A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell.

ABSTRACT: Glucocorticoid receptor (GR?) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GR? resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GR? forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GR?, we constructed the spectrally different fluorescent protein tagged hGR? and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (K_d) of mCherry₂-fused hGR? or EGFP-fused hGR? was determined in vitro. Then, K_d of wild-type hGR? was found to be 3.00 ?M in the nucleus, which was higher than that in vitro. K_d of a DNA-binding-deficient mutant was 3.51 ?M in the nucleus. This similarity indicated that GR? homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GR? mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GR? function both in the cytoplasm and nucleus.

SUBMITTER: Tiwari M 

PROVIDER: S-EPMC5489515 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature