Wallace A D, Cidlowski J A
Molecular Endocrinology Group, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2001 Nov 16;276(46):42714-21. doi: 10.1074/jbc.M106033200. Epub 2001 Sep 12.
Ligand-dependent down-regulation of the glucocorticoid receptor (GR) has been shown to limit hormone responsiveness, but the mechanisms involved in this process are poorly understood. The glucocorticoid receptor is a phosphoprotein that upon ligand binding becomes hyperphosphorylated, and recent evidence indicates that phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover. Because phosphorylation is a key signal for ubiquitination and proteasomal catabolism of many proteins, we evaluated whether the ubiquitin-proteasomal pathway had a role in glucocorticoid receptor down-regulation and the subsequent transcriptional response to glucocorticoids. Pretreatment of COS-1 cells expressing mouse glucocorticoid receptor with the proteasome inhibitor MG-132 effectively blocks glucocorticoid receptor protein down-regulation by the glucocorticoid dexamethasone. Interestingly, both MG-132 and a second proteasome inhibitor beta-lactone significantly enhanced hormone response of transfected mouse glucocorticoid receptor toward transcriptional activation of glucocorticoid receptor-mediated reporter gene expression. The transcriptional activity of the endogenous human glucocorticoid receptor in HeLa cells was also enhanced by MG-132. Direct evidence for ubiquitination of the glucocorticoid receptor was obtained by immunoprecipitation of cellular extracts from proteasome-impaired cells. Examination of the primary sequence of mouse, human, and rat glucocorticoid receptor has identified a candidate PEST degradation motif. Mutation of Lys-426 within this PEST element both abrogated ligand-dependent down-regulation of glucocorticoid receptor protein and simultaneously enhanced glucocorticoid receptor-induced transcriptional activation of gene expression. Unlike wild type GR, proteasomal inhibition failed to enhance significantly transcriptional activity of K426A mutant GR. Together these findings suggest a major role of the ubiquitin-proteasome pathway in regulating glucocorticoid receptor protein turnover, thereby providing a mechanism to terminate glucocorticoid responses.
糖皮质激素受体（GR）的配体依赖性下调已被证明会限制激素反应性，但这一过程涉及的机制仍知之甚少。糖皮质激素受体是一种磷蛋白，在配体结合后会发生过度磷酸化，最近的证据表明，糖皮质激素受体的磷酸化状态在受体蛋白周转中起重要作用。由于磷酸化是许多蛋白质泛素化和蛋白酶体分解代谢的关键信号，我们评估了泛素-蛋白酶体途径是否在糖皮质激素受体下调及随后对糖皮质激素的转录反应中发挥作用。用蛋白酶体抑制剂MG-132预处理表达小鼠糖皮质激素受体的COS-1细胞，可有效阻断糖皮质激素地塞米松对糖皮质激素受体蛋白的下调作用。有趣的是，MG-132和第二种蛋白酶体抑制剂β-内酯均显著增强了转染的小鼠糖皮质激素受体对糖皮质激素受体介导的报告基因表达转录激活的激素反应。MG-132也增强了HeLa细胞中内源性人糖皮质激素受体的转录活性。通过对蛋白酶体受损细胞的细胞提取物进行免疫沉淀，获得了糖皮质激素受体泛素化的直接证据。对小鼠、人和大鼠糖皮质激素受体的一级序列进行分析，确定了一个候选的PEST降解基序。该PEST元件内的赖氨酸-426突变既消除了糖皮质激素受体蛋白的配体依赖性下调，同时又增强了糖皮质激素受体诱导的基因表达转录激活。与野生型GR不同，蛋白酶体抑制未能显著增强K426A突变型GR的转录活性。这些发现共同表明泛素-蛋白酶体途径在调节糖皮质激素受体蛋白周转中起主要作用，从而提供了一种终止糖皮质激素反应的机制。
