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

β-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 β-arrestins to the plasma membrane. However, despite extensive knowledge in this field, it is still disputable whether activation of signaling pathways via β-arrestin recruitment entails paired activation of receptor dimers. To address this question, we investigated the ability of different muscarinic receptor dimers to recruit β-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 β-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 β-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 α 2 /M 3 and M 3 /α 2 heterodimeric receptors to co-immunoprecipitate with β-arrestin-1 following stimulation with adrenergic and muscarinic agonists. β-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 β-arrestin-1. Remarkably, we observed that M 2 /M 3 heterodimers recruit significantly greater amounts of β-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 β-arrestin-1 to muscarinic M 3 receptors requires paired stimulation of two receptor components within the same receptor dimer.