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 an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.
Highlights
The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present
The observed cross-linking patterns indicated that M3 receptor activation leads to structural changes that allow the cytoplasmic ends of transmembrane domain (TM) V and TM VI to move closer to each other and that appear to involve a major change in secondary structure at the cytoplasmic end of TM VI
To examine the potential proximity of the cytoplasmic ends of TM III and TM VI, we recently described a cross-linking protocol involving the use of a series of Cys-substituted mutant M3 muscarinic receptors [30]
Summary
Vol 277, No 3, Issue of January 18, pp. 2247–2257, 2002 Printed in U.S.A. Conformational Changes That Occur during M3 Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy*. Mutational analysis of the rat M3 muscarinic receptor indicated that a limited number of amino acids located within the second (i2) and third intracellular (i3) loops and the adjacent cytoplasmic ends of TM III, V, and VI largely determine the Gq coupling preference of this receptor subtype [13,14,15,16] These findings are in agreement with a large number of studies using other classes of GPCRs [17, 18]. To gain insight into the structural mechanisms involved in M3 receptor activation, we wanted to investigate whether these functionally important residues change their relative positions on the cytoplasmic surface of the receptor protein following agonist-dependent receptor activation To address this issue, we decided to employ Cys substitution mutagenesis followed by disulfide cross-linking of Cys residues that are adjacent to each other in the three-dimensional structure of the receptor. The in situ disulfide cross-linking strategy described here should be highly useful to probe the structural mechanisms involved in GPCR activation in a systematic fashion
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