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The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

ABSTRACT: The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor (FL-GR), which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR’s LBD and suggest many dimeric conformations can coexist within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design.

ORGANISM(S): Mus musculus

PROVIDER: GSE212973 | GEO | 2023/01/11

REPOSITORIES: GEO

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The multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

Project description:The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution and quantitative fluorescence microscopy in live cells. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work unveils likely pathophysiologically relevant quaternary assemblies of the nuclear receptor with important implications for glucocorticoid action and drug design.

2023-03-11 | PXD028039 | Pride

Crosstalk between the glucocorticoid and mineralocorticoid receptor boosts glucocorticoid-induced killing of multiple myeloma cells

Project description:The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR’s action in myeloma. Here we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that culminates in improved myeloma cell killing. We show that the GR agonist Dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist Spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells, while in a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk is arising from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR MR homodimerization but leaves GR-GR homodimerization intact. Unbiased transcriptomics further reveals that c-myc and many of its target genes are downregulated most by Dex and Spi combined, while proteomics analyses identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex+Spi downregulated genes and proteins that may predict prognosis in the CoMMpass patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies towards glucocorticoid-based dose-reduction.

2023-10-24 | PXD043635 | Pride

Structure of the Homodimeric Androgen Receptor Ligand-Binding Domain

Project description:The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the etiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility, and prostate cancer (PCa). Here, we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbors over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the newly unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

2018-10-26 | PXD005575 | Pride

Gene expressions regulated by N/C interacting and non-interactiong GR

Project description:Glucocorticoid receptor (GR) was demonstrated to undergo an N/C interaction between the 26VMDFY30 motif in the N-terminus and C-terminal LBD in the presence of its co-chaperone cytoplasmic constitutive active/androstane receptor retention protein (CCRP). Utilizing the fact that phosphor-mimic mutation of tyrosine 30 to glutamic acid abolished this N/C interaction, CCRP and either GR Y30F or GR Y30E mutant was ectopically expressed in COS-1 cells for subsequent cDNA microarray analysis. Dexamethasone, a potent GR ligand, significantly induced or repressed 673 and 689 genes in Y30F and Y30E, respectively. Among them, only 166 genes were commonly regulated. This analysis strongly suggested that the GR N/C interaction serves to regulate distinct gene sets.

2016-09-13 | GSE77752 | GEO

Profiling primary downstream target of LBD16 in roots of Arabidopsis thaliana

Project description:Lateral roots (LRs) are formed post-embryonically and contribute to root architecture formation in vascular plants. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16) is a key transcription factor to initiate LR formation functioning redundantly with related LBD members. To identify primary downstream targets of LBD16, we engineered a transgenic line with inducible LBD16 activity by expressing a fusion protein of LBD16 and rat glucocorticoid receptor (GR) under the regulation of its own regulatory region (gLBD16-GR) in the lbd16-1 lbd18-1 lbd33-1 mutant. Here we identified primary response genes of LBD16 from transcriptome analysis.

2018-12-31 | GSE115420 | GEO

A new life-saving role for Glucocorticoid Receptor dimers: repressing STAT1-induced IFN response in the gut

Project description:The use of glucocorticoids (GCs), which bind and activate the glucocorticoid receptor (GR), in systemic inflammatory response syndromes (SIRS) is disputed. Mice with a poor transcriptional response of dimer-dependent GR target genes were studied in a model of TNF-induced SIRS. These GR dim/dim mice display a significant increase in TNF sensitivity and a lack of protection by the GC dexamethasone (DEX). Unchallenged GR dim/dim mice have a strong interferon-stimulated gene (ISG) signature at the transcriptional level and this ISG signature is gut specific. Here, we used shotgun proteomics to study the regulation of ISG proteins in the ileum of GR dim/dim mice. Our data showed that unchallenged GR dim/dim mice have a strong interferon-stimulated gene (ISG) signature, along with STAT1 upregulation. Taken together, we show that GR dim/dim mice poorly control ISG expression resulting in excessive necroptosis induction by TNF. Our findings support a critical interplay between gut microbiota, interferons, necroptosis and GR in both the basal response to acute inflammatory challenges and in the pharmacological intervention by GCs.

2019-04-01 | PXD008608 | Pride

Glucocorticoid Receptor Quaternary Structure Drives Chromatin Occupancy and Transcriptional Outcome

Project description:Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor (GR) forms tetramers on enhancers, due to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. Using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knock-out cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to wild-type receptor, and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher order structure that drives both the activating and repressive actions of glucocorticoids.

2019-04-02 | GSE108634 | GEO

General and sequence-specific roles for DNA in glucocorticoid receptor DNA-binding stoichiometry

Project description:The glucocorticoid receptor (GR) binds to DNA in at least three stoichiometric ratios to regulate gene expression. The dimeric binding of GR to a 15 base pair core sequence has been well studied, and is associated with activation of gene expression. Monomeric GR has been associated with repression. GR has also been shown to form tetramers on DNA in live cells. The role of DNA sequence in directing DNA-binding stoichiometry and subsequent gene regulation is not clear. We took an unbiased approach using SELEX-seq to calculate comprehensive monomeric- and dimeric-binding profiles for GR. The monomeric site is composed of a canonical half-site, but is distinguished from the dimer site by a downstream TTTg motif and the absence of a second half-site. To probe the mechanism of how this motif favors monomeric binding, we performed Spec-/Coop-seq. The TTTg increases monomeric affinity while decreasing cooperative dimeric binding. Crystal structures indicated that TTTg results in a narrowed minor groove bringing residues Y455 and K471 of GR into the proximity of DNA, increasing affinity. Interestingly, although monomeric binding is more often associated with repression of gene expression than dimeric binding, the trend is not significant. Consistent with live-cell imaging, a meta-analysis of GR:DNA structures reveals that, regardless of binding sequence, GR binds to DNA as a dimer of dimers using highly similar interfaces. This suggests that the functional stoichiometry of GR in the cell is tetrameric, and that sequence may modulate GR activity.

2018-12-01 | GSE107584 | GEO

Gene expression profiling of glucocorticoid receptor signaling in a prostate cancer associated fibroblast cell model (PF179TCAF-shGR-1)

Project description:Glucocorticoid receptor (GR) has been recently identified as a candidate for acquired anti-androgen and chemotherapy resistance. In order to identify glucocorticoid receptor (GR) targets and studying stromal GR signaling gene expression profiling was performed in a prostate cancer associated fibroblast cell model (PF179TCAF-shGR-1) using different treatments.

2021-06-01 | GSE150432 | GEO

ARGLU1 RIME experiment in N2a cells

Project description:ARGLU1 is a Transcriptional Coactivator and Splicing Regulator Important for Stress Hormone Signaling and Development Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neuronal cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neuronal cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neuronal cells with consequences for glucocorticoid signaling.

2019-02-15 | PXD012406 | Pride

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