Association of calnexin with wild type and mutant AVPR2 that cause nephrogenic diabetes insipidus

Over 155 mutations within the V2 vasopressin receptor (AVPR2) gene are resp onsible for nephrogenic diabetes insipidus (NDI). The expression and subcel lular distribution of four of these was investigated in transfected cells. These include a point mutation in the seventh transmembrane domain (S315R), a frameshift mutation in the third intracellular loop (804delG), and two n onsense mutations that code for AVPR2 truncated within the first cytoplasmi c loop (W71X) and in the proximal portion of the carboxyl tail (R337X). RT- PCR revealed that mRNA was produced for all mutant receptor constructs. How ever, no receptor protein, as assessed by Western blot analysis, was detect ed for 804delG. The S315R was properly processed through the Golgi and targ eted to the plasma membrane but lacked any detectable AVP binding or signal ing. Thus, this mutation induces a conformational change that is compatible with endoplasmic reticulum (ER) export but dramatically affects hormone re cognition. III contrast, the W71X and R337X AVPR2 were retained inside the cell as determined by immunofluorescence. Confocal microscopy revealed that they were both retained in the ER. To determine if calnexin could be invol ved, its interaction with the AVPR2 was assessed. Sequential coimmunoprecip itation demonstrated that calnexin associated with the precursor forms of b oth wild-type (WT) and mutant receptors in agreement with its general role in protein folding. Moreover, its association with the ER-retained R337X mu tant was found to be longer than with the WT receptor suggesting that this molecular chaperone also plays a role in quality control and ER retention o f misfolded G protein-coupled receptors.