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30 Ali, H., Richardson, R. M., Tomhave, E. D., DuBose, R. A., Haribabu, B. and Snyderman, R. (1994) J. Biol. Chem. 269,24247-24254 31 Alaluf, S., Mulvihill, E. R. and McIlhinney, R. A. J. (1995) FEBS Lett. 367,301-305 32 Sasamura, H., Dzau, V. J. and Pratt, R. E. (1994) Kidney Int. 46, 1499-1501
33 Oppermann, M., Freedman, N. J., Alexander, R. W. and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13266- 13272
Received 14 March 1997
Structure and function of neurohypophysial hormone receptors M. Wheatley*§, J. Howl*, N. J. Yarwood*, S. R. Hawtin*, A. R. L. Daviest, G. MatthewsS and R. A. Parslow* *School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B I 5 2TT, U.K., tSchool of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K., and *Department of Surgery, The Medical School, University of Birmingham, Edgbaston, Birmingham B I 5 2TH, U.K.
Introduction T h e neurohypophysial hormones, [Arg'Jvasopressin (AVP) and oxytocin, exhibit a high degree of structural similarity. They are both composed of a 20-membered ring formed by a disulphide bond and tripeptide tail. Seven of the nine residues in AVP and oxytocin are identical. T h e divergence in sequence occurs at residues 3 and 8, which are located in the ring and tripeptide tail respectively. Despite this homology, AVP and oxytocin fulfil discrete physiological functions. Effects of AVP include increased blood pressure resulting from contraction of vascular smoothmuscle cells, antidiuresis and stimulation of corticotropin (ACTH) release [l]. Many actions of AVP and oxytocin have been reported in the central nervous system, where they have a neuromodulator/neurotransmitter role. However, the best-documented responses to oxytocin are an increase in the frequency and intensity of uterine contraction at parturition and the contraction of myoepithelial cells resulting in milk ejection from the mammary gland [2]. These various effects of AVP and oxytocin are mediated by specific G-protein-coupled receptors (GPCRs) expressed by target tissues. T o date, three subtypes of vasopressin receptor (VPR) have been identified. V,, receptors (VlaRs) are widely distriAbbreviations used: AVP, [arginine*]vasopressin;AVT, [arginines]vasotocin;EC domain, extracellular domain; GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; OTR, oxytocin receptor; VI,R, V1, vasopressin receptor; VlbR,Vlb vasopressin receptor; V2R, V, vasopressin receptor; VPR, vasopressin receptor; VTR, vasotocin receptor; r, rat; h, human; TM' domain, transmembrane domain. $To whom correspondence should be addressed.
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buted and generate the pressor effect of AVP. In contrast, Vlb receptors (VlbRs) have a more restricted distribution. In particular, they are expressed by corticotrophs where they regulate corticotropin secretion. Extrapituitary expression of VlbRs has also been reported, with VlbR mRNA detected in several tissues including kidney, spleen, uterus [3] and adrenal medulla [4]. T h e antidiuretic effect of AVP is mediated by V, receptors (V2Rs) in the kidney medulla. Although some pharmacological studies have indicated the possible existence of oxytocin receptor (OTR) subtypes [5,6], low-stringency hybridization studies of human genomic DNA with human (h)OTR cDNA revealed the existence of only one OTR [7]. This implies that one protein, functioning in a wide range of cellular contexts, is responsible for mediating all of the actions of oxytocin. V1,R, VlbR and OTR all stimulate phosphoinositidase C via coupling to Gqill,leading to an inositol 1,4,5-trisphosphate-induced rise in intracellular Ca2+ and protein kinase C activation. V2R stimulates adenylate cyclase via Gs. OTR and all three VPRs have now been cloned from several species and shown to display the characteristic structural motifs of GPCRs [8- 131, including seven putative transmembrane domains (TMs). Overall amino acid sequence homology between the neurohypophysial hormone receptors is about 35-6096, and consequently they are considered to be a subfamily of GPCRs. In the lower vertebrates, the mammalian hormones AVP and oxytocin are replaced by closely related analogues [Arg']vasotocin (AVT) and isotocin. Receptors for AVT and isotocin have been cloned from teleost fish and shown to be members of the VPR/OTR family [14,15], as has the receptor for the invertebrate AVP-related
G-Protein-Coupled Receptors for Peptide Hormones
peptide Lys-conopressin [16]. T o date, the AVT receptor is unique amongst the neurohypophysial hormone receptor family in possessing an additional domain at the C-terminus, which is composed of five repeats of a 12-residue motif. This is somewhat reminiscent of the C-terminal multiple-repeat domain reported for squid rhodopsin [ 171, except that the rhodopsin repeat is prolinerich whereas the AVT receptor repeat provides multiple putative phosphorylation sites.
Glycosylation of the rat (r)V,,R In general, a characteristic structural feature of GPCRs is that they possess at least one consensus sequence for N-linked glycosylation (N-X-S/T) in their N-terminal domain, although exceptions do occur. For example, the A2 adenosine receptor lacks sites at the N-terminus but does have sites in the second extracellular loop [ 181. Furthermore the a 2 b adrenergic receptor does not contain any putative glycosylation sites [18]. All of the VPR/OTR family have putative glycosylation sites at the N-terminus (Figure 1). However, V1,R is unusual in this family in possessing additional conserved glycosylation sites in the second and third extracellular loops AS^'^^ and respectively in the rV,,R; see Figure 1). The rVzR also has one additional site
but this is not conserved in either porcine or human VzR. For membrane proteins in general, it has been found that not all putative sites are actually glycosylated. For glycosylation to occur, the tripeptide acceptor sequence must be able to form a favourable conformation to provide the correct hydrogen-bonding and accessibility for the oligosaccharidetransferase [ 191. In addition, because of the co-translation nature of the modification, the period for which a peptide is accessible for glycosylation may be quite brief [20]. For the vast majority of GPCRs, it is not known if the putative glycosylation sites are actually modified. An exception to this is the gonadotropin-releasing hormone (GnRH) receptor, which, like VI,R, has putative N-glycosylation sites on both the N-terminus and an extracellular loop. Interestingly, only the sites at the N-terminus are actually modified [21]. This study was to determine (i) how many of the four putative glycosylation sites of the rV1,R are actually utilized and (ii) to establish if the glycosylation of the extracellular loops contributes to Vl,R-specific pharmacology or cell-surface expression. A rabbit reticulocyte lysate preparation is often used as a general translation system but this does not support glycosylation. The Xenopus oocyte provides an appropriate cellular context
Location of putative N-glycosylation sites on members of the VPR/OTR family of receptors Putative glycosylation sites (N-X-Sn) are indicated by ?.
P
?
?
plasma membrane
I hVlbR
plasma membrane
hV2R
hOTR
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for expressing GPCRs. This has enabled these oocytes to be widely used for expression cloning and characterization of GPCRs. T o study rVIaR glycosylation, we used a cell-free extract of unfertilized Xenopus laeois eggs, treated with nuclease to remove endogenous mRNA and supplemented with the S-100 function of rabbit reticulocyte lysate. This translation system has been characterized in detail [22] and found to be dependent on exogenous mRNA, capable of segregation and suitable for qualitative analysis of glycosylation. Using [35S]methionineto generate a labelled translation product, SDS/PAGE revealed that rVIaR was glycosylated by the Xenopus extract, resulting in a product with reduced mobility compared with the reticulocyte lysate product (Figure 2). Efficient segregation of rVIaR into membrane was observed, with no receptor detected in the supernatant. Interestingly, the tripeptide acetyl-Asn-Tyr-Thr-amide (NYT) which represents an N-glycosylation consensus site, was able to completely inhibit rVIaR glycosylation, generating a product that co-migrated with that from the reticulocyte lysate (Figure 2). Titration with increasing concentrations of the
peptide NYT produced a gradual inhibition of N-glycosylation, revealing the existence of four products representing four different glycosylation states. When analysed by SDSIPAGE, each had a different apparent mass resulting from a difference in the number of sites modified, such that bands were detected at 35 kDa (unglycosylated), 39 kDa (one glycosylation site utilized), 43 kDa (two sites utilized) and 46 kDa (three sites). These data establish that only three of the four putative N-glycosylation sites in rVIaR are utilized. As to the identity of the non-glycosylated site, it was reasoned that in the third extracellular loop was the most likely candidate. The consensus sequence (N-X-S/T) for this site has Pro334 at position X. Often N-P-S/T sequences do no act as glycosyl acceptors as they cannot adopt the necessary conformation [23]. VIaR also has a proline residue distal to the cognate asparagine in this domain, so it too would be conformationally constrained. Further support is provided by the sheep VIaR sequence, which has only three putative sites (corresponding to Asn14, Asn" and Ami9*).An Asn is conserved in the third loop of the sheep VIaR (corresponding to but it cannot be glycosylated, as the
In vitro translation of the rV,,R Translation products from the reticulocyte lysate system after addition of water (RLO) or V,,R mRNA (RLV) were analysed by SDVPAGE. The translation products from the Xenopus egg extract system were separated into a membrane and supernatant fraction by centrifugation (5 min at low speed, 10 min at high speed in a benchtop Microfuge). Membranes from the translation reactions in the absence (XM -) or presence (XM +) of the competing tripeptide (N-Y-T) were analysed separately from the supernatants from the reactions without (XS -) or with (XS+) tripeptide. Molecular-mass markers are indicated. The position of the dye front is marked DF.
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N-glycosylation consensus site is disrupted by an Ala replacing the necessary Ser/Thr [24]. It is reasonable to assume that the two putative sites in the N-terminus (AsnI4 and AsnZ7)are glycosylated, as (i) they are a common feature of GPCRs in general and (ii) all of the VPR/OTR family possess such sites (Figure 1). Indeed, glycosylation of this domain has been demonstrated for VzR [25] and OTR [26]. Our data would therefore indicate that, unlike the GnRH receptor [21], rVI,R is glycosylated on an extracellular loop in addition to the N-terminal domain. T o confirm that the putative glycosylation site in the second extracellular loop of V1,R is modified, it was disrupted by mutating to GlnI9* using PCR protocols. This also enabled the role of this glycosylation site in rVl,R-specific pharmacology to be addressed. Wild-type and N198Q rVI,R were translated by Xenopus egg extract and analysed by SDSlPAGE. The major glycosylated translation product of the mutant exhibited increased mobility compared with the wild-type receptor (apparent mass of 43 and 46 kDa for N198Q and wild-type respectively). In the presence of NYT peptide, the unglycosylated product of the two constructs was identical (35 kDa). The decrease in apparent molecular mass of the N198Q mutant is entirely consistent with the loss of a single oligosaccharide modification site, confirming that the second extracellular loop of rVI,R is glycosylated. It has been established by peptide-mimetic [27], mutagenesis [28,29] and photoaffinity-labellinglradiosequencing [30] strategies that the extracellular (EC) domains of VPRs have a function in ligand recognition. Consequently, it was important to address whether glycosylation of the second extracellular loop contributed to rVI,Rspecific pharmacology or function. After subcloning into the eukaryote expression vector pcDNA3 (Invitrogen), N198Q mutant and wildtype rV1,Rs were expressed in COS-7 cells. Radioligand-binding studies utilized four different classes of ligand: agonist, cyclic peptide antagonist, linear peptide antagonist and non-peptide antagonist. These revealed that the pharmacological profile and expression of the glycosylation-defective mutant were not significantly different from wild-type rVI,R (results not shown). This is in contrast with the GnRH receptor, where disruption of any one of the two N-glycosylation sites does not affect ligand binding, but results in a marked decrease (about 60%) in the B,,, [21]. VlaR-Gqill coupling was
also not modified by the N198Q mutation, as AVP-stimulated production of inositol phosphates was unaffected (results not shown). Our data establish that N-glycosylation of the extracellular loops of rVI,R is not important for expression, correct processinglmembrane insertion, ligand recognition or intracellular signalling. Interestingly, N-glycosylation also has no known function for histamine Hz [31] and Mz muscarinic receptors [321.
Defining the ligand-binding site The formation of receptor-ligand complex is the primary event in receptor activation by agonist or therapeutic intervention by antagonist. Consequently, localizing this site of interaction within the neurohypophysial hormone receptors is of fundamental interest. After our cloning of VI,R from rat testicular myoid cells using PCR primers based on the sequence of the rat liver VI,R published by Morel et al. [8], we identified errors in the published sequence [ 131. Correction of these errors revealed a higher degre of homology for the ECII domain between members of the VPR/OTR family than was originally thought [33]. The conservation of structure suggested a relevance to function and in particular ligand binding. The extracellular surface of VI,R was reproduced by a series of overlapping mimetic peptides corresponding to individual EC domains. These peptide mimetics were then used in radioligandbinding studies to compete for ligand binding to rat liver V1,R. From these studies, it was possible to map the interaction of agonist and antagonists to subdomains of the extracellular surface of rVI,R [27]. This revealed that the first extracellular loop (ECII domain) provides a major binding-site epitope. Although corresponding to only 3% of the receptor, the peptide, DITYRFRGPDWL, recognized agonists and antagonists with submicromolar affinity. The binding characteristics of the peptide mimetics reflected the pharmacology of the receptors. However, the high-affinity binding and full subtype selectivity of ligands required epitopes located in T M domains. We suggested that the extracellular surface facilitates the initial ‘capture’ of ligands before final ‘docking’ [27]. The importance of the ECII domains to agonist binding has also been demonstrated by photoaffinity labelling of bovine VzR using an (azidophenyl) amidino analogue of [3H]deamino[8-lysine]vasopressin followed by radio-
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sequencing. Label was incorporated into ECII, predominantly at R” but also at TIo2[30]. Further support for a role for ECII in ligand recognition was provided by the site-directed mutagenesis studies, which revealed that Tyr1I5 of rVI,R contributes to the pharmacological profile exhibited by the receptor [28]. In addition, the cognate residue in VzR (Asp’03)was shown to be important for high-affinity binding of deamino[~-arginine~]vasopressin [29]. Computer modelling of neurohypophysial hormone receptors has been used to locate/predict the ligand-binding site [34,35]. The T M domains are based on the structure of bacteriorhodopsdrhodopsin, but as yet the connecting loop domains cannot be modelled. As an alternative strategy, chimaeric constructs of VzR/OTR have been used to identify OTR domains contributing to high-affinity OTR-selective binding [36]. The conclusions of these various studies are presented in Table 1. Although agonists and antagonists are competitive in binding assays, they appear to have different final ‘docked’ positions (Table 1). It can be seen from Table 1 that, as yet, there is no consensus binding site common to all members of the WR/OTR family. Indeed it is possible that, although the various receptors and ligands share a high degree of homology, the binding-site (s) topography is different throughout the family of receptors. In this regard, it is noteworthy that the T M domains of hOTR and rVI,R share about 68% identity including an Asp in T M 11, which is conserved throughout the GPCR family. Mutation of this residue in rVI,R decreased the affinity of the agonist but had no effect on antagonist binding [34]. In contrast, mutation of the corresponding
residue in hOTR decreased both agonist and antagonist binding [37]. This perhaps suggests that the helical packing is different in the two homologous receptors and that extrapolation of structure-function data throughout the W R / OTR family is not valid. The hOTR clone was a gift from Dr. Tadashi Kimura, Osaka University Medical School, Osaka, Japan. We are grateful for financial support from the BBSRC, Merck Sharp & Dohme, the British Heart Foundation, the Wellcome Trust and the Medical Research Council.
1 Howl, J. and Wheatley, M. (1995) Gen. Pharmacol. 26, 1143-1 152 2 Soloff, M. S., Alexandrova, M. and Fernstrom, M. J. (1979) Science 204, 1313-1315 3 Lolait, S. J., O’Carroll, A. M., Mahan, L. C. Felder, C. C., Button, D. C., Young, W. S., Mezey, E. and Brownstein, M. J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6783-6787 4 Grazzini, E., Lodboerer, A. M., Perez-Martin, A., Joubert, D. and Guillon, G. (1996) Endocrinology 137,3906-3914 5 El Alj, A., Bonoris, E., Cynober, E. and Germain, G. (1990) Eur. J. Pharmacol. 186,231-238 6 Chan, W. Y., Chen, D. L. and Manning, M. (1993) Endocrinology 132, 1381-1386 7 Kimura, T. and Saji, F. (1995) Endocrine J. 42, 607-615 8 Morel, A., O’Carroll, A. M., Brownstein, M. J. and Lolait, S. J. (1992) Nature (London) 356, 523-526 9 Birnbaumer, M., Seibold, A., Gilbert, S., Ishido, M., Barberis, C., Antaramian, A., Brabet, P. and Rosenthal, W. (1992) Nature (London) 357, 333-335 10 Lolait, S. J., O’Carroll, A. M., McBride, 0. W., Konig, M., Morel, A. and Brownstein, M. A. (1992) Nature (London) 357, 336-339
Domains of neurohypophysial hormone receptors important for binding agonists and antagonists Receptor
Localization of binding-site epitopes
Reference
ViaR
AVP buried 15-20 8, in cleft of TM domains Antagonist: different site (ill defined)
[341
V,,R
Agonists+antagonists: subdomains of ECI, ECII, ECIII+TM domains
~271
OTR
Agonist: ECI, ECII, EClll Antagonist: top TM7; ECI, ECII, ECIII, EClV NOT involved ECI, TM4-ECIII-TM5: aftinity, binds common structure TM6-ECIV: selectivity
~271
VTR
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[35]
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1 1 Sugimoto, T., Masayuki, S., Shinobu, M., Watanabe, Y., Hashimoto, S. and Kawashima, H. (1994) J. Biol. Chem. 269,27088-27099 12 Kimura, T., Tanizawa, O., Mori, K., Brownstein, M. J. and Okayama, 0. (1992) Nature (London) 356, 526-629 13 Howl, J., Rudge, S. A., Lavis, R. A., Davies, A. R. L., Parslow, R. A., Hughes, P. J., Kirk, C. J., Michell, R. H. and Wheatley, M. (1995) Endocrinology 136,2206-221 3 14 Mahlmann, S., Meyerhof, W., Hausmann, H., Heierhorst, J., Schonrock, C., Zwiers, H. and Richter, D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 1342- 1345 15 Hausmann, H., Meyerhof, W., Zwiers, H., Lederis, K. and Richter, D. (1995) FEBS Lett. 370, 227-231) ___
16 Van Kesteren, R. E., Tensen, C. P., Smit, A. B., Van Minnen, J., Van Soest, P. F., Kits, K. S., Meyerhof, W., Richter, D., Van Heerikhuizen, H., Vreugdenhill, E. and Geraerts, W. P. M. (1995) Neuron 15, 897-908 17 Hall, M. D., Hoon, M. A., Ryba, N. J. P., Pottinger, J. D. D., Keen, J. N., Saibil, H. R. and Findlay, J. B. C. (1991) Biochem. J. 274,35-40 18 Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Vansande, J., Maenhant, C., Simons, M.-J., Dumont, J. E. and Vassart, G. (1989) Science 244, 569-572 19 Kornfield, R. and Kornfield, S. (1985) Annu. Rev. Biochem. 54,631-664 20 Pless, D. D. and Lennarz, W. J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 134-138 21 Davidson, J. S., Flanagan, C. A., Zhou, W., Becker, I. I., Elario, R., Emeran, W., Sealfon, S. C. and Miller, R. P. (1995) Mol. Cell. Endocrinol. 107, 241-245 22 Matthews, G. and Colman, A. (1991) Nucleic Acids Res. 19,6405-6412 23 Bause, E. (1983) Biochem. J. 209, 331-336
24 Hutchins, A.-M., Phillips, P. A., Veuter, D. J., Burell, L. M. and Johnston, C. I. (1995) Biochim. Biophys. Acta 1263, 266-270 25 Innamorati, G., Sadeghi, H. and Birnbaumer, M. (1996) Mol. Pharmacol. 50,467-473 26 Kojro, E., Hackenberg, M., Szigo, J. and Fahrenholz, F. (1991) J. Biol. Chem. 266, 21 416-21421 27 Howl, J. and Wheatley, M. (1996) Biochem. J. 317, 577-582 28 Chini, B., Mouillac, B., Ma, Y., Balestre, M.-N., Trump-Kallmeyer, S., Hoflack, J., Elands, J., Hibert, M., Manning, M., Jard, S. and Barberis, C. (1995) EMBO J. 14,2176-2182 29 Ufer, E., Postina, R., Gorbulev, V. and Fahrenholz, F. (1995) FEBS Lett. 362, 19-23 30 Kojro, E., Eich, P., Gimpl, G. and Fahrenholz, F. (1993) Biochemistry 32, 13537-13544 31 Fukushima, Y., Oka, Y., Saitoh, T., Kataguri, H., Asano, T., Matsuhashi, N., Takata, K., Van Breda, E., Yazaki, Y. and Sugano, K. (1995) Biochem. J. 310, 553-558 32 Van Koppen, C. J. and Nathanson, N. M. (1990) J. Biol. Chem. 265,20887-20892 33 Wheatley, M., Howl, J., Morel, A. and Davies, A. R. L. (1993) Biochem. J. 296,519 34 Mouillac, B., Chini, B., Balestre, M.-N., Elands, J., Trump-Kallmeyer, S., Hoflack, J., Hibert, M., Jard, S. and Barberis, C. (1995) J. Biol. Chem. 270, 25771-25777 35 Hausmann, H., Richters, A., Kreienkamp, H.-J., Meyerhof, W., Mattes, H., Lederis, K., Zwiers, H. and Richter, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93,6907-6912 36 Postina, R., Kojro, E. and Fahrenholz, F. (1996) J. Biol. Chem. 271,31593-31601 37 Yanvood, N. J. and Wheatley, M. (1995) Adv. Exp. Med. Biol. 395, 343-344 Received 15 April 1997
Discovery and development of non-peptide antagonists of peptide hormone receptors D. J. Pettibone and R. M. Freidinger Departments of Pharmacology and Medicinal Chemistry, Merck Research Laboratories,West Point, PA I 9486, U.S.A.
Introduction The last 10-15 years has seen an explosion of research aimed at the identification and development of non-peptide antagonists of receptors Abbreviations used: GPC, G-protein-coupled; CCK, cholecystokinin; Avp, vasopressin; MI, angiotensin 11; ET, endothelin.
whose natural ligands are peptides. The success of these efforts is exemplified by the large numbers of antagonists, particularly for the G-protein-coupled (GPC) class of receptors, that are now available as novel research tools and potential therapeutic agents- Although the development of peptide ligands for the various receptors has been extremely important in eluci-
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