Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gq and -Arrestin through BRET Assays and Molecular Docking

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. 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 proportional to q and beta-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of G proportional to q and beta-arrestin2. Pilocarpine preferentially promoted the recruitment of beta-arrestin2 (Delta Delta RAi = 0.5), while McN-A-343 (Delta Delta RAi = 1.5), Xanomeline (Delta Delta RAi = 0.6), and Iperoxo ( Delta Delta RAi = 0.3) exhibited a preference for the recruitment of G proportional to q. 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 proportional to q signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to beta-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 proportional to q and beta-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.