title: Paired activation of two components within muscarinic M3 receptor dimers is required for recruitment of beta-arrestin-1 to the plasma membrane.
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.
authors.affiliation: Department of Neurosciences, University of Pisa, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Novi
authors.forename: Laura
authors.initials: L
authors.lastname: Stanasila
authors.forename: Franco
authors.initials: F
authors.lastname: Giorgi
authors.forename: Giovanni U
authors.initials: GU
authors.lastname: Corsini
authors.forename: Susanna
authors.initials: S
authors.lastname: Cotecchia
authors.forename: Roberto
authors.initials: R
authors.lastname: Maggio
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001693:Biological Transport, Active; D019556:COS Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003000:Clonidine / Q000494:pharmacology; D019281:Dimerization; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D043587:Receptor, Muscarinic M3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; D019390:Arrestins; D043587:Receptor, Muscarinic M3; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D002217:Carbachol; D003000:Clonidine