Identification and molecular characterization of m3 muscarinic receptor dimers

Several studies suggest, but do not prove directly, that muscarinic recepto rs may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments u sing a modified version of the rat m3 muscarinic receptor (referred to as m 3') as a model system. When membrane lysates prepared from m3' receptor-exp ressing COS-7 cells were subjected to Western blot analysis under non-reduc ing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers, However, unde r reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfid e-linked m3 receptor complexes. Similar results were obtained when native m 3 muscarinic receptors present in rat brain membranes were analyzed. Contro l experiments carried out in the presence of high concentrations of the SH group alkylating agent, N-ethylmaleimide, suggested that disulfide bond for mation did not occur artifactually during the preparation of cell lysates, The formation of m3' receptor dimers/multimers was confirmed in coimmunopre cipitation studies using differentially epitope-tagged m3' receptor constru cts. In addition, these studies showed that m3' receptors were also able to form non-covalently associated receptor dimers and that m3' receptor dimer formation was receptor subtype-specific, Immunological studies also demons trated that m3' receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserve d extracellular Cys residues (Cys-140 and Cys-220) play key roles in the fo rmation of disulfide-linked m3' receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and h ighlight the specificity and molecular diversity of G protein-coupled recep tor dimerization/oligomerization.