FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists

• Published in: PloS one Vol. 7; no. 1; p. e29946
• Main Authors: Markovic, Danijela, Holdich, Jonathan, Al-Sabah, Suleiman, Mistry, Rajendra, Krasel, Cornelius, Mahaut-Smith, Martyn P, Challiss, R A John
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 2012; Public Library of Science (PLoS)
• Subjects: Acetylcholine receptors (muscarinic); Activation; Allosteric properties; Alzheimer's disease; Alzheimers disease; Animals; Arecoline - metabolism; Arecoline - pharmacology; Atropine; Atropine - metabolism; Atropine - pharmacology; Binding sites; Binding, Competitive - drug effects; Biology; C-Terminus; Cell Membrane - metabolism; Dose-Response Relationship, Drug; Energy transfer; Extracellular signal-regulated kinase; Fluorescence resonance energy transfer; Fluorescence Resonance Energy Transfer - methods; G protein-coupled receptors; HEK293 Cells; Humans; Internalization; Intracellular; Kinases; Kinetics; Laboratory animals; Ligands; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Male; Methacholine; Methacholine Chloride - metabolism; Methacholine Chloride - pharmacology; Mice; Microscopy, Confocal; Muscarinic Agonists - metabolism; Muscarinic Agonists - pharmacology; Mutagenesis; Mutation; Neurosciences; Pharmacology; Pharmacy; Phosphorylation; Physics; Physiology; Pilocarpine; Piperidines - metabolism; Piperidines - pharmacology; Proteins; Receptor mechanisms; Receptor, Muscarinic M1 - agonists; Receptor, Muscarinic M1 - genetics; Receptor, Muscarinic M1 - metabolism; Receptors; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Rodents; Shear stress; Shear stresses; Transfection; Whooping cough; United Kingdom > UK; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0029946

Muscarinic 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 M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET). Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells. The 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 Gα(q/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 M(1) FRET (F(EYFP)/F(ECFP)) 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. The M(1) 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 M(1) mAChR activation.