We investigated the biochemical and functional properties of five vasopressin V2 receptor mutants(L44F, L44P, W164S, Si 67L, and S167T) that were recently described in families with a history of X-Iinked nephrogenic diabetes insipidus. COS. M6 cells transfected with cDNA encoding these mutants acquired< 4% specific [H] arginine vasopressin(AVP) binding sites on the cell surface in comparison with cells transfected with cDNA coding for the wild-type receptor. Membrane preparations from COS. M6 cells or human embryonic kidney 293 cells expressing these mutants did not respond with an increase in adenylyl cyclase activity in response to AVP, which is in contrast to membranes from cells expressing the wild-type. By analyzing fusion proteins of the V2 receptor and Escherichia co/i alkaline phosphatase attached to the carboxyl terminus of the receptor moiety, we found that the mutants L44P, Wi 64S, Si 67L, and Si 67T lacked complex glycosylation and were expressed at low levels. The data suggest that the mutants L44P, W164S, S167T, and S167L are misfolded and therefore retained within the endoplasmic reticulum and degraded. In contrast, the fusion proteins carrying the mutant L44F and the in vitro mutant Si 67A were expressed in their mature form at wild-type levels; however, only the mutant Si 67A was functionally active. Site-directed mutagenesis of Si 67 revealed that elimination of the endogenous hydroxyl group (Si 67A) yielded a protein with properties identical to those of the wild-type receptor, whereas both the introduction of a methyl group (Si 67T) and the re-placement of the hydroxyl group by an isopropyl group (Si 67L) profoundly disturbed receptor processing. The data show that minute changes at codon i 67 nearly abolish expression of a mature protein, thus defining structural requirements of this codon.

The antidiuretic action of the hormone AVP is mediated by the vasopressin V2 receptor, which is expressed in epithelial cells ofrenal collecting ducts. The receptor is a member of the superfamily of GPCRs(1). Its activation leads to stimulation of adenylyl cyclase via G8. The subsequent rise in intracellulan cAMP induces the insertion of the vasopressin-sensitive water channel aquaponin-2 into the luminal membrane(2).