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Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing G\u03b1q and \u03b2-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of G\u03b1q and \u03b2-arrestin2. Pilocarpine preferentially promoted the recruitment of \u03b2-arrestin2 (\u2206\u2206RAi = \u22120.5), while McN-A-343 (\u2206\u2206RAi = 1.5), Xanomeline (\u2206\u2206RAi = 0.6), and Iperoxo (\u2206\u2206RAi = 0.3) exhibited a preference for the recruitment of G\u03b1q. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in G\u03b1q signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to \u03b2-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards G\u03b1q and \u03b2-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.<\/jats:p>","DOI":"10.3390\/ijms24087356","type":"journal-article","created":{"date-parts":[[2023,4,17]],"date-time":"2023-04-17T03:14:09Z","timestamp":1681701249000},"page":"7356","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between G\u03b1q and \u03b2-Arrestin through BRET Assays and Molecular Docking"],"prefix":"10.3390","volume":"24","author":[{"given":"Dongxue","family":"Wang","sequence":"first","affiliation":[{"name":"Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"},{"name":"Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunjin","family":"Yao","sequence":"additional","affiliation":[{"name":"Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"},{"name":"Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shiqi","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"},{"name":"Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifei","family":"Hou","sequence":"additional","affiliation":[{"name":"Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"},{"name":"Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lanxue","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China"},{"name":"Shanghai Universities Collaborative Innovation Center for Translational Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongzhuan","family":"Chen","sequence":"additional","affiliation":[{"name":"Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"},{"name":"Shanghai Frontiers Science Center of TCM Chemical Biology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2168-2296","authenticated-orcid":false,"given":"Jianrong","family":"Xu","sequence":"additional","affiliation":[{"name":"Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"},{"name":"Shanghai Frontiers Science Center of TCM Chemical Biology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1016\/j.cels.2021.02.001","article-title":"Diversity of the Gbetagamma complexes defines spatial and temporal bias of GPCR signaling","volume":"12","author":"Masuho","year":"2021","journal-title":"Cell Syst."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1164\/rccm.202009-3686OC","article-title":"Targeting the P2Y(13) Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma","volume":"205","author":"Werder","year":"2022","journal-title":"Am. 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