Determinants of subcellular distribution of the glucocorticoid receptor.
Glucocorticoid receptor (GR) exchanges between an active nuclear form and a complexed inactive, steroid-sensitive cytoplasmic form. Using a semi-quantitative indirect immunofluorescence assay to measure the kinetics of subcellular redistribution of GR in response to challenge during G(o), we have found that the ability to bind DNA is an important determinant for localization and tight binding of GR to the nucleus. The transfer of GR DNA-binding mutants to the nucleus after treatment with hormone agonists and antagonists was markedly reduced. Further, mutant receptors localized to the nucleus were only weakly associated with the nuclear compartment as they were released into cytosol upon hypotonic lysis of the cell membrane. Moreover, after agonist withdrawal, GR redistributed to the cytoplasm more rapidly when unable to bind DNA. By contrast, withdrawal of the hormone antagonist RU486 was found to yield a form of wild type GR that was completely unable to redistribute to the cytoplasm. However, this did not appear to result from a block in nuclear export as selective inactivation of nuclear import with energy inhibitor released RU486-withdrawn GRs from the nucleus at the same rates as agonist-withdrawn receptors. In addition, GR mutants unable to bind DNA, which retained a significant presence in the cytoplasm both during and after antagonist treatment, also failed to redistribute. The effect of RU486 treatment did not appear to be mediated through a block in reassociation of GR into a steroid-responsive form as RU486-withdrawn wild type receptors retained full potential to activate transcription from a glucocorticoid-responsive promoter after a second challenge with hormone. Therefore, reassociation of GR into a steroid-responsive form appears to be independent of signals important for the retention of GR in the cytoplasm.
Sackey
F N
FN
Department of Medicine, University of Ottawa, Ontario, Canada.
Haché
R J
RJ
Reich
T
T
Kwast-Welfeld
J
J
Lefebvre
Y A
YA
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D015398:Signal Transduction
D011965:Receptors, Glucocorticoid