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

Tomoshige Kino, Roland H. Stauber, James H. Resau, George N. Pavlakis, George P. Chrousos

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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) GRαI559N 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-hGRα. Furthermore, hGRαI559N inhibited nuclear import of the wild-type GFP-hGRα, 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 β (GFP-hGRβ), 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-hGRαI559N (250 min) and GFP-hGRβ (300 min) was drastically impaired compared with that of GFP-hGRα (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 β is exerted at the level of transcription.

Original languageEnglish
Pages (from-to)5600-5608
Number of pages9
JournalJournal of Clinical Endocrinology and Metabolism
Volume86
Issue number11
DOIs
Publication statusPublished - 2001
Externally publishedYes

Fingerprint

Green Fluorescent Proteins
Ligands
Glucocorticoids
Cell Nucleus Active Transport
Fusion reactions
Cushing Syndrome
Protein Isoforms
Cytoplasm
Christianity
Cell Fusion
Transcription
Transcriptional Activation
Proteins
Transcription Factors
Mutation

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Endocrinology
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

@article{066531d27af9462bba17177349d41f59,
title = "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) GRαI559N 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-hGRα. Furthermore, hGRαI559N inhibited nuclear import of the wild-type GFP-hGRα, 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 β (GFP-hGRβ), 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-hGRαI559N (250 min) and GFP-hGRβ (300 min) was drastically impaired compared with that of GFP-hGRα (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 β is exerted at the level of transcription.",
author = "Tomoshige Kino and Stauber, {Roland H.} and Resau, {James H.} and Pavlakis, {George N.} and Chrousos, {George P.}",
year = "2001",
doi = "10.1210/jc.86.11.5600",
language = "English",
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pages = "5600--5608",
journal = "Journal of Clinical Endocrinology and Metabolism",
issn = "0021-972X",
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TY - JOUR

T1 - Pathologic human GR mutant has a transdominant negative effect on the wild-type GR by inhibiting its translocation into the nucleus

T2 - Importance of the ligand-binding domain for intracellular GR trafficking

AU - Kino, Tomoshige

AU - Stauber, Roland H.

AU - Resau, James H.

AU - Pavlakis, George N.

AU - Chrousos, George P.

PY - 2001

Y1 - 2001

N2 - 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) GRαI559N 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-hGRα. Furthermore, hGRαI559N inhibited nuclear import of the wild-type GFP-hGRα, 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 β (GFP-hGRβ), 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-hGRαI559N (250 min) and GFP-hGRβ (300 min) was drastically impaired compared with that of GFP-hGRα (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 β is exerted at the level of transcription.

AB - 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) GRαI559N 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-hGRα. Furthermore, hGRαI559N inhibited nuclear import of the wild-type GFP-hGRα, 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 β (GFP-hGRβ), 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-hGRαI559N (250 min) and GFP-hGRβ (300 min) was drastically impaired compared with that of GFP-hGRα (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 β is exerted at the level of transcription.

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