DOI: 10.4225/03/5897c70bb14e4
title: Structure-function studies of M4 muscarinic acetylcholine receptor allosteric modulation

description: The M4 muscarinic acetylcholine receptor (mAChR) is implicated in many central nervous system disorders, however, due to a highly conserved acetylcholine (ACh) binding orthosteric site, there is a lack of highly selective ligands as therapeutics and experimental probes for this target. There are two classes of functionally selective M4 mAChR ligands, one being the allosteric modulators, typified by the small molecule LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide) (Chan et al., 2008), and the other being the atypical agonists, exemplified by xanomeline and McN-A-343, whose mode of binding at the M4 mAChR is not clear. Challenges to understanding the activity of these ligands include the interplay of binding, efficacy and, when considering allosteric modulation, cooperativity. Thus, to investigate the molecular determinants of allosteric and atypical agonist activity, site-directed mutagenesis was utilised in conjunction with radioligand assays, to determine the role of specific amino acid residues on affinity or binding cooperativity, and M4 mAChR-mediated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, as a measure of efficacy or functional modulation by LY2033298. The endogenous agonist, ACh was used as a control agonist.
	Chapter 2 focused on four different regions of the M4 mAChR; extracellular loops (ECLs) 1, 2 and 3, and transmembrane domain (TM) 7. In the ECL1, we identified Ile93(2.65) and Lys95(2.67) as key residues that specifically governed the signalling efficacy of LY2033298 and its binding cooperativity with ACh, while Phe186(5.29) in the ECL2 was identified as a key contributor to the binding affinity of the modulator for the allosteric site. The highly conserved TM7 residues, Tyr439(7.39) and Tyr443(7.43), were important for both McN-A-343 and xanomeline affinity, while the ECL residues, Ile93(2.65), Phe186(5.29), Ser428(6.63) and Asp432(7.32) were detrimental to McN-A-343 affinity. Ser428(6.63) was exclusively involved in atypical agonist efficacy. In contrast, Tyr439(7.39) and Tyr443(7.43), were identified as contributing to a key activation switch utilized by all classes of agonists, except xanomeline. 
	This initial study highlighted the general importance of aromatic residues for allosteric agonist activity, which led us to perform alanine scanning mutagenesis of selected aromatic residues in the top third the M4 mAChR. Additionally, due to the importance of Phe186(5.29) in allosteric agonist binding in the ECL2, residues lining the proximal and distal ends of ECL2 was also mutated. Results outlined in Chapter 3 showed that, Tyr89(2.61) and Trp435(7.35), on top of TM2 and TM7, respectively, were important for LY2033298 binding. Tyr89(2.61) was exclusively involved in LY2033298 efficacy compared to the other ligands, while other TM2/ECL1 residues also played a large role in LY2033298 efficacy. Multiple residues clustered between the putative allosteric and orthosteric sites, on TM2/ECL1 and TM7, appear to form the conformational link for transmitting ACh-LY2033298 cooperativity. Tyr89(2.61) was particularly important for the positive binding cooperativity between ACh and LY2033298. Only two residues (Tyr89(2.61) and Tyr439(7.39)) were identified to affect the functional modulation of ACh by LY2033298 in the current thesis. Orthosteric binding site residues, Trp164(4.57) and Trp413(6.48), were global activation switches for both allosteric and orthosteric agonists.
	The final study, outlined in Chapter 4, characterised the activity of the atypical agonists at the second set of mutant M4 mAChRs. It revealed that Trp413(6.48) is a critical contact residue for xanomeline, while playing a smaller role in ACh and McN-A-343 binding. In the distal ECL2, Ile187(5.30), may play a role in both xanomeline and McN-A-343 binding, while Ile187(5.30), Gln188(5.31) and Phe189(5.32) played a large role in McN-A-343 efficacy. Trp164(4.57) and Trp413(6.48), like Tyr439(7.39) and Tyr443(7.43), were global activation switches for all three classes of agonists. These results provide new insights into the existence of multiple binding pockets and activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric and atypical agonists for future development of selective M4 mAChR ligands, whereas others represent global activation mechanisms for all classes of ligand.