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A somatic cell genetic method for identification of untargeted mutations in the glucocorticoid receptor that cause
1Department of Pathology, University of Southern California Health Sciences Campus, Los Angeles 90033, USA.
Abstract:
Mouse T lymphoma cell line W7MG1, which is killed by physiological concentrations of glucocorticoid agonists, was used as a convenient genetic system for isolating sublines containing mutant glucocorticoid receptors (GR) with hormone-binding deficiencies. Partially hormone-resistant cell clones were derived from chemically mutagenized cell populations by selecting for growth in moderate concentrations of dexamethasone (Dex) and then screening for failure to grow in high Dex concentrations. Such clones are likely to have mutant GR. In GR cDNA clones from the partially resistant cell sublines, three different functionally significant mutations in the hormone-binding domain were identified: Leu-569 changed to Phe (L569F), Leu-670 to Phe (L670F), and Met-672 to Ile (M672I). Dose-response analyses with Dex and affinity labeling studies with dexamethasone 21-mesylate in transiently transfected cells indicated that all three mutant GR species had hormone-binding deficiencies. However, at saturating Dex concentrations the mutant and wild type GR activated a hormone-inducible reporter gene to the same extent; thus, these three mutations did not affect the ability of GR to activate transcription of the reporter gene after hormone was bound. In dose-response curves conducted with several glucocorticoid agonists, mutations L670F and M672I caused no change in ligand-binding specificities, while mutation L569F caused a modest change in specificity. Quantitative hormone-binding studies conducted with mutant GR synthesized in cell-free reactions showed that mutant GR species L569F and M672I had reduced Dex-binding affinities both at 0 C and at 22 C in the presence of molybdate. In contrast, for the L670F mutant, which exhibited the severest deficiency in vivo, the hormone-binding deficiency in the cell-free system was evident only at 26-37 C and primarily in the absence of molybdate. We propose that the L670F GR is an activation-labile type of mutant, which binds hormone normally in the presence of heat shock protein 90 but loses hormone rapidly after dissociation from heat shock protein 90. These three mutations define two new subregions of the GR polypeptide that are important for hormone binding.
Insights
Mutations in the glucocorticoid receptor (GR) hormone-binding domain can cause resistance to steroid hormones. Three specific mutations were identified, affecting hormone binding but not transcriptional activation.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- Glucocorticoid agonists are crucial for cellular regulation.
- Mutations in the glucocorticoid receptor (GR) can lead to hormone resistance.
- The W7MG1 mouse T lymphoma cell line is sensitive to glucocorticoids.
Purpose of the Study:
- To isolate and characterize glucocorticoid receptor (GR) mutants with impaired hormone-binding.
- To identify specific mutations within the GR hormone-binding domain responsible for altered ligand affinity.
- To investigate the functional consequences of these mutations on GR-mediated gene transcription.
Main Methods:
- Chemical mutagenesis of W7MG1 cells to select for hormone-resistant clones.
- Genetic analysis of mutant GR cDNA to identify specific amino acid substitutions.
- Dose-response assays and affinity labeling in transfected cells to assess hormone binding and transcriptional activity.
- Cell-free synthesis and binding studies to quantify ligand affinity at different temperatures and conditions.
Main Results:
- Three functionally significant mutations in the GR hormone-binding domain were identified: L569F, L670F, and M672I.
- All three mutants exhibited reduced hormone-binding affinities, with L670F showing the most severe deficiency in vivo.
- Mutations did not impair the ability of GR to activate transcription at saturating hormone concentrations.
- L670F represents an activation-labile mutant, unstable after dissociation from heat shock protein 90.
Conclusions:
- The identified mutations (L569F, L670F, M672I) define new subregions critical for glucocorticoid receptor hormone binding.
- GR hormone-binding affinity is temperature-dependent and influenced by co-factors like molybdate and heat shock protein 90.
- Understanding these mutations provides insights into glucocorticoid signaling and potential therapeutic strategies for hormone-related disorders.