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Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Scarselli, Marco and Li, Bo and Kim, Soo-Kyung and Wess, Jürgen (2007) Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation. Journal of Biological Chemistry, 282 (10). pp. 7385-7396. ISSN 0021-9258. doi:10.1074/jbc.M610394200.

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Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys220 residue, Gln207, Gly211, Arg213, Gly218, Ile222, Phe224, Leu225, and Pro228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.

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Li, Bo0000-0002-8019-8891
Kim, Soo-Kyung0000-0002-4498-5441
Additional Information:© 2007 the American Society for Biochemistry and Molecular Biology. Received for publication, November 7, 2006 , and in revised form, January 5, 2007. Originally published In Press as doi:10.1074/jbc.M610394200 on January 9, 2007. This article is dedicated to the memory of Bo Li who died in a tragic traffic accident on October 20, 2006. We thank Dr. Jean-Marc Guettier (NIDDK, National Institutes of Health) for critical reading of the manuscript. This work was supported by the Intramural Research Program of the National Institutes of Health, NIDDK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Issue or Number:10
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ID Code:7603
Deposited By: Archive Administrator
Deposited On:12 Mar 2007
Last Modified:08 Nov 2021 20:44

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