THE EFFECT OF 8 V2 VASOPRESSIN RECEPTOR MUTATIONS ON STIMULATION OF ADENYLYL-CYCLASE AND BINDING TO VASOPRESSIN

We previously identified six V2 vasopressin receptor mutations in five unrelated nephrogenic diabetes insipidus (NDI) families. In order to elucidate the effect of these mutations on the function of the V2 vaso pressin receptor, we introduced these six and two additional, naturall y occurring mutations into the V2 vasopressin receptor gene by in vitr o mutagenesis. Five of the mutants (two frameshift, one nonsense, and two missense) failed to stimulate adenylyl cyclase due to their inabil ity to bind vasopressin under the experimental conditions. In contrast , ligand binding and cAMP accumulation were normal for two other mutat ions, a A61V missense mutation and an in-frame deletion of four amino acids (Arg-247 to Gly-250), suggesting that they are not the cause of NDI in these families. The deletion mutation was found in a family in conjunction with a second mutation, R181C, which yielded a much reduce d ligand binding capacity. The K-D of R181C was at least 26 times high er than that of the wild type. Further characterization by an immunofl uorescent assay showed that the R181C mutant receptor is expressed and distributed on the cell surface in a manner similar to that of the wi ld type. This finding indicates that the inability of this mutant to s timulate adenylyl cyclase is caused by the reduced capacity for vasopr essin binding and that the R181C mutation is responsible for NDI in th is family.