Function of Steroid Receptors in Subcellular Compartments

Division Of Basic Sciences - Nci

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Abstract

1. Multimeric structure of the glucocorticoid receptor (GR) The glucocorticoid receptor (GR) is a leading drug target due to its anti-inflammatory and immunosuppressive roles. The functional oligomeric conformation of full-length GR (FL-GR), which is key for its biological activity, remains disputed. A predominant model in the field holds that GR binds DNA as a dimer to regulate gene responses. This model has been built upon widely reported studies that focus on dead cell genomics. We reported earlier that GR adopts a tetrameric conformation on response elements in living cells (1,2). More recently we demonstrated with colleagues in Barcelona that GR-LBD modules can form crystals with various quaternary arrangements depending on bound agonists/antagonists and other biochemical parameters (3). We identified four GR-LBD oligomerization interfaces (termed front, top, back, and base), the combination of which yielded a catalog of 20 distinct homodimers. We have now identified a new crystal structure of the agonist-bound GR ligand-binding domain (GRLBD) comprising eight copies of a non-canonical dimer (4). The biological relevance of this dimer for receptor multimerization in living cells has been verified by studying single- and double-point mutants of full-length GR in Number and Brightness fluorescence microscopy and transcriptomic analysis. Self-association of this GR-LBD basic dimer in two mutually exclusive assemblies reveals clues for full-length GR multimerization and activity in cells. We propose (4) a new tetrameric model for the structure of multidomain GR based on our new data and suggest a detailed oligomerization pathway. This model reconciles all currently available structural and functional information. 2. A second binding pocket on the glucocorticoid receptor Glucocorticoids (GCs) activate the GR upon occupying the internal ligand-binding pocket (LBP) of its ligand-binding domain (GR-LBD), which has been the focus of most previous structure-function studies. Synthetic GCs such as dexamethasone are widely used to treat inflammatory diseases, but their chronic use results in major side effects, whose molecular underpinnings remain unresolved. We have now examined the topography of the GR-LBD and its ability to bind small-molecule compounds, especially cholesterol derivatives. We discovered that one important class of steroids, bile acids, bind to previously unidentified and highly conserved, surface-exposed cavities on the GR-LBD (5). We demonstrated that bile acids affect GR turnover and self-assembly in living cells, modulating receptor transcriptional activity. These findings reveal a previously unrecognized mechanism of GR regulation, with implications for the design of GCs with novel mechanisms of action. 3. Functional interactions of the mineralocorticoid and glucocorticoid receptors The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are members of the steroid receptor subfamily of nuclear receptors. Their main function is to act as ligand-activated transcription factors, transducing the effects of corticosteroid hormones (aldosterone and glucocorticoids) by modulating gene expression. Corticosteroid signaling is essential for homeostasis and adaptation to different forms of stress. GR responds to glucocorticoids by regulating genes involved in development, metabolism, immunomodulation and brain function. MR is best known for mediating the effects of aldosterone, a key hormone controlling electrolyte and water homeostasis. In addition to aldosterone, MR binds glucocorticoids (cortisol and corticosterone) with equally high affinity. This ligand promiscuity has important repercussions to understand MR function, as well as glucocorticoid signaling. MR and GR share significant sequence and structural similarities, regulate overlapping sets of genes, and are able to interact forming heteromeric complexes. However, the precise role of these heteromers in regulating corticosteroid-regulated transcriptional outcomes remains an open question. We have analyzed the impact of GR coexpression on MR genome-wide transcriptional responses and chromatin binding upon activation by aldosterone and glucocorticoids, both physiological ligands of this receptor. Transcriptional responses of MR in the absence of GR result in fewer regulated genes. In contrast, coexpression of GR potentiates MR-mediated transcription, particularly in response to aldosterone, both in cell lines and in the more physiologically relevant model of mouse colon organoids (6,7). MR chromatin binding is altered by GR coexpression in a locus- and ligand-specific way. Single-molecule tracking of MR suggests that the presence of GR contributes to productive binding of MR/aldosterone complexes to chromatin (8). Together, our data indicate that coexpression of GR potentiates aldosterone-mediated MR transcriptional activity, even in the absence of glucocorticoids. 4 Characterization of a human GR DNA binding site mutation Dysregulation of GR function is implicated in numerous human disorders, including severe depressive disorder, where hypothalamic-pituitary-adrenal (HPA) axis hyperactivity and elevated cortisol levels suggest impaired GR-mediated feedback, and primary generalized glucocorticoid resistance, a rare condition characterized by partial or complete insensitivity to glucocorticoids, leading to compensatory HPA axis activation. We have characterized a novel heterozygous T437I mutation within the GR DNA-binding domain in a patient exhibiting partial glucocorticoid resistance accompanied by metabolic derangements. Functional analysis in patient-derived fibroblasts demonstrated impaired glucocorticoid-induced transcription. To assess the in vivo consequences of the mutation, we engineered a knock-in mouse model using CRISPR-Cas9 in Nr3c1+/T444I (murine ortholog of the human T437I mutation) representing the first reported mouse model of a patient-derived point mutation in GR. Heterozygous mice exhibited partial glucocorticoid resistance, dysregulated HPA axis activity and impaired dexamethasone suppression, closely recapitulating the patient phenotype. Homozygous Nr3c1T444I/T444I embryos were recovered at embryonic day 12.5 (E12.5) but failed to survive to term, indicating mid-gestational lethality. Transcriptomic profiling of primary mouse embryonic fibroblasts revealed dosage-dependent effects: heterozygotes showing an intermediate response to dexamethasone compared to wild-type, while homozygotes showed markedly blunted response. Our results challenge prior assumptions by demonstrating that GR DNA-binding is essential for embryogenesis, and we introduce a novel preclinical model for exploring glucocorticoid resistance mechanisms and treatments. 1. Presman, D.M. and G.L. Hager, Transcription, 2017. 8: p. 32. 2. Presman, D.M., et al., DNA PNAS, 2016. 113(29): p. 8236. 3. Jiménez-Panizo, A., et al., Nucleic Acids Res, 2022. 50: p. 13063. 4. Jiménez-Panizo, A., et. al., Hager, G. L., Estébanez-Perpiñá, E.: bioRxiv 2025. doi:https://doi.org/10.1101/2024.12.12.628195 5. Alba Jiménez-Panizo, et al., Hager, G. L., Estébanez-Perpiñá, E. bioRxiv 2025. doi:https://doi.org/10.1101/2025.05.13.653693 6. Johnson, T.A., et al., Hager, G., Alvarez de la Rosa, D.L. PNAS, 2024. 121: p. e2413737121. 7. Alvarez de la Rosa, D., et al. Hager, G. L., Mol Cell Endocrinol, 2024: p. 112389. 8. Fettweis, G., et al., Hager, G., Alvarez de la Rosa, D. L. Protein Sci, 2023: p. e4890.