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FASEB J. 2019 Mar;33(3):3237-3251. doi: 10.1096/fj.201800936R. Epub 2018 Nov 8.

Direct SUMOylation of M1 muscarinic acetylcholine receptor increases its ligand-binding affinity and signal transduction.

Xu J1, Tan P1, Li H1, Cui Y1, Qiu Y1, Wang H1, Zhang X1, Li J1, Zhu L1, Zhou W1,2,3,4,5,6, Chen H1,7.

Author information

1
Department of Pharmacology and Chemical Biology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
2
Laboratory of Oral Microbiota and Systemic Disease, Shanghai Ninth People's Hospital, Shanghai, China.
3
College of Stomatology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
4
National Clinical Research Center for Oral Diseases, Shanghai, China.
5
Shanghai Key Laboratory of Stomatology, Shanghai, China.
6
Shanghai Research Institute of Stomatology, Shanghai, China; and.
7
Institute of Interdisciplinary Integrative Biomedical Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

Abstract

SUMOylation is a significant post-translational modification (PTM) by the small ubiquitin-related modifier (SUMO). Increasing evidence shows SUMOylation regulates GPCR signaling; however, very few GPCRs have been shown to be SUMOylation targets to date. In this study, we identified M1 muscarinic acetylcholine receptor (M1 mAChR), a member of the GPCRs, as a new SUMO substrate. When the mAChR was activated by the agonist carbachol, the colocalization of the M1 mAChR and SUMO-1 protein markedly decreased in immunoprecipitation and immunofluorescence assays. SUMOylation of the M1 mAChR played an important role in increasing the ligand-binding affinity to M1 mAChR, signaling efficiencies, and receptor endocytosis. Through the site-directed mutagenesis approach, K327 was identified as the SUMOylation site of the M1 mAChR. Mutation of the consensus SUMOylation site of the M1 mAChR reduces not only the colocalization of SUMO-1, but also the ligand-binding affinity and signal transduction. The function of M1 mAChR was regulated by SUMOylation through the stabilization of active-state conformation revealed by molecular dynamics simulations. Our results provide evidence that M1 SUMOylation is an important PTM involved in regulation of the affinity for agonists and for activation of signaling pathways.-Xu, J., Tan, P., Li, H., Cui, Y., Qiu, Y., Wang, H., Zhang, X., Li, J., Zhu, L., Zhou, W., Chen, H. Direct SUMOylation of M1 muscarinic acetylcholine receptor increases its ligand-binding affinity and signal transduction.

KEYWORDS:

GPCR; agonist; radio-ligand binding; site-directed mutagenesis

PMID:
30407877
DOI:
10.1096/fj.201800936R

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