Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation. Export

@article{citeulike:1244687, abstract = {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 M(3) 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 Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) 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.}, address = {Molecular Signaling , Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.}, author = {Scarselli, M. and Li, B. and Kim, S. K. and Wess, J.}, citeulike-article-id = {1244687}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M610394200}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/17213190}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=17213190}, day = {9}, doi = {10.1074/jbc.M610394200}, issn = {0021-9258}, journal = {J Biol Chem}, keywords = {activation, amine, binding, ecl2, loops, mutation}, month = {March}, number = {10}, pages = {7385--7396}, posted-at = {2007-04-23 10:28:29}, priority = {4}, title = {Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation.}, url = {http://dx.doi.org/10.1074/jbc.M610394200}, volume = {282}, year = {2007} }

TY - JOUR ID - citeulike:1244687 L3 - citeulike-article-id:1244687 N2 - 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 M(3) 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 Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) 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. IS - 10 JF - J Biol Chem SN - 0021-9258 AD - Molecular Signaling , Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA. EP - 7396 TI - Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation. VL - 282 SP - 7385 KW - activation KW - amine KW - binding KW - ecl2 KW - loops KW - mutation AU - Scarselli, M AU - Li, B AU - Kim, SK AU - Wess, J PY - 2007/03/09/ UR - http://dx.doi.org/10.1074/jbc.M610394200 ER -