Characterization and cloning of an estrogen receptor coactivator

Studies with multiple activators including the sceroid receptors have suggested that activated transcription is mediated by limiting cellular factors, or coactivators. The sum of these results prompted the hypothesis that the activity of AF-2 may be mediated by ligand-dependent estrogen receptor (ER)-coactivators.;Applying the ligand-dependent nature of the protein-protein interaction, we employed a far-western approach to screen a cDNA library for inserts encoding potential ERAPs. One such cDNA encoded a novel highly charged protein of predicted molecular weight 110 kDa.;To study the biological function of ERAP140, the cloned cDNA was cotransfected with plasmids expressing the wild type ER cDNA and an ERE-driven reporter into mammalian cells. The ERAP140 cDNA conferred to the cotransfected cells an enhanced response to estrogen, demonstrating that it encodes a protein with coactivator activity.;The HBD/AF-2 region of ER binds to a number of proteins in a ligand-specific and AF-2 dependent manner. These proteins include at least one ERAP of molecular weight 140 kDa, which cDNA we have isolated. In addition, the AF-2 domain binds to at least one ERAP of molecular weight 160 kDa, and to a number of smaller and larger proteins. (Abstract shortened by UMI.).;In order to identify such factors, we employed an ER-affinity purification protocol to capture ER-associated proteins from mammalian cell extracts. We identified two estrogen receptor-associated proteins, ERAP140 and ERAP160, which bind to the AF-2 domain of ER in a manner that is ligand-dependent and which discriminate estrogenic from non-estrogenic ligands. These proteins were able to bind to endogenous ER in vivo in a ligand-dependent manner. Further, the ERAPs were unable to bind in vitro to mutant ER proteins whose AF-2 activity was impaired in vivo. The correlation between the ability of ER to activate via AF-2 in vivo and to bind ERAPs in vitro provides strong evidence that these ERAPs mediate the ligand-specific AF-2 activity in vivo. The ERAPs were also able to associate with other nuclear receptors such as RXR...