Two serines in the distal C-terminus of the human ß1-adrenoceptor determine ß-arrestin2 recruitment.

Arun Shukla,Laura Hinz,Bernhard Küster,Benjamin Ruprecht,Andrea Ahles,Stefan Engelhardt

doi:10.1371/journal.pone.0176450

Abstract

G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor’s third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.

Highlights

G protein-coupled receptors (GPCRs) form the largest family of mammalian surface receptors and account for more than 30% of all known drug targets [1]
A key event in receptor desensitization is the phosphorylation of the receptor by downstream serine/threonine kinases, predominantly by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA)
Phosphorylation of GPCRs is a key regulatory step in receptor signal transduction. It is a prerequisite for the binding of arrestins, which mediates receptor desensitization i.e. uncoupling of the receptor from its cognate G protein and termination of canonical G protein-dependent signal transduction

Summary

Introduction

G protein-coupled receptors (GPCRs) form the largest family of mammalian surface receptors and account for more than 30% of all known drug targets [1]. Among the three members of the β-adrenoceptor family, the β2-adrenoceptor (ADRB2) is the best studied and has served as a prototype GPCR, at which many fundamental principles of receptor biology have been discovered [5]. SS1-adrenoceptor phosphorylation response that regulates receptor signaling upon an activating stimulus. The extent to which β-arrestin is bound and signals depends on the specific GPCR [13,14]

Methods

Results

Conclusion