Pronounced conformational changes following agonist activation of the M3 muscarinic acetylcholine receptor

The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M-3 muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M-3 muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M-3 receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M-3 receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M-3 receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.