title: Structural basis of M3 muscarinic receptor dimer/oligomer formation.
abstract: Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.
authors.affiliation: Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health,Bethesda, Maryland 20892, USA.
authors.forename: Sara M
authors.initials: SM
authors.lastname: McMillin
authors.forename: Moritz
authors.initials: M
authors.lastname: Heusel
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Stefano
authors.initials: S
authors.lastname: Costanzi
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wess
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D016296:Mutagenesis; D010449:Peptide Mapping; D017510:Protein Folding; D055503:Protein Multimerization / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein
chemical_list: D043587:Receptor, Muscarinic M3