Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus: importance of the ligand-binding domain for intracellular GR trafficking
Abstract
The syndrome of familial or sporadic glucocorticoid resistance is characterized by hypercortisolism without the clinical stigmata of Cushing syndrome. This condition is usually caused by mutations of the human GR, a ligand-activated transcription factor that shuttles between the cytoplasm and the nucleus. A pathological human mutant receptor, in which Ile was replaced by Asn at position 559, had negligible ligand binding, was transcriptionally extremely weak, and exerted a transdominant negative effect on the transactivational activity of the wild-type GR, causing severe glucocorticoid resistance in the heterozygous state. To understand the mechanism of this mutant's trans-dominance, we constructed several N-terminal GR fusion chimeras to green fluorescent protein (GFP) and demonstrated that their transactivational activities were similar to those of the original proteins. The GFP-human (h) GRalphaI559N chimera was predominantly localized in the cytoplasm, and only high doses or prolonged glucocorticoid treatment triggered complete nuclear import that took 180 vs. 12 min for GFP-hGRalpha. Furthermore, hGRalphaI559N inhibited nuclear import of the wild-type GFP-hGRalpha, suggesting that its trans-dominant activity on the wild-type receptor is probably exerted at the process of nuclear translocation. As the ligand-binding domain (LBD) of the GR appears to play an important role in its nucleocytoplasmic shuttling, we also examined two additional GR-related fusion proteins. The natural hGR isoform beta (GFP-hGRbeta), containing a unique LBD, was transactivation-inactive, moderately trans-dominant, and localized instantaneously and predominantly in the nucleus; glucocorticoid addition did not change its localization. Similarly, GFP-hGR514, lacking the entire LBD, was instantaneously and predominantly localized in the nucleus regardless of presence of glucocorticoids. Using a cell fusion system we demonstrated that nuclear export of GFP-hGRalphaI559N (250 min) and GFP-hGRbeta (300 min) was drastically impaired compared with that of GFP-hGRalpha (50 min) and GFP-hGR514 (50 min), suggesting that an altered LBD may impede the exit of the GR from the nucleus. We conclude that the trans-dominant negative effect of the pathological mutant is exerted primarily at the translocation step, whereas that of the natural isoform beta is exerted at the level of transcription.
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Heterozygous point mutations with incomplete dominant negative activity or multiple effects on GRα action have been described.
“Heterozygous point mutations with incomplete dominant negative activity or multiple effects on GR␣ action have also been described (638).”
From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [638]
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- Authors
- Kino, T., Stauber, R. H., Resau, J. H., Pavlakis, G. N., & Chrousos, G. P.
- Affiliations
- National Institutes of Health (US)
- Frederick National Laboratory for Cancer Research (US)
- Corresponding author
- Kino, Tomoshige
- Venue
- The Journal of Clinical Endocrinology & Metabolism
- Publisher
- The Endocrine Society
- Published
- 2001-11
- Volume
- 86
- Issue
- 11
- Pages
- 5600-5608
- ISSN
- 0021-972X (ISSN-L)
- 1945-7197
- OpenAlex
- W2077716901
- Cited by
- 105
- References
- 34
- Citations by year
- 2026: 2 · 2023: 1 · 2022: 1 · 2021: 5 · 2020: 1 · 2019: 3 · 2018: 4 · 2017: 3 · 2016: 4 · 2015: 5 · 2014: 6 · 2013: 5 · 2012: 4
- FWCI
- 4.00
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Clinical Endocrinology & Metabolism (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
- Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Keywords
- glucocorticoid receptor, glucocorticoid resistance, ligand-binding domain, nuclear translocation, nucleocytoplasmic shuttling, nuclear export, transcriptional activity, nuclear localization
- MeSH terms
- Cell Fusion, Cell Line, Cell Nucleus, Dexamethasone, Genes, Dominant, HeLa Cells, Humans, Ligands, Mutation, Protein Binding, Receptors, Glucocorticoid, Subcellular Fractions, Transfection, Translocation, Genetic, Transcriptional Activation, Genes, Reporter
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