scholarly journals Probing Dominant Negative Behavior of Glucocorticoid Receptor β through a Hybrid Structural and Biochemical Approach

2018 ◽  
Vol 38 (8) ◽  
pp. e00453-17 ◽  
Author(s):  
Jungki Min ◽  
Lalith Perera ◽  
Juno M. Krahn ◽  
Christine M. Jewell ◽  
Andrea F. Moon ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Pocket ◽  
Dominant Negative ◽  
Ru 486 ◽  
Dominant Negative Inhibitor ◽  
Helix 12 ◽  
Binding Energy Analysis ◽  
Biochemical Approach ◽  
Biochemical Analyses

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

scholarly journals Human Glucocorticoid Receptor β Binds RU-486 and Is TranscriptionallyActive

2007 ◽  
Vol 27 (6) ◽  
pp. 2266-2282 ◽  
Author(s):  
Laura J. Lewis-Tuffin ◽  
Christine M. Jewell ◽  
Rachelle J. Bienstock ◽  
Jennifer B. Collins ◽  
John A. Cidlowski
Keyword(s):  
Confocal Microscopy ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Orphan Receptor ◽  
Computational Docking ◽  
Ru 486

ABSTRACT Human glucocorticoid receptor (hGR) is expressed as two alternately spliced C-terminal isoforms, α and β. In contrast to the canonical hGRα, hGRβ 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 hGRα. Here we used confocal microscopy to examine the cellular localization of transiently expressed fluorescent protein-tagged hGRβ in COS-1 and U-2 OS cells. Surprisingly, yellow fluorescent protein (YFP)-hGRβ 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 hGRβ. Confocal microscopy of coexpressed YFP-hGRβ and cyan fluorescent protein-hGRα in COS-1 cells indicated that the receptors move into the nucleus independently. Using a ligand binding assay, we confirmed that hGRβ bound RU-486 but not the hGRα ligand dexamethasone. Examination of the cellular localization of YFP-hGRβ in response to a series of 57 related compounds indicated that RU-486 is thus far the only identified ligand that interacts with hGRβ. The selective interaction of RU-486 with hGRβ was also supported by molecular modeling and computational docking studies. Interestingly, microarray analysis indicates that hGRβ, expressed in the absence of hGRα, can regulate gene expression and furthermore that occupation of hGRβ with the antagonist RU-486 diminishes that capacity despite the lack of helix 12 in the ligand binding domain.

Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm

Development ◽  
2000 ◽  
Vol 127 (4) ◽  
pp. 791-800 ◽  
Author(s):  
M. Kishi ◽  
K. Mizuseki ◽  
N. Sasai ◽  
H. Yamazaki ◽  
K. Shiota ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Dominant Negative ◽  
Gastrula Stage ◽  
Blastula Stage ◽  
Sensory Epithelia ◽  
Neural Tissues

From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.

scholarly journals Associations between promoter usage and alternative splicing of the glucocorticoid receptor gene

2007 ◽  
Vol 38 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Henk Russcher ◽  
Virgil A S H Dalm ◽  
Frank H de Jong ◽  
Albert O Brinkmann ◽  
Leo J Hofland ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Splice Variants ◽  
Receptor Gene ◽  
Dominant Negative ◽  
Multiple Promoters ◽  
Glucocorticoid Receptor Gene ◽  
Dominant Negative Inhibitor ◽  
Exon 1 ◽  
Promoter Usage ◽  
Different Tissues

The glucocorticoid receptor (GR) is widely expressed in various tissues throughout the human body. At least three different 3′-splice variants of the GR have been reported: GR-α, which is functionally active; GR-β, which is a dominant negative inhibitor of GR-α function; and GR-P, which is thought to activate the function of GR-α. At least seven different variants for exon 1 exist, 1A–1F and 1H, each with its own promoter. In this study, we explored if tissue-specific splicing of the 3′-end variants of the GR is influenced by alternative promoter usage. cDNAs of different tissues and cell lines were used to investigate which part of transcripts carrying each of the three major variants for exons 1, 1A, 1B, or 1C, encodes for the splice variants GR-α, GR-β, and GR-P. Our data demonstrate that the expression of GR-α is preferentially regulated by promoter 1C and that for the expression of GR-P promoter 1B is predominantly used. This indicates that regulation of GR splice variants could partly occur through selective use of the multiple promoters, and that this is another way to sensitize cells and tissues to the different activities of the GR isoforms.

scholarly journals Molecular Origins for the Dominant Negative Function of Human Glucocorticoid Receptor Beta

2003 ◽  
Vol 23 (12) ◽  
pp. 4319-4330 ◽  
Author(s):  
Matthew R. Yudt ◽  
Christine M. Jewell ◽  
Rachelle J. Bienstock ◽  
John A. Cidlowski
Keyword(s):  
Glucocorticoid Receptor ◽  
Dominant Negative ◽  
Critical Feature ◽  
Negative Function ◽  
Helix 12 ◽  
Sequential Mutagenesis ◽  
Dominant Negative Activity ◽  
Glucocorticoid Receptor Beta ◽  
Receptor Beta ◽  
Negative Phenotype

ABSTRACT This study molecularly elucidates the basis for the dominant negative mechanism of the glucocorticoid receptor (GR) isoform hGRβ, whose overexpression is associated with human glucocorticoid resistance. Using a series of truncated hGRα mutants and sequential mutagenesis to generate a series of hGRα/β 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 hGRβ 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 hGRα and hGRβ provides structural insight and a potential physical explanation for the lack of hormone binding and the dominant negative actions of hGRβ.

scholarly journals Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

2007 ◽  
Vol 28 (6) ◽  
pp. 1915-1923 ◽  
Author(s):  
Kelly Suino-Powell ◽  
Yong Xu ◽  
Chenghai Zhang ◽  
Yong-guang Tao ◽  
W. David Tolbert ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Binding Pocket ◽  
Affinity Binding ◽  
Receptor Ligand ◽  
Ligand Binding Pocket ◽  
Steroid Receptor Coactivator ◽  
High Affinity ◽  
Binding Domains ◽  
Lxxll Motif

ABSTRACT A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 Å3, effectively doubling the size of the GR dexamethasone-binding pocket of 540 Å3 and yet leaving the structure of the coactivator binding site intact. DAC occupies only ∼50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

scholarly journals Discovery of a Functional Glucocorticoid Receptor β-Isoform in Zebrafish

Endocrinology ◽  
2007 ◽  
Vol 149 (4) ◽  
pp. 1591-1599 ◽  
Author(s):  
Marcel J. M. Schaaf ◽  
Danielle Champagne ◽  
Ivo H. C. van Laanen ◽  
Diane C. W. A. van Wijk ◽  
Annemarie H. Meijer ◽  
...  
Keyword(s):  
Animal Model ◽  
Glucocorticoid Receptor ◽  
Developmental Stages ◽  
Splice Variants ◽  
The Body ◽  
Dominant Negative ◽  
Mrna Levels ◽  
Receptor Isoforms ◽  
Asthma Patients ◽  
Dominant Negative Inhibitor

In humans, two glucocorticoid receptor (GR) splice variants exist: GRα and GRβ, which are identical between amino acids 1–727 and then diverge. Whereas GRα (the canonical GR) acts as a ligand-activated transcription factor, GRβ does not bind traditional glucocorticoid agonists, lacks GRα’s transactivational activity, and acts as a dominant-negative inhibitor of GRα. It has been suggested that this receptor isoform is involved in the induction of glucocorticoid resistance in asthma patients. Unfortunately, a GR β-isoform has been detected in only humans, and therefore, an animal model for studies on this isoform is lacking. In the present study, we demonstrate that in zebrafish a GR isoform exists that diverges from the canonical zebrafish GR at the same position as human GRβ from human GRα. The zebrafish GR β-isoform acts as a dominant-negative inhibitor in reporter assays, and the extent of inhibition and the effective GRα/GRβ ratio is similar to studies performed with the human GR isoforms. In addition, the subcellular localization of zebrafish GRβ is similar to its human equivalent. Finally, expression levels of GRα and GRβ were determined in adult zebrafish tissues and at several developmental stages. Both receptor isoforms were detected throughout the body, and GRβ mRNA levels were relatively low compared with GRα mRNA levels, as in humans. Thus, for the first time, a GR β-isoform has been identified in a nonhuman animal species, shedding new light on the relevance of this GR splice variant and providing a versatile animal model for studies on the GR system.

scholarly journals Identification of potential regulatory domains within the MreC and MreD components of the cell elongation machinery

2021 ◽  
Author(s):  
Patricia D. A. Rohs ◽  
Jeanna M. Qiu ◽  
Grasiela Torres ◽  
Mandy D. Smith ◽  
Elayne M. Fivenson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Ligand Binding ◽  
Cell Shape ◽  
Genetic System ◽  
Binding Pocket ◽  
Dominant Negative ◽  
Wall Structure ◽  
Regulatory Domains ◽  
C Terminus ◽  
Rod System

The bacterial peptidoglycan (PG) cell wall maintains cell shape and prevents osmotic lysis. During growth of rod-shaped cells, PG is incorporated along the cell cylinder by the RodA-PBP2 synthase of the multi-protein Rod system (elongasome). Filaments of the actin-like MreB protein orient synthesis of the new PG material. They are connected to the RodA-PBP2 synthase in part through the RodZ component. MreC and MreD are other conserved components of the system, but their function is not well understood. Amino acid changes in RodA-PBP2 were recently identified that bypass a requirement for MreC and MreD function, suggesting the Mre proteins act as activators of the synthase. To further investigate their function, we developed a genetic strategy to identify dominant-negative alleles of mreC and mreD in Escherichia coli. Residues essential for Rod system function were identified at the junction of two subdomains within MreC and in a predicted ligand-binding pocket of MreD. Additionally, we found that although the proline-rich C-terminal domain of MreC is non-essential, substitutions within this region disrupt its function. Based on these results, we propose that the C-terminus of MreC and the putative ligand-binding domain of MreD play regulatory roles in controlling Rod system activity. IMPORTANCE: Cell shape in bacteria is largely determined by the cell wall structure that surrounds them. The multi-protein machine called the Rod system (elongasome) has long been implicated in rod-shape determination in bacilli. However, the functions of many of its conserved components remain unclear. Here, we describe a new genetic system to dissect the function of these proteins and how we used it to identify potential regulatory domains within them that may modulate the function of the shape-determining machinery.

X-ray crystal structure analysis of elacestrant (RAD1901), a novel selective estrogen receptor degrader (SERD), bound to estrogen receptor alpha ligand binding domain.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15647-e15647
Author(s):  
Sean W. Fanning ◽  
Geoffrey Greene ◽  
Maureen G. Conlan
Keyword(s):  
Breast Cancer ◽  
Crystal Structure ◽  
Estrogen Receptor ◽  
Ligand Binding ◽  
Binding Pocket ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Ligand Binding Pocket ◽  
X Ray ◽  
Helix 12

e15647 Background: Antiestrogens are a mainstay of treatment for estrogen receptor positive (ER+) breast cancer in both the adjuvant and the advanced/metastatic settings. Elacestrant is a mixed activity selective estrogen receptor (SER) alpha (ERα) antagonist, acting as a SER modulator (SERM) at low doses and a SER degrader (SERD) at high doses. It has shown activity in hormone sensitive wild type (WT) ERα and insensitive estrogen receptor gene 1 (ESR1) mutation-harboring (Y537S and D538G) ERα breast cancer, both in preclinical models and in clinical studies. It also possesses a unique pharmacology compared to other competitive ER antagonists in its ability to cross the blood brain barrier. Competitive ERα antagonists are typically comprised of a core that sits in the ligand binding pocket and an arm that manipulates the structure to achieve SERM or SERD activities. In these molecules, the arm is attached in the same position as the triphenylethylene core of tamoxifen. However, elacestrant possesses a novel site of attachment. As such, we hypothesized that elacestrant adopts an alternative binding orientation in the ERα ligand binding pocket to achieve its unique pharmaceutical profiles. Methods: X-ray crystallography was used to solve a co-crystal structure of elacestrant in complex with WT ERα ligand binding domain to 2Å. Results: Overall, elacestrant promotes the formation of a canonical ERα ligand binding domain antagonist conformation, whereby helix 12 (H12) is docked into the activating function-2 cleft. However, elacestrant adopts a novel vector in the ERα ligand binding pocket that places it in close proximity to helix 12. As a result, it forms a bifurcated hydrogen bond that is not observed in other competitive antiestrogens and samples a chemical space known to increase H12 mobility and induce SERD activity. This novel vector also places it near positions 537 and 538, the two most common sites of somatic mutation. Conclusions: The high-resolution x-ray crystal structure of elacestrant suggests that the unique binding mode it adopts enables novel pharmacology and positions it to achieve potency in the WT and activating somatic ERα mutated breast cancer setting.

scholarly journals The Human Glucocorticoid Receptor (GR) Isoform β Differentially Suppresses GRα-Induced Transactivation Stimulated by Synthetic Glucocorticoids

2005 ◽  
Vol 90 (6) ◽  
pp. 3505-3509 ◽  
Author(s):  
Oren Fruchter ◽  
Tomoshige Kino ◽  
Emmanouil Zoumakis ◽  
Salvatore Alesci ◽  
Massimo De Martino ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Suppressive Effect ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Compound A ◽  
Pathological Conditions ◽  
Dominant Negative Inhibitor ◽  
Negative Effect ◽  
Synthetic Glucocorticoids ◽  
Selection Of

The β-isoform of human glucocorticoid receptor β (hGRβ) acts as a natural dominant negative inhibitor of hGRα-induced transactivation of glucocorticoid-responsive genes. We determined hGRβ ability to suppress hGRα transactivation that was induced by commonly used synthetic glucocorticoids. HepG2/C3A cells were transiently cotransfected with GR cDNA and a glucocorticoid-responsive promoter, luciferase (MMTV-luc). Transfected cells were incubated for 16 h with glucocorticoid and luciferase. For each compound, a dose-response curve was constructed, and half-maximal effective concentrations and maximal transcriptional activities were compared. hGRβ, at a 1:1 ratio to hGRα, differentially suppressed hGRα-induced maximal transcriptional activity stimulated by triamcinolone, dexamethasone, hydrocortisone, and betamethasone (by 96, 68, 62, and 49%, respectively) but not by methylprednisolone. The suppressive effect of hGRβ on hGRα-induced transactivation was stronger at lower concentrations of all tested glucocorticoids, whereas it was blunted at higher concentrations. We conclude that the potency of the dominant negative effect of hGRβ on hGRα-induced transactivation depends on both the type and the dose of the synthetic glucocorticoids in use. These results may provide helpful information concerning the selection of synthetic glucocorticoids for treatment of pathological conditions in which hGRβ modulates the sensitivity of tissues to glucocorticoids.

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