Use of an in Situ Disulfide Cross-Linking Strategy To Map Proximities between Amino Acid Residues in Transmembrane Domains I and VII of the M₃Muscarinic Acetylcholine Receptor

Abstract

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII. Cysteine residues were substituted, one at a time, into 10 consecutive positions of TM I (Ala71-Val80) of a modified version of the M(3) muscarinic receptor that lacked most endogenous cysteine residues and contained a factor Xa cleavage site within the third intracellular loop. Following their expression in COS-7 cells, the 10 resulting cysteine mutant receptors were oxidized in their native membrane environment, either in the absence or in the presence of muscarinic ligands. Disulfide cross-link formation was monitored by examining changes in the electrophoretic mobility of oxidized and factor Xa-digested receptors on SDS gels. When molecular iodine was used as the oxidizing agent, the L77C receptor (position 1.42) was the only mutant receptor that displayed significant disulfide cross-linking, either in the absence or in the presence of muscarinic agonists or antagonists. On the other hand, when the Cu(II)-(1,10-phenanthroline)(3) complex served as the redox catalyst, muscarinic ligands inhibited disulfide cross-linking of the L77C receptor, probably because of impaired access of this relatively bulky oxidizing agent to the ligand binding crevice. The iodine cross-linking data suggest that M(3) muscarinic receptor activation is not associated with significant changes in the relative orientations of the outer and/or central segments of TM I and VII. In bovine rhodopsin, the residues present at the positions corresponding to Cys532 and Leu77 in the rat M(3) muscarinic receptor are not located directly adjacent to each other, raising the possibility that the relative orientations of TM I and VII are not identical among different class I GPCRs. Alternatively, dynamic protein backbone fluctuation may occur, enabling Cys532 to move within cross-linking distance of Leu77 (Cys77).

Identification of an Agonist-induced Conformational Change Occurring Adjacent to the Ligand-binding Pocket of the M3 Muscarinic Acetylcholine Receptor

“…1). In agreement with a recently developed three-dimensional model of the M 3 muscarinic receptor (27), we demonstrated previously that Cys-532 is located adjacent to the ligand-binding pocket projecting into the center of the TM receptor core (29). Specifically, we examined whether Cys-532 is able to form activity-dependent crosslinks with Cys residues introduced into the exofacial segment of TM III (Leu-142 3.27 -Asn-152 3.37 ) (see Fig.…”

“…Transfected cells were incubated with atropine (1 M) for the last 24 h of culture. Previous studies have shown that this atropine incubation step significantly increases the expression levels of Cys-substituted mutant M 3 muscarinic receptors (26,27,29).…”

“…Cells were transfected with 4 g of receptor plasmid DNA/dish using the Lipofectamine Plus kit (Invitrogen) according to the manufacturer's instructions. To increase receptor expression levels, 1 M atropine was routinely added to the incubation medium for the last 24 h of culture as described previously by us (26,27,29).…”

C5a Receptor Oligomerization

“…Disulfide trapping has been used to demonstrate the spatial arrangement of dimers of the E. coli aspartate receptor (26), bacterial Trg chemoreceptor (27), and the above-mentioned dopamine D2 receptor (18); this technique has also been applied to the analysis of intramolecular contacts between transmembrane domains of GPCRs, such as the M 3 muscarinic receptor (28). In this study, we demonstrate that the oxidation of endogenous C5aR molecules in human neutrophils results in disulfide-linked dimers.…”

Constitutive Activation of the Angiotensin II Type 1 Receptor Alters the Spatial Proximity of Transmembrane 7 to the Ligand-binding Pocket

“…Ala 291(7.42) and Phe 301(7.52) lie parallel to each other on the same helix face mostly facing the same area of the binding pocket. Ala 291(7.42) is predicted to face the protein interior, a feature that seems to be highly conserved among GPCRs, because this position also points inside the binding pocket of the D2 dopamine receptor, M3 muscarinic receptor, A2 adenosine receptor, and rhodopsin (21)(22)(23)(24). Phe 301(7.52) is part of a classical ␣-helical segment underlying the conserved NPXXY motif following the helical deviation.…”