Liu Y, Takeshita A, Nagaya T, Baniahmad A, Chin W W, Yen P M
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Endocrinol. 1998 Jan;12(1):34-44. doi: 10.1210/mend.12.1.0046.
We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor beta ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-beta (TRbeta), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRbeta derepressed transcription to basal level. Using this system and a battery of TRbeta mutants, we found that TRbeta/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRbeta ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRbeta DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRbeta from achieving transcriptional activation above basal level in this chimeric receptor system.
我们采用了一种嵌合受体系统，将酵母GAL4 DNA结合结构域/视黄酸X受体β配体结合结构域嵌合受体（GAL4RXR）、甲状腺激素受体-β（TRβ）以及上游激活序列-报告基因质粒共转染到CV-1细胞中，以研究抑制、去抑制和转录激活。在没有T3的情况下，未结合配体的TR将转录抑制至基础水平的20%，而在有T3的情况下，结合配体的TRβ将转录去抑制至基础水平。利用该系统和一系列TRβ突变体，我们发现TRβ/RXR异二聚体的形成对于该系统中的基础抑制和去抑制是必要且充分的。此外，一个AF-2结构域突变体（E457A）介导基础抑制但不介导去抑制，这表明在此位点与假定的共激活因子相互作用可能对去抑制至关重要。有趣的是，一个仅包含TRβ配体结合结构域（LBD）的突变体不仅介导去抑制，还刺激转录激活比基础水平高10倍。使用缺失和结构域交换突变体的研究将一个抑制区域定位到TRβ DNA结合结构域。滴定研究进一步表明，促进与共激活因子相互作用的变构变化可能解释了LBD增强的转录活性。总之，我们的研究结果表明，TR与RXR形成异二聚体对于抑制和去抑制很重要，并且在这个嵌合系统中，共激活因子与AF-2结构域的相互作用可能是去抑制所必需的。此外，DNA结合结构域中可能存在一个抑制区域，它减少了TR与共激活因子的相互作用，并阻止全长野生型TRβ在这个嵌合受体系统中实现高于基础水平的转录激活。
