β-Arrestin Binding to the β2-Adrenergic Receptor Requires Both Receptor Phosphorylation and Receptor Activation

Cornelius Krasel,Kristina Lorenz,Moritz Bünemann,Martin J. Lohse

doi:10.1074/jbc.m413078200

Abstract

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.

Highlights

Homologous desensitization of ␤2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of ␤-arrestins to the phosphorylated receptor
We show that (a) the initial kinetics of ␤-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind ␤-arrestin2 very rapidly; and (c) the interaction of ␤-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-␤-arrestin2 complex
In this article we show how the kinetics of complex formation between the ␤2-adrenergic receptor and ␤-arrestin2 can be measured in single living cells using fluorescence resonance energy transfer (FRET) between fluorescently labeled proteins

Summary

Introduction

It was shown previously that HEK293 cells transiently transfected with ␤Arr2-GFP fusion proteins and ␤2ARs will translocate the fluorescent protein to the plasma membrane upon stimulation of the receptor [10]. Time-lapse confocal microscopy with HEK293 cells transiently transfected with ␤2AR and ␤Arr2-YFP showed that stimulation of the receptors with an agonist caused a translocation of ␤-arrestin2 to the cell surface (Fig. 1A and supplemental movie S1).

Results

Conclusion