title: A fluorescent indicator to visualize ligand-induced receptor/coactivator interactions for screening of peroxisome proliferator-activated receptor gamma ligands in living cells.
abstract: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of nuclear receptors (NRs) superfamily and plays an important role for modulation of insulin sensitivity in type 2 diabetes. Ligand-dependent protein-protein interactions between NRs and NR coactivators are critical in regulation of transcription. To visualize the ligand-induced coactivator recruitment to PPARgamma in live cells, we developed a genetically encoded fluorescent indicator in which PPARgamma ligand binding domain (PPARgamma LBD) was connected to a steroid receptor coactivator peptide that contains LXXLL motif (L=leucine and X=any amino acid) through a flexible linker. This fusion protein was inserted between cyan and yellow fluorescent proteins (CFP and YFP, donor and acceptor fluorophore, respectively). Monitoring real-time ligand-induced conformational change in the PPARgamma LBD to interact with the coactivator allowed screening of natural and synthetic ligands (drugs against type 2 diabetes) in single living cells using intramolecular fluorescence resonance energy-transfer (FRET) microscopy. The high sensitivity of the present indicator made it possible to distinguish between strong and weak affinity ligands for PPARgamma in a dose-dependent fashion, immediately after adding a ligand to live cells. The indicator can discriminate agonist from antagonist compounds efficiently within a few minutes. The present system may be promising in the development of PPARgamma-targeted drugs against type 2 diabetes and inflammation.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo and Japan Science and Technology Agency, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Awais
authors.forename: Moritoshi
authors.initials: M
authors.lastname: Sato
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Umezawa
mesh_terms: D000818:Animals; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D007668:Kidney / Q000378:metabolism*; D008024:Ligands; D047495:PPAR gamma / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D013552:Swine
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D047495:PPAR gamma; D018160:Receptors, Cytoplasmic and Nuclear