Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

Abstract

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...

Conserved Helix 7 Tyrosine Acts as a Multistate Conformational Switch in the 5HT2C Receptor

“…The hydrogen bonding network identified in the rhodopsin structure suggests that helix 7 is likely to be a principal player in the helical movement underlying activation. The predominant changes in the relative configurations of the helixes during receptor activation is a movement of helix 6 away from helix 3 (8) and closer, at its cytoplasmic side, to helix 5 (12,13). A model of the hydrogen bonding network connecting the rho- dopsin helices in the ground state suggests that the only helix with which helix 6 is connected is helix 7 (7).…”

“…Biophysical studies indicate that activation of rhodopsin and other GPCRs causes a displacement and rotation of helix 6 (8 -11) and a reduction in the distance between the cytoplasmic ends of helices 5 and 6 (12,13). The degree of helix movement is relatively slight as a "straightjacketed" rhodopsin with four engineered disulfide links between adjacent helices can still achieve an active state (14).…”

Homology modeling of opioid receptor-ligand complexes using experimental constraints

“…89 , 130 Accuracy of models of different functional states can also be improved by iterative distance geometry refi nement with experimental interhelical restraints appropriate for only the active or inactive conformation derived from mutagenesis, crosslinking studies and design of metal binding sites together with ligand-receptor distance restraints. 70 , 88 For example, the important interhelical distance constraints for the positioning of TM6 in the activated receptor state can be deduced from recent data on the formation of disulfi des between TM5 and TM6 in the m 3 muscarinic receptor upon agonist binding 131 and from the existence of an intrinsic allosteric Zn 2+ binding site at the interface of TM5 and TM6 of the b 2 -adrenergic receptor that facilitates agonist binding. 132 Additional constraints for adjusting helix packing in the activated state can be taken from the engineering of an activating metal-coordination center between TM3 and TM7 in b 2 -adrenergic 133 and tachykinin receptors, 134 and also between TM2 and TM3 of the MC4 melanocortin receptor.…”

Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function

“…A conserved interhelical movement of TM6 relative to TM3 and TM5 accompanies the activation of rhodopsin (8,24,38), the ␤ 2 -adrenergic receptor (32), and the M 3 muscarinic receptor (40). The high-resolution crystal structure of bovine rhodopsin shows that this molecule is stabilized by a panel of interhelical hydrogen bonds and hydrophobic interactions, most of which are contributed by the side chains of highly conserved GPCR residues (30).…”