TY - JOUR
T1 - Structural determinants of allosteric agonism and modulation at the M 4 muscarinic acetylcholine receptor: Identification of ligand-specific and global activation mechanisms
AU - Nawaratne, Vindhya
AU - Leach, Katherine
AU - Felder, Christian C
AU - Sexton, Patrick M
AU - Christopoulos, Arthur
PY - 2010
Y1 - 2010
N2 - The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M(4) mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M(1)/M(4) mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M(4) mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile(93) and Lys(95) as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe(186) in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp(432) in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr(439) and Tyr(443), were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.
AB - The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M(4) mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M(1)/M(4) mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M(4) mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile(93) and Lys(95) as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe(186) in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp(432) in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr(439) and Tyr(443), were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.
UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20406819
M3 - Article
VL - 285
SP - 19012
EP - 19021
JO - The Journal of Biological Chemistry
JF - The Journal of Biological Chemistry
SN - 1083-351X
IS - 25
ER -