Activated estrogen receptor alpha (ERalpha) modulates transcription triggered by the transcription factor activator protein-1 (AP-1), which consists of Jun-Jun homodimers and Jun-Fos heterodimers. Previous studies have demonstrated that the interference occurs without binding of ERalpha to DNA but probably results from protein.protein interactions. However, involvement of a direct interaction between ERalpha and AP-1 is still debated. Using glutathione S-transferase pull-down assays, we demonstrated that ERalpha bound directly to c-Jun and JunB but not to FOS family members, in a ligand-independent manner. The interaction could occur when c-Jun was bound onto DNA, as shown in a protein-protein-DNA assay. It implicated the C-terminal part of c-Jun and amino acids 259-302 present in the ERalpha hinge domain. ERalpha but not an ERalpha mutant deleted of amino acids 250-303 (ER241G), also associated with c-Jun in intact cells, in the presence of estradiol, as shown by two-hybrid and coimmunoprecipitation assays. We also show that ERalpha, c-Jun, and the p160 coactivator GRIP1 can form a multiprotein complex in vitro and in intact cells and that the ERalpha.c-Jun interaction could be crucial for the stability of this complex. VP16-ERalpha and c-Jun, which both interact with GRIP1, had synergistic effect on GAL4-GRIP1-induced transcription in the presence of estradiol, and this synergistic effect was not observed with the ERalpha mutant VP16-ER241G or when c-Fos, which bound GRIP1 but not ERalpha, was used instead of c-Jun. Finally, ER241G was inefficient for regulation of AP-1 activity, and an ERalpha truncation mutant encompassing the hinge domain had a dominant negative effect on ERalpha action. These results altogether demonstrate that ERalpha can bind to c-Jun in vitro and in intact cells and that this interaction, by stabilizing a multiprotein complex containing p160 coactivator, is likely to be involved in estradiol regulation of AP-1 responses.
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