A fluorescent indicator to visualize ligand-induced receptor/coactivator interactions for screening of peroxisome proliferator-activated receptor γ ligands in living cells

Department of Chemistry, School of Science, The University of Tokyo and Japan Science and Technology Agency, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Biosensors & Bioelectronics (Impact Factor: 6.41). 06/2007; 22(11):2564-9. DOI: 10.1016/j.bios.2006.10.013
Source: PubMed


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.

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    • "The PPARc agonists were ranked based on their potency to induce PPARc-mediated luciferase activity: rosiglitazone > troglitazone = pioglitazone > netoglitazone > ciglitazone. This ranking is in line with results found by Willson and coworkers [52], Lehmann and coworkers [62], Giaginis and coworkers [63], and Henke and coworkers [49], but it differs from the results found by Young and coworkers [64] and Awais and coworkers [65], who reported that troglitazone is less potent than pioglitazone and ciglitazone. "
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    ABSTRACT: Activation of peroxisome proliferator-activated receptor γ (PPARγ) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement of PPARγ agonist and antagonist activity. Two reporter gene assays, PPARγ1 CALUX and PPARγ2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPARγ1 or PPARγ2 and a pGL3-3xPPRE-tata-luc or pGL4-3xPPRE-tata-luc reporter construct, respectively. PPARγ1 CALUX and PPARγ2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPARγ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Δ(12,14)-prostaglandin J(2). The potency to induce luciferase decreased in the following order: rosiglitazone>troglitazone=pioglitazone>netoglitazone>ciglitazone. A concentration-dependent decrease in the response to 50nM rosiglitazone was observed on the addition of PPARγ antagonist GW9662 or T0070907 in both PPARγ1 CALUX and PPARγ2 CALUX cells. The PPARα agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPARγ agonists. In conclusion, PPARγ1 CALUX and PPARγ2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPARγ agonist or antagonist activity.
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    ABSTRACT: This article describes the use of nanobiologic techniques in diagnosis and preventive medicine. It discusses the engineering of fluorescent and bioluminescent proteins to visualize biologic functions; single-molecule measurements of protein structure and function; two-photon microscopy for molecular imaging of the structure and function in living cells and tissues; and imaging ligand-induced protein conformations and interactions by fluorescence in single living cells.
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