The M 1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes.
Sara MarsangoLaura JenkinsJohn D PedianiSophie J BradleyRichard J WardSarah HesseGabriel BienerMichael R StonemanAndrew B TobinValerica RaicuGraeme MilliganPublished in: Proceedings of the National Academy of Sciences of the United States of America (2022)
The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M 1 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-M 1 and anti-green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M 1 -mEGFP was expressed at levels equal to the M 1 receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M 1 -mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M 1 -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 M 1 -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.
Keyphrases
- binding protein
- wild type
- fluorescence imaging
- spinal cord
- poor prognosis
- quantum dots
- high resolution
- spinal cord injury
- transcription factor
- bone marrow
- skeletal muscle
- endothelial cells
- single cell
- amino acid
- functional connectivity
- risk assessment
- resting state
- insulin resistance
- high glucose
- smoking cessation
- protein protein
- fluorescent probe