Endocrine Abstracts

Diabetes insipidus (DI) is a rare disorder which is characterized with inability to concentrate urine because of severe liquid-balance impairment. Mutations in AVPR2 gene is one of the causes of DI. AVPR2 is a G protein coupled receptor (GPCR) and its specific agonist is arginine vasopressin (AVP). When AVP binds to the AVPR2, which locates on the basolateral side of collecting duct principal cells of the kidney, it triggers accumulation of cAMP in the cell as a seconder messenger. If AVPR2 has a mutation, receptor could loose its function which is important for liquid balance of the body. The aim of this study is making functional characterization of G12E mutation of AVPR2 gene. G12E mutation was seen in three cases in our study. Functional characterization of this mutant is important for the therapeutic studies. G12E mutation was generated by site-directed mutagenesis strategy. For this purpose, pLV2R (a mammalian expression vector) was used and the mutant construct was checked with DNA sequencing. Totally expression in the cell and cell surface expression of the mutant receptor was analyzed with ELISA experiments. In addition to this, cAMP accumulation assay was performed after stimulation of mutant receptor with different concentrations of AVP. According to the ELISA results, G12E mutation showed reduced expression in total and also on cell surface. The results of cAMP accumulation assay supported our ELISA results. Mutant receptor showed reduced E_max and it also has a shift for EC₅₀ value according to the wild type receptor. In conclusion, in vitro studies revealed that this mutation yields partial expression in the cell surface.