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)

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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:Milligan, Professor Graeme and Pediani, Dr John and Ward, Dr Richard and Tobin, Andrew and Jenkins, Mrs Laura and Hesse, Ms Sarah and Raicu, Professor Valerica and Marsango, Dr Sara and Bradley, Dr Sophie
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

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
170430The organisational structure of class A GPCRs: Implications for pharmacology, function and therapeutic regulationGraeme MilliganMedical Research Council (MRC)MR/L023806/1MCSB - Molecular Pharmacology