The study of antagonist-binding domains of the human V1a vasopressin receptor was performed using a radioiodinated photoreactive peptide antagonist. This ligand displayed a high affinity for the receptor expressed in Chinese hamster ovary cell membranes, and specifically labeled two protein bands with apparent molecular mass at 85-90 and 46 kDa. Our results clearly show that the V1a receptor is degraded during incubation with the ligand and that the 46-kDa species is probably the result of the 85-90-kDa species proteolytic cleavage. Truncation of the receptor was then confirmed by deglycosylation with N-glycosidase F. A monoclonal antibody directed against a c-Myc epitope added at the receptor NH 2 terminus allowed immunoprecipitation of the 85-90-kDa photolabeled species. The 46-kDa photolabeled protein never immunoprecipitated, indicating that the truncated form of the receptor lacks the NH 2 terminus region. To localize photolabeled domains of the receptor, the 46-kDa protein was cleaved with V8 and/or Lys-C endoproteinases. The identity of the smallest photolabeled fragment, observed at approximately 6 kDa, was then confirmed by mutation of the potential V8 cleavage sites. Our results indicate that covalent labeling of the vasopressin V1a receptor with the photoreactive antagonist occurs in a region including transmembrane domain VII (residues Asn 327 -Lys 370 ).Neurohypophysial hormones, arginine-vasopressin (AVP) 1 and oxytocin, exert a wide range of physiological effects through binding to specific membrane receptors belonging to the G protein-coupled receptor (GPCR) superfamily. To date, three AVP receptor subtypes and one oxytocin receptor have been pharmacologically and functionally described (1). V1a, V1b, and oxytocin receptors activate phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate and diacylglycerol, mobilization of intracellular calcium and activation of protein kinase C. V2 receptors stimulate adenylyl cyclase, resulting in the accumulation of cyclic AMP and activation of protein kinase A. All receptor subtypes from several mammalian species have been recently cloned (2-5), as well as closely related receptors from bony fishes and invertebrates (6, 7).Analysis of the primary sequence of these receptors suggests that they possess the same general architecture with seven transmembrane helices as other well characterized G proteincoupled receptors. Moreover, the comparison of their amino acid sequence reveals significant homology within the putative transmembrane regions (TM) and within the first and second extracellular loops as well. The natural ligands for the receptors of the AVP/oxytocin family are also closely structurally related. All are nonapeptides composed of a 6-amino acid disulfide-linked ring and a COOH terminus tripeptide.Peptides of the AVP/oxytocin series were subjected to an extensive analysis of structure-activity relationships. These studies (for review, see Refs. 8 -11) led to the production of a profusion of valuable pharmacological probes for asse...

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d (

A series of new linear photoactivatable and iodinatable antagonists of the neuropeptidic hormone vasopressin was designed and synthesized by a combination of PyBOP-mediated Boc/solid-phase peptide synthesis and solution synthesis approaches. These were based on modifications of a previously reported potent and selective antagonist of the vasopressor response (V1a receptor) to [arginine]vasopressin, phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2. (Azidophenyl)alkyl substitutions, of the general structure N3-C6H4(CH2)nCO (n = 0, 1, 2, or 3), were employed in position 1. The seven new analogues are 4-N3-C6H4CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (3), 3-N3-C6H4CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (12), 4-N3-C6H4CH2-CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (13), 3-N3-C6H4CH2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (14), 4-N3-C6H4(CH2)2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (15), 3-N3-C6H4(CH2)2CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (16), 4-N3-C6H4-(CH2)3CO-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (17). All analogues were tested for their affinity of the rat hepatic V1a receptor. Analogues 3 and 12 have a low affinity (Ki approximately 20 nM) and analogues 13-17 show a high affinity (Ki between 0.04 and 0.3 nM). The affinity values appear to be mainly a function of the alkyl chain length and to a lesser extent of the meta or para position of the azido group on the aromatic ring. Analogues 13-17 were iodinated on the Tyr-9 residue, giving compounds 18-22. All these five iodinated derivatives exhibited Ki values of 0.2-1 nM for rat liver membranes. Their affinities for oxytocin and renal V2 vasopressin receptors were much lower. Moreover, all analogues completely antagonized the vasopressin-stimulated inositol phosphates production in WRK1 cells and were devoided of any agonistic potency. Preliminary covalent binding studies showed improved covalent yields as compared to any previously reported results. They are very promising candidates as potential high-affinity, highly selective, photosensitive ligands for the V1a receptor. They could serve as a useful pharmacological tools for studies on the vasopressin binding site.

BACKGROUND AND PURPOSEUsing an in-house bioinformatics programme, we identified and synthesized a novel nonapeptide, H-Pro-Pro-Thr-Thr-Thr-LysPhe-Ala-Ala-OH. Here, we have studied its biological activity, in vitro and in vivo, and have identified its target in the brain. EXPERIMENTAL APPROACHThe affinity of the peptide was characterized using purified whole brain and striatal membranes from guinea pigs and rats . Its effect on behaviour in rats following intra-striatal injection of the peptide was investigated. A photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor allowed identification of its target. KEY RESULTSThe peptide bound with high affinity to a single class of binding sites, specifically localized in the striatum and substantia nigra of brains from guinea pigs and rats. When injected within the striatum of rats, the peptide stimulated in vitro and in vivo dopamine release and induced dopamine-like motor effects. We purified the target of the peptide, a~151 kDa protein that was identified by MS/MS as angiotensin converting enzyme (ACE I). Therefore, we decided to name the peptide acein. CONCLUSION AND IMPLICATIONSThe synthetic nonapeptide acein interacted with high affinity with brain membrane-bound ACE. This interaction occurs at a different site from the active site involved in the well-known peptidase activity, without modifying the peptidase activity. Acein, in vitro and in vivo, significantly increased stimulated release of dopamine from the brain. These results suggest a more important role for brain ACE than initially suspected.