Characterization of Three Vasopressin Receptor 2 Variants: An Apparent Polymorphism (V266A) and Two Loss-of-Function Mutations (R181C and M311V)

Stephen P Armstrong,Brian J Feldman,Ruth M Seeber,Kevin D G Pfleger,Mohammed Akli Ayoub

doi:10.1371/journal.pone.0065885

Abstract

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.

Highlights

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water balance homeostasis [1,2,3]
AVP was 29- or 427fold less potent in cells transfected with M311V or R181C HAV2R respectively (Table 1)
We tested for the ability of Rluc8-tagged V2 receptors (V2Rs) to couple to Gs in the same cell line (Figure 1B), as it is important to check that the addition of a BRET tag does not compromise normal receptor function [34,35]

Summary

Introduction

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water balance homeostasis [1,2,3]. AVP, binding to arginine vasopressin V2 receptors (V2R) located on the basolateral membrane of kidney collecting duct epithelial cells, triggers activation of Gs proteins, leading to increased cAMP levels, and activation of Protein Kinase A [1,2,3]. This in turn causes trafficking of aquaporin-2 water channels to the apical membrane of collecting duct cells, resulting in increased water permeability and antidiuresis [1,2,3]. A key feature is the absence of detectable levels of AVP in serum, whereas the closely related syndrome of inappropriate antidiuretic hormone secretion (SIADH) is typically associated with measurably elevated AVP levels and consequent V2R hyperactivity [6,7]

Methods

Results

Conclusion