Altered agonist sensitivity of a mutant V2 receptor suggests a novel therapeutic strategy for nephrogenic diabetes insipidus.

Loss of function mutations of the type 2 vasopressin receptor (V2R) in kidney can lead to nephrogenic diabetes insipidus (NDI). We studied a previously described, but uncharacterized mutation of V2R (N321K missense mutation) of an NDI patient. The properties of the mutant receptor were evaluated. We constructed a highly sensitive Epac based BRET (bioluminescence resonance energy transfer) biosensor to perform real-time cAMP measurements after agonist stimulation of transiently transfected HEK293 cells with V2Rs. beta-arrestin binding of the activated receptors was examined with luciferase-tagged beta-arrestin and mVenus-tagged V2Rs using BRET technique. Cell surface expressions of HA-tagged receptors were determined with flow cytometry using anti-HA-Alexa488 antibodies. Cellular localization examinations were implemented with fluorescent tagged receptors visualized with confocal laser-scanning microscopy. The effect of various vasopressin analogues on V1R was tested on mouse arteries by wire myography. N321K mutant V2R showed normal cell surface expression but the potency of AVP for cAMP generation was low, while the clinically used desmopressin (dDAVP) was not efficient. The beta-arrestin binding and internalization properties of the mutant receptor were also different compared to the wild type. Function of the mutant receptor can be rescued with administration of V2R receptor agonist dVDAVP, which had no detectable side effects on V1R in the effective cAMP generating concentrations. Based on the findings we could propose a therapeutical strategy for NDI patients carrying the N321K mutation, since our in vivo experiments suggest that dVDAVP could rescue the function of the N321K-V2R without significant side effect on V1R