Résumé
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 withN-glycosidase F. A monoclonal antibody directed against a c-Myc epitope added at the receptor NH2 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 NH2 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 Asn327-Lys370).