Full-length Glucocorticoid Receptor Research Articles

Human neutrophils are a key target of glucocorticoids

Abstract Neutrophils play central roles in immunity and inflammation. Glucocorticoids (GCs) are the cornerstone of anti-inflammatory and immunosuppressive therapy. While GCs trigger an acute rise in circulating neutrophils, little is known about the mechanisms by which GCs modulate neutrophil biology. We studied the response of 9 primary human cells to in vitro treatment with GCs. Surprisingly, neutrophils were transcriptionally the most responsive cell type, and they express proportionally the most glucocorticoid receptor (GR) transcripts. Long-read RNA-seq revealed 42 full-length GR transcript isoforms in neutrophils, twice as many as had been reported previously in all other cell types. At the protein level, neutrophils also have a unique and diverse set of GR isoforms. In contrast to the widely accepted model of GR signaling, in which GR binds GC molecules in the cytoplasm and then translocates to the nucleus, GR localization in neutrophils is almost exclusively nuclear, even in the absence of GC treatment. To validate our findings in vivo, we then performed a study of healthy volunteers, with blood collected before and serially after GC infusion. We generated a pathway-level map of the human neutrophil transcriptome at baseline and in response to GCs. The in vivo transcriptional response of human neutrophils to GCs was as strong as that observed in vitro, with a predominance of upregulation. Among the most strongly induced genes are several that are either not expressed, or expressed at very low levels, in human neutrophils at baseline. Some of the proteins encoded by these genes may have putative effects on neutrophil function, including SAMSN1 and ZBTB16. These findings indicate that human neutrophils are a key target for GC action.

Measuring glucocorticoid receptor expression in vivo with PET.

The glucocorticoid receptor (GR) is an emerging drug target for several common and deadly solid tumors like breast and prostate cancer, and clinical trials studying the antitumor effects of GR antagonists are beginning. Since GR expression can be variable in tumor cells, and virtually all normal mammalian tissues express some GR, we hypothesized that an imaging tool capable of detecting GR positive tumors and/or measuring GR occupancy by drug in tumor and normal tissues could improve the precision application of anti-GR therapies in the clinic. To this end, we developed a fluorine-18 labeled corticosteroid termed GR02 that potently binds the endogenous ligand binding pocket on full length GR. Binding of 18F-GR02 was suppressed in many normal tissues by co-treatment with mifepristone, a GR antagonist in human use, and was elevated in many normal tissues among mice lacking circulating corticosteroids due to adrenalectomy. 18F-GR02 also accumulated in GR positive subcutaneous and subrenal capsule prostate cancer models, and uptake in tumors was competed by mifepristone. Combined with a straightforward and high yielding radiosynthesis, these data establish the foundation for near-term clinical translation of 18F-GR02.

Evolution of corticosteroid specificity for human, chicken, alligator and frog glucocorticoid receptors

We investigated the evolution of the response of human, chicken, alligator and frog glucocorticoid receptors (GRs) to dexamethasone, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol and aldosterone. We find significant differences among these vertebrates in the transcriptional activation of their full length GRs by these steroids, indicating that there were changes in the specificity of the GR for steroids during the evolution of terrestrial vertebrates. To begin to study the role of interactions between different domains on the GR in steroid sensitivity and specificity for terrestrial GRs, we investigated transcriptional activation of truncated GRs containing their hinge domain and ligand binding domain (LBD) fused to a GAL4 DNA binding domain (GAL4-DBD). Compared to corresponding full length GRs, transcriptional activation of GAL4-DBD_GR-hinge/LBD constructs required higher steroid concentrations and displayed altered steroid specificity, indicating that interactions between the hinge/LBD and other domains are important in glucocorticoid activation of these terrestrial GRs.

Nucleotide Dependency of Endogenous Hsp90 Chaperone Protein Machinery and Its Association with Baculovirally Expressed Glucocorticoid Receptor

Hsp90 and hsp70 are essential eukaryotic chaperone proteins required for facilitating functional activity of mammalian transcription factors, including the glucocorticoid receptor (GR). Both chaperones exist in ATP‐ and ADP‐bound states, and understanding the relationship between nucleotide‐bound forms of hsp90 and hsp70, their affiliated cochaperones, and GR activation has significant pharmacological implications. The goal of this study was to investigate the extent to which hsp90 and hsp70 nucleotide interactions affect production of a GR‐hsp90 heterocomplex capable of steroid binding activity. Reticulocyte lysate (RL) containing high endogenous concentrations of hsp90 and hsp70 was incubated in the presence or absence of an ATP regenerating system and fractionated by size exclusion chromatography (SEC). ATP incubation did not appear to alter total global protein elution, as detected by A280 spectrophotometry. However, SDS‐PAGE and western blot analysis identified nucleotide‐associated alterations in hsp90 SEC elution profiles. In order to further compare nucleotide dependency and chaperone effects, full‐length GR, produced in baculovirally infected insect cells, was immunoadsorbed, stripped of endogenous chaperones, and reassembled into a heterocomplex with hsp90 ± ATP. SDS‐PAGE and western blotting showed hsp90 derived from RL−ATP samples were tightly bound to the GR, while hsp90 from RL+ATP failed to stay bound to the GR. The difference in SEC elution profiles and co‐immunoadsorption imply a notable role of nucleotide alterations in the hsp90 chaperone machinery and functional interactions with the GR.

Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in vitro Reveal Exchange on the Second Timescale

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that GR interactions with chromatin are highly dynamic, with receptor residence times on the order of seconds. These findings were surprising since early in vitro studies suggested that GR-DNA interactions were more static, with receptor-DNA exchange occurring on the order of minutes to hours. However, these latter analyses were carried out without the modern-day advantages of recombinant receptor expression, established purification protocols or rigorous characterization methods. Here we examine the DNA dissociation kinetics of full-length human GR, using highly purified and homogenous receptor preparations. We find that in vitro dissociation kinetics are not slow as previously reported, but are quite fast, with calculated receptor-DNA residence times of seconds to tens of seconds. These findings are observed at both isolated response elements and the multisite mouse mammary tumor virus (MMTV) promoter used in live cell imaging. However, we also find that dissociation kinetics are identical for all response elements and are biphasic in character. These results indicate that differences in GR affinity toward these sequences are not due to differences in off-rate but on-rate, and further suggest that GR exists in multiple and interconverting states when bound to DNA. Since we previously established that GR-DNA binding affinity is a primary determinant of transcriptional activity at these sequences, the current findings implicate DNA sequence in controlling the extent of receptor interconversion and hence extent of transcriptional activation.

Investigating Allosteric Coupling Mechanism in Constitutively Active Human Glucocorticoid Receptor Two Domain Construct

Glucocorticoid receptor (GR) is a hormone dependent nuclear transcription factor. It plays key roles in organ development, metabolite homeostasis, and stress and inflammatory response. GR is composed of three major domains, the intrinsically disordered (ID) N-terminal domain (NTD), the DNA binding domain (DBD) and the ligand binding domain (LBD). Several human GR translational isoforms differing only in the lengths of their ID NTDs with finely tunable transcription activity have been identified.In my project, through thermodynamic analysis of the ID NTD constructs of different translational isoforms, we found that full length GR NTD can be viewed as having at least two thermodynamically coupled regions, a functional (F) region, indispensable for GR transcriptional activity, and a regulatory (R) region, whose length serves to regulate the stability of NTD, and thus the activity of GR. F region and R region are negatively coupled to each other [1].Truncation of LBD from the full length receptor removes the hormone dependence of GR transcription activity, and generates the constitutively active GR NTD-DBD two domain constructs. From investigating the NTD-DBD two domain constructs of different translational isoforms by in vitro biophysics assays and in vivo luciferase activity assay, we are on the way figuring out the coupling mechanism between the DBD and ID NTD.Allosteric coupling mechanisms derived from our experimental data makes it possible to build the Ensemble Allosteric Coupling Model (EAM) to explain the finely tunable activity of different GR translational isoforms, which cannot be accounted for by the binding energetics between the different isoforms and the GR response element.[1] Jing Li, et al. Thermodynamic dissection of the intrinsically disordered N-terminal domain of human glucocorticoid receptor. J Biol Chem. 2012.

Molecular cloning and characterization of the corticoid receptors from the American alligator

Steroid hormones are essential for health in vertebrates. Corticosteroids, for example, have a regulatory role in many physiological functions, such as osmoregulation, respiration, immune responses, stress responses, reproduction, growth, and metabolism. Although extensively studied in mammals and some non-mammalian species, the molecular mechanisms of corticosteroid hormone (glucocorticoids and mineralocorticoids) action are poorly understood in reptiles. Here, we have evaluated hormone receptor-ligand interactions in the American alligator (Alligator mississippiensis), following the isolation of cDNAs encoding a glucocorticoid receptor (GR) and a mineralocorticoid receptor (MR). The full-length alligator GR (aGR) and aMR cDNAs were obtained using 5’ and 3’ rapid amplification cDNA ends (RACE). The deduced amino acid sequences exhibited high identity to the chicken orthologs (aGR: 83%; aMR: 90%). Using transient transfection assays of mammalian cells, both aGR and aMR proteins displayed corticosteroid-dependent activation of transcription from keto-steroid hormone responsive, murine mammary tumor virus promoters. We further compared the ligand-specifity of human, chicken, Xenopus, and zebrafish GR and MR. We found that the alligator and chicken GR/MR have very similar amino acid sequences, and this translates to very similar ligand specificity. This is the first report of the full-coding regions of a reptilian GR and MR, and the examination of their transactivation by steroid hormones.

Ligand Binding Domain Mutations of the Glucocorticoid Receptor Selectively Modify the Effects with, but Not Binding of, Cofactors

We previously reported that several point mutations in the ligand binding domain (LBD) of glucocorticoid receptors (GRs) marginally affect the binding affinity of the synthetic glucocorticoids dexamethasone (Dex) and deacylcortivazol (DAC). However, these mutations dramatically alter the efficacy (A(max)) and potency (EC(50)) of agonists, along with the partial agonist activity (PAA) of the antisteroid Dex-mesylate (DM), for gene induction and repression in a steroid-dependent manner. This was proposed to result, in part, from altered protein-protein interactions in the complex of GR with the coactivator TIF2 despite normal TIF2 binding. To explore the generality of this phenomenon, we now ask whether these mutations also affect the transactivation properties, but not binding, of other GR-bound factors. We find that an elevated concentration of GR, to probe unidentified cofactors, or of the comodulator Ubc9 does not reverse the effects of GR LBD mutations that increase the EC(50) and lower the PAA with the GREtkLUC reporter in both CV-1 and U2OS cells. This behavior is more dramatic with Ubc9 and the isolated GR LBD fused to the GAL4 DNA binding domain, despite normal binding of Ubc9 to the mutant GRs. Similar effects, albeit gene, steroid, and transcriptional property-specific, are seen with full-length GRs and three endogenous genes in U2OS cells. Thus, changes in simple steady-state binding capacities of mutant receptors for factors cannot account for the modified transcriptional properties. In all cases, the nuclear translocation of Dex- and DAC-bound wild-type and mutant receptors is the same. These results are consistent with the earlier results with TIF2 and support the hypothesis that small changes in the GR LBD can alter the activities of the bound cofactor without modifying cofactor binding. We propose that this separation of binding and the modulation of transactivation parameters occurs for a wide variety of GR-associated cofactors.

Familial Glucocorticoid Resistance Caused by a Novel Frameshift Glucocorticoid Receptor Mutation

ABSTRACT Context Familial glucocorticoid resistance is a rare condition with a typical presentation of women with hirsutism and hypertension, with or without hypokalemia. Objective The aim was to determine the cause of apparent glucocorticoid resistance in a young woman. Patients and Methods We studied a family with a novel glucocorticoid receptor (GR) mutation and a surprisingly mild phenotype. Their discovery resulted from serendipitous measurement of serum cortisol with little biochemical or clinical evidence for either hyperandrogenism or mineralocorticoid excess. Results The causative mutation was identified as a frameshift mutation in exon 6. Transformed peripheral blood lymphocytes were generated to analyze GR expression in vitro. Carriers of the mutation had less full-length GR, but the predicted mutant GR protein was not detected. However, this does not exclude expression in vivo, and so the mutant GR (D612GR) was expressed in vitro. Simple reporter gene assays suggested that Δ612GR has dominant negative activity. Δ612GR was not subject to ligand-dependent Ser211 phosphorylation or to ligand-dependent degradation. A fluorophore-tagged construct showed that Δ612GR did not translocate to the nucleus in response to ligand and retarded translocation of the wild-type GR. These data suggest that Δ612GR is not capable of binding ligand and exerts dominant negative activity through heterodimerization with wild-type GR. Conclusion Therefore, we describe a novel, naturally occurring GR mutation that results in familial glucocorticoid resistance. The mutant GR protein, if expressed in vivo, is predicted to exert dominant negative activity by impairing wild-type GR nuclear translocation.

Identification of a Potential Molecular Link Between the Glucocorticoid and Serotonergic Signaling Systems

Glucocorticoid receptor (GR) and serotonin (5-hydroxytryptamine (5-HT)) signaling systems play a pivotal role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, but the molecular nature of interactions between these two systems remain largely unidentified. We used computational and experimental approaches to evaluate if DNA-protein interactions would provide a molecular link for the interaction between 5-HT and GR systems. Bioinformatic analysis identified nine binding sites in various serotonin receptors (HTR1D, HTR1F, HTR2A, HTR3A, and HTR6) for transcription factors in the GR family. Electrophoretic mobility shift assays (EMSA) using HeLa nuclear extract and purified full-length GR verified most of the predicted DNA-protein interactions. Six binding sites verified by EMSA results were evolutionarily conserved in multiple species. Multiple lines of evidence from computational and experimental analyses in this study support the potential of a molecular link between 5-HT and GR signaling systems. This finding provides new approaches to studies directed at mechanisms for glucocorticoid negative feedback regulation of the HPA axis involving 5-HT and interventional studies directed to neuropsychiatric diseases.

Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

Glucocorticoid receptors (GRs) are ubiquitous, nuclear hormone receptors residing in cell types of both cancer and noncancerous origin. It is not known whether cancer cell-associated GR alone can be selectively manipulated for delivery of exogenous genes to its nucleus for eliciting anticancer effect. We find that GR ligand, dexamethasone (Dex) in association with cationic lipoplex (termed as targeted lipoplex) could selectively manipulate GR in cancer cells alone for the delivery of transgenes in the nucleus, a phenomenon that remained unobserved in normal cells. The targeted lipoplex (i) showed GR-targeted transfections in all cancer cells experimented (P < 0.01), (ii) significantly diminished transfection in cancer cells when GR is downregulated (P < 0.01), and (iii) elicited specific nuclear translocation of targeted lipoplex in cancer cells, followed by upregulated transactivation of glucocorticoid response element (GRE)- promoted gene. Using anticancer gene, targeted lipoplex induced significant tumor growth retardation in mice in comparison to different control groups (P < 0.05). Interestingly, cell surface-associated Hsp90 in cancer cells assisted the intracellular uptake of GR-targeted lipoplex. Moreover, selective inhibition of Hsp90 in noncancer cells resulted in cancer cell-like, aberrant, GR activation. The current study discovers a therapeutically important, unique property of cancer cell associated-GR that may be linked to a compromised role of Hsp90.Molecular Therapy (2009) 17 4, 623-631 doi:10.1038/mt.2009.4.

Interaction and Functional Interference of Glucocorticoid Receptor and SOCS1

Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNgamma and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNgamma treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling.

Targeted Killing of Glioblastoma Multiforme In Vivo by IL-13 Zetakine Redirected CTLs Made Glucocorticoid Resistant with Zinc Finger Nucleases.

Genetic modification of cytolytic T-lymphocytes (CTL) for enhancing their functional immunobiology is a promising immunotherapeutic approach for the treatment of cancer and infectious disease. CTLs modified to express a chimeric antigen receptor comprising an extracellular IL13 domain and cytoplasmic CD3 domain (IL13-zetakine) can be re-directed both in vitro and in animal models to target glioblastoma multiforme (GBM), which is characterized by high expression of IL13Ralpha2. Patient-derived IL13-zetakine/HyTK expressing CD8+ CTL clones have entered early stage clinical trials. However, their clinical application is frequently limited in this patient population by the pervasive use of dexamethasone, a potent glucocorticoid analogue employed in the management of cerebral edema. Thus iatrogenic dexamethasone-mediated T-cell functional anergy and apoptosis in these patients is a barrier to realizing the full clinical utility of this adoptive therapy strategy. We hypothesized that knocking out the expression of the glucocorticoid receptor would render therapeutic CTLs resistant to the effects of synthetic glucocorticoids, including dexamethasone. We therefore developed engineered zinc finger nucleases (ZFNs) to specifically disrupt the glucocorticoid receptor (GR) locus in the human genome. ZFNs include the cleavage domain of the restriction enzyme FokI linked to an engineered zinc finger DNA-binding domain and can be designed to cleave a predetermined site in the genome. Natural repair of such DNA breaks via the error-prone non-homologous end joining pathway results in the inactivation of the target gene at frequencies which permit the isolation of knock out clones. Employing adenovirally delivered and transiently expressed ZFNs targeting exon 3 of the human GR gene, we isolated IL13-zetakine+ CD8+T-cells containing a biallelically mutated GR locus. These cells were characterized by the absence of full length GR protein, lack of glucocorticoid hormone-induced gene regulation and resistance to glucocorticoid hormone-mediated immunosupression and apoptosis. Importantly, the ZFN-modified, glucocorticoid-resistant CTLs demonstrated zetakine re-directed cytolytic activity and tumor cell specificity in chromium release assays in vitro and in an orthotopic mouse model of GBM in vivo. These results indicate that glucocorticoid-resistant IL13-zetakine targeted CTLs should retain function in cancer patients receiving glucocorticoids. A clinical trial to test this hypothesis is currently under development.

Geldanamycin, an inhibitor of Hsp90, Blocks cytoplasmic retention of progesterone receptors and glucocorticoid receptors via their respective ligand binding domains

Steroid hormone receptors (SHRs), such as glucocorticoid receptors (GR) and progesterone receptors (PR), are shuttling proteins that undergo continuous nuclear import and export. Various mechanisms have been proposed to explain the localization of SHRs. It has been suggested that the ligand-binding domain (LBD) of SHRs is important in determining the subcellular localization. We have studied the localization of GR-LBD and PR-LBD alone, as well as of full-length GR and PR in the presence of geldanamycin (GA), a benzoquinoid ansamycin that specifically inhibits heat shock protection (Hsp90), using transient transfections and fluorescent microscopy. Our studies have indicated that GR-LBD and PR-LBD are retained in the cytoplasm via interaction with Hsp90. It was observed that in the unliganded state, treatment with GA translocates these LBDs to the nucleus. Similar results were obtained for full-length PR and GR. Additionally, it was found that after ligand induction, GA accelerated reexport of SHRs after ligand washout, implicating Hsp90 in nuclear retention of SHRs in the washout state. We also propose that a recently found "export" signal present in the LBD of SHRs is involved in interactions with Hsp90 and hence cytoplasmic retention of these receptors. After ligand induction, Hsp90 also may play a role in nuclear retention of SHRs following hormone washout.

Cloning and characterization of cDNAs for hormones and/or receptors of growth hormone, insulin-like growth factor-I, thyroid hormone, and corticosteroid and the gender-, tissue-, and developmental-specific expression of their mRNA transcripts in fathead minnow ( Pimephales promelas)

Growth hormone (GH), insulin-like growth factor-I (IGF-I), thyroid hormones, and corticosteroids play central roles in a wide range of body functions but, in fish, information on their interactions is limited. These axes of the endocrine system are also potential targets for disruption of signaling pathways by hormone-mimicking chemicals, but have received little study. Molecular approaches offer an effective way to help unravel these endocrine interactions but require the appropriate gene-specific assays to do so. In this study, the cDNAs for a suite of hormones and/or receptors involved in signaling for the effects of GH and IGF-I [GH, GH receptor (GHR), IGF-I, IGF-I receptor (IGF-IR)], thyroid hormones [thyroid hormone receptor α (TRα) and β (TRβ)], and corticosteroids [glucocorticoid receptor (GR)] were cloned from the fathead minnow ( Pimephales promelas; fhm), and the tissue-, developmental-, and gender-related expression of their mRNA transcripts established. By polymerase chain reaction (PCR) strategy, we obtained full-length 1123-bp GH, 817-bp IGF-I, 1584-bp TRβ, and 2571-bp GR cDNAs, coding for 210 amino acid (aa) GH, 161 aa IGF-I, 378 aa TRβ, and 745 aa GR putative proteins, and partial-length 158-bp GHR, 811-bp IGF-IR, and 446-bp TRα cDNAs. Real-time PCR analyses revealed broad tissue expression for the target mRNAs; all targets were expressed in brain, pituitary, gill, liver, gonad, intestine, and muscle, with the exception of GH that was expressed only in the pituitary and gonad. Expression patterns in both juvenile and adult fhm were complex, with both temporal-, tissue-, and sex-specific characteristics. For example, hepatic expressions of GHR, IGF-I, and IGF-IR were far higher in males than in females, possibly reflecting the sex-related dimorphism in growth that occurs in this species, and TRα and TRβ showed divergent expression patterns during development (where TRβ predominated) and in adult tissues implying some distinct roles for the two TR subtypes.

Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 "client" protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601-607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR.hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR.hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR.hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR.hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR.hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a approximately 100-fold shift to the right in the steroid dose response for gene activation.

Gene regulation by the glucocorticoid receptor: Structure:function relationship

The glucocorticoid receptor (GR) belongs to the superfamily of ligand-activated transcription factors, the nuclear hormone receptors. Like other members of the family, the GR possesses a modular structure consisting of three major domains—the N-terminal (NTD), DNA binding (DBD), and ligand binding (LBD). Although the structures of independently expressed GR DBD and LBD are known, the structures of the NTD and of full-length GR are lacking. Both DBD and LBD possess overall globular structures. Not much is known about the structure of the NTD, which contains the powerful AF1/tau1/enh2 transactivation region. Several studies have shown that AF1 region is mostly unstructured and that it can acquire folded functional conformation under certain potentially physiological conditions, namely in the presence of osmolytes, when the GR DBD is bound to glucocorticoid response element (GRE), and when AF1 binds other transcription factor proteins. These conditions are discussed here. The functions of the GR will be fully understood only when its working three-dimensional structure is known. Based on the available data, we propose a model to explain data which are not adequately accounted for in the classical models of GR action. In this review, we summarize and discuss current information on the structure of the GR in the context of its functional aspects, such as protein:DNA and protein:protein interactions. Because of the close similarities in modular organization among the members of the nuclear hormone receptors, the principles discussed here for the GR should be applicable to many other receptors in the family as well.

A Serine/Threonine-rich Motif Is One of Three Nuclear Localization Signals That Determine Unidirectional Transport of the Mineralocorticoid Receptor to the Nucleus

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.

Importin 7 and importin alpha/importin beta are nuclear import receptors for the glucocorticoid receptor.

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin alpha selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin alpha-importin beta heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin alpha bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.

Dissociation of steroid receptor coactivator 1 and nuclear receptor corepressor recruitment to the human glucocorticoid receptor by modification of the ligand-receptor interface: the role of tyrosine 735.

Within the human glucocorticoid receptor (GR) steroid binding pocket, tyrosine 735 makes hydrophobic contact with the steroid D ring. Substitution of tyrosine735 selectively impairs glucocorticoid transactivation but not transrepression. We now show, using both mammalian two-hybrid and glutathione-S-transferase pull downs, that such substitutions reduce interaction with steroid receptor coactivator 1, both basally and in response to agonist binding. Using a yeast two-hybrid screen we identified one of the three nuclear receptor interacting domains (NCoR-N1) of nuclear receptor corepressor (NCoR) as interacting with the GR C terminus in an RU486-specific manner. This was confirmed in mammalian two-hybrid experiments, and so we used the NCoR-N1 peptide to probe the GR C-terminal conformation. Substitution of Tyr735phe, Tyr735val, and Tyr735 ser, which impaired steroid receptor coactivator 1 (SRC1) interaction, enhanced NCoR-N1 recruitment, basally and after RU486. RU486 did not direct SRC1 recruitment to any of the GR constructs, and dexamethasone did not allow NCoR-N1 recruitment. Using a glutathione-S-transferase pull-down approach, the NCoR-N1 peptide was found to bind the full-length GR constitutively, and no further induction was seen with RU486, but it was reduced by dexamethasone. As both SRC1 and NCoR are predicted to recognize a common hydrophobic cleft in the GR, it seems that changes favorable to one interaction are detrimental to the other, thus identifying a molecular switch.