biostudies-literatureUnknown14(1)Xu JThe M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestNature communications376https://www.ebi.ac.uk/biostudies/studies/S-EPMC9870890biostudies-literatureStructural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.PMC9870890Hu YMaeda SHubner HDu YWang HXu JNiu XWang QInoue ATao YJin CGmeiner PKobilka BKfalseStructural and dynamic insights into supra-physiological activation and allosteric modulation of a muscarinic acetylcholine receptor.The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrest2023-01-01T00:00:00Z2023 Jan2026-05-27T10:51:16.098Z2025-04-04T11:26:21.724ZS-EPMC98708903669061310.1038/s41467-022-35726-z