title: Using ligand-induced conformational change to screen for compounds targeting G-protein-coupled receptors.
abstract: The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.
authors.affiliation: Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada. brian.odowd@utoronto.ca
authors.forename: Brian F
authors.initials: BF
authors.lastname: O'Dowd
authors.forename: Mohammad
authors.initials: M
authors.lastname: Alijaniaram
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Ji
authors.forename: Tuan
authors.initials: T
authors.lastname: Nguyen
authors.forename: Richard M
authors.initials: RM
authors.lastname: Eglen
authors.forename: Susan R
authors.initials: SR
authors.lastname: George
mesh_terms: D002460:Cell Line; D004298:Dopamine / Q000494:pharmacology; D015259:Dopamine Agents / Q000494:pharmacology*; D018491:Dopamine Agonists / Q000494:pharmacology*; D018492:Dopamine Antagonists / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D008968:Molecular Conformation; D019913:Nuclear Localization Signals; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D015259:Dopamine Agents; D018491:Dopamine Agonists; D018492:Dopamine Antagonists; D008024:Ligands; D019913:Nuclear Localization Signals; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004298:Dopamine