Characterization of the physical interaction between estrogen receptor alpha and JUN proteins.
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.
Teyssier
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C
Institut National de la Santé et de la Recherche Médicale, Endocrinologie Moléculaire et Cellulaire des Cancers (U 540), 60 Rue de Navacelles, Montpellier 34090, France.
Belguise
K
K
Galtier
F
F
Chalbos
D
D
D000818:Animals; D019556:COS Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D005799:Genes, Dominant; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D009154:Mutation; D051958:Nuclear Receptor Coactivator 2; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D047628:Estrogen Receptor alpha; D008024:Ligands; C498001:NCOA2 protein, human; D051958:Nuclear Receptor Coactivator 2; D016755:Proto-Oncogene Proteins c-jun; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004958:Estradiol; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase