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.
