Structural requirements for V-2 vasopressin receptor proteolytic cleavage

The ligand-induced proteolytic cleavage of the Vt vasopressin receptor tran siently expressed in COS cells was investigated. After incubation of the ce ll membranes with a photoreactive ligand possessing full agonistic properti es for Vt receptors, approximately 90% of the porcine and bovine V-2 vasopr essin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptor s. The oxytocin receptor was completely resistant to proteolysis after bind ing the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V-2/oxytocin receptors obtained by transfer of extracel lular domains of the oxytocin receptor into the Vt receptor showed an incre ase in binding affinity for oxytocin versus vasopressin and a diminished cl eavage. The proteolysis-resistant chimeric V-2/oxytocin receptor, which con tains the first three extracellular domains of the oxytocin receptor, stimu lated cAMP accumulation to a larger extent in response to vasopressin than the wild-type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V-2 receptor requires a defined conformation, especially of the first two extr acellular domains that is induced by agonist binding. Furthermore, the resu lts suggest that the proteolytic V-2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.