G protein–coupled receptor (GPCR) oligomers have been proposed to play critical roles in cell signaling, but confirmation of their existence in a native context remains elusive, as no direct interactions between receptors have been reported. To demonstrate their presence in native tissues, we developed a time-resolved FRET strategy that is based on receptor labeling with selective fluorescent ligands. Specific FRET signals were observed with four different receptors expressed in cell lines, consistent with their dimeric or oligomeric nature in these transfected cells. More notably, the comparison between FRET signals measured with sets of fluorescent agonists and antagonists was consistent with an asymmetric relationship of the two protomers in an activated GPCR dimer. Finally, we applied the strategy to native tissues and succeeded in demonstrating the presence of oxytocin receptor dimers and/or oligomers in mammary gland.

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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...