title: Synergism between ERalpha transactivation function 1 (AF-1) and AF-2 mediated by steroid receptor coactivator protein-1: requirement for the AF-1 alpha-helical core and for a direct interaction between the N- and C-terminal domains.
abstract: The transcriptional activity of ERalpha (or NR3A1) after binding of ligand is mediated through synergistic action between activation functions (AFs) AF-1 and AF-2 and the transcriptional machinery. This is functionally achieved by bridging coactivators such as CEBP binding protein/p300 and members of the p160 subfamily such as steroid receptor coactivator protein-1 (SRC-1). We previously identified a conserved potential alpha-helical structure within the AF-1 functional core, and by evaluating point mutants of human ERalpha (hERalpha) within this region, we show that in transfection experiments this structure is required for synergism between SRC-1 and hERalpha. We report that the transcriptional synergism between AF-1 mutants and SRC-1 was abolished in AF-1-sensitive cells such as HepG2, whereas it was reduced by 50% in CHO-K1 cells, which have a mixed context that is sensitive to both the AF-1 and AF-2 regions of hERalpha. Glutathione-S-transferase pulldown assays demonstrate that the AF-1 core is able and sufficient for the hERalpha N-terminal region to interact with SRC-1. Interestingly, an enhancement of this recruitment in the presence of the hERalpha ligand-binding domain was observed, which was found to be dependent on a direct interaction between the N-terminal B domain and the ligand-binding domain. Another functional consequence of this physical interaction, which is promoted by both partial and full agonists of hERalpha, was an increase in the phosphorylation state of the N-terminal domain. Binding of 4-hydroxytamoxifen (OHT) to the hERalpha C-terminal region induced a functional AF-1 conformation in vitro through this N- and C-terminal interaction. The involvement of an SRC-1-mediated pathway in transactivation mediated by hERalpha AF-1 was further substantiated by transfection experiments using the OHTresponsive human C3 promoter, which showed that OHT-induced hERalpha AF-1 activity was enhanced by SRC-1 and required the AF-1 alpha-helical structure. In conclusion, we demonstrate that the synergism between AF-1 and AF-2 is mediated in part by a cooperative recruitment of SRC-1 by both the AF-1 alpha-helical core and AF-2 regions and that it is stabilized by a direct interaction between the B and C-terminal domains. This interaction of SRC-1 with the AF-1 alpha-helical core is essential for both E2- and OHT-induced ERalpha activity.
authors.affiliation: Equipe d'Endocrinologie Moléculaire de la Reproduction, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6026, Université de Rennes I, 35042 Rennes Cedex, France.
authors.forename: R
authors.initials: R
authors.lastname: Métivier
authors.forename: G
authors.initials: G
authors.lastname: Penot
authors.forename: G
authors.initials: G
authors.lastname: Flouriot
authors.forename: F
authors.initials: F
authors.lastname: Pakdel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003065:Coenzyme A / Q000378:metabolism; D006224:Cricetinae; D053487:DEAD-box RNA Helicases; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D051548:Histone Acetyltransferases; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D056920:Nuclear Receptor Coactivator 1; D010766:Phosphorylation; D053284:Polyunsaturated Alkamides; D011487:Protein Conformation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D008024:Ligands; D053284:Polyunsaturated Alkamides; D011960:Receptors, Estrogen; D015534:Trans-Activators; D014157:Transcription Factors; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; C051781:ICI 164384; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D011494:Protein Kinases; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D003065:Coenzyme A