Pronounced Conformational Changes following Agonist Activation of the M3 Muscarinic Acetylcholine Receptor

Abstract

The conformational changes that convert G proteincoupled 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 crosslinks 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.

A chemical-genetic approach to study G protein regulation of β cell function in vivo

“…Cells were then incubated at 37°C for 24 h with 1 Ci/mL myo-[ 3 H]inositol. Receptor-mediated increases in intracellular [ 3 H]-IP1 levels were then determined as described (24). In a similar fashion, receptor-mediated increases in intracellular cAMP assays were measured via ELISA according to the manufacturer's protocol (cAMP; Biotrak; Amersham Biosciences).…”

“…COS-7 cells were grown as monolayers as described (24). For transfections, cells were plated at a density of 1 ϫ 10 6 cells per 10-cm dish.…”

“…[ 3 H]-NMS saturation and inhibition binding assays were performed using membranes prepared from transfected COS-7 cells as described previously (24). ACh and CNO IC50 values were converted to Ki values using the Cheng-Prusoff equation.…”

Functional Analysis of Transmembrane Domain 2 of the M1 Muscarinic Acetylcholine Receptor

“…(SLACAD in the M 1 mAChR) within which Asp 2.50 (in standard nomenclature (10)) is one of the most highly conserved residues in the GPCR superfamily. In rhodopsin, it is directly hydrogen bonded to Asn 1.50 (corresponding to Asn 43 in the M 1 mAChR), and indirectly, via a water-mediated network, to Asn 7.49 (Asn 414 , M 1 ) (6,11,12). In the M 1 mAChR, Asn substitution first established the critical role of Asp 71 in signal transduction (13), a finding subsequently confirmed in the M 2 (14) and M 3 (15) subtypes as well as a host of other receptor types (16).…”

“…Additionally, we have substituted Asn 43 (1.50). The replacement of an amino acid by alanine deletes the side chain beyond the ␤-carbon atom, ablating any interactions that it makes, leaving a small hole in the three-dimensional structure without introducing new contacts to confuse the functional effect (28).…”