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Query: UNIPROT:P06889 (Mol)
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A rapid procedure for the purification of the hepatic glucocorticoid receptor has been developed which exploits the observation that "activation" of this complex enables it to bind to anionic substances such as DNA and phosphocellulose. The procedure consists of two phosphocellulose columns operated in sequence. The first column removes from unfractionated cytosol all basic proteins which adhere to the immobilized phosphate residues; the steroid - receptor complex elutes in the flow-through of this first column. This complex is then thermally activated and applied to a second phosphocellulose column where it is retained, washed, and eluted by a salt gradient. This simple procedure is capable of purifying the steroid - receptor complex over 1000-fold.
Mol Cell Endocrinol
PMID:Partial purification of the activated hepatic glucocorticoid - receptor complex. 18 73

The binding of the glucocorticoid receptor of rat liver to chromatin and DNA has been studied with crude and partially purified preparations of cytosol receptor labelled with [3H]-triamcinolone acetonide in vitro. The use of crude preparations of receptor and increasing protein concentrations leads to an apparent saturation of chromatin and DNA, suggesting a limited number of high affinity nuclear acceptor sites for the receptor. Appropriate controls indicate that the observed saturability of chromatin acceptor sites is due to the presence in crude receptor preparations of heat-stable protein factors which interfere with the binding of the receptor to the genome; whereas the apparent saturation of DNA is due to contamination with deoxyribonucleases. If the activated complex of receptor and triamcinolone acetonide (R-TA) is partially purified to a step where it is free from nucleases and inhibitors, its binding to both chromatin and DNA is linearly dependent on the concentration of free (R-TA) in the incubation medium. There is no absolute specificity with respect to the source of DNA or chromatin, although liver chromatin has considerably higher receptor binding capacity than chromatin from avian erythrocytes. The rate kinetics of association and dissociation for the binding of (R-TA) to DNA and chromatin are very similar, but DNA exhibits a 10-fold higher receptor binding capacity than chromatin. These data, in conjunction with the effect of poly-(D)-lysine and and NaCl on the binding of (R-TA) to chromatin and DNA, suggest that most of the receptor molecules bound to chromatin in vitro interact with the "accessible" DNA stretches. Although a small population of receptor molecules may bind specifically to target tissue genome, the detection of these specific sites against the background of unspecific binding is not possible with unfractionated chromatin or DNA preparations.
Mol Cell Endocrinol 1977 Mar
PMID:Binding of the partially purified glucocorticoid receptor of rat liver to chromatin and DNA. 85 47

Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event.
Mol Cell Biol 1992 Feb
PMID:Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis. 131 Jan 48

The effects of okadaic acid (OA), a protein phosphatase inhibitor, on transcriptional enhancement activity of rat glucocorticoid receptor (GR) were examined in transiently transfected cells. In the absence of hormone, GRs expressed in CV-1 and COS-1 fibroblasts were capable of enhancing transcription from cotransfected chloramphenicol acetyltransferase reporter plasmids in response to OA treatment. Synergistic enhancement resulted from combined hormone and OA treatment. The effects of OA on GR-mediated enhancement required the presence of linked glucocorticoid response elements and were observed with reporter plasmids that contained different promoters and glucocorticoid response elements. Since OA did not affect nuclear translocation of the receptor, enhancement mediated by unliganded GR was most likely accounted for by the accumulation of some unliganded GRs within nuclei of transfected CV-1 and COS-1 cells. Deletion of individual GR transactivation domains and point mutations within DNA- and hormone-binding domains severely reduced the response of receptors to OA, although some mutant receptors retained the capacity to elicit a synergistic response when exposed to OA and hormone. The effects of OA on transcriptional enhancement did not appear to correlate with major changes in GR phosphorylation, as visualized by two-dimensional tryptic mapping of in vivo 32P-labeled GRs. Thus, phosphorylation of various components of the GR signal transduction pathway, and not necessarily the receptor itself, may influence its transcriptional enhancement activity.
Mol Endocrinol 1992 Jan
PMID:Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptor-mediated enhancement. 131 Jul 97

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocorticoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocorticoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half "Zn fingers" of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, preceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Glucocorticoids in malignant lymphoid cells: gene regulation and the minimum receptor fragment for lysis. 131 75

The glucocorticoid receptor (GR) is a regulable transcription factor which can bind to DNA response elements in the vicinity of inducible gene promoters and enhance the rate of transcription initiation. The concentration of endogenously expressed GR has been shown to limit the magnitude of the transcriptional induction response in cultured cells. We have investigated the consequence of increased GR expression on the transcriptional activity of a hormone responsive promoter, the MMTV LTR, and on three non-responsive promoters, the RSV LTR, the SV40 early promoter and the c-fos promoter in transiently transfected cells. Low receptor concentrations allow a slight hormonal induction of the MMTV LTR while maximal inducibility can be observed at intermediate GR concentrations. High GR expression reduces the hormonal induction on the MMTV LTR and also adversely effects transcription from the RSV LTR, the SV40 and c-fos promoters. This repression effect is dependent on GR activation. These experiments suggest that the GR interacts with a nuclear factor that is required for the activation of all four promoters. It is probable that "squelching", i.e. protein-protein complex formation in the nucleus leads to the sequestration of interacting proteins(s) essential for the transcription machinery, causing a limitation for the initiation events.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Overexpression of the glucocorticoid receptor represses transcription from hormone responsive and non-responsive promoters. 131 76

We have identified a novel member of the steroid hormone receptor superfamily by cDNA cloning from a human osteosarcoma SAOS-2/B10 cell library. Sequence analysis predicts a protein of 441 amino acids, which includes the conserved amino acid residues characteristic of the DNA- and ligand-binding domains of nuclear receptors. Amino acid sequence alignment and transcriptional activation experiments revealed that the new protein is closely related to the mouse peroxisome proliferator activated receptor. The overall homology is 62%, and the highest similarity is seen in the DNA- and ligand-binding domains, 86% and 71%, respectively. Northern blot analysis showed that in mature rats, the receptor is highly expressed in heart, kidney, and lung as a transcript of approximately 3500 nucleotides. In human cells, the size of the mRNA is approximately 4000 nucleotides. Transcription assays using hybrid receptors consisting of the ligand-binding domain of the new protein and the DNA-binding domain of the glucocorticoid receptor showed weak stimulation by the peroxisome proliferator activator WY14643, suggesting a relationship to that receptor. Similar stimulation was observed with arachidonic and oleic acid (100-250 microM).
Mol Endocrinol 1992 Oct
PMID:Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids. 133 51

The duration of the antagonizing activity of RU486 on tyrosine aminotransferase (TAT) induction and the glucocorticoid receptor in rat liver was studied. A single dose of RU486 (10 mg/kg) caused occupation of the cytosol glucocorticoid receptor in rat liver at 1 h. During this time no nuclear binding of [3H]dexamethasone ([3H]Dex) receptor complex was recorded, and TAT induction was completely blocked. TAT inducibility recovery parallelled receptor binding in both the cytosol and the nuclei, reaching maximum at 12 h. In contrast, nuclear binding recovered in 24 h, and [3H]Dex receptor binding in cytosol 48 h after RU486 application. It is concluded that the inhibitory effect of a single dose of RU486 on TAT induction is of rather short duration. At concomitant presence of agonist and antagonist in vivo, no direct correlation between agonist receptor occupancy and TAT induction could be observed.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Duration of antagonizing effect of RU486 on the agonist induction of tyrosine aminotransferase via glucocorticoid receptor. 134 26

We have investigated the ability of dehydroepiandrosterone (DHEA) to alter the production of interleukin-2 (IL-2) and to bind to a specific binding complex in antiCD3 epsilon activated T cells. Binding activity correlated with the presence of a specific DHEA binding complex in the cytosol and nuclei of DHEA-responsive T-cell hybridomas, as well as in CD4+ and CD8+ cells isolated from peripheral lymph nodes of normal mice. Scatchard analysis determined that intact lymphocytes and cytosolic fractions contained high affinity binding for [3H]DHEA (approx. 2.6 nM) with 1000-7000 binding sites existing per cell. Five of the T-cell hybridomas tested both responded to DHEA treatment with increased production of IL-2 and also contained specific high affinity [3H]DHEA binding. Four additional T-cell hybridomas were found to contain no specific [3H]DHEA binding and were also unresponsive to DHEA influences on IL-2 production. Sucrose density gradients demonstrated a 3-4s [3H]DHEA binding complex in high salt and a 7-8s binding complex in low salt. Specific binding was inhibited by preincubation of the cytosol fractions with either trypsin or chymotrypsin, or by heating to 60 degrees C for 1 h (less than 15% of control). [3H]DHEA binding was unaffected by preincubation of the cytosol fractions with ribonuclease, deoxyribonuclease, or phospholipase A. The DHEA-protein complexes bound to DNA-cellulose with the amount of binding being slightly increased by preincubation at 25 degrees C as compared to 4 degrees C. As expected, [3H]DHEA binding was inhibited by the addition of unlabeled DHEA, but was also modestly inhibited by dihydrotestosterone and cortisol. Binding of DHEA was unaffected by progesterone, dexamethasone, estradiol, androsterone, DHEAS, and beta-etiocholanolone at all concentrations tested. DHEA was incapable of inhibiting the binding of [3H]DHT to the androgen receptor or [3H]dexamethasone to the glucocorticoid receptor. Collectively, these findings suggest that murine T cells contain a specific DHEA receptor. We believe that DHEA is a steroid hormone that is directly involved in the regulation of IL-2 production by both normal and some T-cell hybridomas.
J Steroid Biochem Mol Biol 1992 May
PMID:The presence of a dehydroepiandrosterone-specific receptor binding complex in murine T cells. 135 1

Obesity is likely to be a multifactorial disease with an important genetic component. Animal models of genetic and experimentally induced obesity suggest that glucocorticoid receptor (GR) activity plays a role in the aetiology and maintenance of the obese state. Glucocorticoid activity appears to be essential for the development of hyperinsulinaemia and subsequent fat deposition. In humans, glucocorticoid excess is associated with central fat distribution. We have therefore investigated the restriction fragment length polymorphisms of the human GR gene locus (GRL) and have sought associations of specific alleles with anthropometric measurements and indices of insulin secretion and resistance in obesity. Fifty-six extremely obese, unrelated, nondiabetic premenopausal British Caucasian females and 43 age-matched, normal weight controls were studied. The obese subjects were characterized by fat distribution (waist to hip ratio), insulin secretion and insulin resistance (fasting insulin (FI)), an index of insulin resistance (HOMA), stimulated insulin secretion during an oral glucose tolerance test and insulin-mediated glucose disposal, steady-state plasma glucose). A Bc1I polymorphism (fragments of 4.5 and 2.3 kb) demonstrated significant association with indices of glucose metabolism in obesity; those subjects homozygous for the 4.5 kb fragment had elevated FI (Pc = 0.012) and HOMA (Pc = 0.012) values. The genotypic and allelic frequencies of the GRL Bc1I polymorphism were otherwise similar in obese and normal weight subjects. We postulate that the GRL Bc1I polymorphism may directly affect GR gene expression, or be in linkage disequilibrium with a possible mutation within one of three exons of the GR gene, and thereby modulate GR transcriptional activity on target genes involved in glucose and insulin homeostasis.
J Mol Endocrinol 1992 Dec
PMID:An association between a Bc1I restriction fragment length polymorphism of the glucocorticoid receptor locus and hyperinsulinaemia in obese women. 136 60


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