Paired Activation of Two Components within Muscarinic M3 Receptor Dimers Is Required for Recruitment of β-Arrestin-1 to the Plasma Membrane

Franco Giorgi,Roberto Maggio,Giovanni U. Corsini,Laura Stanasila,Francesca Novi,Susanna Cotecchia

doi:10.1074/jbc.m411281200

Franco Giorgi, Roberto Maggio

Open Access

https://doi.org/10.1074/jbc.m411281200

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Abstract

beta-Arrestins regulate the functioning of G protein-coupled receptors in a variety of cellular processes including receptor-mediated endocytosis and activation of signaling molecules such as ERK. A key event in these processes is the G protein-coupled receptor-mediated recruitment of beta-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via beta-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit beta-arrestin-1 using both co-immunoprecipitation and fluorescence microscopy in COS-7 cells. Experimentally, we first made use of a mutated muscarinic M(3) receptor, which is deleted in most of the third intracellular loop (M(3)-short). Although still capable of activating phospholipase C, this receptor loses almost completely the ability to recruit beta-arrestin-1 following carbachol stimulation in COS-7 cells. Subsequently, M(3)-short was co-expressed with the M(3) receptor. Under these conditions, the M(3)/M(3)-short heterodimer could not recruit beta-arrestin-1 to the plasma membrane, even though the control M(3)/M(3) homodimer could. We next tested the ability of chimeric adrenergic muscarinic alpha(2)/M(3) and M(3)/alpha(2) heterodimeric receptors to co-immunoprecipitate with beta-arrestin-1 following stimulation with adrenergic and muscarinic agonists. beta-Arrestin-1 co-immunoprecipitation could be induced only when carbachol or clonidine were given together and not when the two agonists were supplied separately. Finally, we tested the reciprocal influence that each receptor may exert on the M(2)/M(3) heterodimer to recruit beta-arrestin-1. Remarkably, we observed that M(2)/M(3) heterodimers recruit significantly greater amounts of beta-arrestin-1 than their respective M(3)/M(3) or M(2)/M(2) homodimers. Altogether, these findings provide strong evidence in favor of the view that binding of beta-arrestin-1 to muscarinic M(3) receptors requires paired stimulation of two receptor components within the same receptor dimer.

Highlights

Receptors [1, 2]
In COS-7 cells transfected with FLAG-␤-arrestin-1, the same antibody recognized a broad band corresponding to the FLAG-␤arrestin-1
In COS-7 cells transfected with FLAG-␤-arrestin-1, the anti-FLAG antibody recognized the same band as the anti␤-arrestin-1 antibody, whereas it did not detect any signal in mock-transfected COS-7 cells (Fig. 2B)

Summary

Introduction

Receptors [1, 2]. the mechanism(s) by which receptors may undergo dimerization has yet to be elucidated in detail, it is becoming ever more clear that the phenomenon is playing a key role in receptor maturation, G protein coupling, and downstream signaling besides regulating such processes as internalization and desensitization. In cells transfected with 3HA-M3 or M3 (results not shown), this antibody recognizes two bands that most likely correspond to the monomeric and dimeric forms of the muscarinic receptor (Fig. 2C). No co-localization of ␤-arrestin-1-GFP and muscarinic 3HA-M3 receptor could be revealed in endocytotic vesicles of cells co-expressing 3HA-M3, M3-short, and ␤-arrestin-1-GFP after 30 min of carbachol stimulation (Fig. 6).

Results

Conclusion