title: Pirenzepine promotes the dimerization of muscarinic M1 receptors through a three-step binding process.
abstract: Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.
authors.affiliation: Département Biotechnologies des Interactions Moléculaires, Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, FRE 3211, France. ilien@esbs.u-strasbg.fr
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ilien
authors.forename: Nicole
authors.initials: N
authors.lastname: Glasser
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Clamme
authors.forename: Pascal
authors.initials: P
authors.lastname: Didier
authors.forename: Etienne
authors.initials: E
authors.lastname: Piemont
authors.forename: Raja
authors.initials: R
authors.lastname: Chinnappan
authors.forename: Sandrine B
authors.initials: SB
authors.lastname: Daval
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Galzi
authors.forename: Yves
authors.initials: Y
authors.lastname: Mely
mesh_terms: D000465:Algorithms; D001667:Binding, Competitive; D001896:Boron Compounds / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D021381:Protein Transport; D043643:Receptor, Muscarinic M1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: C088337:Bodipy-pirenzepine; D001896:Boron Compounds; D008024:Ligands; D043643:Receptor, Muscarinic M1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010890:Pirenzepine