BIOCHEMICAL BASIS OF PARTIAL NEPHROGENIC DIABETES-INSIPIDUS PHENOTYPES

Biochemical properties of mutant type 2 vasopressin receptors (V2Rs) c ausing a partial phenotype of nephrogenic diabetes insipidus were inve stigated in transiently transfected HEK 293 cells. Cell surface expres sion of the V2R was not altered by substituting Asp(85) in the second transmembrane region by Asn as determined by saturation binding assays . Although the affinity of the mutant V2R for arginine vasopressin (AV P) was reduced only 6-fold, the response of adenylyl cyclase activity to AVP revealed a 50-fold right shift in EC50 and a decreased maximum response for the mutant V2R. These data indicated that replacement of Asp(85) by Asn affected coupling of the receptor to G(s), a conclusion substantiated by a 20-fold decrease in the calculated coupling effici ency of this receptor. The Gly(201)Asp mutation in the second extracel lular loop, also found associated with an NDI partial phenotype, decre ased cell surface expression of the V2R with minor reduction in ligand -binding affinity and coupling efficiency to G(s). A pronounced differ ence was observed for this mutant V2R between the stimulation of adeny lyl cyclase activity promoted by AVP and the V2 vasopressin receptor a gonist deamino[Cys(1),D-Arg(8)]-vasopressin, suggesting an involvement of Gly(201) in the selectivity of the receptor for different ligands. These data demonstrated that while decreased ligand-binding affinity and decreased coupling to G(s) are responsible for the attenuation of response to ligand in the Asp(85)Asn mutant V2R, cell surface expressi on of the V2R is the major factor reducing cellular responses to ligan d for the Gly(201)Asp mutant V2R.