Endocrine Abstracts

Background: Loss of function mutations of the type-2 vasopressin receptor (V2R) in kidney lead to nephrogenic diabetes insipidus (NDI). The function of mutant receptors can be rescued using ligands resulting sufficient amount of plasma membrane localized V2Rs in a conformation which are able to generate cAMP signal. We examined a previously described, not characterized mutation. In order to determine personalized therapy for the individual the properties of the mutant receptor were characterized. Based on the findings we could frame a therapeutical strategy.

Materials and methods: We constructed a highly sensitive Epac-based BRET (bioluminescence resonance energy transfer) biosensor to perform real-time cAMP measurments. The β-arrestin binding of the receptors was examined with luciferase-tagged β-arrestin and mVenus-tagged V2Rs using BRET technique. The BRET measurements were performed on transiently transfected HEK293 cells using 96-well plates and Berthold Mithras LB 940 multilabel reader. Cell surface expression and localization examinations were implemented with fluorescent tagged receptors visualised with with Zeiss LSM510 confocal laser-scanning microscope.

Results: The previously described N321K mutation is in the 7th transmembrane helix of the V2R. Determination of the ligand induced cAMP generation of the mutant receptor showed increased EC50 in arginine-vasopressin (AVP) stimulation and lack of signal in desmopressin stimulation. BRET experiments revealed decreased β-arrestin binding of N321K-V2R. The mutant receptor also showed different sensitivity for V2R antagonists compared to wild-type receptor.

Conclusions: N321K mutant V2R showed cell surface expression, the physiologically essential cAMP generation of the receptor can be rescued with elevated dose of AVP, while with clinically used desmopressin was not efficient. Different internalization of the receptor may occur through altered β-arrestin binding. Administration of vasopressin receptor antagonists may be useful to avoid the side effects of high dose AVP in an NDI patient.

Supported by TÁMOP (4.2.1.B-09/1/KMR-2010-0001).

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.