Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS* |
| |
Authors: | Vindhya Nawaratne Katie Leach Christian C Felder Patrick M Sexton Arthur Christopoulos |
| |
Institution: | From the ‡Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Monash University, Parkville 3052, Victoria, Australia and ;§Eli Lilly and Co., Indianapolis, Indiana 46285 |
| |
Abstract: | The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno2,3-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 M4 mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M1/M4 mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M4 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 Ile93 and Lys95 as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe186 in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp432 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, Tyr439 and Tyr443, 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. |
| |
Keywords: | Allosteric Regulation Drug Action G Protein-coupled Receptors (GPCR) Receptor Structure-Function Receptors Acetylcholine Muscarinic Receptor |
|
|