The allosteric core region of the M₂ muscarinic acetylcholine receptor

Abstract

Muscarinic acetylcholine receptors (mAChRs) are seven transmembrane domain-spanning proteins belonging to the family A of G protein-coupled receptors (GPCRs). In addition to the orthosteric binding site (i.e. the site where the endogenous ligand acetylcholine (ACh) binds and activates the receptor), mAChRs possess a common allosteric binding site which is located directly above the orthosteric binding site. The amino acids Y177, W422 and T423 located in the extracellular loop 2 and the upper part of transmembrane domain 7, respectively have been previously found to line the core region of the allosteric binding site of the M<strong>₂</strong> mAChR1 <br /> Here, the aim was to investigate the consequences caused by the loss of the three core amino acids located in the “bottleneck-region” (CHO-hM<strong>₂</strong> W422A, T423A, Y177A), between the orthosteric and the allosteric part of the ligand binding cavity of the M<strong>₂</strong> mAChRs on ligand binding, receptor activation and receptor-ligand selectivity.<br /> Radioligand binding experiments with [3H]NMS in M<strong>₂</strong> wt and triple mutant receptors showed that the triple mutant strongly reduces the binding affinities of the M<strong>₂</strong> muscarinic full agonists. Interestingly, the partial agonist pilocarpine´s binding to M<strong>₂</strong> receptor was observed to be completely insensitive to the triple mutation. The [35S]GTPγS binding assay showed that the M<strong>₂</strong> muscarinic full agonists lose their potencies at the triple mutant but their efficacies remain unchanged compared to M<strong>₂</strong> wt receptor in Gi-signalling. However, the partial agonist pilocarpine loses efficacy at the triple mutant but its potency remains unchanged compared to M<strong>₂</strong> wt receptors. The cAMP and dynamic mass redistribution (DMR) assays showed that the full agonists acetylcholine and iperoxo displayed reduced efficacy in Gs-signalling at the M<strong>₂</strong> triple mutant. The allosteric ligand 6-naph which is a positive allosteric modulator of [3H]NMS binding at the M<strong>₂</strong> wt receptor4,5,6 becomes a negative allosteric modulator at the M<strong>₂</strong> triple mutant. Dualsteric ligands which consist of both the allosteric and orthosteric pharmacophores, e.g. iper-6-naph, switch to a predominantly dualsteric binding pose at the M<strong>₂</strong> triple mutant compared to the M<strong>₂</strong> wt receptors. <br /> Hence, the studies show that not only is the M<strong>₂</strong> allosteric binding site important for allosteric ligand binding, but also regulates orthosteric and dualsteric ligand activity.