Kost S L, Smith D F, Sullivan W P, Welch W J, Toft D O
Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905.
Mol Cell Biol. 1989 Sep;9(9):3829-38. doi: 10.1128/mcb.9.9.3829-3838.1989.
The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.
通过对受体复合物进行免疫分离以及对分离出的蛋白质进行凝胶电泳，分析了禽类孕酮受体的蛋白质组成。如先前所述，未活化的胞质溶胶受体与90千道尔顿（kDa）的热休克蛋白hsp90结合在一起被分离出来。还观察到一种70-kDa的蛋白质，通过蛋白质免疫印迹法表明它能与针对70-kDa热休克蛋白的特异性抗体发生反应。因此，两种与孕酮受体相关的蛋白质与热休克蛋白相同或密切相关。当在没有激素的情况下分别分离出两种孕酮受体亚型A和B时，两者都与hsp90和70-kDa蛋白质结合在一起。然而，从输卵管核提取物中分离出的活化受体与70-kDa蛋白质结合，但不与hsp90结合。在体内用孕酮处理的第一个小时内，观察到hsp90从受体复合物的胞质形式上发生激素依赖性解离，这可以解释核受体复合物中缺乏hsp90的现象。在无细胞体系中，钼酸盐能稳定hsp90与受体的结合，但高盐会破坏这种结合。然而，这些处理并没有改变70-kDa蛋白质与受体的结合。在升高的温度（23摄氏度）下添加ATP可破坏70-kDa蛋白质与受体的结合。如用叠氮基[32P]ATP进行亲和标记所证明 的，与受体相关的70-kDa蛋白质是一种ATP结合蛋白。这些结果表明，两种与受体相关的蛋白质通过不同机制与孕酮受体相互作用，并且它们可能以不同方式影响受体的结构或功能。
