Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking
Abstract
:1. Introduction
2. Results
2.1. Efficacy of M1mAChR Agonists in Gαq-Binding Revealed by BRET Assays
2.2. Kinetics of Gαq-M1mAChR Coupling Assessed by BRET Assays
2.3. Efficacy of Agonists in β-Arrestin2-Binding to M1mAChR Revealed by BRET Assays
2.4. Kinetics of β-Arrestin2-M1mAChR Coupling Assessed by BRET Assays
2.5. Determination of Gαq and β-Arrestin2 Pathway Activation by Commercial Methods
2.6. Evaluation of the Signaling Bias of M1mAChR Agonists
2.7. The Interaction Underlying the Signaling Bias Observed in Molecular Docking
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. cDNA Constructs
4.3. BRET Assays for Gαq and β-Arrestin Coupling
4.4. Fluo-4-AM
4.5. β-Arrestin Recruitment
4.6. Molecular Docking
4.7. Data Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
GPCRs | G-protein-coupled receptors |
M1mAChR | M1 muscarinic acetylcholine receptor |
BRET | Bioluminescence resonance energy transfer |
FRET | Fluorescence resonance energy transfer |
ACh | Acetylcholine |
CCh | Carbachol |
RAi | Relative intrinsic activity |
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Ligand | Gαq (BRET) (Δlog (τ/KA)) | β-Arrestin (BRET) (Δlog (τ/KA)) | Gαq (Fluo4-AM) (Δlog (τ/KA)) | β-Arrestin (FRET) (Δlog (τ/KA)) |
---|---|---|---|---|
McN-A-343 | 1.85 ± 0.010 | 0.34 ± 0.040 | 1.20 ± 0.005 | −0.67 ± 0.020 |
Iperoxo | 1.56 ± 0.090 | 1.27 ± 0.020 | 0.98 ± 0.003 | −0.03 ± 0.040 |
Xanomeline | 1.58 ± 0.050 | 0.94 ± 0.030 | 0.32 ± 0.002 | −0.29 ± 0.020 |
CCh | 0.26 ± 0.020 | 0.26 ± 0.040 | −1.09 ± 0.030 | −1.12 ± 0.040 |
Pilocarpine | −3.20 ± 0.001 | −2.70 ± 0.002 | −2.61 ± 0.002 | −2.08 ± 0.030 |
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Wang, D.; Yao, Y.; Wang, S.; Hou, Y.; Zhao, L.; Wang, H.; Chen, H.; Xu, J. Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking. Int. J. Mol. Sci. 2023, 24, 7356. https://doi.org/10.3390/ijms24087356
Wang D, Yao Y, Wang S, Hou Y, Zhao L, Wang H, Chen H, Xu J. Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking. International Journal of Molecular Sciences. 2023; 24(8):7356. https://doi.org/10.3390/ijms24087356
Chicago/Turabian StyleWang, Dongxue, Yunjin Yao, Shiqi Wang, Yifei Hou, Lanxue Zhao, Hao Wang, Hongzhuan Chen, and Jianrong Xu. 2023. "Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and β-Arrestin through BRET Assays and Molecular Docking" International Journal of Molecular Sciences 24, no. 8: 7356. https://doi.org/10.3390/ijms24087356