Structural requirements for V2 vasopressin receptor proteolytic cleavage.
The ligand-induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis-resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated 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 V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.
Kojro
E
E
Institut für Biohemie, Johannes Gutenberg-Universität, Mainz, Germany.
Postina
R
R
Gilbert
S
S
Bender
F
F
Krause
G
G
Fahrenholz
F
F
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D018045:Receptors, Oxytocin / Q000737:chemistry; D017483:Receptors, Vasopressin / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; D011506:Proteins; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D000242:Cyclic AMP; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases