Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506
Differential control of glucocorticoid receptor hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immunosuppressive ligand FK506
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DOI：
10.1021/bi048503v
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发表时间：
2005-02-15
期刊：
影响因子：
2.9
通讯作者：
Sánchez, ER
中科院分区：
文献类型：
--
作者：
Davies, TH;Ning, YM;Sánchez, ER
许多实验室已经证明，在无激素类固醇受体复合物中存在四肽重复（TPR）蛋白（也称为亲免疫蛋白）。然而，这些蛋白质在类固醇受体作用中的独特作用尚不清楚。在这项工作中，我们研究了四种TPR蛋白（FKBP52, FKBP51， Cyp40和PP5）对哺乳动物L929细胞内源性表达的糖皮质激素受体（GR）激素结合功能的影响。作为第一步，我们用选择的亲免疫素配体[FK506，雷帕霉素，环孢素a （CsA）和环孢素H (CsH)]处理L929细胞，这些配体通常被认为通过抑制膜基类固醇输出物来增加GR对激素的反应。正如预期的那样，所有四种亲免疫蛋白配体都增加了MMTV-CAT报告细胞内地塞米松浓度和GR活性。为了确定这些配体是否能够独立于类固醇输出机制靶向GR功能，我们在产生相同细胞内激素浓度的亲免疫素配体和地塞米松处理条件下进行了GR报告基因检测。发现FK506刺激GR的活性超出了这种配体对激素保留的影响。相比之下，CsA仅通过上调激素潴留来影响GR。通过Scatchard分析，FK506增加了GR激素的结合亲和力，同时减少了激素的总结合位点。这一结果与gr相关的FKBP51缺失和PP5替代相关。有趣的是，在这些细胞中未发现GR相关的Cyp40，这与CsA配体仅通过激素输出机制影响GR的能力一致。为了测试FKBP52独立于FK506的作用，将FKBP52置于四环素诱导启动子的控制下。即使在FK506缺失的情况下，FKBP52的上调也会导致GR激素结合亲和力和交易性的增加。这些结果表明，免疫抑制配体可以通过改变受体复合物的TPR蛋白组成来改变GR激素结合功能，并且TPR蛋白对GR功能的影响顺序为：FKBP52 > PP5 > FKBP51。
Many laboratories have documented the existence of tetratricopeptide repeat (TPR) proteins (also known as immunophilins) in hormone-free steroid receptor complexes. Yet, the distinct roles of these proteins in steroid receptor action are poorly understood. In this work, we have investigated the effects of four TPR proteins (FKBP52, FKBP51, Cyp40, and PP5) on hormone-binding function of glucocorticoid receptor (GR) endogenously expressed in mammalian L929 cells. As a first step, we treated L929 cells with select immunophilin ligands [FK506, rapamycin, cyclosporin A (CsA), and cyclosporin H (CsH)], which are commonly thought to increase the GR response to hormone by inhibiting membrane-based steroid exporters. As expected, all four immunophilin ligands increased both the intracellular concentration of dexamethasone and GR activity at the MMTV-CAT reporter. To determine whether these ligands could target GR function independent of steroid export mechanisms, we performed GR reporter gene assays under conditions of immunophilin ligand and dexamethasone treatment that yielded equal intracellular hormone concentrations. FK506 was found to stimulate GR transactivity beyond the effect of this ligand on hormone retention. In contrast, CsA only affected the GR through upregulation of hormone retention. By Scatchard analysis, FK506 was found to increase GR hormone-binding affinity while decreasing total binding sites for hormone. This result correlated with loss of GR-associated FKBP51 and replacement with PP5. Interestingly, no GR-associated Cyp40 was found in these cells, consistent with the ability of CsA ligand to only affect GR through the hormone export mechanism. To test the role of FKBP52 independent of FK506, FKBP52 was placed under the control of a tetracycline-inducible promoter. Upregulation of FKBP52 caused an increase in both GR hormone-binding affinity and transactivity, even in the absence of FK506. These results show that immunosuppressive ligands can alter GR hormone-binding function by changing the TPR protein composition of receptor complexes and that TPR proteins exert a hierarchical effect on this GR function in the following order: FKBP52 > PP5 > FKBP51.