Characterization of the physical interaction between estrogen receptor alpha and JUN proteins

Activated estrogen receptor alpha (ER alpha) modulates transcription trigge red by the transcription factor activator protein-1 (AP-1), which consists of Jun-Jun homodimers and Jun-Fos heterodimers. Previous studies have demon strated that the interference occurs without binding of ER alpha to DNA but probably results from protein-protein interactions. However, involvement o f a direct interaction between ER alpha and AP-I is still debated. Using gl utathione S-transferase pull-down assays, we demonstrated that ER alpha bou nd directly to c-Jun and JunB but not to FOS family members, in a ligand-in dependent 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 par t of c-Jun and amino acids 259-302 present in the ER alpha hinge domain. ER alpha but not an ER alpha mutant deleted of amino acids 250-303 (ER241G), also associated with c-Jun in intact cells, in the presence of estradiol, a s shown by two-hybrid and coimmunoprecipitation assays. We also show that E R alpha, c-Jun, and the p160 coactivator GRIP1 can form a multiprotein comp lex in vitro and in intact cells and that the ER alpha -c-Jun interaction c ould be crucial for the stability of this complex. VP16-ER alpha and c-Jun, which both interact with GRIP1, had synergistic effect on GAL4-GRIP1-induc ed transcription in the presence of estradiol, and this synergistic effect was not observed with the ER alpha mutant VP16-ER241G or when c-Fos, which bound GRIP1 but not ER alpha, was used instead of c-Jun. Finally, ER241G wa s inefficient for regulation of AP-1 activity, and an ER alpha truncation m utant encompassing the hinge domain had a dominant negative effect on ER al pha action. These results altogether demonstrate that ER alpha can bind to c-Jun in vitro and in intact cells and that this interaction, by stabilizin g a multiprotein complex containing p160 coactivator, is likely to be invol ved in estradiol regulation of AP-1 responses.