Mapping Peptide-binding Domains of the Human V1a Vasopressin Receptor with a Photoactivatable Linear Peptide Antagonist

Abstract

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...

Docking of Linear Peptide Antagonists into the Human V1a Vasopressin Receptor

“…14). At the present time, very few photoaffinity labeling studies have led to the direct determination of labeled amino acid residues in peptide G protein-coupled receptor; remarkable results with bovine V 2 receptor (15), human NK1 tachykinin receptor (16), and rat type A cholecystokinin receptor (17) allowed identification of covalently labeled residues with photoactivatable agonist analogues of AVP, substance P, and cholecystokinin, respectively.Very recently, a first radioiodinated photoreactive linear peptide antagonist has been used in our laboratory to photolabel the human and rat V 1a receptors (12,18,19). Our results have clearly indicated that covalent attachment of the [ 125 I]3N 3 Phpa-LVA occurs in a restricted domain of the human receptor including TMR VII.…”

“…Very recently, a first radioiodinated photoreactive linear peptide antagonist has been used in our laboratory to photolabel the human and rat V 1a receptors (12,18,19). Our results have clearly indicated that covalent attachment of the [ 125 I]3N 3 Phpa-LVA occurs in a restricted domain of the human receptor including TMR VII.…”

“…Photoaffinity Labeling Experiments-These experiments were conducted as described before for the previous photoactivatable antagonist [ 125 I]3N 3 Phpa-LVA (12). Briefly, membranes (500 g) were resuspended in 4 ml of binding Buffer A containing bovine serum albumin (0.5 mg/ml) and were incubated for 1 or 3 h in the dark in the presence of […”

“…Radioligand Binding Assays-As described in previous papers (7,12) ]HO-LVA (50 -100 pM) as the radioligands. The concentrations of the unlabeled ligands varied from 1 pM to 10 M. AVP (10 M) and HO-LVA (400 nM) were used to determine nonspecific binding, respectively.…”

“…Cell Culture and Membrane Preparation-The human V 1a receptor cDNA was a generous gift of Dr. M. Thibonnier (24), and CHO cells were transfected as described before (12). CHO cells stably expressing the human V 1a , V 1b , V 2 , or OT receptor were maintained in culture in Petri dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 4 mM L-glutamine, 500 units/ml penicillin and streptomycin, 0.25 g/ml amphotericin B in an atmosphere of 95% air and 5% CO 2 at 37°C.…”