Neurokinin 1

Neurokinin 1 Joel V. Weinstock* Division of Gastroenterology-Hepatology, Department of Medicine, University of Iowa, 4607 JCP, Iowa City, IA 52242, USA * corresponding author tel: 319-354-2250, fax: 319-353-6399, e-mail: [email protected] DOI: 10.1006/rwcy.2001.23011.

SUMMARY The NK1 receptor is a G protein-coupled, seven transmembrane receptor. The gene and mRNA encoding the NK1 receptor have been cloned from several species. Substance P is the natural high-affinity ligand for this receptor. The receptor desensitizes then gradually resensitizes following repeat substance P exposure. The NK1 receptor is displayed extensively throughout the body. It appears prominently in various regions of the brain, spinal cord, lungs, and intestines. It is expressed on neurons, vascular endothelium, intestinal epithelium, lymphocytes and other cell types of the immune system. In the immune system, various cytokines as well as T cell receptor engagement can induce NK1 expression. Stimulation of the NK1 receptor excites several distinct intracellular second messenger systems. Activation of the receptor affects immunoregulation, cardiorespiratory physiology, neurotransmission, and intestinal secretion and absorption. It also influences neuronal survival and helps regulate the emetic reflex, various behavioral responses, vascular dilatation, and vascular permeability. There are several nonpeptide NK1 receptor antagonists undergoing clinical evaluation for management of emesis, depression, and other pathophysiological processes.

BACKGROUND

Discovery Substance P is the natural high-affinity ligand of the NK1 receptor. Substance P was purified and sequenced in 1971 (Chang et al., 1971). In the late

Cytokine Reference

1970s, reports suggested that rat synaptic membranes displayed high-affinity binding sites for substance P. The amino acid sequence of the rat NK1 receptor was deduced by molecular cloning in 1989 (Yokota et al., 1989). The structure of the first highly specific, nonpeptide NK1 antagonist was published in 1991 (Snider et al., 1991).

Alternative names Substance P receptor.

Structure The NK 1 receptor is a member of the superfamily of G protein-coupled seven transmembrane receptors (Ohkubo and Nakanishi, 1991). Hydrophobic domains form  helices that span the cell membrane seven times. Figure 1 shows the potential N-glycosylation sites (triangles) and conserved amino acids (solid circles) among the three tachykinin receptors (NK1, NK2, NK3) and a possible palmitoylation site represented by a zigzag line. The intracytoplasmic C-terminal conformation presumably determines the type of biological activity displayed. The actual threedimensional structure is unknown.

Main activities and pathophysiological roles The wide distribution of the NK1 receptor and substantial additional evidence suggest that this receptor has many functions (Quartara and Maggi, 1997). The NK1 receptor is involved in pain transmission in

Copyright # 2001 Academic Press

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Joel V. Weinstock Figure 1 Model of the NK1 receptor.

system inflammation in response to Trypanosoma brucei infection (Kennedy et al., 1997). Mouse models demonstrate that activation of the NK1 receptor can significantly upregulate immune inflammatory reactions. However, the importance of NK1 receptors in human disease is unknown. There are no known diseases directly attributed to loss or overexpression of the NK1 receptor. Published Assays and Other Research Tools for Studying the NK1 Receptor

peripheral nerves and the spinal cord (Mantyh et al., 1997), and in expression of neurogenic inflammation (McDonald et al., 1996). In the CNS, it influences neuronal survival and helps to regulate the emetic reflex, cardiovascular, and respiratory functions, and various behavioral responses. Other functions include stimulation of vasodilatation and enhancement of vascular permeability. In addition, it has a role in intestinal secretion, motility, and neuro-neuronal communication. While substance P has various immunoregulatory functions (Maggi, 1997), the role of the NK1 receptor is less clearly defined because substance P may bind to other receptors in addition to NK1. However, recent publications clearly implicate the NK1 receptor in immune modulation and susceptibility to infection. Studies in vivo and in vitro using specific NK1 receptor antagonists and mice deficient in NK1 receptors have shown the importance of this receptor in mediating the T cell IFN response in murine schistosomiasis (Weinstock and Elliott, 1998). This regulation is via a NK1 receptor located on the T cell. Mice pretreated with a substance P receptor antagonist are more susceptible to intestinal salmonellosis, showing a decreased IFN response in the intestine (Kincy-Cain and Bost, 1996). Clostridium difficile is a bacterium that can release toxins, which induces colitis in humans. The NK1 receptor helps to mediate the inflammatory diarrhea and mucosal injury induced by C. difficile toxin A (Castagliuolo et al., 1998). Mice with disruption of the NK1 receptor gene are less susceptible to immune complex-induced, pulmonary injury (Bozic et al., 1996) and IL-1-induced, neutrophil migration (Ahluwalia et al., 1998). Mice receiving an NK1 receptor antagonist develop less severe central nervous

1. Monoclonal and polyclonal antibodies for NK1 receptor identification via immunohistochemistry. 2. Autoradiographic localization of NK1 receptors in tissue sections. 3. Polyclonal antibody for Western blot identification of receptor protein. 4. PCR (Blum et al., 2000; murine) and RNase protection (Gautreau and Kerdelhue, 1998; rat) assays for quantification of receptor mRNA. The PCR assay can detect and measure <100 NK1 receptor mRNA transcripts in 0.3 g of total RNA. 5. Highly selective human and murine nonpeptide NK1 receptor antagonists. 6. NK1 receptor knockout mouse on 129C57BL/6, C57BL/6, and CBA backgrounds. 7. Various human and murine cell lines (i.e. lymphocytes, macrophages) that express the NK1 receptor.

GENE

Accession numbers Human gene: X65177, X65178, X65179, X65180, X65181 Human mRNA: M76675 Rat gene: M64232, M64233, M64235, M64236, M34751, M34752, M34754, M34755 Rat mRNA: M31477 Mouse mRNA: X62934

Sequence See Figures 2, 3, 4, 5, and 6. Sequence data beyond that shown here is available at: www.ncbi.nlm.nih. gov/.

Chromosome location and linkages A single gene encodes the NK1 receptor. In humans, it is located on chromosome 2 (Gerard et al., 1991). Reports suggest that mammalian tissue also may

Neurokinin 1

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Figure 2 Human gene sequence. The complete sequences of the five exons and partial sequences of the four introns are shown. The TATA box is in bold (1339±1342) and the exons are underlined (Takahashi et al., 1992).

produce a truncated isoform of the NK1 receptor that is of unknown physiological significance.

PROTEIN Human, rat, and mouse NK1 receptors all contain 407 amino acids. The deduced amino acid sequence of the rat is 99% identical to that of the mouse, but differs by 22 residues from that of human. These

variations do not affect substance P binding or signaling. However, they do explain species-dependent variation in potency of various pharmacological receptor antagonists.

Accession numbers SwissProt: Human: P25103 Mouse: P30548

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Joel V. Weinstock

Sequence See Figure 7. Figure 3 Human mRNA sequence (Gerard et al., 1991).

Relevant homologies and species differences The mammalian tachykinin system consists of three distinct peptides called substance P, neurokinin A, and neurokinin B. Their receptors are designated NK1, NK2, and NK3, respectively. The three receptors share homology (Table 1). The substance P receptor (NK1) also can bind neurokinin A and neurokinin B.

Affinity for ligand(s) Substance P binds the NK1 receptor with high affinity (Ki < 10ÿ9 M). The rank order of affinity for the various tachykinins for the substance P NK1 binding site is substance P  neurokinin Figure 4 Rat gene sequence (Hershey et al., 1991). Complete sequences of the five exons and partial sequences of the four introns. Exons are underlined. Source: rat (strain Fisher) DNA. The chromosome origin is unknown.

Neurokinin 1 Figure 5 Rat mRNA sequence (Hershey and Krause, 1990).

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Figure 7 Amino acid sequences for NK1 receptor of human (protein ID: CAA46292.1, PID: g825721, GI: 8 25 72 1, S w i s s P r o t: P 25 10 3) , r a t ( p ro t e i n ID : AAB59726.1, PID: g1196819, GI: 1196819), and mouse (protein ID: CAA44707.1, PID: g54207, GI: 54207, MGD:MGI: 98475, SwissProt: P30548). The seven transmembrane domains are underlined.

Figure 6 Mouse mRNA sequence (Sundelin et al., 1992). The chromosome origin is unknown.

Table 1 Homology among the tachykinin receptor subtypes

A > neurokinin B. However, it recently has been suggested that the NK1 receptor actually has two distinct high-affinity binding sites, one for substance P and one for neurokinin A. The latter site is located at least in part at the distal end of the second extracellular loop (Wijkhuisen et al., 1999). The binding site for substance P appears to require regions of transmembrane domains I, II, and VII, and the N-terminus (Berthold and Bartfai, 1997).

Cell types and tissues expressing the receptor In many instances, the precise location and cellular distribution of NK1 receptors remain controversial. Many of the techniques used and reagent employed

NK1

NK2

NK3

Amino acid residues

407

390

452

Molecular weight

46,364

43,851

51,104

Homology in amino acid sequence

54% to NK2 66% to NK3

55% to NK2

could not fully differentiate NK1 receptor binding from among the other tachykinin receptors. There is strong evidence, however, that the NK1 receptor is displayed extensively in various regions of the brain and spinal cord. It also is expression on vascular endothelial cells (Greeno et al., 1993). In various regions of the intestines, there are NK1-binding sites in the smooth muscle layers, submucosa, epithelium and ganglia of the enteric plexuses. The renal pelvis, ureter, bladder, and pulmonary microvasculature (Bowden et al., 1996) are other probable sites of NK1 receptor expression. The salivary glands also have NK1 receptors. Lymphoid organs and immunocytes can express NK1 receptors. There are NK1 receptors in germinal centers of mesenteric lymph nodes and in intestinal lymphoid tissue. Reports suggest that lymphocytes and macrophages can express this receptor in both

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Joel V. Weinstock

human and other mammalian species (Cook et al., 1994; Ho et al., 1997; Goode et al., 1998).

Regulation of receptor expression At least in some cells, desensitization followed by gradual re-sensitization is the physiological response to repeat substance P exposure. Studies using Chinese hamster ovary (CHO) cells and other receptor-transfected cell lines suggest that NK1 receptor desensitization following substance P engagement requires receptor phosphorylation. G protein-coupled receptor kinases like GRK2 (Nishimura et al., 1998) phosphorylate the receptor, while -arrestins uncouple the phosphorylated receptor from the heterotrimeric G proteins (McConalogue et al., 1999; Barak et al., 1999). This terminates signal transduction. There are reports of a second isoform of the NK1 receptor that is truncated at the C-terminus. When transfected into CHO cells, this receptor does not undergo rapid and prolonged desensitization upon exposure to substance P (Li et al., 1997). Substance P also stimulates clathrin-mediated endocytosis and recycling of the NK1 receptor (Garland et al., 1996), which is an important part of the process of receptor re-sensitization. Multiple domains in the receptor's intracellular C-tail and transmembrane domain VII are important for this endocytosis (Sasakawa et al., 1994; Bohm et al., 1997). The substance P dissociates from its receptor in acidified endosomes (Grady et al., 1995), and the refurbished, dephosphorylated receptor is brought back to the cell surface. T cells can express the NK1 receptor. T cell receptor engagement, IL-12, IL-18, and TGF all can trigger and upregulate T cell NK1 receptor mRNA expression and display (Blum et al., 2000). NK1 display is more prominent at sites of inflammation.

Release of soluble receptors The NK1 receptor anchors to the plasma membrane and is not released in a soluble form.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases The receptor is coupled to one or more G proteins (Gq/11, Gs and Go) (Roush and Kwatra, 1998). Stimulation of NK1 receptors expressed in several

experimental cell systems can excite various second messenger pathways. Ligand binding can activate phospholipase C and D (Torrens et al., 1998). This generates I(1,4,5)P3 and increases [Ca2+]. Also activated are arachidonic acid release, adenylyl cyclase and phospholipase A2(1,2,3) (Grady et al., 1995).

THERAPEUTIC UTILITY

Effect of treatment with soluble receptor domain There are several nonpeptide NK1 receptor antagonists undergoing clinical evaluation. Reports suggest that they may prove useful for controlling depression (Kramer et al., 1998), emesis (Navari et al., 1999) and exercise-induced bronchoconstriction in asthma (Ichinose et al., 1996).

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substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization. Proc. Natl Acad. Sci. USA 94, 9475±9480. McConalogue, K., Dery, O., Lovett, M., Wong, H., Walsh, J. H., Grady, E. F., and Bunnett, NW (1999). Substance P-induced trafficking of beta-arrestins. The role of beta-arrestins in endocytosis of the neurokinin-1 receptor. J. Biol. Chem. 274, 16257± 16268. McDonald, D. M., Bowden, J. J., Baluk, P., and Bunnett, N. W. (1996). Neurogenic inflammation. A model for studying efferent actions of sensory nerves. Adv. Exp. Med. Biol. 410, 453±462. Maggi, C. A. (1997). The effects of tachykinins on inflammatory and immune cells. Regul. Pept. 70, 75±90. Mantyh, P. W., Rogers, S. D., Honore, P., Allen, B. J., Ghilardi, J. R., Li, J., Daughters, R. S., Lappi, D. A., Wiley, R. G., and Simone, D. A. (1997). Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278, 275±279. Navari, R. M., Reinhardt, R. R., Gralla, R. J., Kris, M. G., Hesketh, P. J., Khojasteh, A., Kindler, H., Grote, T. H., Pendergrass, K., Grunberg, S. M., Carides, A. D., and Gertz, B. J. (1999). Reduction of cisplatin-induced emesis by a selective neurokinin-1-receptor antagonist. L-754,030 Antiemetic Trials Group. N. Engl. J. Med. 340, 190±195. Nishimura, K., Warabi, K., Roush, E. D., Frederick, J., Schwinn, D. A., and Kwatra, M. M. (1998). Characterization of GRK2catalyzed phosphorylation of the human substance P receptor in Sf9 membranes. Biochemistry 37, 1192±1198. Ohkubo, H., and Nakanishi, S. (1991). Molecular characterization of the three tachykinin receptors. Ann. N.Y. Acad. Sci. 632, 53±62. Quartara, L., and Maggi, C. A. (1997). The tachykinin NK1 receptor. Part I: ligands and mechanisms of cellular activation. Neuropeptides 31, 537±563. Roush, E. D., and Kwatra, M. M. (1998). Human substance P receptor expressed in Chinese hamster ovary cells directly activates G(alpha q/11), G(alpha s), G(alpha o). FEBS Lett. 428, 291±294. Sasakawa, N., Ferguson, J. E., Sharif, M., and Hanley, M. R. (1994). Attenuation of agonist-induced desensitization of the rat substance P receptor by microinjection of inositol pentakis-and hexakisphosphates in Xenopus laevis oocytes. Mol. Pharmacol. 46, 380±385. Snider, R. M., Constantine, J. W., Lowe, J. A., Longo, K. P., Lebel, W. S., Woody, H. A., Drozda, S. E., Desai, M. C., Vinick, F. J., and Spencer, R. W. (1991). A potent nonpeptide antagonist of the substance P (NK1) receptor. Science 251, 435±437. Sundelin, J. B., Provvedini, D. M., Wahlestedt, C. R., Laurell, H., Pohl, J. S., and Peterson, P. A. (1992). Molecular cloning of the murine substance K and substance P receptor genes. Eur. J. Biochem. 203, 625±631. Takahashi, K., Tanaka, A., Hara, M., and Nakanishi, S. (1992). The primary structure and gene organization of human substance P and neuromedin K receptors. Eur. J. Biochem. 204, 1025±1033. Torrens, Y., Beaujouan, J. C., Saffroy, M., Glowinski, J., and Tence, M. (1998). Functional coupling of the NK1 tachykinin receptor to phospholipase D in chinese hamster ovary cells and astrocytoma cells. J. Neurochem. 70, 2091±2098. Weinstock, J. V., and Elliott, D. (1998). The substance P and somatostatin interferon-gamma immunoregulatory circuit. Ann. N.Y. Acad. Sci. 840, 532±539.

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ACKNOWLEDGEMENTS Grants from the National Institutes of Health (DK38327, DK07663, DK25295), the Crohn's and Colitis Foundation of America, Inc. and the Veterans Administration supported this research.