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A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation
Jesus Banuelos1, Soon Cheon Shin1, Nick Z Lu1
1Division of Allergy-Immunology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, United States.
Abstract:
Glucocorticoids (GCs) are used to treat a variety of inflammatory disorders and certain cancers. However, GC resistance occurs in subsets of patients. We found that EL4 cells, a GC-resistant mouse thymoma cell line, harbored a point mutation in their GC receptor (GR) gene, resulting in the substitution of arginine 493 by a cysteine in the second zinc finger of the DNA-binding domain. Allelic discrimination analyses revealed that the R493C mutation occurred on both alleles. In the absence of GCs, the GR in EL4 cells localized predominantly in the cytoplasm and upon dexamethasone treatment underwent nuclear translocation, suggesting that the ligand binding ability of the GR in EL4 cells was intact. In transient transfection assays, the R493C mutant could not transactivate the MMTV-luciferase reporter. Site-directed mutagenesis to revert the R493C mutation restored the transactivation activity. Cotransfection experiments showed that the R493C mutant did not inhibit the transcriptional activities of the wild-type GR. In addition, the R493C mutant did not repress either the AP-1 or NF-κB reporters as effectively as WT GR. Furthermore, stable expression of the WT GR in the EL4 cells enabled GC-mediated gene regulation, specifically upregulation of IκBα and downregulation of interferon γ and interleukin 17A. Arginine 493 is conserved among multiple species and all human nuclear receptors and its mutation has also been found in the human GR, androgen receptor, and mineralocorticoid receptor. Thus, R493 is necessary for the transcriptional activity of the GR and a hotspot for mutations that result in GC resistance.
Insights
A specific mutation in the glucocorticoid receptor (GR) gene, R493C, causes resistance to glucocorticoids (GCs) by impairing transcriptional activity. This finding identifies a key mutation site for GC resistance in various conditions.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- Glucocorticoids (GCs) are vital therapeutics for inflammatory diseases and cancers.
- GC resistance limits treatment efficacy in a subset of patients.
- Understanding the molecular basis of GC resistance is crucial for improving patient outcomes.
Purpose of the Study:
- To investigate the molecular mechanism underlying glucocorticoid resistance in EL4 cells.
- To identify specific mutations in the glucocorticoid receptor (GR) gene associated with GC resistance.
- To assess the functional impact of identified GR mutations on transcriptional activity.
Main Methods:
- Allelic discrimination analysis to detect GR gene mutations.
- Transient transfection assays to evaluate GR transactivation and repression activity.
- Site-directed mutagenesis to confirm the role of specific mutations.
- Stable expression of wild-type (WT) GR in resistant cells to assess functional restoration.
Main Results:
- EL4 cells, a GC-resistant line, possess a homozygous R493C point mutation in the GR gene.
- The R493C mutant GR cannot transactivate reporter genes, while reverting the mutation restores activity.
- The R493C mutant GR shows impaired repression of AP-1 and NF-κB, and does not inhibit WT GR activity.
- Stable expression of WT GR in EL4 cells re-sensitizes cells to GCs, restoring gene regulation.
Conclusions:
- The R493C mutation in the GR DNA-binding domain is responsible for GC resistance in EL4 cells.
- Arginine 493 is essential for GR transcriptional activity and is a mutation hotspot for GC resistance.
- This finding has implications for understanding and potentially overcoming GC resistance in clinical settings.
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