Signaling Diversity Mediated by Muscarinic Acetylcholine Receptor Subtypes and Evidence for Functional Selectivity

Challiss, R. A. J.; Thomas, Rachel L.

doi:10.1007/978-1-60327-335-0_8

Cited by 1 publication

(1 citation statement)

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“…mAChR subtypes are encoded by 5 distinct genes in mammals, referred to as M 1 –M 5 . M 2 and M 4 mAChR subtypes couple predominantly through pertussis toxin-sensitive G i/o proteins to inhibit adenylyl cyclase, whereas M 1 , M 3 and M 5 mAChR subtypes preferentially couple via pertussis toxin-insensitive G q/11 proteins to activate phospholipase C-β, mobilize intracellular Ca 2+ , regulate protein kinase C, and modulate a variety of Ca 2+ and K + -channels [1] , [2] .…”

Section: Introductionmentioning

confidence: 99%

FRET-Based Detection of M1 Muscarinic Acetylcholine Receptor Activation by Orthosteric and Allosteric Agonists

et al. 2012

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Background and ObjectiveMuscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M1 mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).MethodsVariants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M1 mAChR, respectively. The optimized FRET receptor construct (M1-cam5) was expressed stably in HEK293 cells.ResultsThe variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gαq/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M1 FRET (FEYFP/FECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.ConclusionThe M1 FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M1 mAChR activation.

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Section: Introductionmentioning

confidence: 99%