Characterization of three vasopressin receptor 2 variants: an apparent polymorphism (V266A) and two loss-of-function mutations (R181C and M311V)

PLoS One. 2013 Jun 6;8(6):e65885. doi: 10.1371/journal.pone.0065885. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aquaporin 2 / genetics
  • Aquaporin 2 / metabolism
  • Arginine Vasopressin / metabolism
  • Arrestins / genetics
  • Arrestins / metabolism
  • COS Cells
  • Chlorocebus aethiops
  • Cyclic AMP / metabolism
  • Diabetes Insipidus, Nephrogenic / genetics*
  • Diabetes Insipidus, Nephrogenic / metabolism
  • Diabetes Insipidus, Nephrogenic / pathology
  • GTP-Binding Protein alpha Subunits, Gs / genetics
  • GTP-Binding Protein alpha Subunits, Gs / metabolism
  • Gene Expression Regulation
  • Genetic Diseases, X-Linked / genetics*
  • Genetic Diseases, X-Linked / metabolism
  • Genetic Diseases, X-Linked / pathology
  • HEK293 Cells
  • Humans
  • Inappropriate ADH Syndrome / genetics*
  • Inappropriate ADH Syndrome / metabolism
  • Inappropriate ADH Syndrome / pathology
  • Inositol Phosphates / metabolism
  • Mutation*
  • Polymorphism, Genetic*
  • Receptors, Vasopressin / genetics*
  • Receptors, Vasopressin / metabolism
  • Signal Transduction
  • beta-Arrestins

Substances

  • Aquaporin 2
  • Arrestins
  • Inositol Phosphates
  • Receptors, Vasopressin
  • beta-Arrestins
  • Arginine Vasopressin
  • Cyclic AMP
  • GTP-Binding Protein alpha Subunits, Gs

Supplementary concepts

  • Nephrogenic Syndrome of Inappropriate Antidiuresis

Grant support

This research was funded by the Australian Research Council (www.arc.gov.au) Future Fellowship (FT100100271) and a Priming Grant from the Raine Medical Research Foundation (www.raine.uwa.edu.au), both awarded to KDGP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.