Glucocorticoid Receptor Hormone Binding Domain Research Articles

Analysis of glucocorticoid and androgen receptor gene fusions delineates domains required for transcriptional specificity.

Androgen receptor (AR) and glucocorticoid receptor (GR) influence distinct physiologic responses in steroid-responsive cells despite their shared ability to selectively bind in vitro to the same canonical DNA sequence (TGTTCT). While the DNA-binding domains (DBDs) of these receptors are highly conserved, the amino N-terminal domain (NTD) and hormone-binding domain (HBD) are evolutionarily divergent. To determine the relative contribution of these functional domains to steroid-specific effects in vivo, we constructed a panel of AR/GR gene fusions by interchanging the NTD, DBD, and HBD regions of each receptor and measured transcriptional regulatory activities in transfected kidney and prostate cell lines. We found that GR was approximately 10-fold more active than AR when tested with the mouse mammary tumor virus promoter, and that this difference in activity was primarily owing to sequence divergence in the NTDs. We also tested transcriptional activation of the androgen-dependent rat probasin promoter, and in this case, AR was at least twofold more active than GR. Analysis of the chimeric receptors revealed that this difference mapped to the DBD region of the two receptors. Transcriptional repression functions of the wild-type and chimeric receptors were measured using an activator protein 1 (AP-1) transrepression assay and identified the GR HBD as a more potent transrepressor of AP-1 transcriptional activation than the AR HBD. Taken together, our analyses reveal that evolutionary sequence divergence between AR and GR functional domains results in unique promoter-specific activities within biologic systems in which both AR and GR are normally expressed.

Nuclear localization of NPR1 is required for activation of PR gene expression.

Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes. NPR1 is a key regulator in the signal transduction pathway that leads to SAR. Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance. We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR. An NPR1-green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR. To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain. Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.

Technical advance: transcriptional activator TGV mediates dexamethasone-inducible and tetracycline-inactivatable gene expression

A chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone-inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA-box. Tetracycline interferes with induction by negatively regulating the DNA-binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV-driven PTop10 reach from undetectable levels of the reporter enzyme beta-glucuronidase in the absence of dexa- methasone to induced levels reaching 15-20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.

An Additional Region of Coactivator GRIP1 Required for Interaction with the Hormone-binding Domains of a Subset of Nuclear Receptors

Transcriptional coactivators of the p160 family (SRC-1, GRIP1, and p/CIP) associate with DNA-bound nuclear receptors (NRs) and help the NRs to recruit an active transcription initiation complex to the promoters of target genes. Previous studies have demonstrated the importance of the NR interaction domain (NID) of p160 proteins containing three NR box motifs (LXXLL) for the interaction with the hormone-binding domains of NRs. Here we report that, in addition to NID, another region of coactivator GRIP1 (amino acids 1011-1121), called the auxiliary NID (NIDaux), is required in vitro and in vivo for efficient interaction with a subset of NRs, including the glucocorticoid receptor (GR), androgen receptor, and retinoic acid receptor alpha. A second group of NRs, which includes the progesterone receptor, retinoid X receptor alpha, thyroid hormone receptor beta1, and vitamin D receptor, required only NID for efficient interaction. For binding to GR, the NID and NIDaux of GRIP1 must act in cis, but deletion of up to 144 amino acids between the two regions did not reduce binding efficiency. Amino acids 1011-1121 of GRIP1 also contain a p300 interaction domain, but mutational analysis indicated that the p300 interaction function within this region is separable from the ability to contribute to GR hormone-binding domain binding. SRC-1 lacks an NIDaux activity equivalent to that in GRIP1.

Identification of Glucocorticoid Receptor Domains Necessary for Transcriptional Activation of the Mouse Mammary Tumor Virus Promoter Integrated in the Genome

It has previously been determined that the mouse mammary tumor virus (MMTV) promoter when integrated in the genome assumes a defined chromatin structure which is disrupted upon addition of glucocorticoids. In contrast, a transiently introduced MMTV promoter has a random nucleoprotein structure. To reveal glucocorticoid receptor (GR) domains necessary for transcriptional activation of the MMTV promoter we compared the effects of mutations of the GR on transcriptional activation of the stably integrated versus transiently introduced MMTV promoter. For this purpose we generated a GR-negative cell line which has an MMTV promoter/reporter construct integrated in the genome and studied the transcriptional activation of this construct by different GR mutants introduced into the cells. Transcriptional activation of the integrated and transiently introduced promoter was achieved by the wild-type GR or a chimeric receptor in which the MR hormone-binding domain (HBD) replaced the GR HBD. In contrast, we found that deletion of the HBD of the GR or replacement of this region with the equivalent domain of the estrogen receptor produced receptors that were unable to activate the MMTV promoter integrated in the genome although these receptors efficiently activated the transiently introduced MMTV promoter. The HBD was not the sole determinant of MMTV transcriptional activation when integrated in the genome. Chimeric receptors which harbored the MR amino terminal domain or the wild-type MR were also unable to activate the integrated MMTV promoter. Taken together, these data indicate a rigid requirement for sequences in both the GR amino and the carboxy terminal domains for transcriptional activation of a hormone response element in the defined chromatin context of the MMTV promoter.

Folding of the Glucocorticoid Receptor by the Reconstituted hsp90-based Chaperone Machinery: THE INITIAL hsp90·p60·hsp70-DEPENDENT STEP IS SUFFICIENT FOR CREATING THE STEROID BINDING CONFORMATION

Rabbit reticulocyte lysate contains a multiprotein chaperone system that assembles steroid receptors into a complex with hsp90. The glucocorticoid receptor (GR) is bound to hsp90 via its hormone binding domain (HBD), which must be associated with hsp90 to have a steroid binding conformation. Recently, we have reconstituted a receptor.hsp90 heterocomplex assembly system with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). In this work we show that when the GR is incubated with hsp90, hsp70, and p60, steroid binding sites are generated despite the absence of p23. In this minimal reconstituted system, the GR is incubated with the chaperones in the presence of [3H]triamcinolone acetonide ([3H]TA), which binds to the receptor as GR.hsp90 complexes are formed. When molybdate or p23 is also present during the incubation with chaperones at 30 degrees C, the formation of steroid binding sites can be assayed by incubating the washed GR with [3H]TA after heterocomplex assembly at 30 degrees C. However, in the absence of p23 or molybdate, rapid disassembly of GR.hsp90 complexes apparently occurs simultaneously with assembly, such that [3H]TA must be present during the assembly process to trap evidence of conversion of the GR HBD from a non-steroid binding to a steroid binding conformation. Mixture of purified rabbit hsp90 and hsp70 with bacterial lysate containing human p60 results in spontaneous formation of an hsp90.p60.hsp70 complex that can be adsorbed with anti-p60 antibody, and the resulting immune complex converts the GR HBD to a steroid binding state in an ATP-dependent and K+-dependent manner. When the GR is incubated with hsp90, hsp70, and p60 in the presence of the hsp90-binding antibiotic geldanamycin, GR.hsp90.p60. hsp70 complexes are formed, but they have no steroid binding activity. Our data suggest that hsp90, hsp70, and p60 work together as a chaperone complex that possesses all of the folding/unfolding activity necessary to generate the high affinity steroid binding conformation of the receptor.

Use of the Thiol-specific Derivatizing Agent N-Iodoacetyl-3-Iiodotyrosine to Demonstrate Conformational Differences between the Unbound and hsp90-bound Glucocorticoid Receptor Hormone Binding Domain

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GR.hsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The 125I-labeled IAT derivative N-iodoacetyl-3-[125I]iodotyrosine ([125I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an approximately 2-fold increase in [125I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [125I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [125I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GR*hsp90 complex become buried and are no longer accessible to the [125I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.

Genetic analysis of the N-terminal end of the glucocorticoid receptor hormone binding domain

Four site-directed missense mutations were constructed at the N-terminal end of the mouse glucocorticoid receptor (GR) hormone binding domain. This small subdomain is highly conserved among the steroid hormone receptors and is within a larger subregion believed to be important for hormone binding, transcriptional activation, and hsp90 binding. The ability of mutant and wild type GR to activate a recepter gene in response to various concentrations of dexamethasone was examined in transiently transfected COS-7 cells. Mutant GR species V544G (valine-544 changed to glycine) and V549G activated the reporter gene to approximately the same extent as wild type GR, but required approx. 7 and 23 times greater hormone concentrations, respectively. In contrast, double mutant LL541/2GG (leucines changed to glycines) could not activate transcription even at 10 μM dexamethasone or deacylcortivazol, while E543A (glutamic acid to alanine) was functionally indistinguishable from wild type GR. GR mutants LL541/2GG and V549G had reduced abilities to bind covalently to affinity label dexamethasone 21-mesylate. The partially and fully functional mutant GR species had no deficiency in transcriptional transactivation activity in the presence of saturating concentrations of agonist.

Two Point Mutations in the Hormone-Binding Domain of the Mouse Glucocorticoid Receptor That Dramatically Reduce Its Function

Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional.