RRC ID 51310
Author Tiwari M, Oasa S, Yamamoto J, Mikuni S, Kinjo M.
Title A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell.
Journal Sci Rep
Abstract Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα 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 GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (Kd) of mCherry2-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, Kd of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. Kd of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα 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 GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.
Volume 7(1)
Pages 4336
Published 2017-6-28
DOI 10.1038/s41598-017-04499-7
PII 10.1038/s41598-017-04499-7
PMID 28659593
PMC PMC5489515
MeSH Cell Line Cell Nucleus / metabolism Cytoplasm / metabolism Fluorescent Antibody Technique Humans Mutation Protein Binding Protein Multimerization* Protein Transport Receptors, Glucocorticoid / chemistry Receptors, Glucocorticoid / genetics Receptors, Glucocorticoid / metabolism* Spectrometry, Fluorescence*
IF 3.998
Times Cited 14
DNA material pmCherry2-hGRalpha1 (RDB15749) pEGFP-hGRalpha (RDB15750) pmCherry2-hGRalpha/C421G (RDB15751) pEGFP-hGRalpha/C421G (RDB15752) pmCherry2-hGRalpha/A458T (RDB15753) pEGFP-hGRalpha/A458T (RDB15754) pmCherry2-hGRalpha/C421G-A458T (RDB15755) pEGFP-hGRalpha/C421G-A458T (RDB15756) pmCherry2-hGRalpha/deltaNLS (RDB15757) pEGFP-hGRalpha/deltaNLS (RDB15758) pmCherry2-hGRalpha/A458T-deltaNLS (RDB15759) pEGFP-hGRalpha/A458T-deltaNLS (RDB15760) p50-mCherry2/NLS (RDB15761) p50-EGFP/NLS (RDB15762) p50-mCherry2 (RDB15763) p50-EGFP (RDB15764).