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Functional Characterization of the Natural Human Glucocorticoid Receptor (hGR) Mutants hGRR477H and hGRG679S Associated with Generalized Glucocorticoid Resistance

Pediatric Endocrinology Section, Reproductive Biology and Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Evangelia Charmandari, M.D., Department of Pediatric Endocrinology, Great Ormond Street Hospital for Children, 9th Floor, Southwood Building, Great Ormond Street, London, WC1N 3JH, United Kingdom. E-mail: charmane{at}mail.nih.gov.

Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor (hGR) gene, which impair one or more of hGR’s functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRR477H and hGRG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction.

Methods and Results: In transient transfection assays, hGRR477H displayed no transcriptional activity, whereas hGRG679S 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 hGR. Neither hGRR477H nor hGRG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRR477H preserved normal affinity for the ligand, whereas hGRG679S displayed a 2-fold reduction compared with hGR. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRR477H (25 min) and hGRG679S (30 min) into the nucleus than the wild-type hGR (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRR477H did not bind to glucocorticoid-response elements, whereas hGRG679S preserved its ability to bind to glucocorticoid-response elements. Finally, in glutathione-S-transferase pull-down assays, hGRG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRR477H and hGR, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2.

Conclusions: The natural mutants hGRR477H and hGRG679S 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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