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

Background: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor alpha(hGR alpha) gene, which impair one or more of hGR alpha's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGR alpha R477H and hGR alpha G679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. Methods and Results: In transient transfection assays, hGR alpha R477H displayed no transcriptional activity, whereas hGR alpha G679S 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 alpha. Neither hGR alpha R477H nor hGR alpha G679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGR alpha R477H preserved normal affinity for the ligand, whereas hGR alpha G679S displayed a 2-fold reduction compared with hGR alpha. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGR alpha R477H ( 25 min) and hGR alpha G679S ( 30 min) into the nucleus than the wild-type hGR alpha ( 12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGR alpha R477H did not bind to glucocorticoid-response elements, whereas hGR alpha G679S preserved its ability to bind to glucocorticoid-response elements. Finally, in glutathione-S-transferase pull-down assays, hGR alpha G679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGR alpha R477H and hGR alpha, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. Conclusions: The natural mutants hGR alpha R477H and hGR alpha G679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.