title: The molecular basis of oligomeric organization of the human M3 muscarinic acetylcholine receptor.
abstract: G protein-coupled receptors, including the M3 muscarinic acetylcholine receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved fluorescence resonance energy transfer studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI, and VII as well as intracellular helix VIII to the overall organization. Molecular modeling studies based on both these results and an X-ray structure of the inactive state of the M3 receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M3 receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally resolved, multiphoton fluorescence resonance energy transfer. Modeling studies furthermore suggest an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, which is consistent with the presence of cholesterol at key locations in many G protein-coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (M.J.V.L., G.C., R.J.W., E.A.-C., S.M., G.M.), and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (G.C.).
authors.forename: María José Varela
authors.initials: MJ
authors.lastname: Liste
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (M.J.V.L., G.C., R.J.W., E.A.-C., S.M., G.M.), and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (G.C.) Graeme.Milligan@glasgow.ac.uk gianluigicaltabiano@gmail.com.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Caltabiano
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (M.J.V.L., G.C., R.J.W., E.A.-C., S.M., G.M.), and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (G.C.).
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (M.J.V.L., G.C., R.J.W., E.A.-C., S.M., G.M.), and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (G.C.).
authors.forename: Elisa
authors.initials: E
authors.lastname: Alvarez-Curto
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (M.J.V.L., G.C., R.J.W., E.A.-C., S.M., G.M.), and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (G.C.).
authors.forename: Sara
authors.initials: S
authors.lastname: Marsango
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (M.J.V.L., G.C., R.J.W., E.A.-C., S.M., G.M.), and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (G.C.) Graeme.Milligan@glasgow.ac.uk gianluigicaltabiano@gmail.com.
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D001285:Atropine / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry; D054314:Drug Inverse Agonism; D031541:Fluorescence Resonance Energy Transfer; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D018721:Muscarinic Agonists / Q000737:chemistry / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D021381:Protein Transport; D011869:Radioligand Assay; D043587:Receptor, Muscarinic M3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000737:chemistry; D000069447:Tiotropium Bromide
chemical_list: D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D043587:Receptor, Muscarinic M3; D012602:Scopolamine Derivatives; D001285:Atropine; D002784:Cholesterol; D000069447:Tiotropium Bromide