|Budget Amount *help
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,700,000 (Direct Cost: ¥1,700,000)
Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type2 receptor gene mutations in unrelated X-linked NDI families, including R143P, DELTA V278, R202C, and 8O4insG.All these mutations reduced ligand binding activity to <10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized and intracellular proteins, similar to the wild-type AVPR2 clone, whereas the R143P and DELTA V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 8O4insG mutant cell produced no proteins despite adequate mRNA level. Immunofluorescence staining confirmed that R202C mutants reached cell surface, whereas the R143P and A V278 are retained within the cytoplasmic compartment. Thus, R202C, R143P/DELTA V278, and 8O4insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI.