He Yuanzheng, Szapary Daniele, Simons S Stoney
Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD 20892, USA.
J Biol Chem. 2002 Dec 20;277(51):49256-66. doi: 10.1074/jbc.M205536200. Epub 2002 Oct 9.
Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonist-bound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes. These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors. We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity. Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities. DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities. The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved. The ability of a TIF2.4 fragment (i.e. TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2. Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids. These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes. In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s).
共激活因子如TIF2和SRC-1可调节激动剂结合型糖皮质激素受体（GRs）的剂量反应曲线位置以及抗糖皮质激素复合物的部分激动剂活性。共激活因子的这些特性不同于其最初定义的与激动剂结合型类固醇受体结合并增加转录激活总水平的活性。我们现在报告，缺乏两个激活域（AD1和AD2）的TIF2和SRC-1构建体增加转录激活的能力明显降低，但保留了大部分调节剂量反应曲线和部分激动剂活性的能力。哺乳动物双杂交实验表明，具有调节活性的最小TIF2片段（TIF2.4）不与p300、CREB结合蛋白或也调节GR活性的PCAF相互作用。DRIP150和DRIP205与共激活因子作用有关，但无法调节GR活性。PCAF或DRIP150分别与SRC-1或TIF2缺乏协同作用，进一步表明这些其他因子不参与其中。一个未知与蛋白质相互作用的TIF2.4片段（即TIF2.37）能够阻断TIF2.4的作用，这表明一种未知的结合蛋白介导了TIF2的调节活性。用GST/TIF2.4进行的下拉实验表明，TIF2与GR以激素依赖的方式直接相互作用，这需要TIF2的受体相互作用结构域，并且与激动剂和大多数抗糖皮质激素的相互作用同样强烈。这些在哺乳动物双杂交实验中得到证实的观察结果表明，诸如TIF2之类的共激活因子调节抗类固醇部分激动剂活性的能力是由共激活因子与GR-拮抗剂复合物的结合介导的。总之，共激活因子与GR-激动剂和-拮抗剂复合物的调节活性在机制上不同于共激活因子增加转录激活总水平的能力，并且似乎涉及与GR-类固醇复合物以及一种未知的TIF2相关因子的结合。
