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

Abstract

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 Gαq and β-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and β-arrestin2. Pilocarpine preferentially promoted the recruitment of β-arrestin2 (∆∆RAi = -0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. 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 Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to β-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 Gαq and β-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.

Keywords: BRET; Gαq; M1 muscarinic acetylcholine receptor; molecular docking; signaling bias; β-arrestin.

Figure 1

The 2D chemical structures of the six agonists.

Figure 2

Efficacy of agonists in Gαq-binding to M1mAChR revealed by BRET assays. (A) Schematic illustration of bioluminescent resonance energy transfer (BRET) and dose-response curves for ACh- (B), CCh- (C), Pilocarpine- (D), Iperoxo- (E), McN-A-343- (F), and Xanomeline-induced (G) Gαq interactions with M1mAChR. (H) EC50 of agonists and (I) Emax of agonists (means ± S.E.M., n = 3). The statistical significance of the differences between the indicated groups and the ACh was assessed by one-way ANOVA, where *** p < 0.001, ** p < 0.01 is ns, not significant.

Figure 3

Analysis of antagonist effects of Gq-M1mAChR binding assessed by BRET assays. (A) ACh, (B) CCh, (C) Pilocarpine, (D) Iperoxo, (E) McN-A-343, (F) Xanomeline, (G) antagonistic potency of Atropine to agonists, and (H) antagonistic potency of Scopolamine to agonists. Data are means ± S.E.M. of 3–8 independent experiments performed in duplicate. The statistical significance of differences between the indicated groups was assessed by two-way ANOVA, where ** p < 0.01, *** p < 0.001.

Figure 4

Efficacy of agonists in β-arrestin2 binding to M1mAChR revealed by BRET assays. (A) Schematic illustration of bioluminescent resonance energy transfer (BRET) and dose-response curves for ACh- (B), CCh- (C), Pilocarpine- (D), Iperoxo- (E), McN-A-343- (F), and Xanomeline-induced (G) β-arrestin2 interactions with M1mAChR. (H) EC50 of agonists and (I) Emax of agonists (means ± S.E.M., n = 3). The statistical significance of differences between the indicated group and the ACh was assessed by one-way ANOVA, where ** p < 0.01, *** p < 0.001 is ns, not significant.

Figure 5

Analysis of antagonist effects on β-arrestin2-M1mAChR binding by BRET. (A) ACh, (B) CCh, (C) Pilocarpine, (D) Iperoxo, (E) McN-A-343, (F) Xanomeline, (G) antagonistic potency of Atropine to agonists, and (H) antagonistic potency of Scopolamine to agonists. Data are means ± S.E.M. of 3–8 independent experiments performed in duplicate. The statistical significance of differences between the indicated groups was assessed by two-way ANOVA, where *** p < 0.001.

Figure 6

Determination of Gαq and β-arrestin2 recruitment to M1mAChR using commercial methods. (A) Schematic illustration of Fluo4-AM and dose-response curves for (B) ACh, (C) CCh, (D) Pilocarpine, (E) Iperoxo, (F) McN-A-343, and (G) Xanomeline, verified by Fluo4-AM. (H) Schematic illustration of FRET and dose-response curves for (I) Ach, (J) CCh, (K) Pilocarpine, (L) Iperoxo, (M) McN-A-343, and (N) Xanomeline, verified by FRET. Data are means ± S.E.M. of 3 independent experiments performed in duplicate.

Figure 7

Evaluating the signaling bias of M1mAChR agonists. (A) Bias plots for ligands of M1mAChR using BRET assays. (B) Bias plots for ligands of M1mAChR obtained by commercial methods. (C) Comparing BRET assays and commercial methods in order identifying which agonist signal is preferred (relative intrinsic activity of each method). Data are means ± S.E.M. of 3–8 independent experiments performed in duplicate.

Figure 8

Interaction sites of agonists with M1mAChR. Binding site and interaction of McN-A-343 with inactive M1mAChR (A), Binding site and interaction of McN-A-343 with active M1mAChR (B), Binding site and interaction of Pilocarpine with inactive M1mAChR (C), and binding site and interaction of Pilocarpine with inactive M1mAChR (D). Dotted lines indicate the interaction between the ligand and the corresponding residues; while the line colours indicate the interaction types.