hurk WH, Bui C et al. Enzymatic characterization of immunopurified prohormone convertase 2: Potent inhibition by a 7B2 peptide fragment. Biochemistry 1995; 34:5486-5493. 87. Van Horssen AM, Van den Hurk WH, Bailyes EM et al. Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2. J Biol Chem 1995; 270:14292-14296. 88. Zhu X, Lindberg I. 7B2 facilitates the maturation of proPC2 in neuroendocrine cells and is required for the expression of enzymatic activity. J Cell Biol 1995; 129:1641-1650. 89. Lamango NS, Zhu X, Lindberg I. Purification and enzymatic characterization of recombinant prohormone convertase 2: Stabilization of activity by 21 kDa 7B2. Arch Biochem Biophys 1996; 330:238-250. 90. Braks JAM, Martens GJM. The neuroendocrine chaperone 7B2 can enhance in vitro POMC cleavage by prohormone convertase PC2. FEBS Lett 1995; 371:154-158. 91. Zhu X, Lamango NS, Lindberg I. Involvement of a polyproline helix-like structure in the interaction of 7B2 with prohormone convertase 2. J Biol Chem 1996; 271:23582-23587. 92. Benjannet S, Lusson J, Hamelin J et al. Structure-function studies on the biosynthesis and bioactivity of the precursor convertase PC2 and the formation of the PC2/7B2 complex. FEBS Lett 1995; 362:151-155. 93. Ellis RJ, Van der Vies SM. Molecular chaperones. Annu Rev Biochem 1991; 60:321-347. 94. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992. 355:33-45. 95. Hendrick JP, Hartl F-U. Molecular chaperone functions of heat shock proteins. Annu Rev Biochem 1993; 62:349-384. 96. Herbert DN, Simons JF, Peterson JR et al. Calnexin, calreticulin, and BiP/Kar2p in protein folding. Cold Spring Harb Symp Quant Biol 1995; 60:405-415. 97. Haas IG, Wabl M. Immunoglobulin heavy chain binding protein. Nature 1983; 306:387-389 98. Munro S, Pelham HRB. An HSP70-like protein in the ER: Identity with the 78 kD glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell 1986; 46: 291-300. 99. Koch G, Smith M, Macer D et al. Endoplasmic reticulum contains a common, abundant calcium-binding glycoprotein, endoplasmin. J Cell Sci 1986; 86:217-232. 100. Lee AS. Coordinated regulation of a set of genes by glucose and calcium ionophores in mammalian cells. Trends Biochem Sci 1987; 12:20-23. 101. Degen E, Williams DB. Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibility molecule. J Cell Biol 1991; 112:1099-1115. 102. Wada I, Rindress D, Cameron PH et al. SSRα and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J Biol Chem 1991; 266: 9599-19610.

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103. Höchstenbach F, David V, Watkins S et al. Endoplasmic reticulum resident protein of 90 kilodalton associates with T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly. Proc Natl Acad Sci USA 1992; 89:4734-4738. 104. Fliegel L, Burns K, MacLennan DH et al. Molecular cloning of the high affinity calciumbinding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 1989; 264:21522-21528. 105. Smith MJ, Koch GLE. Multiple zones in the sequence of calreticulin (CRP55, calregulin, HACBP), a major calcium binding ER/SR protein. EMBO J 1989; 8:3581-3586. 106. Nakai A, Satoh M, Hirayoshi K et al. Involvement of the stress protein HSP47 in procollagen processing in the endoplasmic reticulum. J Cell Biol 1992; 117:903-914. 107. Sauk JJ, Smith T, Norris K et al. Hsp47 and the translation-translocation machinery cooperate in the production of α 1(I) chains of type I procollagen. J Biol Chem 1994; 269:3941-3946. 108. Neefjes JJ, Ploegh HL. Intracellular transport of MHC class II molecules. Immunol Today 1992; 13:179-183. 109. Willnow TE, Rohlmann A, Horton J et al. RAP, a specialized chaperone, prevents ligandinduced ER retention and degradation of LDL receptor-related endocytic receptors. EMBO J 1996; 15:2632-2639. 110. Bu G, Remke S. Receptor-associated protein is a folding chaperone for low density lipoprotein receptor-related protein. J Biol Chem 1996; 271:22218-22224. 111. Matthews G, Shennan K, Seal AJ et al. Autocatalytic maturation of the prohormone convertase PC2. J Biol Chem 1994; 269:588-592. 112. Benjannet S, Rondeau N, Paquet L et al. Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: Glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2. Biochem J 1993; 294:735-743. 113. Shen F-S, Seidah NG, Lindberg I. Biosynthesis of the prohormone convertase PC2 in Chinese Hamster Ovary cells and in rat insulinoma cells. J Biol Chem 1993; 268:24910-24915. 114. Milgram SL, Mains RE. Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins. J Cell Sci 1994; 107:737-745. 115. Zhou A, Mains RE. Endoproteolytic processing of proopiomelanocortin and prohormone convertases 1 and 2 in neuroendocrine cells overexpressing prohormone convertases 1 or 2. J Biol Chem 1994; 269:17440-17447. 116. Lindberg I. Evidence for cleavage of the PC1/PC3 pro-segment in the endoplasmic reticulum. Mol Cell Neurosc 1994; 5:263-268. 117. Shinde U, Inouye M. The structural and functional organization of intramolecular chaperones: The N-terminal propeptides which mediate protein folding. J Biochem 1994; 115:629-636. 118. Zhu X, Ohta Y, Jordan F et al. Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature 1989; 339:483-485. 119. Power SD, Adams RM, Wells JA. Secretion and autoproteolytic maturation of subtilisin. Proc Natl Acad Sci USA 1986; 83:3096-3100. 120. Ikemura H, Inouye M. In vitro processing of pro-subtilisin produced in Escherichia coli. J Biol Chem 1988; 263:12959-12963. 121. Silen JL, Frank D, Fujishige A et al. Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J Bacteriol 1989; 171:1320-1325. 122. Baker D, Sohl J, Agard DA. A protein folding reaction under kinetic control. Nature 1992; 356:263-265. 123. Li Y, Hu Z, Jordan F et al. Functional analysis of the propeptide of subtilisin E as an intramolecular chaperone for protein folding. Refolding and inhibitory abilities of propeptide mutants. J Biol Chem 1995; 270:25127:25132. 124. Molloy SS, Thomas L, VanSlyke JK. Intracellular trafficking and activation of the furin proprotein convertase: Localization to the TGN and recycling from the cell surface. EMBO J 1994; 13:18-33.

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125. Vey M, Schäfer W, Berghöfer S et al. Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal, substrate cleavage, and COOH-terminal truncation. J Cell Biol 1994; 127:1829-1842. 126. Creemers JWM, Vey M, Schäfer W et al. Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of furin out of the endoplasmic reticulum. J Biol Chem 1995; 270:2695-2702. 127. Anderson ED, VanSlyke JK, Thulin CD et al. Activation of the furin endoprotease is a multi-step process: Requirements for acidification and internal propeptide cleavage. EMBO J 1997; 16:1508-1518. 128. Laskowski Jr M, Kato I. Protein inhibitors of proteinases. Annu Rev Biochem 1980; 49:593-626. 129. Ferri G-L, Possenti R. VGF a neurotrophin-inducible gene expressed in neuroendocrine tissues. Trends Endocrinol Metab 1996; 7:8-13. 130. Ischia R, Lovisetti-Scamihorn P, Hoque-Angeletti R et al. Molecular cloning and characterization of NESP55, a novel chromogranin-like precursor of a peptide with 5-HT1B receptor antagonist activity. J Biol Chem 1997; 272:11657-11662. 131. Chaudhuri B, Huijbregts RPH, Coen J et al. The neuroendocrine protein 7B2 contains unusually potent transcriptional activating sequences. Biochem Biophys Res Comm 1995; 216:1-10. 132. Braks JAM. The Role of the Neuroendocrine Polypeptide 7B2: A Molecular Chaperone for Prohormone Convertase PC2 in the Secretory Pathway. PhD Thesis, University of Nijmegen, 1995:134.

CHAPTER 9

Neuroendocrine α1-Antichymotrypsin as a Possible Regulator of Prohormone and Neuropeptide Precursor Processing Shin-Rong Hwang and Vivian Y.H. Hook

Introduction

P

eptide hormones and peptide neurotransmitters are initially synthesized as inactive precursors that require proteolytic processing to generate active neuropeptides.1-3 The processing proteases are critical for the specific production of bioactive peptides. Prohormone processing is known to be regulated and is controlled by endogenous cellular mechanisms. It is known that the extent of prohormone processing may vary in a tissue-specific manner. For example, proenkephalin in adrenal medulla is incompletely processed,4 but is much more completely processed in brain.5 Differential processing of prodynorphin occurs in anterior and posterior pituitary,6 as well as in brain.7 In addition, processing of the neuroendocrine-specific chromogranin peptides varies extensively in adrenal medulla compared to brain and other regions.8,9 These observations suggest a role for endogenous protease inhibitors that may regulate proteolytic steps of the prohormone processing pathway. Proteases required for key biological functions are nearly always regulated by endogenous protease inhibitors.10-12 For this reason, identification of cellular protease inhibitors that regulate proneuropeptide processing is essential for elucidating possible control mechanisms for the production of potent peptide hormones and neurotransmitters. Studies from numerous laboratories indicate that proteases of different mechanistic classes are involved in prohormone processing. These processing proteases include the subtilisin-like prohormone convertases PC1/3 and PC2,13-15 the novel cysteine protease known as ‘prohormone thiol protease’ (PTP),3,16,17 and a 70 kDa aspartyl protease18-21 (also known as ‘POMC converting enzyme’ or PCE).18-20 Additional candidate processing enzyme activities have also been identified.22-24 These neuroendocrine-specific processing proteases function primarily in the regulated secretory pathway. These processing proteases of different mechanistic classes—serine, cysteine, and aspartyl—cleave prohormones and proneuropeptides at paired basic residue processing sites. Processing of some prohormones, such as prodynorphin, also occurs at monobasic sites.22,25 The participation of multiple proteases in the processing pathway leads to the prediction that each protease could potentially be regulated independently. Given their differences in mechanistic protease classes, it is possible Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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that endogenous protease inhibitors with specificity for certain proteases may participate in regulating prohormone processing. Recent studies have provided evidence for α 1antichymotrypsin,26-29 7B2 (see chapter 8),30-33 and propeptide regions of the subtilisin-like prohormone convertases34 as regulators of processing enzymes. In this chapter, we summarize the biochemical and molecular evidence for a role of the protease inhibitor α1antichymotrypsin (ACT), a member of the serpin (serine protease inhibitor) family, as an endogenous regulator of prohormone and proneuropeptide processing. Protease inhibitors that regulate prohormone processing enzymes in vivo should satisfy several criteria. Firstly, the inhibitor should be colocalized with prohormones and processing proteases in neuroendocrine tissues. Prohormones and processing proteases are colocalized in the regulated secretory vesicle pathway, primarily in the maturing secretory vesicle. Some of the processing events occur in the trans-Golgi. It is, therefore, predicted that endogenous protease inhibitors of prohormone processing should be colocalized with processing proteases within secretory vesicles, or in the regulated secretory pathway. The second criterion is that the protease inhibitor and target protease should interact in a manner that prevents substrate binding and proteolysis. Alternatively, inhibitor and protease interactions may distort the active site to indirectly reduce proteolytic activity. Thirdly, removal or addition of protease inhibitor(s) in cells, through expression of sense and antisense constructs of the inhibitor, should reduce or enhance prohormone processing, respectively. Fourth, since the processing enzyme and cognate protease inhibitor have opposing actions for production of biologically active neuropeptides, it is likely that the protease and inhibitor may be differentially regulated by distinct factors or elements. These criteria will require biochemical, molecular, and cellular studies to define the biological functions and molecular mechanisms of endogenous inhibitors as regulators of prohormone processing.

Biochemical Evidence for α1-Antichymotrypsin (ACT) as an Endogenous Regulator of the ‘Prohormone Thiol Protease’ (PTP) and Other Prohormone Processing Proteases ACT in Brain and Plasma Alpha1-antichymotrypsin (ACT) is a member of the mammalian serpins (serine protease inhibitors), a family of protease inhibitors with varying degrees of homology.11,35,36 Other serpins include α1-antitrypsin, antithrombin, and others.35-38 Serpins typically form SDS-stable complexes with their target proteases that involve interactions (covalent or tetrahedral intermediates) between the P1 residue of the reactive site loop (RSL) domain of the serpin (Fig. 9.1) and active site residues of target proteases. The nomenclature for amino acids at the protease cleavage site, termed P1-P1', indicates residues on the N-terminal side of the cleaved peptide bond as residues P1, P2, P3, and so forth. Residues on the C-terminal side of the cleaved peptide bond are referred to as P1', P2', and so forth. Amino acid residues within the reactive site loop (RSL) of the serpin that are distal to either P1 or P1' residues (such as P11 to P20 or P11' to P20'), are also important for serpin/protease complex formation.36,39 ACT/protease complexes, as well as serpin/protease complexes in general, are often stable under SDS (sodium dodecyl sulfate) denaturing conditions;36,40 thus, these complexes can be detected by differences in electrophoretic mobilities compared to serpin or protease alone. In the plasma of mammalian systems, ACT is an acute phase glycoprotein that is secreted into the plasma from the liver in response to inflammation and other conditions.41 However, the target protease inhibited by ACT in the plasma is unknown. ACT is known as an effective inhibitor of chymotrypsin, or chymotrypsin-like proteases.11,36,40 Presumably, chymotrypsin-like proteases may be inhibited by ACT in the periphery.

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Fig. 9.1. Characteristics of α1-antichymotrypsin and serpins. ACT and serpins possess a characteristic reactive site loop (RSL) within the COOH-terminal regions of these proteinase inhibitors. The RSL contains P1-P1' residues which often mimic the natural cleavage specificity of the target protease. The serpin functions as an inhibitor by recognition of its reactive site by the target protease. The serpin also serves as a pseudosubstrate since the target protease cleaves (slowly) the inhibitor at the reactive site P1-P1' residues.

In the central nervous system, ACT is present in many brain regions,42 including regions abundant in enkephalin and other neuropeptides.43,44 Importantly, ACT is a significant component of brain amyloid plaques in Alzheimer’s Disease.45-47 Moreover, neuronal ACT is developmentally regulated.42 However, endogenous target protease(s) regulated by ACT in brain have not been examined. The neuroendocrine tissue distribution of ACT is consistent with processing of neuropeptide precursors in neuronal and endocrine regions.26 We have, therefore, characterized ACT as a candidate protease inhibitor of the novel cysteine protease ‘prohormone thiol protease’ (PTP) that is involved in proenkephalin processing.3,16,17 Preliminary results also indicate that ACT inhibits the subtilisin-like PC1/3 and PC2 proteases.27

Secretory Vesicle ACT-Like Protein Inhibits the ‘Prohormone Thiol Protease’ (PTP) Prohormones and proteolytic processing enzymes are colocalized in secretory vesicles, where the majority of prohormone processing occurs. In adrenal medulla, the secretory vesicles (also known as chromaffin granules) contain high levels of enkephalin opioid peptides,48 as well as the neuropeptides galanin,49 somatostatin,48 neuropeptide Y,50 vasoactive intestinal polypeptide,51 and others. Chromaffin granules, therefore, contain relevant processing enzyme(s) for proenkephalin and other neuropeptide precursors. Studies of proenkephalin processing in chromaffin granules show that the novel cysteine protease ‘prohormone thiol protease’ (PTP) represents the major processing enzyme activity for production of (Met)enkephalin in chromaffin granules.3,16,17 Similar to proenkephalin processing in vivo,53,54 PTP in vitro converts recombinant enkephalin precursor to multiple 15-22 kDa intermediates that contain the NH2-terminal segment of the precursor.16,55 PTP produces the peptide product (Met)enkephalin by cleaving at dibasic, as well as at monobasic, arginine sites.16,55-57 Lesser proteolytic activities in chromaffin granules that are involved in processing proenkephalin are represented by the subtilisin-like PC1/3 and PC2 proteases,27 and a 70 kDa aspartyl protease21,58 that resembles the pituitary ‘POMC converting enzyme’ (PCE).18-20

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Limited proteolytic processing of proenkephalin in adrenal medulla is indicated by the observation that less that 10% of (Met)enkephalin in this tissue is present as free (Met)enkephalin.4 The majority of (Met)enkephalin in this tissue is present as high molecular weight intermediates derived from proenkephalin. The limited processing suggests that endogenous protease inhibitor(s) may regulate proenkephalin processing enzymes. Since the cysteine protease PTP is the major proenkephalin cleaving activity, it was predicted that endogenous protease inhibitors exist for the PTP processing enzyme. Colocalization of ACT immunoreactivity (58-60 kDa) with PTP in chromaffin granules is consistent with participation of ACT as an inhibitor of a secretory vesicle processing enzyme.26 ACT immunoreactivity is also present in isolated bovine pituitary secretory vesicles. ACT (human liver) was effective in inhibiting PTP. It was important, however, to characterize the endogenous ACT-like protein to confirm that it, indeed, represents a potent inhibitor of PTP. Immunoblots indicated higher levels of the ACT-like protein in pituitary; therefore, purification and characterization of the pituitary ACT-like protein was achieved.26 The bovine pituitary ACT-like protein was purified by ion exchange on DEAESepharose, chromatofocusing, butyl-Sepharose, and Sephacryl S-200.26 PTP was potently inhibited by the purified 60 kDa bovine pituitary ACT-like protein and human liver ACT with Ki(app) values of 2.2 and 8.4 nM, respectively. PTP is, indeed, potently inhibited by ACT. Furthermore, the purified ACT-like protein resembles authentic ACT, as demonstrated by potent inhibition of chymotrypsin (Ki(app) of 2.3 nM). The purified pituitary ACT-like protein appears to be a genuine protease inhibitor related to ACT. ACT and other inhibitors of the serpin family typically form SDS-stable complexes with their target protease(s) at a 1:1 molar ratio of inhibitor to protease.11,36,40 The bovine pituitary ACT-like protein forms SDS-stable complexes with chymotrypsin, thus demonstrating that the pituitary ACT-like protein resembles ACT.26 A significant finding was that PTP forms SDS-stable complexes with ACT (human liver) (Fig. 9.2), which can be detected at inhibitor:enzyme molar ratios of 1:1, 2:1, and 4:1.26 The formation of ACT/PTP complexes indicates that PTP possesses the property of a target protease inhibited by a serpin. Formation of SDS-stable complexes suggests parallel P1 residue cleavage specificities of PTP and reactive site P1 residues of ACT. PTP cleaves at the NH2-terminal side of paired basic residues, as well as between the two basic residues that flank (Met)enkephalin within the enkephalin-containing peptide substrates peptide F and BAM-22P (Fig. 9.3).55-57 When PTP cleaves at the NH2-terminal side of the dibasic residues, the primary sequence of peptide F and BAM-22P indicates methionine or leucine as the P1 residue (Fig. 9.3). Natural ACT (human liver) that contains leucine as the P1 residue, or mutant ACT with methionine at the P1 position, inhibits chymotrypsin.40 The parallel P1 residue specificities of PTP and ACT for methionine or leucine provide an explanation for their interactions as SDS-stable complexes, which is typical of serpins and their target proteases. Consideration of interactions other than parallel P1 residues are also possible. Serpins typically inhibit serine proteases. However, ACT inhibition of PTP indicates cross-class inhibition of a cysteine protease by a serpin. Interactions between cysteine proteases and serpins have been reported for the interleukin-1β-converting enzyme (ICE) that is inhibited by the serpin encoded by the CrmA gene of the cowpox virus.59,60 Parallel specificities of ICE and CrmA for aspartate as the P1 residue are proposed as a basis for their interactions. Our demonstration of PTP inhibition by ACT,26 and evidence for CrmA inhibition of ICE,59,60 indicate cross-class inhibition of certain cysteine proteases by serpins. Additionally, a squamous cell carcinoma antigen (SCCA) noncompetitively inhibits the cysteine protease cathepsin L; however, SCCA does not inhibit serine proteases.61,62 Thus, serpin inhibition of cysteine proteases appears to be a characteristic of certain serpins.

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Fig. 9.2. SDS-stable ACT:PTP complexes. Formation of SDS-stable ACT:PTP complexes is detected by SDS-PAGE gels. PTP (0.3 pmol) was incubated with ACT (0.6 pmol human liver ACT) to achieve a molar ratio of inhibitor to PTP of 2:1, and complexes were detected by immunoblots with anti-ACT sera. The position of standard ACT (0.6 pmol) is shown by an arrow. The ACT:PTP complex is detected as a 110 kDa band (arrow).

Fig. 9.3. PTP cleavage sites within enkephalin-containing peptides. PTP cleavage sites within peptide F and BAM-22P are indicated by arrows. Peptide F and BAM-22P are intermediate products of proenkephalin processing. (Met)enkephalin (Tyr-Gly-Gly-Phe-Met) pentapeptide sequences are underlined, and basic residues (K, R) are shown as bold letters.

These results suggest PTP as a target protease regulated by ACT in neuroendocrine cells. ACT meets several criteria expected of an endogenous protease inhibitor of prohormone processing. ACT is colocalized with processing proteases within neuroendocrine secretory vesicles. Characterization of the endogenous inhibitor shows that it potently inhibits PTP. Parallel P1 residue specificities of protease and inhibitor are consistent with the observed inhibition of PTP by ACT. Future studies to examine the effects of recombinant ACT, as well as reduction of endogenous ACT through antisense expression, on cellular prohormone processing will be important to define the role of ACT in regulating PTP and prohormone processing.

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Evidence for Serpin Inhibition of the Subtilisin-Like PC1/3 and PC2 Processing Proteases The subtilisin or serine protease nature of the PC1/3 and PC2 processing proteases suggest that these proteases may be inhibited by ACT. Indeed, PC1/3 and PC2 are both inhibited by micromolar concentrations of ACT.27 PC1/3 is also inhibited (by 50%) by α1-antitrypsin (750 µg/ml, or 12 µM), as well as the variant α1-antitrypsin Pittsburgh (250 µg/ml, or 4 µM).63 Wild-type α1-antitrypsin and the mutant α1-antitrypsin Pittsburgh contain Met and Arg residues at the reactive site center, respectively. While the P1 Arg residue of α1-antitrypsin Pittsburgh is consistent with the cleavage specificity of PC1/3 for basic residues, inhibition of PC1/3 by α1-antitrypsin with Met at the reactive site indicates that interactions other than P1 residues are involved in serpin inhibition of PC1/3 or other proteases. Studies of subtilisin Carlsberg (SCARL),64 which is related to the prohormone convertases, indicate that SCARL is inhibited by the serpin α1-antiproteinase (same as α1-antitrypsin) but not by α1-antichymotrypsin. Thus, the mammalian PC enzymes and SCARL resemble one another with respect to inhibition by certain serpins. However, the mammalian PC enzymes differ from SCARL with respect to inhibition by ACT. It will be important in future studies to obtain biological and molecular characterization of endogenous serpins for possible regulation of PC enzymes in vivo.

Molecular Cloning Reveals Multiple Isoforms of Bovine ACT Expressed in Neuroendocrine Tissues Isoforms of Bovine ACTs Our studies showing different inhibitory potencies of the bovine pituitary ACT-like protein and human plasma ACT against the ‘prohormone thiol protease’ (PTP) processing enzyme suggests that the bovine pituitary form of ACT may not be identical to human liver ACT.26 Furthermore, the anti-human liver ACT antiserum is less reactive towards the bovine pituitary ACT-like protein, since anti-ACT immunoblots detect 1-5 ng human liver ACT, but only 50-100 ng bovine pituitary ACT-like protein is detected by the same antiACT antibody (Hook et al, unpublished observations). These results suggest species differences in ACT, or neuroendocrine specific isoforms of ACT. To distinguish between these possibilities, molecular cloning of bovine liver and neuroendocrine (adrenal medulla and pituitary) ACT cDNAs28,29 was conducted. Importantly, results revealed the presence of neuroendocrine-specific isoforms of ACT in bovine adrenal medulla and pituitary. Screening of a bovine liver cDNA library with the human ACT cDNA as probe resulted in the isolation of two clones encoding isoforms of bovine liver ACT-like proteins.28 The L2 (liver-2) ACT cDNA of 1.5 kb is a nearly full-length clone, and the other L1 (liver-1) ACT cDNA represents a partial 0.9 kb clone. These two forms of bovine liver ACT possess homology to human liver ACT. Both bovine ACT cDNAs encode a serpin reactive site loop domain, yet they also differ in their deduced amino acid sequences. These two forms of ACT share 68% homology in primary sequence, and their Kyte-Doolittle hydropathy plots are virtually identical. The L2 ACT cDNA encodes an NH2-terminal hydrophobic signal sequence, similar to human liver ACT.40,65 The reactive site loop (RSL) domain near the COOH-termini of serpins, however, differs between the L2 and L1 ACT cDNAs. The 1.5 kb L2 ACT cDNA contains a predicted Ser as the predicted P1 residue, while the partial 0.9 kb L1 ACT cDNA contains Arg as the predicted P1 residue. These different predicted P1 residues suggest that the L1 and L2 ACTs may each inhibit a different target protease. These results demonstrate isoforms of bovine liver ACT that vary in predicted reactive site P1 residues.

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Fig. 9.4. Alignment of predicted reactive site domains among bovine ACT isoforms, human ACT, and mouse contrapsin. Alignment of the reactive site domains of the bovine ACT isoforms, human ACT, and mouse contrapsin. The reactive site domains from P1 to P6' are compared for bovine adrenal medulla ACT (b. AM ACT), bovine pituitary ACT (b. Pit ACT), the two liver L1 and L2 forms of ACT (b. L-1 ACT and b. L-2 ACT), bovine trypsin inhibitor (b. TI), bovine elastase inhibitor (b. EI), human liver ACT (h. L ACT), and mouse contrapsin (m. contrapsin). The alignments are illustrated as groups possessing optimum homology. The identical residues within the same group are underlined. The arrow indicates predicted cleavage sites between P1-P1'.

Further studies identified cDNA clones representing endogenous forms of ACT expressed in bovine neuroendocrine tissues.29 Screening of bovine adrenal medulla (AM) and pituitary (Pit) cDNA libraries with the bovine liver L1 ACT cDNA as probe resulted in the isolation of partial ACT-like cDNAs that possess the reactive site domain. The 1.0 kb AM ACT cDNA contains an opening reading frame of 252 amino acids, and the 0.5 kb Pit ACT cDNA contains an open reading frame of 93 residues. The AM, Pit, and L1 ACT isoforms share high degrees of homology with one another of 88-98%. However, these three ACT isoforms share a lower degree of homology with the L2 ACT of 67%, 53%, and 58%, respectively. The reactive site domains of ACT isoforms were compared by aligning the residues corresponding to predicted P6 to P6' positions of the reactive site loop (RSL) domain (Fig. 9.4). These comparisons indicated two subgroups of bovine ACT reactive sites that differ from human ACT and mouse contrapsin. The first group consists of the AM, Pit, and L1 ACTs, and the bovine plasma trypsin inhibitor (TI). These serpins possess a consensus sequence for P6 to P6' residues similar to IGIERTILRII. The second group consists of the L2 ACT and the bovine plasma elastase inhibitor (EI), possessing VVMATXSXLLHT as a proposed consensus sequence. The bovine ACT reactive site sequences clearly differ from human ACT and mouse contrapsin. Notably, the bovine ACT isoforms possess differences in proposed P1 residues (Fig. 9.4). The ACT isoforms from bovine adrenal medulla and pituitary, as well as the L1 ACT from

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liver, possess Arg as the predicted P1 residue, which is consistent with possible inhibition of prohormone processing enzymes cleaving at basic residues. In contrast, the L2 ACT differs from the other forms of bovine ACT, since the L2 ACT contains Ser as the predicted P1 residue. Furthermore, the predicted P1 residues of bovine ACT isoforms differ from ACTlike proteins in other species. Human liver ACT and mouse contrapsin possess Leu and Lys as P1 residues, respectively. Differences in the hydrophilic and hydrophobic nature of the reactive sites of the two groups of bovine ACT-like cDNAs are illustrated by Kyte-Doolittle hydropathy plots (Fig. 9.5). The region at residues P5 to P2' of the bovine L2 ACT and human liver ACT is very hydrophobic. In contrast, the AM, Pit, and L1 ACT isoforms show much less hydrophobicity compared to this same region within the L2 ACT or human liver ACT. The charged residues within the P5 to P2' segment, especially Arg as the predicted P1 residue, of the AM, Pit, and L1 forms of bovine ACT increase the hydrophilicity of the reactive region. These differences in hydrophobic/hydrophilic nature of the reactive sites suggest that these variant forms of ACT bind to their target proteases with different serpin/protease interactions.

Tissue Distribution RT-PCR and Southern blots compared the tissue distribution of the four bovine ACT isoforms known as L1, L2, AM, and Pit forms of ACT.28,29 Results indicated tissue-specific expression of each ACT isoform. Importantly, the AM ACT mRNA is expressed in a neuroendocrine-specific manner, since it is expressed in adrenal medulla and pituitary, but not in liver. The Pit ACT mRNA is expressed in pituitary, as well as liver. The L2 ACT mRNA is more widely distributed in neuroendocrine (adrenal medulla, pituitary, and pancreas) and liver tissues (Hwang and Hook, unpublished observations). Since the AM and Pit ACT isoforms in neuroendocrine tissues predict P1 as Arg, it will be of interest to test whether AM and Pit ACTs inhibit prohormone processing proteases. The bovine L2 ACT28 shares homology with the SCCA (squamous cell carcinoma anti62,63 serpin in the RSL (reactive site loop) region. Both L2 ACT and the SCCA possess gen) Ser-Ser as predicted P1-P1' residues. SCCA, however, does not inhibit any serine proteases tested. However, SCCA potently inhibits cathepsin L, a papain-like cysteine protease, with a Ki of less than 1 nM. Therefore, based on homology to SCCA in the RSL region, bovine L2 ACT may inhibit a cysteine protease. It will be of interest to test whether L2 ACT inhibits the cysteine protease PTP (‘prohormone thiol protease’) that is involved in prohormone processing.

Multiple Bovine ACT Genes Further evidence for heterogeneity in bovine ACTs was indicated by genomic blots. Probing of genomic blots with the bovine liver partial L1 ACT cDNA as probe demonstrated multiple copies of the ACT gene.28 This is consistent with the isolation of two isoforms of bovine liver ACTs that may be encoded by separate genes. In other species, mouse66 and rat67 ACT-related cDNAs (known as contrapsins) are represented by multicopy genes. However, human ACT appears as primarily a single copy gene.37,68 It will be important to compare the organization of the ACT gene in several species.

Conclusions and Future Perspectives The studies described in this chapter indicate emergence of a family of neuroendocrine isoforms of the protease inhibitor α1-antichymotrypsin that may be involved in regulating prohormone processing enzymes. Demonstration of ACT inhibition of the prohormone processing protease PTP (‘prohormone thiol protease’) has been demonstrated.

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Fig. 9.5. Comparison of the KyteDoolittle hydropathy plots of the reactive site domains of bovine isoforms of ACT and serpins. Kyte-Doolittle hydropathy plots for the isoforms of bovine ACT and human liver ACT are illustrated. Panels (a)-(f) represent hydropathy plots of the reactive sites of the: (a) bovine AM ACT; (b) bovine Pit ACT; (c) bovine L1 (liver #1) ACT; (d) bovine L2 (liver #2) ACT; (e) human liver ACT; and (f) human α1-antitrypsin. The predicted P1-P1' residues are indicated by arrows.

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An endogenous bovine pituitary ACT-like protein is a potent inhibitor of PTP. Moreover, molecular cloning reveals unique isoforms of ACT-like proteins—AM, Pit, and L2 forms of ACT—that are expressed in neuroendocrine tissues. Importantly, the predicted Arg P1 residues within the reactive site loop (RSL) domains of the AM and Pit forms of ACT indicate that possible inhibition of prohormone processing enzymes cleaving at basic residues should be tested. Further studies to test whether these ACT isoforms meet the criteria expected of an endogenous protease inhibitor of prohormone processing enzymes will lead to exciting conclusions concerning regulatory mechanisms for the cellular production of neuropeptides. In addition to ACT-like protease inhibitors, other inhibitory mechanisms may be involved in regulating prohormone processing, including the 7B2 protein30-33 (chapter 8) or propeptide regions of zymogen forms of proteases.34 It will, therefore, be important to determine which regulatory mechanisms operate under certain conditions to modify the production of peptide hormones and neurotransmitters.

Acknowledgments. This work was supported by grants from the National Institute of Neurological Disease and Stroke and the National Institute of Drug Abuse of the NIH.

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34. Lazure, C, Boudreault A, Basak A, Gauthier D, Seidah NG, Chretien M. The use of recombinant baculovirus expressed mPC1 and mPC2 and synthetic peptides to probe the role of their respective propeptide. In: Fricker LD, Shields D, Thomas G, eds. On Molecular and Cellular Biology. Silverthorne: Keystone Symposia 1997:50. 35. Marshall CJ. Evolutionary relationships among the serpins. Phil Trans R Soc Lond B 1993; 342:101-119. 36. Gettins PGW, Patston PA, Olson ST. Serpins: Structure, Function and Biology. Austin: RG Landes Co., 1996:1-14. 37. Bao J, Sifers RN, Kidd VJ, Ledley FD, Woo SLC. Molecular evolution of serpins: Homologous structure of the human α1-antichymotrypsin and α1-antitrypsin genes. Biochemistry 1987; 26:7755-7759. 38. Jiang H, Kanost MR. Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta. J Biol Chem 1997; 272:1082-1087. 39. He S, Sim RB, Whaley K. A secondary C1s interaction site on C1-inhibitor is essential for formation of a stable enzyme-inhibitor complex. FEBS Lett 1997; 406:42-46. 40. Rubin H, Wang A, Nickarg EB, McLarney S, Naidoo N, Schoenberger OL, Johnson JL, Cooperman BS. Cloning, expression, purification, and biological activity of recombinant native and variant human α1-antichymotrypsins. J Biol Chem 1990; 265:1199-1207. 41. Erikson S, Lindmark B, Lilja H. Familial alpha1-antichymotrypsin deficiency. Acta Med Scandia 1986; 220:447-453. 42. Justice DL, Rhodes RH, Tokes ZA J. Immunohistochemical demonstration of proteinase inhibitor alpha-1-antichymotrypsin in normal human central nervous system. Cell Biochem 1987; 34:227-238. 43. Williams RG, Dockray GJ. Distribution of enkephalin-related peptides in rat brain: Immunohistochemical studies using antisera to met-enkephalin and met-enkephalin-Arg6Phe7. Neurosci 1983; 9:563-586. 44. Merchenthaler I, Maderdrut JL, Altschuler A, Petrusz P. Immunocytochemical localization of proenkephalin-derived peptides in the central nervous system of the rat. Neurosci 1986; 17:325-348. 45. Abraham CR, Selkoe DJ, Potter H. Immunochemical identification of the serine protease inhibitor α1-antichymotrypsin in the brain amyloid deposits of Alzheimer’s Disease. Cell 1988; 52:487-501. 46. Abraham CR, Shirahama T, Potter H. Alpha1-antichymotrypsin is associated solely with amyloid deposits containing the beta-protein. Amyloid and cell localization of alpha1antichymotrypsin. Neurobiol. Aging 1990; 11:123-129. 47. Rozemuller JM, Abbink JJ, Kamp AM, Stam FC, Hack CE, Eikelenboom P. Distribution pattern and functional state of alpha1-antichymotrypsin in plaques and vascular amyloid in Alzheimer’s disease. A immunohistochemical study with monoclonal antibodies against native and inactivated alpha1-antichymotrypsin. Acta Neuropathol 1991; 82:200-207. 48. Lundberg JM, Hamberger B, Schultzberg M, Hokfelt T, Granberg PO, Efendie S, Terenis L, Goldstein M, Luft R. Enkephalin- and somatostatin-like immunoreactivities in human adrenal medulla and pheochromocytoma. Proc Natl Acad Sci USA 1979; 76:4079-4083. 49. Rokaeus A, Brownstein MG. Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor. Proc Natl Acad Sci USA 1986; 83:6287-6291. 50. Carmichael WS, Stoddard SL, O’Connor DT, Yaksh TL, Tyce GM. The secretion of catecholamines, chromogranin A and neuropeptide Y from the adrenal medulla of the cat via the adrenolumbar vein and thoracic duct: Different anatomic routes based on size. Neuroscience 1990; 34:433-440. 51. Holzward MA. The distribution of vasocative intestinal peptide in the rat adrenal cortex and medulla. J Auton Nerv Syst 1984; 11:269-283. 52. Hook VYH, Schiller MR, Azaryan AV, Tezapsidis N. Proenkephalin-processing enzyme in chromaffin granules, a model for neuropeptide biosynthesis. Ann NY Acad Sci 1996; 780:121-133.

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53. Spruce BA, Jackson S, Lowry PJ, Lande DP, Glover D. Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. J Biol Chem 1988; 263:19788-19795. 54. Birch NP, Christie DL. Characterization of the molecular forms of proenkephalin in bovine adrenal medulla and rat adrenal, brain, and spinal cord with a site-directed antiserum. J Biol Chem 1986; 261:12213-12221. 55. Schiller MR, Mende-Mueller L, Miller KW, Hook VYH. ‘Prohormone thiol protease’ (PTP) processing of recombinant proenkephalin. Biochemistry 1995; 34:7988-7995. 56. Krieger TK, Mende-Mueller L, Hook VYH. Prohormone thiol protease and enkephalin precursor processing; Cleavage at dibasic and monobasic sites. J Neurochem 1992; 59:26-31. 57. Azaryan AV, Hook VYH. Unique cleavage site specificity of ‘prohormone thiol protease’ related to proenkephalin processing. FEBS Lett 1994; 341:197-202. 58. Azaryan AV, Schiller MR, Hook VYH. Chromaffin granule aspartic proteinase processes recombinant proopiomelanocortin (POMC). Biochem Biophys Res Comm 1995; 215:937-944. 59. Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salvesen GS, Pickup DJ. Viral inhibition of inflammation: Cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 1992; 69:597-604. 60. Komiyama T, Ray CA, Pickup DJ, Howard AD, Thornberry NA, Peterson EP, Salvesen G. Inhibition of interleukin-1β converting enzyme by the cowpox virus serpin CrmA. J Biol Chem 1994; 269:19331-19337. 61. Suminami Y, Kishi F, Sekiguchi K, Kato H. Squamous cell carcinoma antigen is a new member of the serine protease inhibitors. Biochem Biophys Res Commun 1991; 27:51-58. 62. Takeda A, Yamamoto T, Nakamura Y, Takahasi T, Hibino T. Squamous cell carcinoma antigen is a potent inhibitor of cysteine proteinase cathepsin L. FEBS Lett 1995; 359:78-80. 63. Rufaut NW, Brennan SO, Hakes DJ, Dixon JE, Birch NP. Purification and characterization of the candidate prohormone processing enzyme SPC3 produced in a mouse L cell line. J Biol Chem 1993; 268:20291-20298. 64. Komiyama T, Gron H, Pemberton PA, Salvesen GS. Interaction of subtilisins with serpins. Protein Sci 1996; 5:874-882. 65. Chandra T, Stackhouse R, Kidd VJ, Robson KJ, Woo SL. Sequence homology between human alpha1-antichymotrypsin, alpha1-antitrypsin, and antithrombin III. Biochemistry 1983; 22:5055-61. 66. Hill RE, Shaw PH, Boyd PA, Baumann H, Hastie ND. Plasma protease inhibitors in mouse and man: Divergence within the reactive centre regions. Nature 1984; 311:175-177. 67. Ogata OK, Ogata S, Misumi Y, Takami N, Ikehara Y. Molecular cloning and characterization of rat contrapsin-like protease inhibitor and related proteins. J Biochem 1991; 109:243-250. 68. Hwang SR, Steineckert BD, Kohn A, Palkovits M, Hook VYH. Molicular studies define the primary structure of α1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains: Comparison of ACT mRNA's in hippocampus and liver. J Biol Chem 1998; submitted.

CHAPTER 10

Proteolytic Inactivation of Secreted Neuropeptides Eva Csuhai, Afshin Safavi, Michael W. Thompson and Louis B. Hersh

Introduction

N

europeptides can function as neurotransmitters or neuromodulators by binding to specific receptors and activating intracellular signaling pathways. It has generally been accepted that the actions of neuropeptides can be terminated by two general mechanisms: 1. internalization of the peptide-receptor complex followed by intracellular degradation in lysosomal or endosomal compartments or alternatively in the cytosol; or 2. extracellular degradation of the free peptide by ectopeptidases which reduce the concentration of peptide available for receptor binding. Until recently it was believed that neuropeptide action was terminated by either one or the other of these mechanisms, but not both. However, there is an increasing body of evidence for the internalization of at least some neuropeptides into endosomes and subsequently into other intracellular compartments such as the nucleus, secretory granules, cytosol,1 lysosomes, and the rough endoplasmic reticulum.2 Examples of internalized peptides include insulin,3 somatostatin,4 thyrotropin releasing hormone,1 gastrin releasing peptide,5 and neurotensin.6 This pathway could allow for intracellular degradation of neuropeptides in the cytosol, in lysosomes, or in other intracellular compartments.7 Thus, in at least some cases the action of a given peptide may be terminated by both mechanisms, and both ectoenzymes and intracellular enzymes, localized in the proper compartment, could participate in this process. When we consider degradation of secreted neuropeptides we will discuss peptidases anchored in the plasma membrane as well as intracellular and secreted peptidases. Although it was originally believed that peptidases would be specific for a given neuropeptide, with one notable exception, pyroglutamyl peptidase II, this does not appear to be the case. Most of the peptidases that act on neuropeptides do not exhibit a specificity that is directed at a particular linear amino acid sequence, but rather a specificity directed toward classes of amino acid residues, with contributions to specificity from adjacent residues. Neuropeptidases tend to have a broad and overlapping range of substrates, with two or more different peptidases capable of cleaving the same neuropeptide. Whether this represents nature’s built in redundancy system or simply an artifact of in vitro studies has in most cases yet to be determined. However, as described below with the enkephalins, there is clearly redundancy with at least two peptidases functioning as “enkephalinases” in vivo. Peptidases that act on neuropeptides represent an alternative drug target to receptor agonists or antagonists. Inhibition of these peptidases can result in increased levels of Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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endogenous peptides and a concomitant increased or prolonged physiological effect of the target peptide. As noted, the built in redundancy with some neuropeptides will require the use of multiple inhibitors or inhibitors with dual specificity to achieve this effect. With the current advances in molecular cloning it has become increasingly apparent that the peptidases which act on neuropeptides can be grouped into gene families. These include the family of metalloendopeptidases related to neprilysin, with a gluzincin-type HEXXH active site; the aminopeptidase family of Zn-metallopeptidases, which also contain the gluzincin-type active site; the inverzincin family of metalloendopeptidases, which contain an inverted Zn-binding site, HXXEH; and a family of serine proteases whose Ser/Asp/ His catalytic triad shows a primary sequence arrangement distinct from the trypsin/chymotrypsin and subtilisin family of serine proteases.8 In this review we describe a number of peptidases thought to participate in neuropeptide degradation. The action of many of these peptidases is not limited to neuropeptides; however, studies related to these substrates will be the primary focus. It should be noted that in most cases the evidence that in vitro substrates of a given peptidase are actually in vivo substrates is limited at best.

Neprilysin One of the best studied neuropeptidases is neprilysin (EC 3.4.24.11), also known as “enkephalinase”, neutral endopeptidase (NEP), CALLA, or CD10. Neprilysin is a glycosylated, membrane-bound Zn-metallopeptidase with a molecular weight of 87 to 94 kDa.9 A soluble form from serum has recently been characterized.10 The enzyme is a type II integral membrane protein composed of three domains: a short 27 amino acid cytoplasmic domain, a 22 amino acid membrane spanning domain, and a 699 amino acid extracellular domain which contains the catalytic activity. It has recently been found that the intracellular domain can be phosphorylated on serine and threonine residues by casein kinase II.11 This phosphorylation leads to association of the phosphorylated neprilysin with the ~56 kDa Lyn Src-related kinase and two phosphotyrosine proteins. It is suggested that neprilysin phosphorylation and its complex formation with other phosphoproteins may promote internalization of the enzyme, perhaps as a mode of regulating its activity.11 Although neprilysin is a major enkephalin-degrading enzyme in brain, as is the case with a number of other neuropeptide-degrading enzymes, it is widely distributed in the body. The enzyme is present in spinal cord, intestine, kidney and a number of other tissues.12 In neurons neprilysin is specifically targeted to the axonal and synaptic membranes.13 Like other neuropeptidases, neprilysin is not specific for a particular peptide substrate. In addition to acting on enkephalins, neprilysin is capable of cleaving a variety of substrates with Km values in the µM range, including atrial natriuretic factor, substance P and the neurokinins,14 neurotensin, the endothelins,15 and lysylbradykinin. Thiorphan- or phosphoramidon-sensitive cleavage of somatostatin,16 γ-endorphin, atrial natriuretic peptide,17 CGRP,18 neurotensin and neuromedin N,19 in addition to the enkephalins, has been observed with membrane preparations, brain slices or cultured neuronal cells. Insight into the specificity and catalytic mechanism of neprilysin comes from a number of studies utilizing site-directed mutagenesis to identify and characterize active site residues. Neprilysin preferentially cleaves substrates on the amino side of hydrophobic residues (e.g., Met, Leu, Phe),20 but in addition contains an active site arginine which can direct cleavage to C-terminally oriented sites. This residue is not required for activity and does not participate in catalysis with all substrates.21 One of the unique characteristics of neprilysin is its high sensitivity to inhibition by phosphoramidon, N-(α-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp,22 which places the enzyme in the thermolysin-like family of peptidases. Numerous inhibitors of neprilysin

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have since been developed in the hopes of using them to increase endogenous enkephalin levels. One of the first of these inhibitors was thiorphan, a synthetic peptide thiol, exhibiting a high specificity for neprilysin (Ki = 4 nM).23 More recent inhibitors include SQ 28,603 and RB38B.24 Neprilysin inhibitors were effective in producing a nociceptive response in mice,25 but they were considerably more effective if coadministered with an aminopeptidase inhibitor. This redundancy in “enkephalinases”, in which enkephalins are hydrolyzed by aminopeptidases in addition to neprilysin, clearly established the concept that more than one peptidase may act on a target peptide. This finding led to the introduction of dual inhibitors. Thus the prodrug RB10126 links a potent neprilysin inhibitor with a potent aminopeptidase inhibitor through a disulfide bond which is cleaved in vivo. This inhibitor induces a strong analgesic response and potentiates the anti-depressant effect of opioids. A variant of thiorphan, ES37, has been developed as a dual inhibitor of neprilysin (Ki = 5.2 nM) and angiotensin converting enzyme (Ki = 12 nM).22 ES37 has potential use in treating cardiovascular disorders by inhibiting the angiotensin converting enzyme dependent conversion of angiotensin I to angiotensin II and the neprilysin dependent inactivation of atrial natriuretic factor. Recently a mercaptoacetyl inhibitor possessing a peptidomimetric moiety at its carboxy terminus (BMS-182657) was found to be an effective dual neprilysin/ angiotensin converting enzyme inhibitor in the monkey.27 Specific neprilysin and dual neprilysin/ACE and neprilysin/aminopeptidase N inhibitors are playing increasingly important roles for the treatment of asthma,28 depression,29 hypertension and myocardial ischemia.30 The primary amino acid sequence of neprilysin has been deduced from cDNA clones.31 The enzyme is a member of a newly emerging gene family which includes endothelin converting enzymes 1a,32,33 1b,34 and 2,35 KELL blood group protein,36 and PEX,37 the most recently described member of the family. PEX has been implicated in hypophosphatemic rickets, but its function is unknown. Two bacterial peptidases, PepO and pepOx isolated from Lactococcus lactis38,39,40 were shown to belong to the neprilysin gene family.34,40 The members of this neprilysin gene family display 25-37% overall homology to neprilysin; however, in the region surrounding the active site the homology increases to greater than 50%. Isolation of neprilysin cDNAs led to the finding of multiple mRNA species which differed only in their 5' untranslated sequence. Four distinct neprilysin mRNAs are produced in human: type 1, type 2a, type 2b, and type 3,41,42 while only three are expressed in rat.43 The rat does not express a type 2a mRNA. These mRNAs are generated from three noncoding exons: exon 1, exon 2, and exon 3, with the type 2a and 2b mRNAs being derived from alternative splicing of exon 2. Studies in the rat indicate that all three mRNAs are expressed in peripheral tissues, with the type 2 mRNA being the predominant species. However, in brain the type 1 mRNA appears to predominate and cell specific expression of the three mRNA transcripts have been observed in rat brain.44 Each of the three neprilysin noncoding exons appears to contain its own promoter. The type 2 promoter contains multiple putative SP-1 binding sites,45 while studies on the type 1 promoter have led to the identification of a unique 22 base pair cell specific enhancer sequence (Li, Hersh et al; manuscript in preparation). In an attempt to elucidate the in vivo functions of neprilysin, Gerard et al46 generated a neprilysin deficient mouse by targeted disruption of the neprilysin gene. The heterozygous NEP(+/-) and even the homozygous NEP(-/-) mice appeared developmentally and reproductively normal with no gross phenotypic differences from wild type aside from subtle alterations in lymphoid development and slightly lower turnover rates of certain peptides, for example bradykinin and substance P. The heterozygous animals, however, were 25 times more likely to die from endotoxic shock than their wild type litter-mates; this susceptibility increased to 120-fold over wild type in the homozygous mice. It was concluded that neprilysin

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has an important protective role in septic shock and in the inflammatory response.47 Surprisingly, the transgenic NEP(-/-) mice showed decreased (Leu)enkephalin levels in the hypothalamus and spinal cord, which was attributed to a selective feedback regulation of opioid biosynthesis.48 The homozygous animals display an increased hot plate latency due to an opioid-related increase in the thermonociceptive threshold.49

Aminopeptidases Aminopeptidase N As noted above, aminopeptidases also act as “enkephalinases” in vivo. The physiologically most important “amino-enkephalinase” in brain is aminopeptidase N (ApN) E.C. 3.4.11.2; also known as aminopeptidase M or CD13. This exopeptidase is a surface glycoprotein with 109 kDa subunits, reported to exist in various multimeric forms. It is a type II integral membrane protein anchored to the plasma membrane through an N-terminal domain with the catalytic domain facing the extracellular environment.50 Like neprilysin, ApN is a member of the gluzincin family of metallopeptidases.51 ApN exhibits a broad if not ubiquitous tissue distribution and is the source of the major aminopeptidase activity in serum.52 In brain it is ubiquitously expressed but found in great abundance in the cerebral cortex, nucleus accumbens, striatum, ventral tegmental area and substantia nigra53 and is enriched in the microvessels of the brain.54 The enzyme has been cloned from a variety of sources.55 The human ApN gene is encoded by 20 exons.56 Like neprilysin, aminopeptidase N is not specific for the enkephalins, but cleaves a variety of peptides, including CGRP, angiotensin II, angiotensin III,57 the ORL1 orphan receptor ligand nociceptin/orphanin FQ, 58 somatostatin, 59 dynorphin A 1-1760 and substance P.61 Safavi and Hersh found that as the size of the peptide substrate increases there is a tendency for the hydrolytic rate to decrease, but the affinity for the peptide increases.62 Thus ApN cleaves dynorphin A1-13, dynorphin A1-17, and dynorphin B at rates which are only a fraction of those obtained with enkephalins as substrate. However, the affinity for these dynorphins is considerably higher than for the enkephalins. The net effect is that in terms of catalytic efficiency dynorphins are as good substrates of aminopeptidase N as are enkephalins. Aminopeptidase N is probably the enzyme responsible for the formation of des-tyrosine-dynorphins and des-tyrosine-γ-endorphin,62 peptides which exhibit physiological activities distinct from the parent peptide.63 In this respect aminopeptidase N can be considered a “convertase”. ApN does not hydrolyze peptides such as neurotensin,64 bradykinin or neurokinin analogs which contain a modified N-terminus.65 Furthermore, ApN has a low affinity for peptides containing an N-terminal proline or pyroglutamate.64 There are a number of relatively specific aminopeptidase inhibitors which are active toward ApN. These include amastatin (IC50 = 0.2 µM), bestatin (IC50 = 10 µM) and proctolin (IC50 = 0.1 µM).66 In addition to its role as an enkephalinase in brain, which it shares with neprilysin, ApN is the major enkephalinase in macrophages67 and T cells.68 Infusion of ApN into the paraventricular nucleus of the hypothalamus of rats resulted in a hypotensive response which was attributed to increased angiotensin levels, suggesting aminopeptidase N may play a role in regulating the angiotensin system.69 This enzyme has also been implicated in a number of other cellular roles besides the degradation of enkephalins. ApN is believed to have a role in the control of growth and differentiation of hematopoietic and epithelial cells.52 It may also play a role in tumor cell invasion of basement membranes and in the degradation of the extracellular matrix. A monoclonal antibody to ApN or the administration of amastatin or bestatin prevented invasion of melanoma cells into a reconstituted basement membrane.70 Similarly, a mono-

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clonal antibody to ApN inhibited the invasion of SN12M carcinoma, HT1080 fibrosarcoma, and A375M melanoma cells into reconstituted membranes, but did not have any effect on adhesion or migration to the extracellular matrices.71 It was suggested that the invasion was due to the degradation of entactin/nidogen, a 160 kDa protein component of the basement membrane. This represents the first case in which aminopeptidase N can reportedly cleave a large protein. Differences in glycosylation have been shown to exist between ApN isolated from normal tissue and from corresponding cancerous tissues.72 Whether these glycosylation differences account for the change in substrate specificity from peptides to proteins has yet to be established. Interestingly, ApN acts as a receptor for the coronaviruses, which are a family of viruses that infect the respiratory and enteric epithelial lining. This family includes the human coronavirus 229E (HCV-229E), the porcine transmissible gastroenteritis virus (TGEV), canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV). In order to gain entry into the cell, the virus recognizes and binds to a domain on the C-terminus of ApN that is distinct from the active site. Recognition is species-specific for the host homologue.73

Puromycin-Sensitive Aminopeptidase The puromycin-sensitive aminopeptidase (PSA) was originally identified as the most abundant amino-enkephalinase in brain which hydrolyzed (Met)enkephalin and γ-endorphin.74 This aminopeptidase is distinguished from aminopeptidase N and other aminopeptidases based on its sensitivity to inhibition by puromycin (IC50 = 0.2 µM). PSA is found in virtually all tissues examined, but is most highly enriched in brain.75 It is a 99 kDa member of the gluzincin family.51 PSA is largely cytoplasmic; however ~20% of the enzyme is associated with the membrane fraction.74-76 Recent immunocytochemical data confirm the primarily cytoplasmic localization of PSA, showing some accumulation in a perinuclear region and association with the spindle apparatus during mitosis.77 Sequence analysis of PSA cDNA clones77,78 shows the presence of a microtubule binding motif, as well as a motif that is conserved among subunits of the proteosome. The substrate specificity of PSA has been determined using di- and tri-peptides. The enzyme shows a preference for a basic or hydrophobic residues in the P1 and P1' sites and a cooperative subsite interaction between these sites affects binding.79 PSA hydrolyzes dynorphins A and B,62 α- and β-neo-endorphin,80 α-, β- and γ-endorphin,81 CCK-4 and CCK-8, oxytocin, des-Ac-α-MSH and Arg-vasopressin.82 Whether or not any of these peptides are physiological substrates for the enzyme has yet to be established. Although the localization for the membrane associated form of PSA has not been definitively established, it does not appear to function as an ectoenzyme. Thus its importance in regulating the extracellular levels of the above peptides seems unlikely. However, the enzyme could be involved in the metabolism of internalized peptides. Like aminopeptidase N, PSA is inhibited by bestatin (IC50 = 75 nM), amastatin (IC50 = 80 nM)76 and proctolin (IC50 = 0.4 µM),66 but as noted above, it is uniquely sensitive to inhibition by puromycin. Several studies have shown that aminopeptidase inhibitors suppress cell growth and proliferation.83,84 Constam et al77 recently showed that treatment of cells with bestatin and puromycin, at concentrations which did not inhibit protein synthesis, caused a cell cycle block at the G2/M phase border and subsequent apoptosis. Cycloheximide did not elicit these effects. These effects were attributed to inhibition of PSA, leading to the suggestion that PSA is required for the recycling of amino acids and that this is essential for cell viability.85 Another possibility is that PSA regulates the level of a “checkpoint” peptide which prevents transition from the G2 to M phase. Hydrolysis of the peptide by aminopeptidase N

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signals the onset of mitosis. Thus there is a potential novel role for an aminopeptidase in regulating the cell cycle.

Angiotensin Converting Enzyme Angiotensin converting enzyme (ACE, EC 3.4.15.1, also referred to as kininase II, or dipeptidyl carboxypeptidase) is another Zn-metallopeptidase ectoenzyme. ACE generally cleaves dipeptides from the C-terminus of its substrates,86 although, with C-terminally amidated peptides, tripeptides can be released. ACE is a type I integral membrane protein which is anchored to the plasma membrane through its carboxy terminal domain.87 The enzyme exhibits a broad tissue distribution and occurs in two distinct forms, a pulmonary (150-180 kDa) and a testicular (90-110 kDa) form.88 The two forms are synthesized as the result of alternative splicing: The pulmonary form contains two homologous repeated N-terminal domains, which results in two active sites being present.89 Both of these sites are active in peptide cleavage.90 Peptides with a proline, glutamate or aspartate residue at the C-terminus inhibit ACE-activity.91 ACE was originally characterized as the enzyme cleaving the C-terminal dipeptide of angiotensin I, converting it to angiotensin II, and thus it received considerable attention as a target for anti-hypertensive drugs. Captopril is the first highly selective sulfhydryl ACE inhibitor used for the treatment of hypertension. It was quickly followed by the nonsulfhydryl drugs enalapril and lisinopril, all of which were based on proline-analogs. These types of compounds were strategically combined with features of NEP inhibitors to develop a series of dual inhibitors, containing a hydrophobic moiety, a zinc binding site and strategically placed C-terminal amino acid analogs, directed to both NEP and ACE. These dual inhibitors such as RB10692 and RB10593 display optimal inhibition characteristics in vivo and are favored candidates for the treatment of hypertension and heart failure.94 ACE has a broad substrate specificity and, in addition to angiotensin I, will cleave bradykinin, neurotensin, substance P, luteinizing hormone-releasing hormone (LHRH), dynorphin(1-6), (Leu)enkephalin, (Met)enkephalin and β-neo-endorphin80,86 in vitro. Whether any of these peptides are physiologically relevant substrates has yet to be established. The high Km exhibited by the enkephalins makes it unlikely that they are normal physiological substrates for this enzyme. However, the in vivo cleavage of Ac-seryl-aspartyllysyl-proline (a regulatory factor of hematopoiesis) and bradykinin by ACE has been demonstrated.90,95 Aside from its action on angiotensin I, ACE may well serve as a back-up mechanism for other peptidases which are more active on its proposed substrates.

Pyroglutamyl Peptidase II Pyroglutamyl peptidase II (PP II, EC 3.4.19.6; an enzyme distinct from the cytosolic pyroglutamyl peptidase I) is a Zn-metallopeptidase with a neutral pH optimum. It is an ectoenzyme with an apparent molecular weight of 240 kDa, consisting of two identical subunits with a molecular weight of 97 kDa. The difference in the apparent and actual subunit molecular weights of the enzyme is due to extensive glycosylation of the protein.96 PP II exhibits a high specificity for thyrotropin-releasing hormone (TRH) by cleaving the N-terminal pyroglutamic acid.97 The enzyme does not cleave the pyroglutamyl residue from LHRH. The use of the specific PP II inhibitor N-1-carboxy-2-phenylethyl(Nimbenzyl)histidyl-β-naphthylamide proved the significance of the enzyme in extracellular TRH-degradation in the brain. 98 Thyrotropin-releasing hormone selectively downregulates pyroglutamyl peptidase II activity through TRH receptor activation, although the mechanism of the downregulation is unknown.99 PP II has been detected in brain, liver, spinal cord, kidney and adrenal gland based on enzyme activity and mRNA detection.100

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Proline Specific Peptidases Dipeptidyl Peptidase IV In contrast to the metallopeptidases described above, there are two peptidases which exhibit a substrate specificity directed at proline residues. One of these is dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5, also known as adenosine deaminase binding protein and thymocyte activating molecule), a dimeric ectoenzyme with a molecular weight of approximately 260 kDa.101 It is a serine peptidase containing a typical catalytic triad.102 Dipeptidyl peptidase IV activity has been found in the intestine, placenta, lymph nodes, liver, blood vessels and pancreas103 with the highest activity in the kidney and in the intestinal brush-border membrane. Immunostaining detected DPP IV in brush borders of kidney, bile ducts, salivary glands, small intestine, colon, pancreas and also in T-cell areas in thymus, spleen and lymph node.104 Dipeptidyl peptidase IV generates Xaa-Pro(Ala) dipeptides through cleavage at the N-terminal peptide fragments with the proline residue in a trans conformation. It is a proline (or alanine)-specific ectoenzyme105 which appears to be the major enzyme acting on substance P in brush border membranes106 by the sequential removal of Arg-Pro and LysPro. Other potential in vivo substrates include neuropeptide Y and peptide YY.107 In vivo enzyme activity has been confirmed by the finding that rats deficient in the enzyme excrete more proline-containing peptides than control animals.108 In vitro the enzyme cleaves β-casomorphin, prolactin, GRP, hCG, hGHRH and aprotinin.109 Diprotin A and B (Ile-Pro-Ile and Val-Pro-Leu, respectively) have been widely used as inhibitors for DPP IV, but their modest IC50 and potential degradation as substrates by DPP IV and other peptidases limit their use. Potent and selective inhibitors based on peptidylnitrile structures110 as well as proline-boronic acid dipeptides111 have recently been developed and were shown to inhibit dipeptidyl peptidase IV in the submicromolar range. DPP IV is identical to the human T lymphocyte activation antigen CD26: It is implicated as an adhesion molecule in the invasion of cancer cells into the extracellular matrix.112 It is proposed to play a role in the regulation of the proliferation of natural killer cells through PMA-induced hyperphosphorylation of p561ck, which activates a signaling mechanism of T lymphocytes. Although this mechanism is not easily understood based on the substrate specificity of DPP IV, its specific inhibitors strongly suppressed p561ck hyperphosphorylation and the removal of the inhibitors completely restored it.113 These inhibition experiments suggest that enzymatically active dipeptidyl peptidase is essential for the signaling function.

Prolyl Endopeptidase The second proline specific peptidase is prolyl endopeptidase (also known as postproline cleaving enzyme or prolyl oligopeptidase, EC 3.4.21.26). This enzyme is a 70-80 kDa cytoplasmic serine peptidase with a neutral pH optimum that displays specificity toward cleavage after Pro residues but not between Pro-Pro.114 It also functions, with decreased efficiency, as a prolyl carboxypeptidase in the cases of angiotensin II and LHRH.105 The enzyme was found to be similar to dipeptidyl peptidase IV, with conserved sequences including the putative catalytic site. Although their active sites all contain the same catalytic triad, neither prolyl endopeptidase nor dipeptidyl peptidase IV belong to the trypsin/chymotrypsin or the subtilisin families of serine proteases: The three key residues in DPP IV and prolyl endopeptidase are arranged in the order Ser/Asp/His in the primary amino acid sequence of the protein as opposed to His/Asp/Ser for trypsin and an Asp/His/Ser arrangement for subtilisin.115 In vitro substrates of prolyl endopeptidase include substance P, neurotensin, TRH, bradykinin, dynorphin, angiotensin I, oxytocin and vasopressin.109 By the use of Z-Pro-prolinal

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as a specific inhibitor, the in vivo cleavage of angiotensin I by prolyl endopeptidase has been reported in brain.116 The enzyme has been found in the testis, liver, skeletal muscle, lung, brain and kidney.109

Soluble Neuropeptidases N-arginine Dibasic Convertase N-arginine dibasic convertase (NRDc) was first isolated in 1994 by Cohen et al117 based on its ability to cleave somatostatin and its synthetic analogs. It is a 130 kDa metalloenzyme, with a pH optimum of 7 to 8.8, that contains a sensitive thiol residue and the putative active site residues HXXEH. This “inverted” zinc-binding motif places NRDc in the inverzincin family of Zn-metalloproteases along with insulin degrading enzyme and pitrilysin, a bacterial protease.118 The main distinguishing feature of NRDc compared to the other members of this family of peptidases is the presence of an extended acidic stretch of 76 residues, consisting mainly of aspartates and glutamates, immediately prior to the active site.119 This peptidase is capable of cleaving the opioid peptides dynorphin A and B, prodynorphin B and α-neo-endorphin, as well as somatostatin. Cleavage occurs at paired basic residues of the type R-R or R-K, with Km values that are compatible with the physiological concentrations of these peptides. It does not process neurotensin peptides (P-R-R-P) or mastoparan (K-K).120 NRD convertase is most abundant in testis, followed by heart and brain.118 It appears that the enzyme can be secreted from cells and thus could regulate neuropeptide substrates in the extracellular space.120,121

Insulin Degrading Enzyme Endopeptidase 3.4.24.56122 (formerly EC 3.4.99.45 and 3.4.22.11, also known as insulin degrading enzyme {IDE}, insulinase, insulin-specific protease, neutral thiolpeptidase, or glucagon peptidase) is a metallopeptidase123 which, like NRD convertase, contains an inverted zinc binding domain placing it in the inverzincin family.124 This peptidase now joins the family of neuropeptidases. It has recently been shown that the β-endorphin metabolizing enzyme, γ-endorphin generating enzyme (γ-EGE) and insulin degrading enzyme are identical.125 IDE cleaves β-endorphin at the Leu17-Phe18 and Phe18-Lys19 bonds, the former cleavage generating γ-endorphin.125 IDE is found in all tissues and cell lines examined to date. The enzyme has been purified from a variety of sources126 and it is a 220 kDa homodimer exhibiting a pH optimum in the range of 7.0 to 8.6.125 Although IDE was originally characterized based on its high affinity for,123,126 and ability to, hydrolyze insulin,127 it has more recently been shown to cleave a variety of peptides including dynorphin A1-13, dynorphin A1-17, dynorphin B, β-endorphin, pancreastatin1-49, GRF1-29,125 ANP, BNP, and CNP.128 The specificity of IDE is complex, exhibiting a preference for larger peptides as opposed to enzymes such as neprilysin, endopeptidase 24.15, and endopeptidase 24.16, which prefer small peptides. Cleavage preferentially occurs on the amino side of basic and hydrophobic residues.125 Even though IDE contains the peroxisomal targeting sequence SKL at its C-terminus, the majority of the enzyme is cytosolic, with a smaller fraction localized to the peroxisomal compartment.129 Seta and Roth130 have recently shown that a fraction of the enzyme could be labeled by a membrane-impermeable biotinylating agent, indicating that IDE is also present as an ectoenzyme. These findings suggest that IDE could be involved in extracellular processing or degradation of neuropeptides. Of particular interest is the report that IDE/γ-EGE can be crosslinked to amyloid β peptide (Aβ) in crude rat brain and liver extracts, suggesting that Aβ is bound to the en-

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zyme. Purified IDE is capable of degrading Aβ1-40.131 Based on these results, it has been suggested that IDE might be involved in the cellular processing of β-amyloid or the regulation of Aβ levels.131

Endopeptidase 24.15 and Endopeptidase 24.16 Two closely related zinc metallopeptidases are endopeptidase 24.15 (EC 3.4.24.15 formerly EC 3.4.99.31, also referred to as thimet oligopeptidase, endo-oligopeptidase A, Pz-peptidase),132 and endopeptidase 24.16 (EC 3.4.24.16) also referred to as neurolysin or oligopeptidase M.133 Both of these endopeptidases, which exhibit molecular weights of ~75 kDa, have been purified and cloned from several sources.134,135,136 Alignment of their sequences shows over 60% conservation of amino acids, clearly demonstrating that these are related proteins.135 Both enzymes are active at neutral pH and are found in soluble and membrane forms. The soluble forms account for the majority (~80%) of the total activity.133,137 Both endopeptidases 24.15 and 24.16 exhibit a broad and overlapping substrate specificity. Both cleave β-neo-endorphin, bradykinin, neurotensin, somatostatin, VIP, angiotensin II, and substance P. However, even though both will process neurotensin and somatostatin, they exhibit distinct cleavage patterns: endopeptidase 24.15 cleaves neurotensin at the Arg8Arg9 bond, while endopeptidase 24.16 cleaves at this bond as well as at the Pro10-Tyr11 bond. Similarly endopeptidase 24.15 cleaves somatostatin at Asn5-Phe6, Phe6-Phe7, Thr10-Phe11, while only the Asn5-Phe6 and Thr10-Phe11 bonds are cleaved by endopeptidase 24.16.133,138,139 The substrate specificities of these enzymes are complex and not fully understood, but cleavage is believed to be influenced by aromatic residues in the P1, P1', P2', and P3' positions. Inhibitors which distinguish between these enzymes have been developed. The synthetic peptide Cpp-Ala-Ala-Phe-pAb (Ki = 10 to 30 nM) is a specific inhibitor of endopeptidase 24.15,132 whereas the dipeptide Pro-Ile (Ki = 90 µM) is a low affinity, but selective, inhibitor of endopeptidase 24.16.140 It has been reported that Cpp-Ala-Ala-Phe-pAb can undergo proteolytic cleavage141 resulting in the formation of a potent inhibitor for ACE. This limits the usefulness of this compound for in vivo studies. Highly potent and selective phosphinic peptide inhibitors have been developed. Z-Phe(PO2CH2)Ala-Arg-Phe is three orders of magnitude more potent against endopeptidase 24.15 (Ki = 0.16 nM) than endopeptidase 24.16 (Ki = 530 nM) and does not inhibit other neuropeptide degrading enzymes such as endopeptidase 24.11, angiotensin converting enzyme, or aminopeptidase M.142 Several studies suggest that endopeptidases 24.15 and 24.16 can participate in the extracellular metabolism of peptides. However, the amino acid sequences, as deduced from their cDNAs, do not reveal any signal peptide sequences, nor do the enzymes appear to contain a GPI-anchor. Recent evidence has been obtained which indicates that in neurons at least a fraction of the membrane form of endopeptidase 24.16 is on the plasma membrane acting as an ectoenzyme. Interestingly, astrocytes do not contain a plasma membrane form of the enzyme, but are capable of secreting endopeptidase 24.16 through a monensin, brefeldin A, and forskolin-independent mechanism.143 As neurons mature there is an increase in the membrane form of endopeptidase 24.16, thought to represent the plasma membrane associated form of the enzyme.144 Checler and his colleagues have provided considerable evidence that endopeptidase 24.16 is the peptidase responsible for regulating endogenous neurotensin levels. The endopeptidase 24.16 selective inhibitor, Pro-Ile, enhanced the recovery of vascular infused neurotensin in the ileum of the anesthetized dog.145 Intracerebroventricular administration of a phosphinic inhibitor, phosphodiepryl, which inhibits both endopeptidases 24.15 and 24.16 potentiated neurotensin-induced analgesia in mice.146 Also, a restricted population of cultured neurons from mouse cerebral hemisphere which degraded neurotensin expressed

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the neurotensin receptor. Approximately 90% of these neurotensin receptor bearing cells also exhibited immunoreactivity toward endopeptidase 24.16.147 Acker et al have shown that endopeptidase 24.15 is present in rat brain synaptosomal plasma membranes and inhibited this activity by Cpp-AAF-pAB.148 Cpp-AAF-pAB blocks the degradation of intraventricularly administrated dynorphins.149 Davis et al showed that inhibition of endopeptidase 24.15 by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phep-aminobenzoate in rat brain slices resulted in a decrease in the conversion of neurotensin to neurotensin1-8 and neurotensin9-13.64 Evidence that endopeptidase 24.15 is involved in the degradation of LHRH came from the observation that Cpp-AAF-pAB inhibited the degradation of ICV or IV administered peptide. Based on increased 24.15 activity in tissues with high remodeling activity, it has been postulated that this enzyme might be involved in the degradation of collagen.150 Inhibition of 24.15 results in a decrease in blood pressure thought to be a result of decreased bradykinin hydrolysis by the enzyme.151 In addition, in isolated strips of trachea from guinea pig it was demonstrated that endopeptidase 24.15 modulates contraction induced by bradykinin.151

Summary In this review we have pointed out that there are a variety of peptidases which are capable of degrading or interconverting neuropeptides. In many cases these appear to represent overlapping and redundant systems, perhaps indicating the importance of this process in nature. In most cases neuropeptidases exhibit a broad substrate specificity, suggesting that their in vivo specificity may be determined more by substrate availability than by peptide recognition. These neuropeptidases represent a target for pharmacological intervention, as their inhibition can lead to an increase in endogenous peptides available for receptor binding.

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81. Hersh LB, Smith TE, McKelvy JF. Cleavage of endorphins to des-Tyr endorphins by homogeneous bovine brain aminopeptidase. Nature 1980; 286:160-162. 82. McDermott JR, Mantle D, Lauffart B et al. Purification and characterization of a neuropeptide-degrading aminopeptidase from human brain. J Neurochem 1985; 45:752-759. 83. Davidoff AN, Mendelow BV. Unexpected cytokinetic effects induced by puromycin include a G2 arrest, a metaphase-mitotic-arrest, and apoptosis. Leukemia Res 1992; 16:1077-1085. 84. Takahashi S, Ohishi Y, Kato H et al. The effects of bestatin, a microbial aminopeptidase inhibitor, on epidermal growth factor-induced DNA synthesis and cell division in primary cultured hepatocytes of rats. Exp Cell Res 1989; 183:399-412. 85. Takahashi SI, Kato H, Takahashi A et al. Mode of action of bestatin and leupeptin to induce the accumulation of acid soluble peptides in rat liver in vivo and the properties of the accumulated peptides. The important role of bestatin- and leupeptin-sensitive proteases in the protein degradation pathway in vivo. Biochem 1987; 19:401-412. 86. Turner AJ, Hooper NM, Kenny AJ. Metabolism of neuropeptides. In: Kenny AJ, Turner AJ, eds. Mammalian Ectoenzymes. Amsterdam: Elsevier, 1987:211-248. 87. Corvol P, Michaud A, Soubrier F et al. Recent advances in knowledge of the structure and function of the angiotensin I converting enzyme. J Hypertension 1995; 13:S3-S10. 88. Roy SN, Kusari J, Soffer RL et al. Isolation of cDNA clones of rabbit angiotensin converting enzyme: Identification of two distinct mRNAs for the pulmonary and the testicular isozymes. Biochem Biophys Res Commun 1988; 155:678-684. 89. Perich RB, Jackson B, Rogerson F et al. Two binding sites on angiotensin-converting enzyme: Evidence from radioligand binding studies. Mol Pharmacol 1992; 42:286-293. 90. Azizi M, Rousseau A, Ezan E. et al. Acute angiotensin converting enzyme inhibition increases the plasma level of the natural stem cell regulator N-acetyl-seryl-aspartyl-lysyl-proline. J Clin Invest 1996; 97:839-844. 91. Cheung H-S, Wang F-L, Ondetti MA et al. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. J Biol Chem 1980; 255:401-407. 92. Fournié-Zaluski MC, Coric P, Thery V et al. Design of orally active dual inhibitors of neutral endopeptidase and angiotensin-converting enzyme with long duration of action. J Med Chem 1996; 39:2594-2608. 93. Gonzalez W, Beslot F, Laboulandine I et al. Inhibition of both angiotensin-converting enzyme and neutral endopeptidase by S21402 (RB105) in rats with experimental myocardial infarction. J Pharmacol Exp Ther 1996; 278:573-581. 94. Seymour AA, Asaad MM, Abboa-Offei BE et al. In vivo pharmacology of dual neutral endopeptidase/angiotensin-converting enzyme inhibitors. J Cardiovasc Pharmacol 1996; 28:672-678. 95. Ryan JW, Berryer P, Chung AY et al. Characterization of rat pulmonary vascular aminopeptidase P in vivo: Role in the inactivation of bradykinin. J Pharmacol Exp Ther 1994; 269:941-947. 96. Bauer K. Purification and characterization of the thyrotropin-releasing-hormone-degrading ectoenzyme. Eur J Biochem 1994; 224:387-396. 97. Charli J-L, Cruz C, Vargas MA et al. The narrow specificity pyroglutamate aminopeptidase degrading TRH in rat brain is an ectoenzyme. Neurochem Int 1988; 13:237-242. 98. Charli JL, Mendez M, Vargas MA et al. Pyroglutamyl peptidase inhibition specifically increases recovery of TRH released from rat brain slices. Neuropeptides 1989; 14:191-196. 99. Vargas MA, Joseph-Bravo P, Charli J-L. Thyrotropin-releasing hormone downregulates pyroglutamyl peptidase II activity in adenohypophyseal cells. Neuroendocrinology 1994; 60:323-330. 100. Schauder B, Schomburg L, Kohrle J et al. Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone. Proc Natl Acad Sci USA 1994; 91:9534-9538. 101. Misumi Y, Hayashi Y, Arakawa F et al. Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine protease on the cell surface. Biochim Biophys Acta 1992; 1131:333-336.

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102. David F, Bernard A-M, Pierres M et al. Identification of serine 624, aspartic acid 702 and histidine 734 as the catalytic triad residues of mouse dipeptidyl peptidase IV (CD26). J Biol Chem 1993; 268:17247-17252. 103. Kenny AJ, Stephenson SL Turner AJ. Cell surface peptidases. In: Kenny AJ, Turner AJ, eds. Mammalian ectoenzymes, Amsterdam: Elsevier, 1987: 169-210. 104. McCaughan GW, Wickson JE, Creswick PF et al. Identification of the bile canalicular cell surface molecule GP110 as the ectopeptidase dipeptidyl peptidase IV: An analysis by tissue distribution, purification and N-terminal amino acid sequence. Hepatology 1990; 11:534-544. 105. Vanhoof G, Goossens F, de Meester I et al. Proline motifs in peptides and their biological processing. FASEB J 1995; 9:736-744. 106. Wang L, Ahmad S, Benter IF et al. Differential processing of substance P and neurokinin A by plasma dipeptidyl (amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme. Peptides 1991; 12:1357-1364. 107. Medeiros MD, Turner AJ. Processing and metabolism of peptide YY: Pivotal roles of dipeptidylpeptidase-IV, aminopeptidase-P and endopeptidase-24.11. Endocrinology 1994; 134:2088-2094. 108. Watanabe Y, Kojima-Komatsu T, Iwaki-Egawa S et al. Increased excretion of proline-containing peptides in dipeptidyl peptidase IV-deficient rats. Res Commun Chem Pathol Pharmacol 1993; 81:323-330. 109. Yaron A, Naider F. Proline-dependent structural and biological properties of peptides and proteins. Crit Rev Biochem Mol Biol 1993; 28:31-81. 110. Li J, Wilk E, Wilk S. Aminoacylpyrrolidine-2-nitriles: Potent and stable inhibitors of dipeptidyl peptidase IV (CD26). Arch Biochem Biophys 1995; 323:148-154. 111. Coutts SJ, Kelly TA, Snow RJ et al. Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides. J Med Chem 1996; 39:2087-2094. 112. Chen W-T. Proteases associated with invadopodia, and their role in degradation of the extracellular matrix. Enzyme Prot 1996; 49:59-71. 113. Kahne T, Neubert K, Ansorge S. Enzymatic activity of DPPIV/CD26 is involved in PMAinduced hyperphosphorylation of p561ck. Immunol Lett 1995; 46:189-193. 114. Kalwant S, Porter AG. Purification and characterization of human brain prolyl endopeptidase. Biochem J 1991; 276:237-244. 115. Vanhoof G, Goossens F, Hendriks L et al. Cloning and sequence analysis of the gene encoding human lymphocyte prolyl endopeptidase. Gene 1994, 149:363-366. 116. Welches WR, Santos RAS, Chappell MC et al. Evidence that prolyl endopeptidase participates in the processing of brain angiotensin. J Hypertension 1991; 9:631-638. 117. Chesneau V, Pierotti AR, Barré N et al. Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues. J Biol Chem 1994; 269:2056-2061. 118. Pierotti AR, Prat A, Chesneau V. N-arginine dibasic convertase, a metalloendopeptidase as a prototype of a class of processing enzymes. Proc Natl Acad Sci USA 1994; 91:6078-6082. 119. Chesneau V, Pierotti AR, Prat A et al. N-arginine dibasic convertase (NRD convertase): A newcomer to the family of processing endopeptidases. An overview. Biochimie 1994; 76:234-240. 120. Csuhai E, Safavi A, Hersh LB. Purification and characterization of a secreted argininespecific dibasic cleaving enzyme from EL-4 cells. Biochemistry 1995; 34:12411-12419. 121. Chesneau V, Prat A, Segretain D et al. NRD convertase: A putative processing endoprotease associated with the axoneme and the manchette in late spermatids. J Cell Sci 1996; 109:2737-2745. 122. Duckworth WC, Hamel FG, Peavy DE et al. Degradation products of insulin generated by hepatocytes and by insulin protease. J Biol Chem 1988; 263:1826-1833. 123. Roth RA, Mesirow ML, Cassell DJ et al. Characterization of an insulin degrading enzyme from cultured human lymphocytes. Diabetes Res Clin Pract 1985;1:31-39.

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124. Becker AB, Roth RA. An unusual active site identified in a family of zinc metalloendopeptidases. Proc Natl Acad Sci USA 1992; 89:3835-3839. 125. Safavi A, Miller BC, Cottam L et al. Identification of gamma-endorphin generating enzyme as insulin degrading enzyme. Biochem 1996; 35:14318-14325. 126. Yokono K, Imamura Y, Shii K et al. Immunochemical studies on the insulin degrading enzyme from pig and rat skeletal muscles. Diabetes 1980; 29:856-859. 127. Duckworth WC, Hamel FG, Peavy DE et al. Degradation products of insulin generated by hepatocytes and by insulin protease. J Biol Chem 1988; 263(4):1826-1831. 128. Muller D, Schulze C, Baumeister H et al. Rat insulin degrading enzyme: Cleavage pattern of the natriuretic peptide hormones ANP, BNP, and CNP revealed by HPLC and mass spectrometry. Biochem 1992; 31:11138-11143. 129. Kuo WL, Gehm BD, Rosner et al. Inducible expression and cellular localization of insulindegrading enzyme in a stably transfected cell line. J Biol Chem 1994; 269:22599-22606. 130. Seta K, Roth RA. Overexpression of insulin degrading enzyme: Cellular localization and effects on insulin signaling. 1997; Biochem Biophys Res Commun 1997; 231:167-171. 131. Kurochkin IV, Goto S. Alzheimer’s β-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Letters 1994; 345:33-37. 132. Dando PM, Brown MA, Barrett AJ. Human thimet oligopeptidase. Biochem J 1993; 294:451-457. 133. Barelli H, Vincent JP, Checler F. Rat kidney endopeptidase 24.16: Purification, physicochemical characteristics and differential specificity toward opiates, tachykinins and neurotensin-related peptides. Eur J Biochem 1993; 211:79-90. 134. McKie N, Dando PM, Rawling ND et al. Thimet oligopeptidase: Similarity to soluble angiotensin II-binding protein and some correction to the published amino acid sequence of the rat testis enzyme. Biochem J 1993; 295:57-60. 135. Sugiura N, Hagiwara H, Hirose S. Molecular cloning of porcine soluble angiotensin-binding protein. J Biol Chem 1992; 267:18067-18072. 136. Dauch P, Vincent JP, Checler F. Molecular cloning and expression of rat brain endopeptidase 3.4.24.16. J Biol Chem 1995; 270:27266-27271. 137. Orlowski M, Michaud C, Chu TG. A soluble metalloendopeptidase from rat brain: Purification of the enzyme and determination of the specificity with synthetic and natural peptides. J Biochem 1983; 135:81-88. 138. Orlowski M, Reznik S, Ayala J et al. Endopeptidase 24.15 from rat testes: Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin containing peptides. Biochem J 1989; 261:951-958. 139. Dahms P, Mentlein R. Purification of the main somatostatin-degrading proteases from rat and pig brains, their action on other neuropeptides, and their identification as endopeptidases 24.15 and 24.16. Eur J Biochem 1992; 208:145-154. 140. Dauch P, Vincent JP, Checler F. Specific inhibition of endopeptidase 24.16 by dipeptides. Eur J Biochem 1991; 202:269-276. 141. Williams CH, Yamamoto T, Walsh DM et al. Endopeptidase 3.4.24.11 converts N-1(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme. Biochem J 1993; 294:681-684. 142. Jiracek J, Jiotakis A, Vincent B et al. Development of highly potent and selective phosphinic peptide inhibitors of zinc endopeptidase 24.15 using combinatorial chemistry. J Biol Chem 1995; 270:21701-21706. 143. Vincent B, Beaudet A, Dauch P et al. Distinct properties of neuronal and astrocytic endopeptidase 3.4.24.16: A study on differences, subcellular distribution, and secretion process. J Neurosci 1996; 16:5049-5059. 144. Vincent B, Dauch P, Vincent JP et al. Stably transfected human cells overexpressing rat brain endopeptidase 3.4.24.16: Biochemical characterization of the activity and expression of soluble and membrane-associated counterparts. J Neurochem 1997; 68:837-845. 145. Barelli H, Fox-Threlkeld JET, Dive V et al. Role of endopeptidase 3.4.24.16 in the catabolism of neurotensin, in vivo, in the vascularly perfused dog ileum. Br J Pharmacol 1994; 112:127-132.

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CHAPTER 11

Stimulation of Peptidergic Receptors by Peptide Hormones and Neurotransmitters: Studies of Opioid Receptors George Bot, Allan D. Blake and Terry Reisine

Introduction

S

ince ancient times mankind has displayed a remarkable ingenuity in the search for compounds which would relieve pain and produce euphoria with a sense of well-being. The opium poppy (Papaver somniferum) and its derivatives have been used in this regard for centuries. In 1805, Serturner1 reported the isolation of a pure substance from opium, which he named morphine. Isolation of other alkaloids in opium poppy quickly followed, for example, codeine by Robiquet in 1832 and papaverine by Merck in 1848. By the middle of the 19th century the use of pure alkaloids rather than crude opium preparations spread throughout the medical and nonmedical world. However, widespread abuse also developed as the alkaloids produced undesirable effects such as tolerance, dependence and compulsive use. The recognition of this serious liability of morphine stimulated a search for potent analgesics that would not lead to compulsive drug use. Since the isolation of morphine, many structurally similar compounds have been prepared in an effort to minimize the undesirable effects of morphine (for example, heroin in 1898, meperidine in 1939, methadone in 1946). However, the search for potent analgesics that can substitute for morphine continues.

Opioid Receptor Types Despite their long history of use/abuse, the neurochemical systems responsible for the actions of opiates (designates products derived from the juice of the opium poppy and semisynthetic congeners of morphine) and opioids (natural and synthetic drugs with morphine-like actions) have been elucidated only in the past 20 years or so. Basic concepts of pharmacology dictate that the multiple and often paradoxical actions of morphine and related opioid drugs emanate from the same initial event, the in vivo binding of those drugs to their specific receptors. Existence of opioid receptors was first hypothesized in 19542,3 and structure-activity studies as early as 1965 suggested the existence of multiple opioid receptors.4 In an endeavor to explain the actions of morphine and nalorphine, Martin proposed a theory of ‘receptor dualism’, postulating the existence of two classes of opioid receptors, M for morphine and N for nalorphine.5 On the basis of different pharmacological Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, edited by Vivian Y.H. Hook. ©1998 R.G. Landes Company.

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profiles of morphine and its derivatives, this was later extended to three different types of opioid receptors called µ (mu) for morphine, κ (kappa) for ketocyclazocine and σ (sigma) for N-allylnormetazocine or SKF10,047.6 In 1977 a novel enkephalin-preferring receptor, most abundant in the mouse vas deferens, was identified and termed δ (delta).7 Although the σ receptor (and ligands) were originally thought to be opioid in nature6 they appear to lack true opioid pharmacology. Unlike the three major opioid receptors, σ receptors are stereoselective for the (+)-enantiomers of alkaloids, and the opioid antagonist naloxone fails to block the binding and in vivo effects of these substances.8 Since these pioneering studies other receptors such as epsilon (ε), iota (ι), lambda (λ) and zeta (ζ) have been tentatively identified but not clearly differentiated.9,10

Endogenous Opioids When it became clear that the brain could bind morphine and other opioids stereospecifically, the (l)-enantiomer usually being the active form,11-13 the search began for the endogenous morphine-like compounds which normally activate such receptors. In 1975 Hughes et al14 successfully isolated and identified from pig brain two endogenous pentapeptides (Met5)- and (Leu5)enkephalin, both with central opioid activity. Since then, several other natural endogenous ligands, the endogenous opioid peptides, have been identified and found to exist in multiple forms in the central nervous system.15 These endogenous opioids are usually grouped into three major classes, the enkephalins, β-endorphins and related compounds and the dynorphins, members of which have distinct precursors and distinct, though overlapping, distributions in the central nervous system. The mammalian opioid peptides arise from three different precursor molecules in three independent biosynthetic pathways. Proenkephalin, which produces primarily (Leu5)- and (Met5)enkephalin, metorphamide, peptide E, peptide F and BAM peptides, prodynorphin which produces the dynorphin peptides (dynorphin-A and dynorphin-B), leumorphin, α-neo-endorphin and β-neo-endorphin, and proopiomelanocortin, which produces β-endorphin, adrenocorticotrophic hormone and β-lipotrophin following extensive posttranslational processing16-18 (Table 11.1). The extent of peptide processing, and the resulting peptide products that are ultimately released, depend on the cells in which they are localized and the physiological state of these cells. In addition to these known main classes, several other characterized opioids such as morphine, 6-acetylmorphine, sigmaphin, codeine and thebaine as well as uncharacterized opioids, have been detected in mammalian tissue and human cerebrospinal fluid. Whether these alkaloids are the result of biosynthesis or whether they are derived from exogenous sources such as food remains to be determined, as does their possible physiological significance.19,20

Endogenous Peptide Receptor Selectivity It has been well documented that the major opioid receptor types do not correspond in a one-to-one relationship either pharmacologically or anatomically with the three opioid peptide families. Although there appears to be pharmacological selectivity of endogenous opioid peptides and exogenous drugs for certain receptors, there is also considerable crossreactivity, as most endogenous peptides will recognize all receptor types in a descending order of affinity. Thus, several peptides, even those from different families, can also activate the same opioid receptor type and the action of a compound at a particular brain site will depend on the specific receptor effects of peptides derived from the population at the site.21 Hence, of the endogenous opioid peptides, the endorphins, enkephalins and dynorphins bind with low to moderate specificity to the three opioid receptors.21 β-endorphin binds to the µ- and δ receptors with comparable affinity, but because of their selectivity, (Met)- and

Sequence

Y-G-G-F-M Y-G-G-F-L Y-G-G-F-M-R-R-V-NH2 Y-G-G-F-M-R-R-V-G-R-P-E Y-G-G-F-M-R-R-V-G-R-P-E-W-W-M-D-Y-Q Y-G-G-F-M-R-R-V-G-R-P-E-W-W-M-D-Y-Q-K-R-Y-G Y-G-G-F-M-R-R-V-G-R-P-E-W-W-M-D-Y-Q-K-R-Y-G-G-F-L Y-G-G-F-M-K-K-M-D-E-L-Y-P-L-E-V-E-E-E-A-N-G-G-E-V-L-G-K-R-Y-G-G-F-M Y-G-G-F-M-T-S-E-K-S-Q-T-P-L-V-T-L-F-K-N-A-I-I-K-N-A-Y Y-G-G-F-M-T-S-E-K-S-Q-T-P-L-V-T-L-F-K-N-A-I-I-K-N-A-Y-K-K-E-E Y-G-G-F-L-R Y-G-G-F-L-R-R Y-G-G-F-L-R-R-I Y-G-G-F-L-R-R-I-R Y-G-G-F-L-R-R-I-R-P-K-L-K Y-G-G-F-L-R-R-I-R-P-K-L-K-W-D-N-Q Y-G-G-F-L-R-R-I-R-P-K-L-K-W-D-N-Q-K-R-Y-G-G-F-L-R-R-Q-F-K-V-V-T Y-G-G-F-L-R-R-Q-F-K-V-V-T Y-G-G-F-L-R-R-Q-F-K-V-V-T-R-S-G-E-D-P-N-A-Y-Y-E-E-L-F-D-V Y-G-G-F-L-R-K-Y-P Y-G-G-F-L-R-K-Y-P-K Y-P-W-F-NH2 Y-P-F-F-NH2 F-G-G-F-T-G-A-R-K-S-A-R-K-L-A-N-Q

Peptide

(Met)enkephalin (Leu)enkephalin Metorphamide BAM12 BAM18 BAM22 Peptide E Peptide F β-Endorphin1-27 β-Endorphin1-31 DynorphinA1-6 DynorphinA1-7 DynorphinA1-8 DynorphinA1-9 DynorphinA1-13 DynorphinA1-17 DynorphinA1-32 (DynAB) DynorphinB1-13 Leumorphin β-neo Endorphin α-neo Endorphin Endomorphin-I Endomorphin-II Nociceptin

Table 11.1. Endogenous ligands of opioid receptors

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(Leu)enkephalin are considered to be endogenous ligands for the δ receptor and dynorphins for the κ receptor. No known mammalian peptide, however, has both high affinity and selectivity for the µ receptor. Recently, Zadina et al22 identified two peptides, endomorphin I and endomorphin II, which exhibit the highest specificity and affinity for the µ receptor of any endogenous substance so far described and thus have been proposed to be the endogenous ligands for the µ receptor. However, the significance and role of these peptides remains to be determined. Some of the endogenous peptides have also been reported to interact with naloxoneinsensitive binding sites; for example, dynorphin has been reported to bind to the tachykinin and NMDA receptors with high affinity. Hence, their biological effect may not be exclusively opioid in nature.23,24

Opioid Ligands In an endeavor to improve the specificity of opioid compounds, structural analogs of traditional opioid peptides with increased specificity for µ-, δ- or κ receptors were developed (Table 11.2).25 These ligands, and others like them, bind with high affinity and specificity and have been useful in elucidating the in vitro and in vivo anatomical, physiological and pharmacological functions of opioid receptors. Each of the opioid receptor classes possesses a characteristic pharmacology that can be distinguished by receptor subtype selective ligands.26 For example, the µ opioid receptor has high affinity binding for morphine and most of the opiates used clinically in pain management, such as fentanyl, codeine and methadone.27 However, each class of opioid selective compounds is not a functionally homogeneous group of drugs. For example, DPDPE, a δ-selective agonist, enhanced antinociception induced by morphine, normorphine and codeine but not that induced by sufentanil nor DAMGO.28,29 On the other hand, the neuroleptic droperidol enhanced fentanyl- and sufentanil-induced, but not morphine-induced, antinociception.30 In addition, lithium and meptazinol (an opioid agonist/antagonist) differentially antagonized morphine-, sufentaniland DAMGO-induced antinociception.31,32 Cross-tolerance between opioids has also been reported not to be equally expressed between two drugs. For example, patients with cancer-related pain refractory to morphine did not exhibit tolerance to methadone.33 Likewise, morphine-treated rats were found to be cross-tolerant to fentanyl, levorphanol and meperidine, but not to methadone.34 Butorphanol, a partial agonist/antagonist, has been reported to precipitate withdrawal in methadonedependent, but not morphine-dependent subjects.35,36 This suggests differential actions for morphine and methadone, compounds which are generally accepted as µ-preferring agonists. Hence, although these compounds may be interacting with the same opioid receptor type, the µ-opioid receptor, different intracellular effector mechanisms may be induced by them in producing their effects. This may arise from dependence upon different amino acid moieties within the receptor for interaction and subsequent induction of intracellular effects. The in vitro study of opioid receptor interaction and signal transduction has relied on the use of tumor cell lines expressing opioid receptors or brain homogenates. However, the issue of the individual opioid receptor subtype involved in the agonist response is often confounded by the presence of one of more opioid receptor subtypes, or splice variants of the same receptor subtype, in individual cell lines.37,38 A cellular system expressing a single receptor type would allow for the examination of the effects of opioid ligands in the absence of these restrictions. Recent cloning of the opioid receptors (see below) has allowed the study of the pharmacology and biochemistry of these receptors in identifying the receptor domains involved in ligand binding and intracellular effects, and thus has also offered a better understanding of opioid mechanisms with the promise of safer and more effective analgesic agents.39

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Table 11.2. Receptor classification of opiates and opioid peptides Opioid Receptor Subtype

Endogenous Peptide

Agonist

Antagonist

Nonselective

µ

(Leu)enkephalin (Met)enkephalin β-endorphin1-31 Peptide E DynorphinA1-17 DynorphinA1-32 Metorphamide BAM 12, 18, 22 Dermorphin

Morphine Methadone DAMGO DADLE Fentanyl Sufentanyl Ohmefentanyl Etonitazene

CTOP Naloxone Naltrexone Diprenorphine CTAP Naloxonazine

Levorphanol Etorphine Bremazocine Buprenorphine Pentazocine EKC Nalbuphine Nalorphine

δ

(Leu)enkephalin (Met)enkephalin β-endorphin1-31 DynorphinA1-13 DynorphinA1-17 DynorphinA1-32 DynorphinB1-13 Metorphamide BAM 12, 18, 22 Peptide E

DSLET DPDPE DADLE Deltorphin II BW3734U86 SIOM SNC-80 TAN-67

Naltrindole NTB BNTX Diprenorphine TIPP[ψ]

Levorphanol Etorphine Bremazocine Buprenorphine

κ

DynorphinA1-32 DynorphinA1-17 DynorphinA1-7 DynorphinB1-13 BAM 12, 18, 22 Peptide E Leumorphin

Spiradoline U50,488 U69,593 Metorphamide

Diprenorphine norBNI

Levorphanol Etorphine Bremazocine Nalbuphine EKC Nalorphine

Opioid Cellular Activity While three main opioid receptor classes are generally recognized, experimental observations indicate that a common mechanism of opioid action is the cellular inhibition of neuronal activity,40 an effect that was suggested shortly after the discovery of the endogenous opioid peptides.41 Although the opioid inhibitory effects may be cell-dependent, accumulating evidence suggests that opioid receptors regulate many cellular effectors via pertussis toxin-sensitive and insensitive G proteins42,43 and data further suggests that opioids may also act independently of G protein.25 G protein cascades, activated by opioid receptors, can result in hyperpolarization of the cell via stimulation of K+ efflux, in closure of L- and N-type voltage-sensitive Ca2+ channels and in inhibition of adenylyl cyclase, alter inositol triphosphate turnover and activate mitogen-activated protein kinase and arachidonate release.25,44 However, it is noteworthy that opioid effects in sensory neurons have been reported to be both inhibitory and excitatory.25 Furthermore, an increase in free intracellular Ca2+ in NG108-15 cells in response to δ receptor occupation and opioid suppression of a Na+-dependent inward current have also been reported.45 Coordinate changes at the cellular level may contribute to the physiological effect of opioids.

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Opioid Receptor Cloning A recent hallmark of opioid research was the genetic determination of the opioid receptors by molecular cloning, with these receptors identified as members of the superfamily of the G protein-coupled, seven transmembrane class of receptors (Fig. 11.1).39 Since the initial cloning of the δ-opioid receptor46,47 cloning of other rodent opioid receptors, κ and µ, quickly followed. Likewise, human opioid receptors have also been cloned, largely based upon the high degree of sequence conservation with their rodent counterparts (for a review see ref. 25).

Structural Identity The results of the molecular cloning studies demonstrate a high degree of identity between the cloned opioid receptors at the amino acid level, with the highest sequence homology occurring in the transmembrane domains and the connecting intracellular loops.39 Conversely, divergent regions exist between the opioid receptors at the amino terminus, carboxyl terminus and the extracellular loops. It is possible that these divergent sequences are critical for discrimination amongst these receptors by type-selective opioid ligands (Fig. 11.1).

Binding and Functional Aspects of Cloned Opioid Receptors Radioligand binding studies showed that the heterologously expressed cloned opioid receptors had pharmacological characteristics consistent with native tissue preparations (for review see refs. 26, 48, 49). However, pharmacological subtypes of µ, δ and κ opioid receptors have been tentatively identified in native tissues25,50 and, based on this subtype classification, the cloned µ receptor has been determined to be the µ1 subtype, due to the high affinity of the expressed receptor for the µ-selective antagonist, naloxonazine.51 The pharmacological profile of the cloned κ receptor is thought to correspond to the previously characterized κ1 subtype, due to the high affinity binding of U50,48852 and the cloned δ receptor corresponds to the δ2 receptor, as determined by a selective higher affinity for the antagonist NTB, when compared to the δ1 selective antagonist BNTX.53 Heterologous expression studies of the recombinant opioid receptors in surrogate cell lines and Xenopus laevis oocytes, both of which lack endogenous opiate receptors, have demonstrated that the cloned receptors also possess similar functional properties to the receptors in native nervous tissue preparations.27,54 Thus the cloned opioid receptors, by coupling to pertussis toxin-sensitive and/or insensitive G proteins, have been demonstrated to mediate the inhibition of cAMP accumulation, the inhibition of voltage-dependent Ca2+ channels and the activation of an inward rectifying K+ channel in various tissues.46,47,55,56 All three main opioid receptors are capable of interacting with a number of G proteins, namely the Giα1, Giα2, Giα3, Goα, Gsα and Gqα subclasses.57,58 Thus, both the pharmacological and functional characteristics of the cloned and endogenously expressed opioid receptors are similar, and data on the relationship between structure and function in the recombinant proteins may reflect the in vivo situation.

ORL1 and Nociceptin/Orphanin FQ In addition to the three major opioid receptors, recombinant DNA technology has allowed the identification of a fourth class of receptor, the ORL1 (opiate receptor like-1) receptor, which displays substantial sequence homologies with opioid receptors, especially in the putative membrane-spanning domains and intracellular loops.59,60 This receptor, however, has low affinity for most opiate compounds and peptides.61 In the search for a natural endogenous ligand, two groups purified the same heptadecapeptide which has been named both nociceptin62 and orphanin FQ.63 The pep-

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Fig. 11.1. Schematic representation of the predicted G protein sequences for the cloned δ, κ and µ receptors. The predicted G protein sequences of the cloned opioid receptors are presented as a seven transmembrane spanning model. The G proteins are presented as circles, with the open circles representing G proteins that differ between the three cloned receptors and the filled circles representing G proteins that are identical among the three receptors. TM refers to the predicted transmembrane spanning regions, which are indicated by Roman numerals. The three intracellular loops are indicated.

tide has amino acid sequence related, however distantly, to dynorphin A (Table 11.1). However, the N-terminal amino acid in nociceptin is phenylalanine, in contrast to all endogenous opiate peptides which have a tyrosine. Furthermore, in the amino acid sequence position number five, nociceptin has a threonine whereas all opiates have either a methionine or leucine. These amino acid differences may explain the low affinity of nociceptin for the opioid receptors and the low affinity of endogenous opiates for ORL1.61-63 The structural similarity of nociceptin to dynorphin A is consistent with structural similarities between ORL1 and the κ receptor. Both receptors have highly acidic second extracellular loops and both peptides have a highly basic, initial 4-5 amino acid sequence C-terminal. The second extracellular loop of the κ receptor has been speculated to be a recognition site for dynorphin A and is believed to facilitate ligand binding through electrostatic interactions.64 However, in spite of the dual structural homology of ORL1 and the κ receptor on the one hand and their endogenous ligands on the other, binding and functional studies with nociceptin analogs have indicated that the mode of interaction between nociceptin and ORL1 receptor is quite different from that between dynorphin A and κ receptor, as the ORL1 receptor recognizes different parts of the nociceptin molecule and requires the complete peptide structure for biological activity, in contrast to the κ receptor.61,65 Nociceptin has been reported to display nanomolar binding potency to the ORL1 receptor, to mediate adenylyl cyclase inhibition in cells expressing the ORL1 receptor and, when administered intracerebroventricularly or intrathecally in mice, to induce hyperalgesia,61-63 which is opposite to the action of opiates. Such properties of nociceptin have initiated major attempts to develop antagonists of ORL1 which might be expected to induce

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analgesia by blocking the endogenous actions of nociceptin. Analgesia-producing antagonists would hold major advantages over classical opiates since they might not be expected to induce tolerance, a major drawback of opiate use.

Structure-Function Analysis of Cloned Opioid Receptors Site-directed mutagenesis studies on the cloned opioid receptors have demonstrated that there are distinct molecular interactions for each of the ligand categories of nonpeptide agonists, nonpeptide antagonists and peptide agonists which may be reflected in cellular function.48 Two genetic modification approaches have been used to examine the molecular determinants for opioid action within the receptors: receptor chimeras, where domains between opioid receptor subtypes are interchanged; and point mutations, where individual amino acids within the receptors are altered. Both approaches have been useful in delineating specific domains and amino acids critical for receptor binding and selectivity. However, caution must be used in interpreting these results, as the precise three-dimensional structure of the receptor pocket may be altered by either of these structural manipulations. When used in conjunction with the subtype-selective opioid ligands for the δ-, µ- and κ receptors, these studies provide important structural information on the molecular constituents of ligand recognition. Since opioid receptors bind both peptide and nonpeptide ligands (Table 11.2), the determination of regions that confer ligand specificity is critical for the rational development of synthetic opioids with improved therapeutic benefit.48,66 µ Receptor The preferential binding of peptide ligands to distinct domains of the opioid receptors has been reinforced by studies carried out on the µ receptor. Deletion of the N-terminal domain (amino acids 1-66) or the C-terminal 33 amino acids of the µ receptor produced little changes in receptor agonist binding affinity and selectivity, suggesting that the region critical for binding lies within the transmembrane domains and the extracellular loops.67 Wang et al68 have reported that the domain delineated by the second transmembrane region, the first extracellular loop, and the proximal amino acids in the third transmembrane region of the µ receptor are important for DAMGO binding, whereas the third extracellular loop and the surrounding transmembrane spanning regions VI through VII are important for morphine binding. Neither the second or third extracellular loop nor the transmembrane spanning regions V through VII were essential for naloxone binding. These results indicate that opioids may have distinct ligand binding domains in the µ receptor which cause them to interact differently with the µ receptor and induce different intracellular effector systems. Mutations of aspartic residue 114 (Asp-114) in transmembrane domain II (TM2) to asparagine produced a dramatic loss of the receptor binding affinity of agonists such as DAMGO, morphine, methadone, (Met)enkephalin, etorphine, levorphanol and fentanyl with little changes in the binding of bremazocine, EKC, nalbuphine and nalorphine and the µ antagonist naloxone.67,69,70 Likewise, mutation of His-297 to Ala in TM6 resulted in complete loss of binding for DAMGO, bremazocine and EKC.71 On the other hand, mutation of Tyr-326 to Phe in TM7 produced a dramatic loss in DAMGO binding whilst not affecting the binding of the nonselective agonists bremazocine and EKC.71 However, mutation of His-297 to Asn in TM6 of the µ receptor, did not significantly affect the binding of morphine, DAMGO, bremazocine or levorphanol but did decrease the binding of buprenorphine and diprenorphine,69 suggesting that the magnitude of the changes in receptor binding affinity may depend upon the specific mutation that has been introduced. DAMGO, a synthetic µ selective peptide, was found to discriminate between µ and δ receptor chimeras at the amino terminal region, including the first extracellular loop.72 But

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differences in the regions required for ligand selectivity can be affected by the opioid receptor subtypes used in the chimera construction. For example, although some investigators72,73 found the first extracellular loop of the µ receptor to be involved in DAMGO binding, based upon chimeras formed between µ/δ receptors, others found that with µ/κ chimeras that the interaction of DAMGO was dependent on the third extracellular loop of the µ receptor.74,75 Chimeric receptor studies have also suggested that DAMGO may bind to a different region of the µ receptor than morphine and codeine,72 whereas others argue for a common site of interaction.74 A similar phenomenon has been reported for µ/κ and µ/δ chimeras with the µ selective agonist sufentanil.76 In this study,76 the region for sufentanil selectivity in the µ receptor resides in extracellular loop 3 and TM regions 6 and 7 of µ/κ receptors, while the first extracellular loop and TM regions 1 and 3 were involved in the sufentanil selectivity observed for µ/δ receptors. These results suggest that the hydrophilic extracellular loops of the cloned receptors are important for peptide binding, although these regions may be constrained by the receptor conformation that is defined by the transmembrane helices. The results of the mutagenesis studies indicate that differences exist for the ligand recognition domains of the opioid receptors, particularly in terms of the regions involved in peptide binding. While the nonpeptidyl opioids are thought to bind in a hydrophobic pocket that is dependent on the transmembrane domains of the receptor, the extracellular loops of the receptors are also enlisted to give tight and specific peptide binding, with different extracellular loops being preferred by different peptides. Figure 11.2 shows that a common theme has emerged from the mutagenesis studies performed on the opioid receptors, with the peptide ligand binding sites being localized to the extracellular domains of the receptors.48,77 This is consistent with the hypothesis that regions of sequence divergence of the receptors are responsible for the functional differences of the receptors. δ Receptor Initial mutagenesis studies on the cloned opiate δ receptor examined conserved aspartate residues (ASP-95 and ASP-128) in the transmembrane domains II (TM2) and III (TM3)78,79 regions, respectively, that were important in ligand binding in other G proteincoupled receptors. Mutagenesis of these aspartate residues diminished agonist binding in other G protein receptors, either through a conformational change in the receptor or from disrupting anionic ion-pairing between the receptor and ligand.80 These studies78,79 showed that changing the charged aspartate residues in TM2 or TM3 to a neutral asparagine reduced agonist affinity. The TM2 and TM3 aspartate residues possibly play an important role in reducing the dissociation rates of agonist interaction at the receptor, consistent with the observations that neither aspartate residue had a substantial effect on opioid antagonist binding.78,79 Aromatic transmembrane amino acids have also been found to be critical determinants for δ-selective peptide binding,81 with nonpeptidyl ligands being less affected by alterations in these amino acids. These results suggest that the differences exist between the binding of δ-selective peptide and nonpeptide agonists, a result that has been the focus of several laboratories. Wang et al82 used receptor chimeras formed with the µ and δ receptors to establish that δ-selective peptides bind to TM5-TM7, and that the third extracellular loop region was necessary for binding of the δ-selective peptides DSLET and DPDPE, with arginine residues present in the extracellular loop essential for DSLET interaction. Meng et al83 also demonstrated that in the δ receptor the third extracellular loop and TM6 were essential for δ ligand selectivity, which, when taken together with the other studies suggests that the δ selective peptides are primarily dependent upon extracellular domains of the receptor, whereas nonpeptides interact largely with transmembrane regions of the receptor. In contrast, Varga et al84 found that while the third extracellular loop of the human δ receptor,

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Fig. 11.2. Peptide agonist recognition sites are different in the three cloned opiate receptors. The κ selective peptide, dynorphin A, binding site may be at the second extracellular loop of the cloned κ receptor. The δ selective peptides, DSLET and DPDPE, may recognize the third extracellular loop of the cloned δ receptor. DAMGO, a µ selective peptide, may bind to the first and third extracellular loops of the cloned µ receptor.

whilst not being important for diprenorphine or morphine binding, was important in the determination of the selectivity for both the peptides DPDPE and deltorphin II, and the nonpeptide δ agonists such as SNC 80 and (-)TAN67. By using single residue substitutions in the third extracellular loop of the human δ receptor, Valiquette et al85 defined three critical amino acids for δ-selective ligands, indicating that peptides and nonpeptide ligands may recognize overlapping epitopes in the third extracellular loop of the δ receptor. κ Receptor The notion that opioid peptides and nonpeptides interact with distinct receptor domains was originally established for the mouse κ receptor, using receptor chimeras where the amino termini of the δ and κ receptors were exchanged.86 In this study,86 the amino terminus of the κ receptor was found to be necessary for binding the nonselective antagonist naloxone, while agonist binding was unaffected. The binding of dynorphin A, a κ-selective opioid peptide, has been shown to require the second extracellular loop of the κ receptor, as determined by receptor chimeras formed between the κ and µ receptors.87 Also using chimeras formed between the κ and µ receptors, Hjorth et al88 defined a single residue (Glu-297), at the boundary of TM6 and the third extracellular loop, as the point of recognition for the κ-selective antagonist norbinaltorphimine. These authors88 also noted that the lower affinity for norbinaltorphimine binding at the µ and δ receptors was due to the presence of lysine (µ receptor) or tryptophan (δ receptor) residues in the corresponding posi-

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tions, allowing an extrapolation from the results obtained with the κ receptor to the other receptor subtypes.

Intracellular Loops of Opioid Receptors In contrast to the extracellular loops of the opioid receptors, the sequences of the intracellular loops are very similar. Given this structural similarity, all three opioid receptors couple to similar cellular effector systems and modulate them in a similar manner. The only intracellular domain of the opioid receptors that differs in amino acid sequence is the carboxyl terminus.39 However, this region may not be necessary for coupling the receptor to effector systems, since truncation of the µ and δ receptor carboxyl terminus did not prevent the receptor from coupling to adenylyl cyclase.70,89,90

µ Receptor Knockout Mice Model Pharmacological analysis have suggested that µ receptors mediate the analgesic actions of opioids, since most of the clinically relevant opioids used in pain management bind to this receptor with high affinity.27,91 To further investigate the functions of the µ receptor, Matthes et al92 and Sora et al93 disrupted expression of the mouse µ receptor by homologous recombination, producing homologous, recombinant µ receptor knockout mice and tested baseline and morphine-altered pain responses in these animals with deletion of the µ receptor gene. Mice with the µ receptor gene disrupted showed no obvious morphological differences from normal mice. However, investigations of the behavioral effects of morphine revealed that a lack of µ receptors abolished the analgesic effect of morphine, as well as the place-preference and physical dependence. The authors interpreted this as suggesting that the analgesic, rewarding effects and dependence of morphine are exclusively mediated by µ receptors. This result is unusual, since morphine is capable of binding and stimulating δ receptors94,95 and δ receptors are believed in part to mediate spinal analgesic effects of opiates.96,97 Furthermore, blockade of δ receptors by intracerebroventricular administration of the δ-selective antagonist naltrindole inhibited the development of morphine dependence in rats without compromising the antinociceptive actions of morphine.98 In addition, the δ receptor-selective antagonist TIPP[ψ] suppressed the development of morphine tolerance and dependence in rats.99 These results indicate that the activation of δ receptors by morphine may be critical in the development of morphine induced tolerance and dependence. The relevance of knockout studies to these results still has to be further examined.

Agonist Regulation of Cloned Opioid Receptors G protein-coupled receptors have been shown to adapt to the presence of chronic agonist treatment. This adaptation may involve desensitization or functional uncoupling of the receptors from signal-transducing G proteins and subsequent downstream effectors and downregulation or net loss of receptor protein. Both desensitization and downregulation of opioid receptors may be critical adaptive responses in the development of tolerance and dependence to opiates. However, even though the three opioid receptors all couple to similar effector systems, their abilities to be regulated differ in terms of the agonist used and intracellular effector examined.

cAMP Studies µ receptor The µ receptor transiently expressed in COS-7 cells was found to be relatively resistant

to agonist regulation, as morphine treatment resulted in no effect on either radiolabeled agonist or antagonist binding.100 Using stable expression in HEK 293 cells, Arden et al101

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Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

showed that the expressed µ receptor was downregulated by DAMGO treatment and that this downregulation event was correlated with phosphorylation of the receptor. However, it is unclear from this study101 whether the downregulation and phosphorylation events that were observed in HEK 293 cells also correlated with functional desensitization of the µ receptor. Zhang et al102 have reported the phosphorylation of the µ receptor in CHO cells, when the cells were challenged with either morphine or an activator of protein kinase C, although the impact of the phosphorylation of receptor function was unclear. Chakrabarti et al103 observed a time and concentration-dependent effect of morphine and DAMGO on receptor downregulation and receptor desensitization. In contrast, Blake et al104 found that morphine and DAMGO pretreatment sensitized their subsequent actions on µ receptor function in HEK 293 cells and that neither agonist caused receptor desensitization, with only DAMGO causing receptor downregulation. However, methadone and buprenorphine, two opioids used clinically in treating opioid dependence, caused a pronounced receptor desensitization, suggesting that the therapeutic effect of these agents may be linked to the inhibition of receptor function.104 Studies have also shown that those compounds, fentanyl, sufentanil, lofentanil and nalbuphine, that desensitized the µ receptor, were not dependent on the Asp-114 residue for activation of the µ receptor to inhibit cAMP accumulation.70 The necessity of Asp-114 for morphine and levorphanol to stimulate the µ receptor, and the lack of its requirement for the fentanyl analogs and nalbuphine activation, indicate that these compounds have different determinants in the µ receptor for activation. By interacting with the µ receptor differently, the fentanyl analogs and nalbuphine may activate adaptive cellular responses that result in µ receptor/adenylyl cyclase uncoupling. In contrast, morphine may not stimulate these cellular pathways, even though fentanyl and morphine both bind to the same receptor and are equally effective in inhibiting adenylyl cyclase. Studies have also shown that receptor desensitization occurred independently of receptor internalization70,104 and that the internalization induced did not correlate with the magnitude of µ receptor desensitization.70 Binding and immunofluorescent techniques demonstrated that morphine had little effect on receptor internalization, while etorphine induced a rapid receptor sequestration and desensitization.70,105 However, lofentanil, which abolished coupling of the µ receptor to adenylyl cyclase, caused no greater magnitude of internalization than fentanyl, which caused only a small reduction in maximal accumulation of cAMP.70 While differences in receptor regulation may be an inherent property of the surrogate cell lines used in the different studies, these results suggest that opioids differ markedly in their abilities to regulate the µ receptor at the cellular level. A recent study has noted that the agonist regulation effects observed in µ receptor-transfected surrogate cell lines reflect biologically relevant actions that occur at the µ receptor in native tissue preparations.106 κ receptor

Agonist regulation studies on the opioid κ receptor have yielded contrasting results. Raynor et al107 were able to show a role of G protein-coupled protein kinases in the desensitization of the mouse κ receptor and the desensitization appeared to be independent of receptor downregulation. Tallent et al108 observed that agonist pretreatment of the mouse κ receptor resulted in a pronounced homologous desensitization of adenylyl cyclase activity, in addition to desensitizing a receptor-coupled K+ current. On the other hand, Blake et al109 found that agonist regulation of the human κ receptor was dependent on the agonist used, with κ-selective agonists desensitizing and downregulating the receptor, while nonselective agonists were without effect. In contrast, Avidor Reiss et al110 observed no agonist-mediated desensitization or downregulation with the rat κ receptor. At present, the cellular basis for the differences observed in the agonist regulation of the opioid v receptor remains unre-

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solved, although the differences may reflect properties unique to the surrogate cell lines used in the studies, or the species isoforms of the receptors studied. δ receptor

The opioid δ receptor appears to undergo desensitization and downregulation in response to δ-selective peptide and nonpeptide agonists more readily than either the µ or κ receptors.95,111-115 But like the µ and κ receptors, the δ receptor may also be capable of undergoing differential agonist regulation, since pretreatment with δ-selective peptides desensitized the receptor, whereas pretreatment with the δ-selective nonpeptide SIOM did not.95 Agonist-induced desensitization of the δ receptor appears to fit the general paradigm that has been described for G protein-coupled receptors,116 with agonist treatment uncoupling the receptor from G proteins111 and G protein-coupled protein kinases playing a role in attenuating the functional response.112 Agonist mediated sequestration and downregulation of the δ receptor appear to involve the receptor carboxyl terminal domain, with differences in the amino acids required for short-term receptor sequestration and downregulation.113,116 In this regard, agonist regulation of the opioid δ receptor appears to differ from that of the µ receptor, since studies indicate that the carboxyl terminal domain of the µ receptor is not necessary for receptor internalization89 but may be still necessary for desensitization.117

Electrophysiological Studies In Xenopus laevis oocytes expressing the cloned µ, δ or κ receptor and the cloned inward rectifier GIRK1, selective µ, δ and κ agonists increased K+ conductance.118-122 Similar results have been observed in the AtT20 cell line expressing the cloned µ receptor.108 In either systems expressing the µ receptor, the continuous presence of opiates uncoupled the µ receptor from the K+ channel and abolished the subsequent opiate potentiation of K+ conductance in a heterologous manner. In the cell line AtT20 expressing the κ receptor, pretreatment with agonist U50,488 resulted in desensitization which was homologous since AtT20 cells treated with U50,488 still responded to somatostatin agonists with an increase in K+ currents.108 Chen and Yu119 observed a differential regulation, by intracellular protein kinases, of the human µ receptor activation of an inwardly rectifying K+ current, as protein kinase C activation potentiated DAMGO-mediated desensitization of the response, whilst protein kinase A activation abolished the current desensitization. Mestek et al120 were able to demonstrate that intracellular protein kinases known to be dependent on phospholipase C activation were involved in potentiating the K+ current desensitization, as both protein kinase C and calcium\calmodulin-dependent protein kinase accentuated the desensitization elicited by DAMGO. Zhang et al123 confirmed the earlier observations on protein kinase C effects with the µ receptor and found that these effects also occurred with the κ receptor. The latter group proposed that two distinct pathways are involved in K+ current desensitization, based on observed differences in the time course of the agonist-mediated effect and the effects of the protein kinase C inhibitor staurosporine.123 In contrast, Kovoor et al118 suggested a post receptor mechanism for the agonist regulation of the K+ current in oocytes, with the desensitization occurring independently of protein kinase C activation. Hence opiates appear to have a wide and varied effect upon intracellular effector systems. However, it should be noted that adaptive responses occurring during administration of a particular opiate may not be identical in all opiate-sensitive neuronal populations and that a particular opiate may selectively desensitize some, but not all, intracellular functions.

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G Protein Role in Differential Agonist Activity The receptor binding differences exhibited by opioids suggests that different opioids may interact differently with the receptor and induce a myriad of intracellular effector systems. In support of this, there is also reasonable evidence to suggest that various opioid analgesics have different intrinsic efficacies and that these differences are likely due to the different abilities of these agonists to interact with the receptors and differentially activate G proteins.124,125 Hence opioids exhibit different efficacy and/or potency in the activation of different classes of G proteins.43,126 For example, antisense oligodeoxyribonucleotide against the subtype Gi2α protein antagonized morphine but not sufentanil-induced antinociception.124 Evidence also indicates that a single opioid receptor type can interact with several G proteins127 which, in turn, can couple to more than one effector128 and these may integrate coincident signals from different G protein subtypes.129 This coupling may differ in cell lines and neuronal membranes. For example, in neuroblastoma glioma hybrid NG108-15 cells, δ receptors are coupled to inhibition of adenylyl cyclase via Gi2,42,130 whereas in human neuroblastoma SH-SY5Y cells they are coupled mainly via Gi1 and Go.131 The coupling efficacy to G proteins may also differ, as it has been reported that the δ receptor is more efficiently coupled to Gi2 protein than the µ receptor.130,132 Hence multiple G protein subunits are able to influence the actions of a single opioid agonist. This suggests that although these compounds may be interacting with the same opioid receptor type, different intracellular effector mechanisms may be induced by them in producing their effects. Hence the molecular determinants of receptor recognition may be different than for cellular activation. The degree and efficiency of coupling to different cellular effectors in different systems may explain why agonist pretreatment has been shown to desensitize the coupling of the cloned µ, δ and κ receptors to K+ channels,109,133 and the δ and κ receptors from inhibition of adenylyl cyclase activity,95,107,112 whereas morphine has been reported not to uncouple the cloned µ receptor from adenylyl cyclase even though it effectively inhibited the activity of this enzyme.104 It may also explain the reported heterologous/homologous desensitization demonstrated for the opioid receptors. The type of desensitization reported, i.e., homologous or heterologous, seems to be dependent on the opioid receptor type and the intracellular effector under investigation. Interaction of the opioid receptor with the K+ channel may be of different magnitude and involve different G proteins than interaction with adenylyl cyclase. Hence, adaptive responses occurring during opiate administration may not be identical in all opiate-sensitive neuronal populations and opiates may selectively desensitize some, but not all, intracellular functions of the opioid receptor.

Conclusion The use of heterologous expression studies in examining opioid receptor structure and function, in combination with genetic manipulations on the cloned opioid receptors, has revealed receptor domains involved in opioid ligand recognition and in the cellular regulation of receptor function. However, binding of opiates and subsequent activation of intracellular function is agonist dependent. Agonists and antagonists appear to have different determinants for binding to and activating the opioid receptors. Furthermore, opioid compounds which produce tolerance readily, such as morphine, bind and activate the opioid receptor differently than opioids such as methadone and buprenorphine. These differences may be linked to long-term functional consequences associated with their use. Since the effectiveness of opioids in controlling chronic pain is limited by the undesirable side effects associated with long-term treatment, a better understanding of the molecular pharmacology of opioids is necessary. Modeling of the receptor-binding sites, together with the known structure of the synthetic opiates, should facilitate the rational development of new compounds with improved therapeutic profile and limited side effects.

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Abbreviations TIPP[ψ] DPDPE DADLE DSLET EKC SIOM BW373U86 NTB BNTX TAN-67 U50,488 nor-BNI U69,593 NMDA DAMGO CTOP CTAP Deltorphin II

H-Tyr-Ticψ(CH2-NH]-Phe-Phe-OH cyclic [D-Pen2, D-Pen5]enkephalin [D-Ala2, D-Leu5]enkephalin [D-Ser2, D-Leu5]enkephalin-Thr6 ethylketocyclazocine 7-spiroindanyloxymorphone (±)-4-{(α-R*)-α-[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl]-3hydroxybenzyl}-N,N-diethylbenzamide naltriben methanesulfonate 7-benylidenenaltrexone 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aαoctahydroquinolino [2,3,3,-g]isoquinoline trans-(±)-3,4-dichloro-N-methyl-N-[2(pyrrolidinyl)cyclohexyl]benzeneacetemide nor-binaltorphimine (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4.5)dec-8yl]-benzeneacetamide. N-Methyl-D-Aspartic acid [D-Ala2-MePhe4-Gly-ol5]enkephalin D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH2 D-Phe-Cys-Tyr-D-Try-Arg-Thr-Pen-Thr Tyr-D-Ala-Glu-Val-Val-Gly-NH2

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Index A α-melanocyte stimulating hormone (α-MSH)

29, 30, 42, 45, 55, 60, 68, 73, 87 α1-antichymotrypsin (ACT) 160-168 α1-antitrypsin portland (a1-PDX) 66

ACTH (adrenocorticotropin hormone) 10, 29, 30, 32, 34, 36-42, 61, 63, 80, 97, 107, 124, 127, 128, 130, 133, 135, 142 ADAM family of metalloproteases 60, 67, 68 Adrenocorticotropin 29, 30, 107, 124 Alzheimer’s disease 51, 161 Amastatin 133, 176, 177 Aminopeptidase 36, 90, 95, 96, 121, 123, 133-135, 174-178, 181 Aminopeptidase N 175, 176, 177 Angiotensin converting enzyme 175, 178, 181 Antisense 34, 41, 61, 63, 65, 66, 81, 82, 132, 160, 163, 204 Arterial restenosis 65 Aspartyl protease 67, 94, 97, 98, 100, 101, 121, 135, 159, 161 Atherosclerosis 65

Carboxypeptidase Z (CPZ) 130-132 Catalytic domain 51, 53, 55, 65, 77, 78, 148, 149, 176 Chaperone 3, 13, 51, 79, 80, 109, 144, 148, 149, 151, 152 Cholecystokinin 38, 63 Cholesterol metabolism 51 Chromaffin granule 36, 79, 92, 93, 95, 97, 99-101, 123, 127, 128, 133, 134, 161, 162 Chromogranin 9, 16, 32, 52, 81, 141, 159 Constitutive secretory pathway 1, 31, 49, 59, 66, 77 Cysteine protease 92, 93, 99-101, 121, 159, 161, 162, 166

D DAMGO 194, 195, 198-200, 202, 203, 205 Dipeptidyl peptidase IV (DPP IV) 179 Diprotin A and B 179 DPDPE 194, 195, 199, 200, 205 Dynorphin 81, 82, 130, 176, 178-180, 192, 194, 197, 200

B

E

β-endorphin (β-END) 29, 30, 36, 38-42, 50,

Ectoenzyme 173, 177-181 Ectopeptidase 173 Endo-oligopeptidase A 181 Endomorphin 193, 194 Endoprotease 13, 29, 34, 41, 82, 89, 105-107, 110, 112, 134, 146, 147, 149, 174, 179, 180-182 Endoproteolytic processing 34, 89, 90, 121, 123, 133-135 Endothelin converting enzyme 175 Enkephalin 81, 82, 92-94, 97, 99, 100, 121-123, 126-128, 130, 133, 161-163, 174-178, 192-195, 198, 205 Enkephalinase 173-177 Exocytosis 1, 4, 8, 12, 31, 112 Exopeptidase 34, 106, 112, 121, 124, 133, 134, 176

97, 107, 180, 192-194 β-lipotropic hormone (β-LPH) 29, 30, 36, 38-42, 55, 107 Bestatin 133, 176, 177 Biosynthesis 29, 79, 99, 105, 106, 110-116, 127, 129, 142, 147, 152, 179, 198 Buprenorphine 195, 198, 202, 204

C CALLA 174 Captopril 178 Carboxypeptidase D (CPD) 131, 132 Carboxypeptidase E (CPE) 10, 11, 13-15, 32-35, 42, 132 Carboxypeptidase E (CpE) 5, 32, 35, 82, 121, 123 Carboxypeptidase E/H (CPE/H) 29, 121, 123-132, 134, 135 Carboxypeptidase H (CPH) 32, 80, 106, 109, 115, 121, 123

F Furin 5-7, 12, 13, 39, 49, 51, 53, 55, 59-64, 66-68, 77-79, 81, 82, 146-149, 151

214

Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing

G

O

γ-endorphin generating enzyme (γ-EGE) 180 Gene expression 29, 105-109, 127 Glucose 2, 79, 83, 105-107, 110-116, 127, 148 Gluzincin 174, 176, 177 GroEL 144 Growth factor 59, 63, 65, 66, 142, 147 Guanine nucleotide binding protein 108, 195-197, 199, 201-204 Guanine nucleotide binding protein (G protein) 5

Obesity 64, 65, 83, 121, 129, 130 Opiate 29, 191, 194-201, 203, 204 Opioid receptor 191-199, 201, 204 Orphan receptor 176

I Immature secretory granule (ISG) 3-5, 8-16, 29, 31, 35, 49, 51, 67, 146-150 Inactivation 64-66, 175 Insulin degrading enzyme (IDE) 180, 181 Integrin 50, 55, 67, 68, 79 Inverzincin 174, 180

K KELL blood group protein 175 Kexin 49, 51, 53-55, 59, 68

M Metalloprotease 67, 124, 131, 133, 134, 180 Methadone 191, 194, 195, 198, 202, 204 Morphine 191, 192, 194, 195, 198-202, 204 MRNA 38-41, 59-63, 65, 68, 79, 80, 82, 89, 105-113, 115, 116, 126, 127, 131, 142, 144, 166, 175, 178 Mutagenesis 32, 148, 174, 198, 199

N Neprilysin 174-176, 180 Neuroendocrine 9, 10, 34, 39, 77, 89, 90, 101, 105, 121, 122, 124-128, 130, 134, 141, 142, 144-149, 151, 152, 159-161, 163-166, 168 Neurolysin 181 Neuropeptide 29, 38, 89, 90, 92, 94, 101, 121-123, 125-130, 133-135, 141, 147, 149, 152, 159-161, 168, 173, 174, 179-182 Nociceptin 176, 193, 196-198

P Peptide hormone 1, 3, 5, 12, 14, 30, 31, 34, 38, 89, 101, 105, 106, 110, 121-123, 126, 127, 130, 135, 141, 142, 145, 147, 149, 150, 152, 159, 168 Peptide neurotransmitter 1, 159 Pituitary 8, 29, 30, 32-34, 36-42, 59-61, 79, 80, 90, 91, 94, 100, 101, 107-109, 122, 124-134, 141, 142, 144-149, 159, 161, 162, 164-166, 168 POMC sorting signal 32, 33 Posttranslational modification 34, 110, 129, 145 Precursor convertase 49, 51, 68 Precursor convertase (PC) 49-51, 53-55, 57, 59, 60-68, 77-83, 94, 95, 97, 98, 100, 101, 106-112, 114-116, 121, 130, 131, 135, 142, 146-152, 159, 161, 164 Proenkephalin 32, 34, 37, 79, 81, 92-94, 97-101, 121, 122, 142, 159, 161-163, 192 Prohormone 1, 3-5, 7-16, 29, 31, 32, 34, 36-39, 42, 65, 77-83, 89-95, 97, 98, 100, 101, 105-110, 112, 121-125, 129, 130, 132-135, 141, 142, 146-152, 159-161, 163, 164, 166, 168 Prohormone convertase 1, 5, 12, 16, 29, 34, 37-39, 77, 78, 83, 94, 101, 105, 121, 130, 135, 142, 146, 147, 152, 159, 160, 164 Prohormone processing 4, 10, 14, 15, 36, 38, 42, 77, 78, 81, 82, 89-92, 98, 100, 101, 105, 107, 110, 121-124, 129, 130, 133, 134, 135, 146, 149, 159-161, 163, 166, 168 Prohormone thiol protease (PTP) 37, 92-101, 121, 134, 135, 159-164, 166, 168 Proinsulin 3, 5, 7, 9, 10, 32, 34, 36, 41, 42, 50, 65, 83, 90, 91, 97, 101, 106-116, 121, 122, 130, 134, 142, 147 Promoter 63, 80, 82, 92, 107-109, 142, 175 Proneuropeptide 31, 32, 37, 39, 63, 65, 77, 81, 92, 97, 159, 160 Proopiomelanocortin (POMC) 9, 10, 13, 29-42, 50, 55, 63, 65, 80, 81, 94, 97, 98, 100, 101, 106-110, 121, 122, 124, 130, 132-135, 142, 146, 147, 159, 161, 192

Index ProPC2 maturation 13, 147, 149 Proprotein convertase 67, 105, 110, 143, 144, 146, 151 Protease inhibitor 36, 99, 100, 124, 131, 134, 159-163, 166, 168 Proteolysis 12, 13, 49, 65, 89, 90, 105, 129, 160

R Reactive site loop (RSL) 160, 161, 164-166, 168 Receptor desensitization 202, 203 Receptor internalization 173, 202, 203 Receptor mutagenesis 198, 199 Receptor subtype 194-196, 198, 199, 201 Regulated secretory pathway 3, 8, 10, 11, 31, 32, 34, 39, 42, 49, 59, 77, 78, 89, 107, 129, 132, 141, 145, 146, 148, 159, 160 Regulation 1, 4, 12, 34, 39, 40, 42, 51, 59, 60, 68, 80, 83, 99, 105-108, 110-115, 127, 128, 135, 142, 152, 164, 176, 178, 179, 181, 201-204 Renin 50, 52, 67, 81

215 Secretogranin 81, 141 Secretory granule 1, 3, 8, 11, 16, 31-35, 41, 42, 49, 51, 55, 59, 66, 67, 77, 79, 81, 110, 129, 141, 142, 144, 146-150, 173 Secretory vesicle 1, 15, 36, 42, 77, 81, 89-93, 97, 99-101, 122-125, 128, 129, 131-134, 160-163 Serine proteinase 49, 59, 68 Serpin 66, 160-162, 164-167 Seven-transmembrane protein 196, 197 Sorting receptor 3, 5, 10, 32-34, 42, 132 Subtilisin 38, 49, 51, 54, 55, 59, 68, 77, 78, 83, 94, 100, 101, 121, 135, 144, 149, 159, 160, 161, 164, 174, 179

T Trans-Golgi (TGN) 1, 3-16, 29, 31, 32, 34, 35, 49, 51, 55, 59, 66, 67, 145, 147-150, 160 Transcription factor 107-109, 142 Translational regulation 105, 110, 112, 113

V Viral surface glycoprotein 66

S Secretase 52 Secretion 1-5, 11, 12, 16, 32, 34, 36, 55, 59, 78, 80, 89, 105-107, 112, 128, 132-134, 141, 142, 145-147, 149

Z Zymogen 51, 55, 59, 67, 97, 149, 168