INTERACTION OF THE PROGESTERONE-RECEPTOR WITH BINDING-PROTEINS FOR FK506 AND CYCLOSPORINE-A
INTERACTION OF THE PROGESTERONE-RECEPTOR WITH BINDING-PROTEINS FOR FK506 AND CYCLOSPORINE-A
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DOI：
10.1210/me.9.7.838
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发表时间：
1995-07-01
影响因子：
--
通讯作者：
TOFT, DO
中科院分区：
文献类型：
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作者：
MILAD, M;SULLIVAN, W;TOFT, DO
以T47D人乳腺癌细胞和鸡输卵管为材料，研究了非激活孕酮受体(PR)复合体的结构。用针对鸡PR的交叉反应单抗PR6从胞浆中免疫沉淀PR(B型)。用十二烷基硫酸钠凝胶和免疫印迹对PR复合体进行分析，发现存在几种特定的共增蛋白。与以前的报告一致，两种热休克蛋白HSP90和HSP70被证明存在。先前观察到的第三个59千克的道尔顿(KDa)蛋白被证实为P59(也称为hsp56或FKBP52)，它已被证明与免疫抑制药物FK506结合。观察到另外两个与PR相关的蛋白，这两个蛋白以前没有被人类PR识别过。它们的分子质量分别为54 kDa和23 kDa，Western blotting表明它们与与鸡PR相关的蛋白质p54和p23有关。P23是一种新的功能未知的蛋白，最近发现P54或FKBP54是另一种与P59相关的FK506结合蛋白。最后，在分离的鸡PR复合体和体外重组的PR复合体中可以检测到环孢素A结合蛋白CYP-40，但在人PR复合体中检测不到该蛋白，因为人PR复合体在胞浆提取物中的稳定性不如鸡PR复合体。用含有孕激素报告基因MMTV-CAT的T47D细胞株检测FK506和环孢素A结合蛋白对激素作用的功能意义。环孢菌素A处理对CAT表达的基础水平没有影响，但它导致对合成孕激素R5020的反应的敏感度和幅度显著增加。抗孕激素RU486可阻断药物诱导的增强反应，提示这一作用是由受体介导的，而环孢菌素A可增强T47D细胞的孕激素作用，抑制L细胞的PR/报告基因系统。药物FK506和雷帕霉素对T47D细胞的孕激素作用无影响，但对T47D细胞的糖皮质激素作用有刺激作用。因此，这些免疫抑制药物的效果因细胞类型和所测试的激素系统而异。目前尚不清楚这些药物效应是否与受体复合体中结合的免疫亲和素直接相关。
T47D human breast carcinoma cells and the chicken oviduct were used to study the structure of the nonactivated progesterone receptor (PR) complex. Immunoprecipitation of PR (B form) from cytosol extracts was performed using monoclonal antibody PR6, a cross-reactive antibody prepared to chicken PR. Analysis of the PR complex by sodium dodecyl sulfate gels and Western immunoblotting revealed the presence of several specific copurifying proteins. Consistent with previous reports, the two heat shock proteins, hsp90 and hsp70, were shown to be present. A third 59-kilo-dalton (kDa) protein observed previously was confirmed to be p59 (also called hsp56 or FKBP52), which has been shown to bind the immunosuppressant drug FK506. Two additional PR-associated proteins were observed that had not been previously recognized with human PR. These have molecular masses of 54-kDa and 23-kDa and have been shown by Western blotting to be related to the proteins p54 and p23 that are associated with chicken PR. P23 is a novel protein of unknown function and p54 or FKBP54 has been recently shown to be another FK506-binding protein related to p59. Finally, the cyclosporin A-binding protein, CyP-40, could be detected in isolated chicken PR complexes and in PR complexes that were reconstituted in vitro, but this protein was not detected in human PR complexes, which are less stable than chicken PR complexes in cytosol extracts. The functional significance of FK506 and cyclosporin A-binding proteins to hormone action was tested using a T47D cell line that contained a progestin reporter gene, MMTV-CAT. Treatment with cyclosporin A had no effect on the basal level of CAT expression, but it caused a dramatic increase in the sensitivity and magnitude of the response to the synthetic progestin, R5020. The enhanced response elicited by drug treatment was blocked by the antiprogestin RU486 indicating that this effect was receptor-mediated, While cyclosporin A enhanced progestin action in T47D cells, it inhibited a PR/reporter gene system in L cells. The drugs FK506 and rapamycin had no effect on progestin action in T47D cells, but they stimulated glucocorticoid action in T47D cells. Thus, the effects of these immunosuppressant drugs vary with the cell type and hormonal system that is tested. Whether these drug effects relate directly to the immunophilins bound in receptor complexes remains unknown.