Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor alpha (hGRalpha) gene, which impair one or more of hGRalpha's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRalphaR477H and hGRalphaG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction.
Methods and results: In transient transfection assays, hGRalphaR477H displayed no transcriptional activity, whereas hGRalphaG679S 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 hGRalpha. Neither hGRalphaR477H nor hGRalphaG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRalphaR477H preserved normal affinity for the ligand, whereas hGRalphaG679S displayed a 2-fold reduction compared with hGRalpha. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRalphaR477H (25 min) and hGRalphaG679S (30 min) into the nucleus than the wild-type hGRalpha (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRalphaR477H did not bind to glucocorticoid-response elements, whereas hGRalphaG679S preserved its ability to bind to glucocorticoid-response elements. Finally, in glutathione-S-transferase pull-down assays, hGRalphaG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRalphaR477H and hGRalpha, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2.
Conclusions: The natural mutants hGRalphaR477H and hGRalphaG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.