Reference - Detail
|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.|
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.
|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*|
|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).|