Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRαR477H and hGRαG679S associated with generalized glucocorticoid resistance

Evangelia Charmandari, Tomoshige Kino, Takamasa Ichijo, Keith Zachman, Anton Alatsatianos, George P. Chrousos

Research output: Contribution to journalArticle

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Abstract

Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor α (hGRα) gene, which impair one or more of hGRα's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRαR477H and hGRαG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. Methods and Results: In transient transfection assays, hGRαR477H displayed no transcriptional activity, whereas hGRαG679S showed a 55% reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGRα. Neither hGRαR477H nor hGRαG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRαR477H preserved normal affinity for the ligand, whereas hGRαG679S displayed a 2-fold reduction compared with hGRα. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRαR477H (25 min) and hGRαG679S (30 min) into the nucleus than the wild-type hGRα (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRαR477H did not bind to glucocorticoid-response elements, whereas hGRαG679S preserved its ability to bind to glucocorticoidresponse elements. Finally, in glutathione-S-transferase pull-down assays, hGRαG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRαR477H and hGRα, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. Conclusions: The natural mutants hGRαR477H and hGRαG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.

Original languageEnglish
Pages (from-to)1535-1543
Number of pages9
JournalJournal of Clinical Endocrinology and Metabolism
Volume91
Issue number4
DOIs
Publication statusPublished - Apr 2006
Externally publishedYes

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Glucocorticoid Receptors
Glucocorticoids
Nuclear Receptor Coactivator 2
Assays
Dexamethasone
Mouse mammary tumor virus
Chemical activation
Ligands
Viruses
Carcinogens
Tumors
Signal transduction
Chromatin Immunoprecipitation
Response Elements
Amino Acid Substitution
Transcription
Glucocorticoid Receptor Deficiency
Glutathione Transferase
Chromatin
Transfection

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

Cite this

Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRαR477H and hGRαG679S associated with generalized glucocorticoid resistance. / Charmandari, Evangelia; Kino, Tomoshige; Ichijo, Takamasa; Zachman, Keith; Alatsatianos, Anton; Chrousos, George P.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 91, No. 4, 04.2006, p. 1535-1543.

Research output: Contribution to journalArticle

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abstract = "Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor α (hGRα) gene, which impair one or more of hGRα's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRαR477H and hGRαG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. Methods and Results: In transient transfection assays, hGRαR477H displayed no transcriptional activity, whereas hGRαG679S showed a 55{\%} reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGRα. Neither hGRαR477H nor hGRαG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRαR477H preserved normal affinity for the ligand, whereas hGRαG679S displayed a 2-fold reduction compared with hGRα. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRαR477H (25 min) and hGRαG679S (30 min) into the nucleus than the wild-type hGRα (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRαR477H did not bind to glucocorticoid-response elements, whereas hGRαG679S preserved its ability to bind to glucocorticoidresponse elements. Finally, in glutathione-S-transferase pull-down assays, hGRαG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRαR477H and hGRα, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. Conclusions: The natural mutants hGRαR477H and hGRαG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.",
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T1 - Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRαR477H and hGRαG679S associated with generalized glucocorticoid resistance

AU - Charmandari, Evangelia

AU - Kino, Tomoshige

AU - Ichijo, Takamasa

AU - Zachman, Keith

AU - Alatsatianos, Anton

AU - Chrousos, George P.

PY - 2006/4

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N2 - Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor α (hGRα) gene, which impair one or more of hGRα's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRαR477H and hGRαG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. Methods and Results: In transient transfection assays, hGRαR477H displayed no transcriptional activity, whereas hGRαG679S showed a 55% reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGRα. Neither hGRαR477H nor hGRαG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRαR477H preserved normal affinity for the ligand, whereas hGRαG679S displayed a 2-fold reduction compared with hGRα. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRαR477H (25 min) and hGRαG679S (30 min) into the nucleus than the wild-type hGRα (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRαR477H did not bind to glucocorticoid-response elements, whereas hGRαG679S preserved its ability to bind to glucocorticoidresponse elements. Finally, in glutathione-S-transferase pull-down assays, hGRαG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRαR477H and hGRα, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. Conclusions: The natural mutants hGRαR477H and hGRαG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.

AB - Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor α (hGRα) gene, which impair one or more of hGRα's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRαR477H and hGRαG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. Methods and Results: In transient transfection assays, hGRαR477H displayed no transcriptional activity, whereas hGRαG679S showed a 55% reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGRα. Neither hGRαR477H nor hGRαG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRαR477H preserved normal affinity for the ligand, whereas hGRαG679S displayed a 2-fold reduction compared with hGRα. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRαR477H (25 min) and hGRαG679S (30 min) into the nucleus than the wild-type hGRα (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRαR477H did not bind to glucocorticoid-response elements, whereas hGRαG679S preserved its ability to bind to glucocorticoidresponse elements. Finally, in glutathione-S-transferase pull-down assays, hGRαG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRαR477H and hGRα, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. Conclusions: The natural mutants hGRαR477H and hGRαG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.

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