Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist - Comparison with the human V-1a vasopressin receptor

Understanding of the molecular determinants responsible for antagonist bind ing to the oxytocin receptor should provide important insights that facilit ate rational design of potential therapeutic agents for the treatment of pr eterm labor. To study ligand/receptor interactions, we used a novel photose nsitive radioiodinated antagonist of the human oxytocin receptor, d(CH2)(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH29]vasotocin. This ligand h ad an equivalent high affinity for human oxytocin and V-1a vasopressin rece ptors expressed in Chinese hamster ovary cells. Taking advantage of this du al specificity, we conducted photoaffinity labeling experiments on both rec eptors, Photolabeled oxytocin and V-1a receptors appeared as a unique prote in band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, res pectively. To identify contact sites between the antagonist and the recepto rs, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and d igested with Lys-C and Arg-C endoproteinases, The fragmentation patterns al lowed the identification of a covalently labeled region in the oxytocin rec eptor transmembrane domain III consisting of the residues Leu(114)-Val(115) - Lys(116). Analysis of contact sites in the V-1a receptor led to the ident ification of the homologous region consisting of the residues Val(126)-Val( 127)-Lys(128). Binding domains were confirmed by mutation of several CNBr c leavage sites in the oxytocin receptor and of one Lys-C cleavage site in th e V-1a receptor. The results are in agreement with previous experimental da ta and three-dimensional models of agonist and antagonist binding to member s of the oxytocin/vasopressin receptor family.