The Molecular Basis of Oligomeric Organization of the Human M₃Muscarinic Acetylcholine Receptor

Abstract

G protein-coupled receptors, including the M 3 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 sitedirected 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 M 3 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 M 3 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 1 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 Nglycosylation. 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.

Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs

“…For example, studies on the muscarinic M 2 receptor subtype have variously concluded that it may be predominantly monomeric but with a capacity to form dimers ( 4 ), is routinely dimeric ( 5 ), or is predominantly tetrameric ( 6 ). Similar variation has been reported for each of the muscarinic M 1 ( 5 , 7 ) and M 3 ( 8 – 11 ) subtypes. In addition, although certain studies have indicated that addition of muscarinic ligands does not affect the prevalence of receptor dimers/oligomers ( 5 , 12 ), other studies have indicated a capacity for regulation.…”

Analysis of Human Dopamine D3 Receptor Quaternary Structure

“…Recently, mathematical analysis of spectrally resolved multi-photon FRET microscopy data has provided evidence that a substantial proportion of the human M 3 muscarinic acetylcholine receptor is present at the surface of transfected cells as a tetramer with rhombic organization ( 39 ). Moreover, as in-house data had shown a specific role for both TMD VII and TMD VI in organization of the tetramer via cholesterol molecules that bridge a pair (dimer + dimer) of TMD I-helix VIII interface M 3 muscarinic receptor dimers ( 40 ), we generated both additional mutants and models of potential organization of the hD 3 , akin to these muscarinic M 3 models, to explore if these could unify the experimental observations.…”

A Molecular Basis for Selective Antagonist Destabilization of Dopamine D3 Receptor Quaternary Organization

“…This implies that the observed proportions of receptor monomers, dimers and oligomers may well vary between individual cells and tissues and, furthermore, the binding of distinct ligand chemotypes may selectively alter this if they either differentially regulate receptor expression levels or stabilize distinct states of the receptor. Given roles of segments of the seven transmembrane domains (TMDs) of GPCRs that are located close to the extracellular face in controlling class A receptor dimerization 4, 20, 21 it is clearly possible that different antagonist/inverse agonist-bound structures of the same GPCR may alter the dimerization potential or propensity of the receptor and, therefore, the steady-state distribution of monomers, dimers and oligomers.…”