Vasopressin receptor 2 mutations in the nephrogenic syndrome of inappropriate antidiuresis show different mechanisms of constitutive activation for G protein coupled receptors

Vanessa Vezzi,Caterina Ambrosio,Susanna Cotecchia,Maria Cristina Grò,H Ongun Onaran,Tommaso Costa,Gökçe Süral,Paola Molinari

doi:10.1038/s41598-020-65996-w

Abstract

Vasopressin receptor 2 (V2R) mutations causing the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) can generate two constitutively active receptor phenotypes. One type results from residue substitutions in several V2R domains and is sensitive to vaptan inverse agonists. The other is only caused by Arg 137 replacements and is vaptan resistant. We compared constitutive and agonist-driven interactions of the vaptan-sensitive F229V and vaptan-resistant R137C/L V2R mutations with β-arrestin 1, β-arrestin 2, and Gαs, using null fibroblasts reconstituted with individual versions of the ablated transduction protein genes. F229V displayed very high level of constitutive activation for Gs but not for β-arrestins, and enhanced or normal responsiveness to agonists and inverse agonists. In contrast, R137C/L mutants exhibited maximal levels of constitutive activation for βarrestin 2 and Gs, minimal levels for β-arrestin 1, but a sharp decline of ligands sensitivity at all transducer interactions. The enhanced constitutive activity and reduced ligand sensitivity of R137 mutants on cAMP signaling persisted in cells lacking β-arrestins, indicating that these are intrinsic molecular properties of the mutations, not the consequence of altered receptor trafficking. The results suggest that the two groups of NSIAD mutations represent two distinct molecular mechanisms of constitutive activation in GPCRs.

Highlights

Vasopressin receptor 2 (V2R) mutations causing the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) can generate two constitutively active receptor phenotypes
Spontaneous signaling of the vasopressin 2 receptor (V2R) is responsible for the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD), a rare genetic disease characterized by decreased renal water excretion, hyponatremia and undetectable vasopressin circulating levels[1,2,3]
We show that constitutive cAMP signaling in R137 mutants does not depend on β-arrestins

Summary

Introduction

Vasopressin receptor 2 (V2R) mutations causing the nephrogenic syndrome of inappropriate antidiuresis (NSIAD) can generate two constitutively active receptor phenotypes. The first mutations of NSIAD were characterized in two male infants with hyponatremia and involved the substitution of Arg 137 with either Cys or Leu (R137C or R137L) at the cytosolic end of helix 3 in V2R1 This GPCR-wide conserved Arg[4] is part of the well-known consensus motif E/DRY/H and plays a crucial role in the mechanism of receptor activation, as indicated by structural data[5,6]. Another natural substitution of codon 137 identified earlier in V2R studies is R137H7, but unlike NSIAD mutations, it generates a loss-of-function phenotype[8] that occurs in patients with the X-linked form of congenital Nephrogenic Diabetes Insipidus (NDI). Based on biological and pharmacological traits, NSIAD mutants can be divided in two different functional groups

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