Use of an in Situ Disulfide Cross-Linking Strategy To Study the Dynamic Properties of the Cytoplasmic End of Transmembrane Domain VI of the M₃Muscarinic Acetylcholine Receptor

Abstract

The ligand-induced activation of G protein-coupled receptors (GPCRs) is predicted to involve pronounced conformational changes on the intracellular surface or the receptor proteins. A reorientation of the cytoplasmic end of transmembrane domain VI (TM VI) is thought to play a key role in GPCR activation and productive receptor/G protein coupling. Disulfide cross-linking studies with solubilized, Cys-substituted mutant versions of bovine rhodopsin and the M3 muscarinic acetylcholine receptor suggested that the cytoplasmic end of TM VI is conformationally highly flexible, even in the absence of activating ligands (Farrens, D. L., et al. (1996) Science 274, 768-770; Zeng, F. Y., et al. (1999) J. Biol. Chem. 274, 16629-16640). To test the hypothesis that the promiscuous disulfide cross-linking pattern observed in these studies was caused by the use of solubilized receptor proteins endowed with increased conformational flexibility, we employed a recently developed in situ disulfide cross-linking strategy that allows the detection of disulfide bonds in Cys-substituted mutant M3 muscarinic receptors present in their native membrane environment. Specifically, we used membranes prepared from transfected COS-7 cells to analyze a series of double Cys mutant M3 receptors containing one Cys residue within the sequence K484(6.29) to S493(6.38) at the cytoplasmic end of TM VI and a second Cys residue at the cytoplasmic end of TM III (I169C(3.54)). This analysis revealed a disulfide cross-linking pattern that was strikingly more restricted than that observed previously with solubilized receptor proteins, both in the absence and in the presence of the muscarinic agonist, carbachol. Carbachol stimulated the formation of disulfide bonds in only two of the 10 analyzed mutant muscarinic receptors, I169C(3.54)/K484C(6.29) and I169C(3.54)/A488C(6.33), consistent with an agonist-induced rotation of the cytoplasmic end of TM VI. These findings underline the usefulness of analyzing the structural and dynamic properties of GPCRs in their native lipid environment.

Distinct Structural Changes in a G Protein-coupled Receptor Caused by Different Classes of Agonist Ligands

“…Receptors were oxidized with Cu(II)-phenanthroline (25 M) in the absence or the presence of the muscarinic agonist, CCh (1 mM), digested with factor Xa, and subjected to Western blotting analysis (nonreducing conditions), using the anti-M 3 antibody. All bands shown correspond to the ϳ38-kDa full-length receptor species, which is indicative of successful disulfide cross-linking (23)(24)(25)(26). Note that two of the investigated receptors, A91C/T549C and F92C/F550C, gave pronounced cross-linking signals, the intensity of which was significantly reduced in the presence of CCh.…”

“…Cells were transfected with 4 g/dish of receptor plasmid DNA using the Lipofectamine Plus kit (Invitrogen), according to the manufacturer's instructions. To achieve higher receptor expression levels, transfected cells were incubated with 1 M atropine for the last 24 h of culture, as described previously (23)(24)(25)(26).…”

“…1; see "Experimental Procedures" for details). Under these conditions, the appearance of a full-length receptor band (ϳ38 kDa) is indicative of successful disulfide cross-linking (23)(24)(25)(26).…”

“…To monitor ligand-induced changes in receptor structure, we employed an in situ disulfide cross-linking strategy that allows the detection of disulfide bond formation between Cys residues that are adjacent to each other in the three-dimensional structure of the receptor (23)(24)(25)(26). One major advantage of this strategy is that ligand-dependent conformational changes can be detected in receptors present in their native membrane environment, without the need for any receptor purification and reconstitution steps.…”

The First Extracellular Loop of the Saccharomyces cerevisiae G Protein-coupled Receptor Ste2p Undergoes a Conformational Change upon Ligand Binding

“…The disruption of the membrane architecture, which must occur when membranes are prepared, could subtly alter the position of the transmembrane domains and the extracellular loops, making fine mapping of the extracellular-membrane boundaries difficult. Underlying the importance of examining receptors in their native environment is the recent finding that formation of disulfide bonds between transmembrane domains in the M3 muscarinic acetylcholine receptor was much more restricted when examined in its native lipid environment rather than as solubilized reconstituted receptors (41). Based on our observations with cysteine accessibility of residues at the N-terminal end of EL1, we propose that residue Tyr 101 is positioned at the extracellular boundary of TM2, rather than deeper in the transmembrane bundle as has been proposed (27).…”