Differential Hormone-Dependent Transcriptional Activation and -Repression by Naturally Occurring Human Glucocorticoid Receptor Variants (original) (raw)
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Mechanism of gene expression by the glucocorticoid receptor: Role of protein-protein interactions
BioEssays, 1997
The glucocorticoid receptor belongs to an important class of transcription factors that alter the expression of target genes in response to a specific hormone signal. The glucocorticoid receptor can function at least at three levels: (1) recruitment of the general transcription machinery; (2) modulation of transcription factor action, independent of DNA binding, through direct proteinprotein interactions; and (3) modulation of chromatin structure to allow the assembly of other gene regulatory proteins and/or the general transcription machinery on the DNA. This review will focus on the multifaceted nature of protein-protein interactions involving the glucocorticoid receptor and basal transcription factors, coactivators and other transcription factors, occurring at Accepted these different levels of regulation.
Journal of Biological Chemistry, 2004
Mammalian species are well known to differ in their sensitivity to glucocorticoids, but the molecular basis for this difference remains largely uncharacterized. To address this issue, the transcriptional activity of the mouse and human glucocorticoid receptor (GR) was analyzed on two model glucocorticoid-responsive promoters. Mouse GR (mGR) displayed unique promoter discrimination in response to a range of glucocorticoids, with enhanced activity on a simple glucocorticoid response element (GRE)-based promoter and diminished activity on the complex mouse mammary tumor virus promoter compared with human GR (hGR). Promoter discrimination between mGR and hGR was mapped to a single amino acid change at residue 437 (glycine to valine) of mGR and to sequence differences within individual GREs of the different promoters. Mouse GR displayed higher activation on GREs with a guanine rather than a thymine at the ؊6 position. Binding studies indicated mGR (mGR437V) displayed a weaker affinity for GREs containing a thymine at the ؊6 position than a mGR mutant containing a glycine at residue 437 (mGR437G). Despite distinct transcriptional activities, both receptors had similar affinities for response elements that contain a guanine at the ؊6 position. Our findings support a model by which the presence of a valine residue at position 437 of mGR induces a conformational change that leads to alterations in affinity and/or transcriptional activation in a promoter-dependent context.
The Journal of Steroid Biochemistry and Molecular Biology, 2003
Previous reports have suggested that the native hormone-responsive glucocorticoid receptor is a heterocomplex with hsp90 and that the receptor constantly cycles between the hormone-responsive and an inactive state, with complex assembly and turnover being driven by hsp70 and hsp90, respectively. Since hsp70 appears to be titrated in cells that transiently overexpress the receptor, assembly intermediates may accumulate when more receptor is produced than can be assembled to hormone-responsive complex. Comparison of receptor protein and hormone-binding levels in extracts from transiently transfected COS-7 cells revealed the presence of non-hormone-binding receptor forms in addition to the native heterocomplex. The receptor was predominantly nuclear in the majority of the transfected cells even in the absence of hormone, with the DNA-binding domain (DBD) being necessary for nuclear localisation. Moreover, the unliganded receptor exhibited constitutive DNA-binding activity and reactivity towards antibodies against the hinge region where NLS1 is known to reside. By comparing fluorography to immunoblotting of two-dimensional SDS-PAGE of cross-linked [ 3 H]dexamethasone-mesylate-labelled receptor, we detected non-hormone-binding receptor species capable of binding DNA in vitro. In addition, using a constitutively active receptor mutant, we found that the overexpressed wild-type receptor was capable of repressing mutant-activated transcription of transiently and stably transfected reporter genes alike in a DBD-dependent manner.
Modulation of transcription parameters in glucocorticoid receptor-mediated repression
Molecular and Cellular Endocrinology, 2008
Glucocorticoid receptors (GRs) affect both gene induction and gene repression. The disparities of receptor binding to DNA and increased vs. decreased gene expression have suggested significant mechanistic differences between GR-mediated induction and repression. Numerous transcription factors are known to modulate three parameters of gene induction: the total activity (V max ) and position of the dose-response curve with glucocorticoids (EC 50 ) and the percent partial agonist activity with antiglucocorticoids. We have examined the effects on GR-mediated repression of five modulators (coactivators TIF2 [GRIP1, SRC-2] and SRC-1, corepressor SMRT, and comodulators STAMP and Ubc9), a glucocorticoid steroid (deacylcortivazol [DAC]) of very different structure, and an inhibitor of histone deacetylation (trichostatin A [TSA]). These factors interact with different domains of GR and thus are sensitive topological probes of GR action. These agents altered the V max , EC 50 , and percent partial agonist activity of endogenous and exogenous repressed genes similarly to that previously observed for GR-regulated gene induction. Collectively, these results suggest that GR-mediated induction and repression share many of the same molecular interactions and that the causes for different levels of gene transcription arise from more distal downstream steps.
Cancer research, 1989
We have previously described the inhibition of glucocorticoid-dependent transcription from the mouse mammary tumor virus long terminal repeat promoter by products of the H-ras and v-mos oncogenes. We have studied the effects of conditional oncogenes on expression of glucocorticoid-dependent indicator genes. Expression of the glucocorticoid-dependent transcription of the tyrosine aminotransferase gene was monitored in FTO-2B rat hepatoma cells during Mr 21,000 protein (p21) H-ras induction. A strong transcriptional repression of the tyrosine aminotransferase gene followed p21 H-ras expression. The sequences in a glucocorticoid-dependent promoter which are responsible for the oncogene-mediated repression could be localized to the glucocorticoid response element; a construct in which a 15-base pair glucocorticoid response element was inserted 5' of the thymidine kinase promoter exhibited the oncogene-mediated repression of transcription. We observed a strong repression of glucocort...
Molecular Endocrinology, 1991
Steroid receptors have been reported to stimulate transcription in a manner synergistic with other transcription factors. We have examined this synergism or functional cooperativity between glucocorticoid receptors and basal transcription factors in a variety of promoter and reporter gene contexts. A fragment containing a hormone response element from mouse mammary tumor virus was fused to well characterized promoters from the herpes virus thymidine kinase and mouse 0-globin genes and to related mutant promoters altered by inactivation of transcription factor-binding sites through point mutagenesis or deletion. These constructs were transfected into glucocorticoid-sensitive fibroblasts, and reporter gene activity was assessed with or without hormonal stimulation. In contrast to previous studies, we found little indication of synergistic interaction between elements mediating a hormone response and adjacent basal promoters. In fact, we observed that inactivating basal factor-binding sites, thereby decreasing promoter strength, actually increased hormone inducibility. We suggest that the inverse relationship between basal promoter strength and the induction ratio attained upon hormonal stimulation may be due to limitation of a common factor, an "adaptor" through which glucocorticoid receptor and basal transcription factors interact with the components of the RNA polymerase II complex to stimulate rates of transcription.
The Journal of Clinical Endocrinology & Metabolism, 2005
Glucocorticoid resistance is a rare, familial or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition is broad, ranging from completely asymptomatic to severe hyperandrogenism and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance has been ascribed to mutations in the human glucocorticoid receptor-␣ (hGR␣) gene, which impair one or more of the molecular mechanisms of GR action, thus altering tissue sensitivity to glucocorticoids. We identified a new case of generalized glucocorticoid resistance in a young woman who presented with a long-standing history of fatigue, anxiety, hyperandrogenism, and hypertension. The disease was caused by a novel, heterozygous mutation (T3 C) at nucleotide position 2318 (exon 9) of the hGR␣ gene, which resulted in substitution of leucine by proline at amino acid position 773 in the ligand-binding domain of the receptor. We systematically investigated the molecular mechanisms through which the natural hGR␣L773P mutant impaired glucocorticoid signal transduction. Compared with the wild-type hGR␣, hGR␣L773P demonstrated a 2-fold reduction in the ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter, exerted a dominant negative effect on the wild-type receptor, had a 2.6-fold reduction in the affinity for ligand, showed delayed nuclear translocation (30 vs. 12 min), and, although it preserved its ability to bind to DNA, displayed an abnormal interaction with the GR-interacting protein 1 coactivator in vitro. We conclude that the carboxyl terminus of the ligand-binding domain of hGR␣ is extremely important in conferring transactivational activity by altering multiple functions of this composite transcription factor. (J Clin Endocrinol Metab 90: 3696 -3705, 2005)
The Journal of Clinical Endocrinology & Metabolism, 2007
Background: Generalized glucocorticoid resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in adrenocorticotropic hormone and cortisol secretion, and increased production of adrenal steroids with androgenic and/or mineralocorticoid activity. We have identified a new case of glucocorticoid resistance caused by a novel mutation of the human glucocorticoid receptor (hGR) gene and studied the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. Methods and Results: We identified a novel, single, heterozygous nucleotide (T 3 C) substitution at position 2209 (exon 9␣) of the hGR gene, which resulted in phenylalanine (F) to leucine (L) substitution at amino acid position 737 within helix 11 of the ligand-binding domain of the protein. Compared with the wild-type receptor, the mutant receptor hGR␣F737L demonstrated a significant ligand-exposure time-dependent decrease in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone and displayed a 2-fold reduction in the affinity for ligand, a 12-fold delay in nuclear translocation, and an abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. The mutant receptor preserved its ability to bind to DNA and exerted a dominant-negative effect on the wild-type hGR␣ only after a short duration of exposure to the ligand. Conclusions: The mutant receptor hGR␣F737L causes generalized glucocorticoid resistance because of decreased affinity for the ligand, marked delay in nuclear translocation, and/or abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. These findings confirm the importance of the C terminus of the ligandbinding domain of the receptor in conferring transactivational activity.
Proceedings of the National Academy of Sciences, 1981
Activated glucocorticoid receptor protein, purified to 40-60% homogeneity from rat liver extracts, binds selectively in vitro to a cloned fragment of murine mammary tumor virus (MTV) DNA. The DNA fragment tested contains about half of the sequences present in intact MTV DNA, and its rate of transcription, like that of the intact viral element, is strongly stimulated by glucocorticoids when it is introduced into the genome of a receptor-containing cell. In contrast, the receptor fails to bind selectively to DNA restriction fragments from E. coli plasmids pBR322 and RSF2124 or from bacteriophages A and T4. Preliminary experiments to localize regions within MTV DNA responsible for selective binding have revealed thus far one subfragment that fails to bind the receptor and one selectively bound subfragment that maps far downstream from the 5' terminus ofthe normal RNA transcript. These studies are consistent with the notion that steroid receptors may modulate rates of transcription by recognizing specific DNA sequences within or near the regulated genes.