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1.
J Clin Endocrinol Metab ; 103(10): 3630-3639, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30020469

ABSTRACT

Background: The glucocorticoid receptor (GR) consists of two alternatively spliced isoforms: GRα, which activates gene transcription, and GRß, a dominant-negative receptor. Theoretically, inactivating variants of GRß could result in glucocorticoid hypersensitivity. Design: A 46-year-old woman presented for evaluation of adrenal insufficiency prompted by low plasma cortisol levels and multiple unexplained symptoms but without clinical evidence of glucocorticoid insufficiency. To explain these findings, extensive clinical, genetic, and molecular studies were performed. Methods: Standard clinical methods assessed the patient's hypothalamic-pituitary-adrenal axis. Validated molecular techniques were used for receptor sequencing, stable transfections, stimulation of candidate genes, cDNA arrays, Ingenuity Pathway Analysis, volcano analysis, and isolation and analysis of the patient's mononuclear cells. Results: Clinical studies excluded primary or secondary adrenal insufficiency, established consistently low basal cortisol levels, and demonstrated hypersensitivity to ultra-low-dose dexamethasone. Receptor sequencing identified two variants of GR9ß (A3669G and G3134T) as well as the known Bcl1 polymorphism. Reductionist studies using stable osteosarcoma cell lines transfected with the GRß variants demonstrated glucocorticoid hypersensitivity of transcribed genes on cDNA array analysis. The patient's monocytes responded to hydrocortisone with exaggerated stimulation of the candidate genes GILZ and FKBP5. Conclusion: Two variants of the dominant-negative GRß, in conjunction with a common Bcl1 intron variant, resulted in hypersensitivity to endogenous and exogenous glucocorticoids and, as a reflection of severity, low circulating cortisol levels without clinical evidence of glucocorticoid insufficiency. This prismatic case exemplifies the unique effects of variants of a dominant-negative receptor.


Subject(s)
Glucocorticoids/pharmacology , Hypersensitivity/epidemiology , Hypersensitivity/genetics , Mutation , Receptors, Glucocorticoid/genetics , Biomarkers/blood , Female , Humans , Hydrocortisone/blood , Hypersensitivity/drug therapy , Incidence , Middle Aged , Prognosis
2.
Mol Cell Biol ; 38(8)2018 04 15.
Article in English | MEDLINE | ID: mdl-29437838

ABSTRACT

Glucocorticoid receptor ß (GRß) is associated with glucocorticoid resistance via dominant negative regulation of GRα. To better understand how GRß functions as a dominant negative inhibitor of GRα at a molecular level, we determined the crystal structure of the ligand binding domain of GRß complexed with the antagonist RU-486. The structure reveals that GRß binds RU-486 in the same ligand binding pocket as GRα, and the unique C-terminal amino acids of GRß are mostly disordered. Binding energy analysis suggests that these C-terminal residues of GRß do not contribute to RU-486 binding. Intriguingly, the GRß/RU-486 complex binds corepressor peptide with affinity similar to that of a GRα/RU-486 complex, despite the lack of helix 12. Our biophysical and biochemical analyses reveal that in the presence of RU-486, GRß is found in a conformation that favors corepressor binding, potentially antagonizing GRα function. This study thus presents an unexpected molecular mechanism by which GRß could repress transcription.


Subject(s)
Receptors, Glucocorticoid/metabolism , Amino Acid Sequence , Escherichia coli/metabolism , Glucocorticoids/metabolism , Humans
3.
Lab Invest ; 97(11): 1282-1295, 2017 11.
Article in English | MEDLINE | ID: mdl-28759007

ABSTRACT

Alternative splicing of the human glucocorticoid receptor gene generates two isoforms, hGRα and hGRß. hGRß functions as a dominant-negative regulator of hGRα activity and but also has inherent transcriptional activity, collectively altering glucocorticoid sensitivity. Single-nucleotide polymorphisms in the 3' UTR of hGRß have been associated with altered receptor protein expression, glucocorticoid sensitivity, and disease risk. Characterization of the hGRß G3134T polymorphism has been limited to a relatively small, homogenous population. The objective of this study was to determine the prevalence of hGRß G3134T in a diverse population and assess the association of hGRß G3134T in this population with physiological outcomes. In a prospective cohort study, 3730 genetically diverse participants were genotyped for hGRß G3134T and four common GR polymorphisms. A subset of these participants was evaluated for clinical and biochemical measurements. Immortalized human osteosarcoma cells (U-2 OS), stably transfected with wild-type or G3134T hGRß, were evaluated for receptor expression, stability, and genome-wide gene expression. Glucocorticoid-mediated gene expression profiles were investigated in primary macrophages isolated from participants. In a racially diverse population, the minor allele frequency was 74% (50.7% heterozygous carriers and 23.3% homozygous minor allele), with a higher prevalence in Caucasian non-Hispanic participants. After adjusting for confounding variable, carriers of hGRß G3134T were more likely to self-report allergies, have higher serum cortisol levels, and reduced cortisol suppression in response to low-dose dexamethasone. The presence of hGRß G3134T in U-2 OS cells increased hGR mRNA stability and protein expression. Microarray analysis revealed that the presence of the hGRß G3134T polymorphism uniquely altered gene expression profiles in U-2 OS cells and primary macrophages. hGRß G3134T is significantly present in the study population and associated with race, self-reported disease, and serum levels of glucocorticoids. Underlying these health differences may be changes in gene expression driven by altered receptor stability.


Subject(s)
3' Untranslated Regions , Gene Expression Regulation , Glucocorticoids/metabolism , Polymorphism, Single Nucleotide , Receptors, Glucocorticoid/agonists , Signal Transduction , Adult , Amino Acid Substitution , Black People , Cell Line, Tumor , Cells, Cultured , Cohort Studies , Female , Genetic Association Studies , Glucocorticoids/blood , Hispanic or Latino , Humans , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Male , North Carolina , Prospective Studies , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Registries , White People
4.
Am J Physiol Endocrinol Metab ; 311(4): E741-E748, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27600822

ABSTRACT

The glucocorticoid receptor single-nucleotide polymorphism (SNP) N363S has been reported to be associated with metabolic syndrome, type 2 diabetes, and cardiovascular disease. Our aim was to determine how the N363S SNP modifies glucocorticoid receptor signaling in a healthy population of individuals prior to the onset of disease. We examined the function of the N363S SNP in a cohort of subjects from the general population of North Carolina. Eighteen N363S heterozygous carriers and 36 noncarrier, control subjects were examined for clinical and biochemical parameters followed by a low-dose dexamethasone suppression test to evaluate glucocorticoid responsiveness. Serum insulin measurements revealed that N363S carriers have higher levels of insulin, although not statistically significant, compared with controls. Glucocorticoid receptor protein levels evaluated in peripheral blood mononuclear cells from each clinical subject showed no difference between N363S and control. However, investigation of gene expression profiles in macrophages isolated from controls and N363S carriers using microarray, quantitative RT-PCR, and NanoString analyses revealed that the N363S SNP had an altered profile compared with control. These changes in gene expression occurred in both the absence and the presence of glucocorticoids. Thus, our observed difference in gene regulation between normal N363S SNP carriers and noncarrier controls may underlie the emergence of metabolic syndrome, type 2 diabetes, and cardiovascular disease associated with the N363S polymorphism.


Subject(s)
Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Metabolic Syndrome/genetics , Metabolic Syndrome/metabolism , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Adult , Aged , Dexamethasone/pharmacology , Diabetes Mellitus, Type 2/genetics , Female , Genotype , Glucocorticoids/pharmacology , Heterozygote , Humans , Inflammation/genetics , Insulin/blood , Male , Metabolic Syndrome/epidemiology , Middle Aged , Monocytes/drug effects , Monocytes/metabolism , Polymorphism, Single Nucleotide/genetics , Prevalence , RNA/biosynthesis , RNA/genetics , Young Adult
5.
Blood ; 121(9): 1553-62, 2013 Feb 28.
Article in English | MEDLINE | ID: mdl-23297131

ABSTRACT

Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow­derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte­derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms.


Subject(s)
Cell Differentiation/drug effects , Dendritic Cells/drug effects , Drug Resistance/drug effects , Glucocorticoids/pharmacology , Receptors, Glucocorticoid/physiology , Animals , Caspase 3/metabolism , Cell Differentiation/genetics , Cells, Cultured , Dendritic Cells/metabolism , Dendritic Cells/physiology , Drug Resistance/genetics , Female , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/physiology , Humans , Mice , Mice, Inbred BALB C , Protein Biosynthesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Substrate Specificity/drug effects , Substrate Specificity/genetics
6.
Mol Endocrinol ; 26(2): 244-56, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22174376

ABSTRACT

A mutation in the D-loop of the second zinc finger of the DNA-binding domain of the human glucocorticoid receptor (hGR), A458T (GR(dim)), has been suggested to be essential for dimerization and DNA binding of the GR, and genetically altered GR(dim) mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GR(dim) mice were reported to be resistant to glucocorticoid-induced apoptosis. To further evaluate the dim mutations in glucocorticoid-induced apoptosis, we stably expressed either the hGR(dim) (A458T) or the hGR(dim4) (A458T, R460D, D462C, and N454D) mutant receptors in human osteosarcoma (U-2 OS) cells that are devoid of hGR and unresponsive to glucocorticoids. We analyzed these cell lines by comparison with a stable expression hGRα U-2 OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGR(dim) mutation had diminished steroid responsiveness and cells carrying the hGR(dim4) mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor κB repression was unaffected by either mutation. Interestingly, both the hGR(dim) and hGR(dim4) receptors readily formed dimers as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGR(dim) cells were only partially resistant to apoptosis, whereas hGR(dim4) cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGR(dim4) cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants.


Subject(s)
Apoptosis , Glucocorticoids/physiology , Osteocytes/physiology , Receptors, Glucocorticoid/genetics , Amino Acid Substitution , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Dexamethasone/pharmacology , Gene Expression Profiling , Gene Expression Regulation , Genes, Reporter , Glucocorticoids/pharmacology , Humans , Luciferases/biosynthesis , Luciferases/genetics , Oligonucleotide Array Sequence Analysis , Osteocytes/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Receptors, Glucocorticoid/metabolism , Signal Transduction , Transcriptional Activation
7.
Mol Endocrinol ; 25(7): 1087-99, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21527497

ABSTRACT

Glucocorticoids regulate a variety of physiological processes and are commonly used to treat disorders of inflammation, autoimmune diseases, and cancer. Glucocorticoid action is predominantly mediated through the classic glucocorticoid receptor (GR)α isoform. Recent data suggest that the mature GRα mRNA is translated into multiple N-terminal isoforms that have distinct biochemical properties and gene regulatory profiles. Interestingly, osteosarcoma cells stably expressing the GRα-D translational isoform are unique in that they are resistant to glucocorticoid-induced apoptosis. In this study, we investigate whether GRα isoform-specific differences in the regulation of antiapoptotic genes contribute to this resistant phenotype. We now show that GRα-D, unlike the other receptor isoforms, does not inhibit the activity of a nuclear factor κB (NF-κB)-responsive reporter gene and does not efficiently repress either the transcription or protein production of the antiapoptotic genes Bcl-xL, cellular inhibitor of apoptosis protein 1, and survivin. The inability of GRα-D to down-regulate the expression of these genes appears to be associated with a diminished interaction between GRα-D and NF-κB that is observed in cells, but not in vitro, and likely reflects the sequestration of GRα-D in the nucleus. Deletion of the GRα N-terminal amino acids 98-335 also results in a nuclear resident GR, which fails to interact with NF-κB in cells and promote apoptosis in response to glucocorticoids. These data suggest that the N-terminal translational isoforms of GRα selectively regulate antiapoptotic genes and that the GRα-D isoform may contribute to the resistance of certain cancer cells to glucocorticoid-induced apoptosis.


Subject(s)
Apoptosis/genetics , Dexamethasone/pharmacology , Drug Resistance, Neoplasm , Glucocorticoids/pharmacology , Inhibitor of Apoptosis Proteins/genetics , Receptors, Glucocorticoid/metabolism , Amino Acid Motifs , Caspases/metabolism , Cell Line, Tumor , Genes, Reporter , Humans , Inhibitor of Apoptosis Proteins/metabolism , Luciferases, Renilla/biosynthesis , Luciferases, Renilla/genetics , NF-kappa B/metabolism , Osteosarcoma , Promoter Regions, Genetic , Protein Binding , Protein Isoforms/metabolism , Signal Transduction , Survivin , bcl-X Protein/genetics , bcl-X Protein/metabolism
8.
J Cell Mol Med ; 15(2): 339-49, 2011 Feb.
Article in English | MEDLINE | ID: mdl-19874421

ABSTRACT

Airway inflammation is a common condition where glucocorticoids (GC) are a well-established therapy. It has been demonstrated that GC stimulate components of innate immunity. Specifically, GC up-regulate TLR2 expression and activation upon inflammatory stimuli; however, little is known about the signalling involved in this process. To determine the mechanism by which dexamethasone modulates TLR2-induced cytokine production this signalling pathway was monitored in a lung epithelial cell line exposed to the TLR2 synthetic agonist, Pam(3) -Cys-Ser-Lys(4) . These experiments demonstrate that phosphatidylinositol 3-kinase (PI3K) is critical for the TLR2 downstream effects of GC. Cells expressing a PI3K mutant (p85-dominant negative, DN; p85 Δ478-511) and exposed to Pam(3) -Cys-Ser-Lys(4) in the presence or absence of dexamethasone, showed enhanced tumour necrosis factor (TNF)α expression while AP-1 and NF-κB transcriptional activity were repressed. We provide experimental evidence that PI3K physically interacts with the glucocorticoid receptor (GR) through two putative PI3K recruitment consensus YxxM binding motifs in the GR, suggesting that some functions regulated by this receptor might occur through kinase interaction. Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression. However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells. Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.


Subject(s)
Dexamethasone/pharmacology , Phosphatidylinositol 3-Kinase/metabolism , Receptors, Glucocorticoid/metabolism , Toll-Like Receptor 2/immunology , Cell Line , Cytokines/biosynthesis , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Lung/metabolism , NF-kappa B/biosynthesis , Peptides/pharmacology , Phosphatidylinositol 3-Kinase/genetics , Receptors, Glucocorticoid/genetics , Signal Transduction/drug effects , Toll-Like Receptor 2/metabolism , Transcription Factor AP-1/biosynthesis , Transcriptional Activation , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
9.
J Clin Endocrinol Metab ; 92(8): 3268-77, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17535992

ABSTRACT

CONTEXT: A single-nucleotide polymorphism (SNP) in the human glucocorticoid receptor (hGR) N363S (rs6195) has been the focus of several clinical studies, and some epidemiological data link this SNP to increased glucocorticoid sensitivity, coronary artery disease, and increased body mass index. However, molecular studies in vitro using reporter gene expression systems have failed, for the most part, to define a link between this polymorphism and altered glucocorticoid receptor function. OBJECTIVE: The objective of this study was to address the biological relevancy of N363S SNP in GR function by establishing stable U-2 OS (human osteosarcoma) cell lines expressing wild-type hGR or N363S and examining these receptors under a variety of conditions that probe for GR activity including human gene microarray analysis. DESIGN: Functional assays with reporter gene systems, Western blotting, and human microarray analysis were used to evaluate the activity of wild-type and N363S GR in both transiently and stably expressing cells. In addition, quantitative RT-PCR was used to confirm the microarray analysis. RESULTS: Functional assays with reporter gene systems and homologous down-regulation revealed only minor differences between the wild-type hGR and N363S receptors in both transiently and stably expressing cell lines. However, examination of the two receptors by human gene microarray analysis revealed a unique gene expression profile for N363S. CONCLUSIONS: These studies demonstrate that the N363S SNP regulates a novel set of genes with several of the regulated genes supporting a potential role for this GR polymorphism in human diseases.


Subject(s)
Polymorphism, Genetic/genetics , Receptors, Glucocorticoid/genetics , Animals , Blotting, Western , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Dexamethasone/pharmacology , Gene Expression/genetics , Gene Expression/physiology , Genes, Reporter/genetics , Genetic Linkage , Humans , Oligonucleotide Array Sequence Analysis , Osteosarcoma/genetics , Osteosarcoma/metabolism , Polymorphism, Genetic/physiology , Polymorphism, Single Nucleotide/genetics , Receptors, Glucocorticoid/physiology , Reverse Transcriptase Polymerase Chain Reaction , Transfection
10.
Mol Cell Biol ; 27(6): 2266-82, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17242213

ABSTRACT

Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, alpha and beta. In contrast to the canonical hGRalpha, hGRbeta is a nucleus-localized orphan receptor thought not to bind ligand and not to affect gene transcription other than by acting as a dominant negative to hGRalpha. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRbeta in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRbeta was predominantly located in the cytoplasm and translocated to the nucleus following application of the glucocorticoid antagonist RU-486. This effect of RU-486 was confirmed with transiently expressed wild-type hGRbeta. Confocal microscopy of coexpressed YFP-hGRbeta and cyan fluorescent protein-hGRalpha in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRbeta bound RU-486 but not the hGRalpha ligand dexamethasone. Examination of the cellular localization of YFP-hGRbeta in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRbeta. The selective interaction of RU-486 with hGRbeta was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRbeta, expressed in the absence of hGRalpha, can regulate gene expression and furthermore that occupation of hGRbeta with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.


Subject(s)
Mifepristone/chemistry , Mifepristone/pharmacology , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Animals , Cell Line , Cell Nucleus/metabolism , Chlorocebus aethiops , Computational Biology , Gene Expression Profiling , Gene Expression Regulation , Humans , Ligands , Mifepristone/metabolism , Models, Molecular , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary , Protein Transport , Receptors, Glucocorticoid/genetics
11.
J Steroid Biochem Mol Biol ; 102(1-5): 11-21, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17070034

ABSTRACT

Glucocorticoids regulate diverse physiological effects in virtually every organ and tissue in the body. Glucocorticoid actions are mediated through the glucocorticoid receptor (GR), a ligand-dependent transcriptional factor that activates or represses gene transcription. Since, the cloning of the human GR in 1985, research efforts have been focused on describing the mechanism of action exerted by one of the GR isoforms, GRalpha. However, recent studies from our lab and others have suggested that multiple isoforms of hGR are generated from one single gene and one mRNA species by the mechanisms of alternative RNA splicing and alternative translation initiation. These isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcription activities. In addition, this new information predicts that each hGR protein can be subjected to a variety of post-translational modifications, such as phosphorylation, sumoylation and ubiquitination. The nature and degree of post-translational modification, as well as subcellular localization, may differentially modulate stability and function among the GR isoforms in different tissues providing an additional important mechanism for regulation of GR action. We outline the recent advances made in identifying the processes that generate and modify multiple GR isoforms and the post-translational modifications that contribute to the increasing diversity in the glucocorticoid signaling pathway.


Subject(s)
Receptors, Glucocorticoid/metabolism , Alternative Splicing , Animals , Glucocorticoids/pharmacology , Humans , Phosphorylation , Protein Isoforms , Protein Processing, Post-Translational , Ubiquitin/metabolism
12.
J Steroid Biochem Mol Biol ; 101(2-3): 106-17, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16914312

ABSTRACT

Although LXXLL motifs in coactivators mediate binding to liganded nuclear receptors, the roles of comparable motifs within nuclear receptors are less understood. We investigated the role of the LXXLL motifs in the human glucocorticoid receptor both in transcriptional activation and repression of gene expression. The two conserved LXXLL motifs within the ligand binding domain of the receptor, amino acids 532-536 (helix 1) and 718-722 (helix 10), were characterized by evaluating LXXLL mutant receptors as well as comparable mutants in other helices of the ligand binding domain. All mutant receptors were expressed at comparable levels to wild type in COS-1 cells. Mutation of 532-536 LXXLL to LXXAA completely disrupted dexamethasone induced transcription, whereas the 718-722 LXXAA mutant fully activated reporter genes at high ligand concentrations. Ligand binding analysis demonstrated that both LXXLL motif mutations resulted in disruption of ligand binding capacity without altering their association with hsp90. Proteolytic cleavage studies suggested that mutations of the LXXLL motifs introduced changes in the receptor conformation. Interestingly, the 532-536 LXXAA mutant was not able to transrepress NF-kappaB activity, whereas the 718-722 LXXAA mutant did so in the presence of ligand. These data suggest that although LXXLL motifs in helices 1 and 10 appear to lie outside the predicted ligand binding pocket they may contribute to receptor ligand binding affinity.


Subject(s)
Ligands , Mutation , Receptors, Glucocorticoid/genetics , Amino Acid Motifs , Animals , Binding Sites , COS Cells , Cell Nucleus/metabolism , Chlorocebus aethiops , Gene Expression , Glucocorticoids/pharmacology , Green Fluorescent Proteins/metabolism , HSP90 Heat-Shock Proteins/genetics , Models, Molecular , NF-kappa B/metabolism , Protein Structure, Tertiary , Signal Transduction , Transcriptional Activation , Transfection , Triamcinolone Acetonide/pharmacology
13.
Mol Cell Biol ; 23(12): 4319-30, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12773573

ABSTRACT

This study molecularly elucidates the basis for the dominant negative mechanism of the glucocorticoid receptor (GR) isoform hGRbeta, whose overexpression is associated with human glucocorticoid resistance. Using a series of truncated hGRalpha mutants and sequential mutagenesis to generate a series of hGRalpha/beta hybrids, we find that the absence of helix 12 is neither necessary nor sufficient for the GR dominant negative phenotype. Moreover, we have localized the dominant negative activity of hGRbeta to two residues and found that nuclear localization, in addition to heterodimerization, is a critical feature of the dominant negative activity. Molecular modeling of wild-type and mutant hGRalpha and hGRbeta provides structural insight and a potential physical explanation for the lack of hormone binding and the dominant negative actions of hGRbeta.


Subject(s)
Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/physiology , Algorithms , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , Cell Line , Cell Nucleus/metabolism , Chloramphenicol O-Acetyltransferase/metabolism , Crystallography, X-Ray , DNA/metabolism , Dimerization , Drug Resistance , Genes, Dominant , Glutathione Transferase/metabolism , Humans , Immunohistochemistry , Ligands , Models, Molecular , Molecular Sequence Data , Mutation , Phenotype , Plasmids/metabolism , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Receptors, Glucocorticoid/metabolism , Sequence Homology, Amino Acid , Transcriptional Activation , Transfection
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