title: Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties.
abstract: Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.
authors.affiliation: Département Récepteurs et Protéines Membranaires, CNRS UPR 9050, Illkirch, France. ilien@esbs.u-strasbg.fr
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ilien
authors.forename: Christelle
authors.initials: C
authors.lastname: Franchet
authors.forename: Philippe
authors.initials: P
authors.lastname: Bernard
authors.forename: Séverine
authors.initials: S
authors.lastname: Morisset
authors.forename: Claire Odile
authors.initials: CO
authors.lastname: Weill
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Bourguignon
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive / Q000502:physiology; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D008164:Luminescent Proteins; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins