Regulation of Glucocorticoid Receptor Activity by a Stress Responsive Transcriptional Cofactor (original) (raw)
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The Journal of Steroid Biochemistry and Molecular Biology, 1999
The glucocorticoid receptor (GR) regulates target gene expression in response to corticosteroid hormones. We have investigated the mechanism of transcriptional activation by the GR by studying the role of the receptor interacting protein RIP140. Both in vivo and in vitro protein±protein interaction assays revealed a ligand-dependent interaction between the GR and RIP140. The ligand binding domain of the GR was sucient for this interaction, while both the N-and C-terminal regions of RIP140 bound to the receptor. In a yeast transactivation assay RIP140 and SRC-1, a member of the steroid receptor coactivator family of proteins, both enhanced the transactivation activity of a GR protein (GRDt 1 ) in which the potent Nterminal t 1 transactivation domain has been deleted. In contrast, in COS-7 cells increasing amounts of RIP140 signi®cantly inhibited GRDt 1 function. In cotransfection studies in COS-7 cells, RIP140 also inhibited receptor activity in presence of both SRC-1 and the coactivator protein CBP together. Thus, in yeast cells a stimulation of receptor activity was observed, while in mammalian cells RIP140 repressed GR function. Taken together, these data suggest that, (1) RIP140 is a target protein for the GR and (2) RIP140 can modulate the transactivation activity of the receptor. #
Molecular and Cellular Biology, 2011
Glucocorticoids are stress hormones that maintain homeostasis through gene regulation mediated by nuclear receptors. We have discovered that other cellular stressors are integrated with glucocorticoid signaling through a new hormone-independent phosphorylation site, Ser134, on the human glucocorticoid receptor (GR). Ser134 phosphorylation is induced by a variety of stress-activating stimuli in a p38 mitogen-activated protein kinase (MAPK)-dependent manner. Cells expressing a mutant glucocorticoid receptor incapable of phosphorylation at Ser134 (S134A-GR) had significantly altered hormone-dependent genome-wide transcriptional responses and associated hormone-mediated cellular functions. The phosphorylation of Ser134 significantly increased the association of the GR with the zeta isoform of the 14-3-3 class of signaling proteins (14-3-3zeta) on chromatin promoter regions, resulting in a blunted hormone-dependent transcriptional response of select genes. These data argue that the phosp...
Steroids, 2011
The glucocorticoid receptor (GR) is a ligand-activated transcription factor for which a number of endogenous and synthetic ligands exist. A key question in steroid receptor biology is how different ligands elicit different maximal transcriptional responses via the same receptor and on the same promoter. This question was addressed quantitatively for the GR, using a panel of agonists, partial agonists and antagonists, on the endogenous GILZ gene in two different human cell lines. It was found that the extent of GR nuclear localization correlated with the efficacy for GILZ transactivation by the GR in U2OS cells. However, in A549 cells there was no significant correlation, with all ligands resulting in similar levels of GR nuclear localization, despite different levels of transcriptional activation of the GILZ gene. Chromatin immunoprecipitation analysis on the other hand, revealed ligand-specific differences in GILZ promoter occupancy in the A549 cells, which correlated with the transcriptional efficacy of the subset of ligands investigated. This suggests that ligand-specific differences in promoter occupancy by activated GR play a major role in discrimination between agonist, partial agonist and antagonist responses on the endogenous GILZ gene in A549 cells, while differences in nuclear localisation of liganded GR play a role in determining the transcriptional outcome in U2OS cells. These cell line-specific differences were not dependent on the amount of GR present, since transient overexpression of GR in U2OS did not alter the relative ligand-selective nuclear localisation. Our results show that there is a relationship between ligand-specific transactivation efficacy, extent of nuclear translocation and recruitment of GR to the promoter. However, the relative contribution of nuclear translocation and GR promoter recruitment to ligand-specific transactivation efficacy is cell-specific.
Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor
Molecular and cellular biology, 1999
Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin alpha. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent ...
Experimental Cell Research, 2002
We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11-ol-3,20-dione (⌬HOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the ⌬HOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of ⌬HOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and ⌬HOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type.
Steroids, 2013
Glucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10 À6 M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10 À10 -10 À9 M). Loss of dimerization results in a slower observed rate of nuclear import (2.5-to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18-27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4-to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5-to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.
The Journal of Cell Biology, 1999
RAP46 is a eukaryotic cochaperone that associates with several proteins, including the heat shock protein hsp70/hsc70 and the glucocorticoid receptor (GR). Here we show a downregulation of GR-mediated transactivation by RAP46 via a mechanism independent of a cytoplasmic action of this cochaperone. We demonstrate a specific cytoplasmic–nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor. A repeated sequence motif [EEX4]8 at the NH2 terminus of RAP46 or BAG-1L, a larger isoform of RAP46, is responsible for this downregulation of GR activity. BAG-1, a shorter isoform with only a duplication of the [EEX4] sequence, does not inhibit GR activity. The [EEX4]8 motif, when linked to an otherwise unrelated protein, abrogated the inhibitory action of endogenous RAP46 on GR-mediated transactivation. The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected. These stud...