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

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) 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 PPARγ in live cells, we developed a genetically encoded fluorescent indicator in which PPARγ ligand binding domain (PPARγ 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 PPARγ 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 PPARγ 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 PPARγ-targeted drugs against type 2 diabetes and inflammation.