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

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. Fluorescen...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2022-06, Vol.119 (24), p.1-e2201103119
Main Authors: Marsango, Sara, Jenkins, Laura, Pediani, John D, Bradley, Sophie J, Ward, Richard J, Hesse, Sarah, Biener, Gabriel, Stoneman, Michael R, Tobin, Andrew B, Raicu, Valerica, Milligan, Graeme
Format: Article
Language:English
Subjects:
Acetylcholine receptors (muscarinic)
Antisera
Biological Sciences
Brain slice preparation
Fluorescence
G protein-coupled receptors
Green fluorescent protein
Ligands
Mixtures
Monomers
Neuroimaging
Neurons
Physical Sciences
Polypeptides
Proteins
Receptors
Rhodopsin
Spectrometry
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2201103119