title: FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.
authors.forename: Danijela
authors.initials: D
authors.lastname: Markovic
authors.forename: Jonathan
authors.initials: J
authors.lastname: Holdich
authors.forename: Suleiman
authors.initials: S
authors.lastname: Al-Sabah
authors.forename: Rajendra
authors.initials: R
authors.lastname: Mistry
authors.forename: Cornelius
authors.initials: C
authors.lastname: Krasel
authors.forename: Martyn P
authors.initials: MP
authors.lastname: Mahaut-Smith
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D000818:Animals; D001115:Arecoline / Q000378:metabolism / Q000494:pharmacology; D001285:Atropine / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D016210:Methacholine Chloride / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D018721:Muscarinic Agonists / Q000378:metabolism* / Q000494:pharmacology; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C456520:4-n-butyl-1-(4-(2-methylphenyl)-4-oxo-1-butyl)-piperidine hydrogen chloride; D008164:Luminescent Proteins; D018721:Muscarinic Agonists; D010880:Piperidines; D043643:Receptor, Muscarinic M1; D011993:Recombinant Fusion Proteins; D016210:Methacholine Chloride; D001115:Arecoline; D001285:Atropine