An Active Nuclear Retention Signal in the Glucocorticoid Receptor Functions as a Strong Inducer of Transcriptional Activation

Abstract

The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin ␣/␤ karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling. The glucocorticoid receptor (GR)5 is a nuclear hormone receptor of the steroid receptor subfamily whose activity is tightly regulated by glucocorticoids (1). GR is a modular protein that features a central DNA binding domain (DBD) and a C-terminal ligand binding domain (LBD). Two activation functions (AF-1 and AF-2) are located in the N terminus and the LBD of the receptor, respectively (2, 3). GR is directly and indirectly involved in both the activation and suppression of genes that are involved in cell differentiation, glucose uptake and homeostasis, inflammation, response to stress, neuronal function, lipid metabolism, and cancer (4, 5).Transcriptional regulation by GR is tightly controlled. The regulation of function by segregation in subcellular compartment has been proposed to act as an important regulatory checkpoint for a number of transcription factors, including steroid receptors (6, 7). Unlike other steroid receptors, such as estrogen receptor and progesterone receptor, that are nuclear in the absence of steroid, naive GR is largely cytoplasmic (8). In the cytoplasm GR is maintained in an inactive hsp90 cochaperone complex that includes hsp70, immunophilins, p23, and other factors (9). Upon ligand binding, GR dissociates from the chaperone complex, homodimerizes, and rapidly translocates to the nucleus to regulate target gene expression (10 -12). Nuclear import of GR is accomplished through two nuclear localization signals as follows: NL1, located in a hinge-like region of GR that separates the DBD from the LBD; and NL2, which is within the LBD (13). NL1 is a basic NLS that mediates the nuclear import of GR through interaction with importin ␣ and importin 7 (14 -16).NL2 is strictly steroid-dependent, and bot...

Selective Mutations in Estrogen Receptor α D-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms

“…The H2 ER␣ mutant must be counteracted by a nuclear export signal to keep the nucleocentric ER␣ out of the nucleus. The glucocorticoid receptor (GR) has a nuclear retention signal that overlaps the nuclear localization function and is necessary for ligand bound transcriptional activation (41). A similar nuclear retention signal or event for ER␣ is possible and the cytoplasmic versus nuclear distribution of the H1 and H2 ER␣ mutants suggest that this region of ER␣ may be responsible for ligand-dependent nuclear retention of ER␣.…”

Site-specific Androgen Receptor Serine Phosphorylation Linked to Epidermal Growth Factor-dependent Growth of Castration-recurrent Prostate Cancer

“…Human GR hinge region DNA-PK phosphorylation site Ser-508 implicated in GR nuclear retention (63,66,67) is positioned close to human GR nuclear targeting residues 479 -498 and is displaced in position by two residues to the corresponding human AR hinge residue Ser-650 near AR nuclear targeting residues 617-633 (22,68). We and others have shown that a mutation at Ser-650 has relatively little effect on AR transcriptional activity (28,35).…”

Abrogation of Glucocorticoid Receptor Dimerization Correlates with Dissociated Glucocorticoid Behavior of Compound A

“…Within this context the decrease in maximal nuclear import and increase in nuclear export rate for CpdA-bound wtGR, as for Dex-bound GR dim , as compared with Dex-bound wtGR, shown in the current paper, suggests that loss of GR dimerization results in a GR species that exits the nucleus at a significantly faster rate and that GR dimerization contributes to the amount of GR present in the nucleus upon ligand activation. Previous work has suggested that the hinge region of the GR contains a solution dimerization domain (23) involved in cytosolic dimerization of the liganded GR, a nuclear localization signal, NL1 (40), that mediates liganded and unliganded GR nuclear location, and a nuclear retention signal that actively retains the GR in the nucleus (67). Loss of NL1 and nuclear retention signal results in increased nuclear export and reduced nuclear occupancy, which closely resembles the effect found with CpdA and GR dim in the current study.…”