||A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
Tiwari, Manisha ,
Oasa, Sho ,
Yamamoto, Johtaro ,
Mikuni, ShintaroKinjo, Masataka
7p.4336 , 2017-06-28 , Nature Publishing Group
Glucocorticoid receptor (GR alpha) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRa 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 GRa forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GR alpha, we constructed the spectrally different fluorescent protein tagged hGR alpha and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (K-d) of mCherry2-fused hGR alpha or EGFP-fused hGRa was determined in vitro. Then, K-d of wild-type hGRa was found to be 3.00 mu M in the nucleus, which was higher than that in vitro. K-d of a DNA-bindingdeficient mutant was 3.51 mu M in the nucleus. This similarity indicated that GRa 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 GRa mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRa function both in the cytoplasm and nucleus.