The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M 3 muscarinic receptor, a prototypical G q -coupled receptor, as a model system. It is known that a tyrosine residue (Tyr 254 ) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M 3 receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M 3 receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys 484-Ser 493 at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M 3 receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing aninsitudisulfidecross-linkingstrategytoexamineagonistdependent dynamic structural changes in a G proteincoupled receptor. G protein-coupled receptors (GPCRs)1 constitute the largest class of signaling molecules in the mammalian genome (1-3).All GPCRs are predicted to share a conserved molecular architecture consisting of a bundle of seven ␣-helically arranged transmembrane domains (TM I-VII) linked by alternating intracellular and extracellular loops (Fig. 1). Despite the remarkable structural diversity of the ligands that exert their physiological functions via interaction with specific classes of GPCRs, all GPCRs are thought to share a conserved mechanism of activation. Several lines of evidence indicate that the binding of ligands to the extracellular side of the receptor leads to changes in the arrangement of distinct TM helices, which are then propagated to the intracellular surface of the receptor, thus enabling the receptor to recognize and activate specific classes of heterotrimeric G proteins (4 -7).Accumulating evidence suggests that GPCR activation may involve a change in the relative disposition of TM III and VI (4 -11). Elegant site-directed spin labeling studies (9) carried out with t...

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.

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide crosslinking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M 3 muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.