The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

Marsango, S. et al. (2022) The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes. Proceedings of the National Academy of Sciences of the United States of America, 119(24), e220110311. (doi: 10.1073/pnas.2201103119) (PMID:35671422) (PMCID:PMC9214538)

Abstract

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M1 muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M1 and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M1-mEGFP was expressed at levels equal to the M1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M1-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M1-mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M1-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

• Item Type: Articles
• Additional Information: These studies were supported by the Medical Research Council UK (Grant MR/L023806/1 to G.M.). The University of Wisconsin-Milwaukee team was supported by the NSF (Grant DBI 1919670) and the University of Wisconsin–Milwaukee Research Growth Initiative (Grant 101X396).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Ward, Dr Richard and Jenkins, Mrs Laura and Bradley, Dr Sophie and Tobin, Andrew and Hesse, Ms Sarah and Pediani, Dr John and Milligan, Professor Graeme and Raicu, Professor Valerica and Marsango, Dr Sara
• Authors: Marsango, S., Jenkins, L., Pediani, J. D., Bradley, S. J., Ward, R. J., Hesse, S., Biener, G., Stoneman, M. R., Tobin, A. B., Raicu, V., and Milligan, G.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Proceedings of the National Academy of Sciences of the United States of America
• Publisher: National Academy of Sciences
• ISSN: 0027-8424
• ISSN (Online): 1091-6490
• Copyright Holders: Copyright © 2022 The Authors
• First Published: First published in Proceedings of the National Academy of Sciences of the United States of America 119(24):e2201103119
• Publisher Policy: Reproduced under a Creative Commons License

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
170430		The organisational structure of class A GPCRs: Implications for pharmacology, function and therapeutic regulation	Graeme Milligan	Medical Research Council (MRC)	MR/L023806/1	MCSB - Molecular Pharmacology