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Effect of various agonists and antagonists on mobility of glucocorticoid receptors measured by fluorescence correlation spectroscopy Glucocorticoids (GCs) are anti-inflammatory agents. Their receptor, GC receptor (GR), is one of the nuclear receptors and its transcriptional activity is regulated by ligands. Effects of agonists and antagonists on GR have been examined by various assays containing receptor binding and reporter gene assays. Here, we propose a novel parameter, diffusion constant of GR, to examine function of GR ligands. Fluorescence correlation spectroscopy (FCS) is a powerful method used to measure mobility of fluorescent molecules in subfemtoliter observation volume. We transfected EGFP-hGRα cDNA into Hela cells. EGFP-hGRα was translocated from cytoplasm into nucleus by addition of ligands tested. The diffusion constant of EGFP-hGRα in the nucleus was measured by FCS with a LSM510-ConfoCor2 system (Carl Zeiss, Germany). Fluorescence autocorrelation function was calculated according to a two-component fitting model and we selected second diffusion constant for analysis of ligands. High affinity ligands, dexamethasone, prednisolone, deltafludrocortisone and desoxymetasone dramatically decreased the diffusion constant to about 10% of that in the absence of ligands. In contrast, cortisone and progesterone known as low affinity ligands did not affect the mobility of EGFP-hGRα. GR is reported to associated with various cofactors and specific DNA sequence. The difference of the diffusion constant between high and low affinity ligands might reflect different association profile of GR. This work was performed as a part of a research and development projects of the Industrial Science and Technology Program supported by the New Energy and Industrial Technology Development Organization (NEDO). |
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