EXPRESSION STUDIES OF 2 VASOPRESSIN V2 RECEPTOR GENE-MUTATIONS, R202CAND 804INSG, IN NEPHROGENIC DIABETES-INSIPIDUS

Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder assoc iated with renal tubule resistance to arginine vasopressin (AVP). To u nderstand the mechanisms of AVP resistance underlying this disorder, w e have analyzed the vasopressin V2 receptor gene in two unrelated Japa nese kindreds with NDI and expressed the mutants to characterize their functional properties. Direct sequencing revealed two V2 receptor gen e mutations: a missense mutation from Arg202 to Cys in the third extra cellular domain (R202C) and a single base insertion (G) in two consecu tive GGG triplets (nucleotide 804 to 809) in the third cytoplasmic dom ain, resulting in a frame shift with premature termination at codon 25 8 (804insG). Transient expression study with COS-7 cells showed that R 202C mutation reduced both binding affinity (15%) and capacity (30%), while 804insG mutation abolished binding ability. For further evaluati on of the binding ability of the R202C mutant, we expressed the mutant s in Chinese hamster ovary (CHO) cells. Although the mutant cell lines produced V2 receptor mRNA comparable levels to the wild-type receptor cell lines, R202C mutant cell lines had no binding ability. Our resul ts suggest an introduction of a new cysteine residue in the extracellu lar domain and a receptor truncation removing one third of the carboxy l terminus could impair ligand binding activity of the V2 receptor thr ough a post-transcriptional mechanism, thereby causing AVP resistance in the NDI patients.