Homodimerization of glucocorticoid receptor from single cells investigated using fluorescence correlation spectroscopy and microwells

FEBS Lett. 2015 Aug 4;589(17):2171-8. doi: 10.1016/j.febslet.2015.07.003. Epub 2015 Jul 13.

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.

Keywords: Dissociation constant; Fluorescence correlation spectroscopy; Glucocorticoid receptor; Homodimerization; Microwell; Single cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Blotting, Western
  • Dimethylpolysiloxanes / chemistry
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Kinetics
  • Microscopy, Confocal
  • Protein Multimerization*
  • Receptors, Glucocorticoid / chemistry*
  • Receptors, Glucocorticoid / genetics
  • Receptors, Glucocorticoid / metabolism
  • Single-Cell Analysis / instrumentation
  • Single-Cell Analysis / methods*
  • Spectrometry, Fluorescence / methods*

Substances

  • Dimethylpolysiloxanes
  • Receptors, Glucocorticoid
  • Green Fluorescent Proteins
  • baysilon