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 FRET studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of transmembrane domains I, V, VI and VII, as well as intracellular helix VIII, to the overall organization. Molecular modelling studies were then employed 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. The results could be accommodated fully by a model in which the cell surface M3 receptor was a tetramer with rhombic, but not linear, orientation, consistent with previous studies based on spectrally-resolved, multi-photon FRET. Modelling studies suggest, furthermore, a key role for molecules of cholesterol at the dimer + dimer interface, 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 occurs before plasma membrane delivery and may be a key step in cellular quality control assessment.
- Muscarinic cholinergic
- Structure-activity relationships and modeling
- Fluorescence techniques
- Mutagenesis/Chimeric approaches
- The American Society for Pharmacology and Experimental Therapeutics