Charmandari Evangelia, Kino Tomoshige, Ichijo Takamasa, Zachman Keith, Alatsatianos Anton, Chrousos George P
Pediatric Endocrinology Section, Reproductive Biology and Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20895, USA.
J Clin Endocrinol Metab. 2006 Apr;91(4):1535-43. doi: 10.1210/jc.2005-1893. Epub 2006 Jan 31.
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.
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.
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.
糖皮质激素抵抗通常是人类糖皮质激素受体α（hGRα）基因突变的结果，这些突变会损害hGRα的一种或多种功能。我们研究了两种先前描述的突变受体hGRαR477H和hGRαG679S分别在DNA结合域和配体结合域发生氨基酸替换后影响糖皮质激素信号转导的分子机制。
在瞬时转染实验中，hGRαR477H未显示转录活性，而hGRαG679S与野生型hGRα相比，其对糖皮质激素反应性小鼠乳腺肿瘤病毒启动子转录的刺激能力在给予地塞米松后降低了55%。hGRαR477H和hGRαG679S对野生型受体均未发挥显性负效应。地塞米松结合实验表明，hGRαR477H对配体保留正常亲和力，而hGRαG679S与hGRα相比亲和力降低了2倍。核转位研究证实，在无配体时突变受体主要定位于细胞质。给予地塞米松后，hGRαR477H（25分钟）和hGRαG679S（30分钟）转位入核的速度比野生型hGRα（12分钟）慢。在用小鼠乳腺肿瘤病毒启动子稳定转染的细胞中进行的染色质免疫沉淀实验中，hGRαR477H不与糖皮质激素反应元件结合，而hGRαG679S保留了与糖皮质激素反应元件结合的能力。最后，在谷胱甘肽-S-转移酶下拉实验中，hGRαG679S仅通过其激活功能（AF）-1在体外与糖皮质激素受体相互作用蛋白1共激活因子相互作用，这与hGRαR477H和hGRα不同，它们通过AF-1和AF-2与糖皮质激素受体相互作用蛋白1相互作用。
天然突变体hGRαR477H和hGRαG679S通过影响糖皮质激素受体的不同功能导致全身性糖皮质激素抵抗，这些功能贯穿hGR信号系统级联反应。
