Signal Transduction Profiling of Angiotensin II Type 1 Receptor With Mutations Associated to Atrial Fibrillation in Humans.

Sarah C Simões,Michel Bouvier,André L B Bitencourt,Lucas T Parreiras-E-Silva,André L Balico-Silva,Claudio M Costa-Neto

doi:10.3389/fphar.2020.600132

Abstract

The AT1 receptor (AT1R) has a major role in the Renin-Angiotensin System, being involved in several physiological events including blood pressure control and electrolyte balance. The AT1R is a member of the G protein coupled receptors (GPCR) family, classically known to couple Gαq and engage β-arrestin recruitment. Both G protein and arrestin signaling pathways are involved in modulation of different downstream kinases. A previous study reported that mutations in the AT1R (A244S and I103T-A244S) were positively correlated with higher risk of atrial fibrillation in men. Based on that report, we aimed to investigate if these mutations, including I103T only, could affect AT1R signal transduction profile, and consequently, implicate in atrial fibrillation outcome. To address that, we engineered an AT1R carrying the above-mentioned mutations, and functionally evaluated different signaling pathways. Phosphokinase profiler array to assess the mutations downstream effects on kinases and kinase substrates phosphorylation levels was used. Our results show that the I103T-A244S mutant receptor presents decreased β-arrestin 2 recruitment, which could lead to a harmful condition of sustained Gαq signaling. Moreover, the phosphokinase profiler array revealed that the same mutation led to downstream modulation of kinase pathways that are linked to physiological responses such as fibrous tissue formation, apoptosis and cell proliferation.

Highlights

G protein coupled receptors (GPCRs) represent the major family of cell surface receptors (Wise et al, 2004)
angiotensin II (AngII) affinities for the mutant receptors were assessed by competition binding assay using radiolabeled 3H-AngII, and non-radioactive AngII in different concentrations as the competitor ligand
In the present study we aimed to evaluate whether the presence of the I103T-A244S and A244S would affect the downstream signaling of the AT1 receptor (AT1R), which in turn could have possible implications in atrial fibrillation (AF)

Summary

Introduction

G protein coupled receptors (GPCRs) represent the major family of cell surface receptors (Wise et al, 2004). The angiotensin II type 1 receptor (AT1R), a G protein coupled receptor, is the main receptor of the Renin-Angiotensin System, and angiotensin II (AngII) is the main agonist of this system. AT1R activation promotes cell proliferation, inflammation and fibrosis (Forrester et al, 2018), which are related to cardiovascular diseases (de Gasparo et al, 2000). Activation of AT1R by AngII triggers the canonical Gαq protein signaling pathways, which leads to Signal Profiling Fibrosis-Related AT1R Mutations inositol trisphosphate (IP3) and diacylglycerol (DAG) generation, and Ca2+ mobilization from the endoplasmic reticulum, culminating in protein kinase C (PKC) and other downstream kinases activation, including ERK1/2. After AT1R activation by AngII, G protein coupled receptor kinases (GRKs) promote the phosphorylation of the receptor and β-arrestins recruitment, leading to receptor internalization and activation of other signaling pathways (Lima et al, 2014)

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