Negative Regulation of Estrogen Receptors

描述（由申请人提供）：尽管已经花费了大量的精力来研究配体激活雌激素受体α (er α)转录活性的机制，但对于雌激素受体作用被负调控的分子事件知之甚少。很明显，雌激素对许多细胞类型的处理通过多泛素化和26S蛋白酶体对受体的降解导致erα表达下调。然而，与受体结合的配体如何靶向其降解尚不清楚。矛盾的是，即使细胞内存在更多的受体，蛋白酶体抑制剂也会阻断erα依赖性基因的表达，这表明蛋白酶体与基因表达之间存在重要联系。本应用程序中概述的实验的总体目标是提供对配体诱导蛋白酶体降解erα的机制的更详细的了解，如何实现细胞特异性调节，以及这如何有助于erα转录活性的细胞特异性。我们计划的研究基于五个关键观察。首先，erα的配体结合域足以介导配体依赖性下调。其次，雌二醇和ICI 182780诱导的erα降解被蛋白酶体抑制剂阻断。第三，蛋白酶体介导的erα下调是细胞类型特异性的。第四，雌二醇和ICI 182780诱导erα配体结合域和CBP/p300以细胞特异性的方式相互作用，与erα降解相关。最后，抑制CBP/p300功能可阻断配体依赖性erα下调。这些发现支持了一种假设，即CBP和/或p300通过激动剂或纯拮抗剂配体招募到er - α的能力是26S蛋白酶体降解ERa所必需的重要分子事件，因此，细胞特异性ERa与CBP和/或p300的相互作用有助于细胞类型依赖性ERa的下调以及转录活性。具体目的如下：1)检验26S蛋白酶体与配体依赖性erα表达下调和erα依赖性转录活性的关系；2)确定AF2相互作用因子在配体诱导的er - α下调中的作用；3)研究细胞类型特异性受体下调与转录活性之间的关系，特别是关于er - α的AF-1和AF-2结构域的相对转录强度。

DESCRIPTION (provided by applicant): Although great effort has been spent examining the mechanisms through which ligands activate estrogen receptor-alpha (ERalpha) transcriptional activity, relatively little is known about the molecular events through which Estrogen receptor action is negatively regulated. It is clear that estrogen treatment of many cell types leads to the down regulation of ERalpha expression through polyubiquitination and degradation of the receptor by the 26S proteasome. However, it is unknown how ligand binding to receptor targets it for degradation. Paradoxically, proteasome inhibitors block ERalpha-dependent gene expression, even though there is more receptor present within the cell, and this indicates an important link between the proteasome and gene expression. The overall goal of the experiments outlined in this application is to provide a more detailed understanding of the mechanisms utilized by ligands to induce ERalpha degradation by the proteasome, how cell-specific regulation of this is achieved, and how this contributes to the cell specificity of ERalpha transcriptional activity. Our planned studies are based on five key observations. First, the ligand binding domain of ERalpha is sufficient to mediate ligand-dependent down regulation. Second, induction of ERalpha degradation by estradiol and ICI 182,780 is blocked by proteasome inhibitors. Third, proteasome-mediated down regulation of ERalpha is cell-type specific. Fourth, estradiol and ICI 182,780 induce interactions between the ERalpha ligand binding domain and CBP/p300 in a cell-specific manner that correlates with ERalpha degradation. Lastly, inhibition of CBP/p300 function blocks ligand-dependent ERalpha down regulation. These findings support the hypothesis that the ability of CBP and/or p300 to be recruited to ERalpha by agonist or pure antagonist ligands is an important molecular event necessary for ERa degradation by the 26S proteasome, and that cell specific ERa interactions with CBP and/or p300 therefore contribute to cell-type dependent ERa down regulation as well as transcriptional activity. This will be tested in the following specific aims: 1) Examine the relationship of the 26S proteasome with ligand-dependent down regulation of ERalpha expression and ERalpha-dependent transcriptional activity; 2) Determine the role of AF2 interacting factors in ligand-induced down regulation of ERalpha and 3) Examine the relationship between cell-type specific receptor down regulation and transcriptional activity, particularly with respect to the relative transcriptional strength of ERalpha's AF-1 and AF-2 domains.