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A novel photoactivatable linear peptide antagonist selective for the V1avasopressin receptor, [125I][Lys(3N3Phpa)8]HO-LVA, was synthesized, characterized, and used to photolabel the human receptor expressed in Chinese hamster ovary cells. Two specific glycosylated protein species at 85–90 and 46 kDa were covalently labeled, a result identical to that obtained with a previous photosensitive ligand, [125I]3N3Phpa-LVA (Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahe, E., Jard, S., Barberis, C., and Mouillac, B. (1997) J. Biol. Chem.272, 26536–26544). To identify contact sites between the new photoreactive analogue and the V1areceptor, the labeled receptors were digested with Lys-C or Asp-N endoproteinases and chemically cleaved with CNBr. Fragmentation with CNBr, Lyc-C, and Asp-N used alone or in combination, led to the identification of a restricted receptor region spanning the first extracellular loop. The results established that sequence Asp112–Pro120could be considered as the smallest covalently labeled fragment with [125I][Lys(3N3Phpa)8]HO-LVA. Based on the present experimental result and on previous photoaffinity labeling data obtained with [125I]3N3Phpa-LVA (covalent attachment to transmembrane domain VII), three-dimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V1areceptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.