Homodimerization of glucocorticoid receptor from single cells investigated using fluorescence correlation spectroscopy and microwells. Issue 17 (13th July 2015)

• Record Type: Journal Article
• Authors: Oasa, Sho; Sasaki, Akira; Yamamoto, Johtaro; Mikuni, Shintaro; Kinjo, Masataka
• Is Part Of: FEBS letters. Volume 589:Issue 17(2015)
• Page Start: 2171
• Page End: 2178
• Publication Date: 2015-07-13
• Subjects: CPP -- counts per particle -- Dex -- dexamethasone -- EGFP -- enhanced green fluorescence protein -- ESS -- equivalent-single-cell-lysate solution -- FCS -- fluorescence correlation spectroscopy -- GR -- glucocorticoid receptor -- PDMS -- polydimethylsiloxane -- Dissociation constant -- Fluorescence correlation spectroscopy -- Glucocorticoid receptor -- Homodimerization -- Microwell -- Single cell; Biochemistry -- Periodicals; Biophysics -- Periodicals; Molecular biology -- Periodicals; Biochimie -- Périodiques; Biochemistry; Biophysics; Molecular biology; Periodicals; 572.05
• DOI: 10.1016/j.febslet.2015.07.003
• Languages: English
• ISSNs: 0014-5793

Abstract

Abstract : Glucocorticoid receptor α (GR) binds to the promoter regions of target genes as a homodimer and activates its transcriptional process. Though the homodimerization is thought to be the initial and essential process, the dissociation constant for homodimerization of GR remains controversial. To quantify homodimerization of (enhanced green fluorescence protein) EGFP–(glucocorticoid receptor) GR, the particle brightness in lysates from single cell was estimated for the fraction of homodimeric EGFP–GR using fluorescence correlation spectroscopy and microwells. Fitting the data with a bimolecular reaction model, the dissociation constant was determined. Moreover slow‐diffusion complex was observed. These results suggest that EGFP–GR forms not only a monomer–dimer equivalent state but also a large‐molecular‐weight complex. Abstract : FCS can determine a 0.17nM concentration of EGFP with background correction. The dissociation constant for homodimerization of GR was determined in the microwell. GR forms not only a homodimer but also a large‐molecular‐weight complex.