The antagonists RU486 and ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid in vitro and in vivo, but have distinct effects on receptor conformation.
The antagonists RU486 and ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid in vitro and in vivo, but have distinct effects on receptor conformation.
复制标题
DOI：
10.1210/endo.139.4.5944
复制
发表时间：
1998-04
期刊：
影响因子：
4.8
通讯作者：
Elizabeth K. Gass;S. Leonhardt;S. Nordeen;D P Edwards
中科院分区：
文献类型：
--
作者：
Elizabeth K. Gass;S. Leonhardt;S. Nordeen;D P Edwards
通过三种不同类型的转基因实验，检测了不同类型的拮抗剂刺激完整哺乳动物细胞中孕酮受体(PR)与孕酮反应元件(PRE)结合的能力。其中包括启动子干扰实验，其中PR与位于TATA盒和转录开始之间的PreS的结合被检测为结构性活性报告基因的表达减少，PR拮抗剂和糖皮质激素受体激动剂竞争共同的糖皮质激素反应元件/预控报告构建体，以及包含来自单纯疱疹病毒VP16蛋白的结构性反式激活结构域的嵌合受体(PR-VP16)的激活。通过每种方法，所有被测试的拮抗剂在刺激细胞中的PR与PreS结合方面都同样有效。这包括以前指定的类型I(ZK98299)和类型II(RU486、ZK98734和ZK112993)11β-芳基取代的类固醇类似物。ZK98299对PR与PreS结合的刺激作用是有意义的，因为与RU486相比，ZK98299在体外缺乏通过凝胶迁移率改变实验刺激PR-DNA结合的能力，RU486促进PR与PreS的有效结合。为了澄清ZK98299的完整细胞和体外结果之间的明显差异，我们改变了凝胶迁移率变化分析条件，以允许检测不太稳定的DNA复合体。在此条件下，ZK98299诱导形成了特异的PR-Pre复合体。对ZK98299诱导的DNA复合体的进一步分析表明，它们表现出与RU486诱导的复合体不同的电泳迁移率，并且从DNA上的PR的关闭速度比与激动剂结合的PR的关闭速度更快。这表明ZK98299促进了PR内不同于RU486的构象变化。这些结果与ZK98299刺激PR与靶DNA序列结合的结论一致，ZK98299和RU486代表了两类基于诱导PR不同构象变化的机制拮抗剂。
Three types of transfection experiments were used to detect the abilities of different classes of antagonists to stimulate binding of progesterone receptor (PR) to progesterone response elements (PRE) in intact mammalian cells. These included a promoter interference assay, in which PR binding to PREs positioned between the TATA box and the start of transcription is detected as a reduction of expression of a constitutively active reporter gene, competition of PR antagonist and glucocorticoid receptor agonist for a common glucocorticoid response element/PRE-controlled reporter construct, and activation of a chimeric receptor (PR-VP16) containing the constitutive trans-activation domain derived from the VP16 protein of herpes simplex virus. By each approach, all antagonists tested were equally effective in stimulating PR binding to PREs in the cell. This included previously designated type I (ZK98299) and type II (RU486, ZK98734, and ZK112993) 11beta-aryl substituted steroid analogs. Stimulation of PR binding to PREs in the cell by ZK98299 was of interest because this antagonist has been reported to lack the ability to stimulate PR-DNA binding in vitro by electrophoretic gel mobility shift assay compared with RU486, which promotes efficient binding of PR to PREs. To clarify the apparent discrepancy between intact cell and in vitro results with ZK98299, we altered electrophoretic gel mobility shift assay conditions to allow detection of less stable DNA complexes. Under these conditions, ZK98299 induced the formation of specific PR-PRE complexes. Further analysis of the ZK98299-induced DNA complexes revealed that they exhibited an electrophoretic mobility different from that of the complexes induced by RU486, and the off-rate of PR from DNA was faster than that of the PR bound to agonist. This suggests that ZK98299 promotes a conformational change within PR distinct from that induced by RU486. The present results are consistent with the conclusions that ZK98299 stimulates PR binding to target DNA sequences and that ZK98299 and RU486 represent two mechanistic classes of antagonists based on inducing different conformational changes in PR.