Ikeda M, Kawaguchi A, Takeshita A, Chin W W, Endo T, Onaya T
Third Department of Internal Medicine, Yamanashi Medical University Tamaho, Japan.
Mol Cell Endocrinol. 1999 Jan 25;147(1-2):103-12. doi: 10.1016/s0303-7207(98)00211-1.
Full-length of steroid receptor coactivator-1 (F-SRC-1) has been shown to interact with thyroid hormone receptors (TRs) in a ligand-dependent manner and to stimulate receptor-dependent transcription. To identify functional domains of F-SRC-1, several internal deletion mutants of F-SRC-1 were constructed. Although in vitro pull down assay with TR showed interaction of all of these mutants with TR, lack of mid legion (amino acids 398-1172) lost enhancing activity of TR-mediated transcription in a transient transfection assay. However, F-SRC-1 mutant lacking CBP-interacting domain still preserved enhancing activity. Surprisingly, F-SRC-1 mutants also increased basal level of viral promoter activity depending upon their deleted region. Yeast activation function assay revealed that these F-SRC-1 mutants had intrinsic activation function when bound to DNA. Analyses of small fragments of F-SRC-1 identified three separable activation domains. In vitro binding assay showed that TBP and TFIIB bound to C-terminal half of F-SRC-1. These results suggest that F-SRC-1 can function via both CBP-dependent and independent manners using various sets of activation domains and that direct interactions between F-SRC-1 and TBP or TFIIB may not be important for CBP-independent transcription.
全长类固醇受体辅激活因子-1（F-SRC-1）已被证明以配体依赖的方式与甲状腺激素受体（TRs）相互作用，并刺激受体依赖的转录。为了确定F-SRC-1的功能结构域，构建了几个F-SRC-1的内部缺失突变体。尽管用TR进行的体外下拉试验表明所有这些突变体都与TR相互作用，但在瞬时转染试验中，中间区域（氨基酸398-1172）的缺失丧失了TR介导的转录增强活性。然而，缺乏CBP相互作用结构域的F-SRC-1突变体仍保留增强活性。令人惊讶的是，F-SRC-1突变体还根据其缺失区域增加了病毒启动子活性的基础水平。酵母激活功能试验表明，这些F-SRC-1突变体在与DNA结合时具有内在的激活功能。对F-SRC-1小片段的分析确定了三个可分离的激活结构域。体外结合试验表明，TBP和TFIIB与F-SRC-1的C末端一半结合。这些结果表明，F-SRC-1可以通过使用各种激活结构域的CBP依赖和独立方式发挥作用，并且F-SRC-1与TBP或TFIIB之间的直接相互作用对于不依赖CBP的转录可能不重要。
