Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor

Abstract

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/alloste-ric) 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) cooperativ-ity 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. The GPCR 3 superfamily comprises nearly 1000 7-transmem-brane (TM)-spanning proteins that represent the largest target class for all current drugs (1). The mAChRs are prototypical family A GPCRs that mediate, along with the ionotropic nico-tinic receptors, the actions of the neurotransmitter, ACh. As with all biogenic amines, the orthosteric binding pocket for the endogenous agonist is located within the core of the helical TM bundle of these receptors. For the mAChRs, major contact points conserved across all five receptor subtypes include