PMID- 11682626 OWN - NLM STAT- MEDLINE DCOM- 20020321 LR - 20161124 IS - 0888-8809 (Print) IS - 0888-8809 (Linking) VI - 15 IP - 11 DP - 2001 Nov TI - 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. PG - 1953-70 AB - 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. FAU - Metivier, R AU - Metivier R AD - Equipe d'Endocrinologie Moleculaire de la Reproduction, Unite Mixte de Recherche Centre National de la Recherche Scientifique 6026, Universite de Rennes I, 35042 Rennes Cedex, France. FAU - Penot, G AU - Penot G FAU - Flouriot, G AU - Flouriot G FAU - Pakdel, F AU - Pakdel F LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - United States TA - Mol Endocrinol JT - Molecular endocrinology (Baltimore, Md.) JID - 8801431 RN - 0 (Estrogen Antagonists) RN - 0 (Estrogen Receptor alpha) RN - 0 (Ligands) RN - 0 (Polyunsaturated Alkamides) RN - 0 (Receptors, Estrogen) RN - 0 (Trans-Activators) RN - 0 (Transcription Factors) RN - 094ZI81Y45 (Tamoxifen) RN - 17197F0KYM (afimoxifene) RN - 4TI98Z838E (Estradiol) RN - 84LT43726C (ICI 164384) RN - EC 2.3.1.48 (Histone Acetyltransferases) RN - EC 2.3.1.48 (NCOA1 protein, human) RN - EC 2.3.1.48 (Nuclear Receptor Coactivator 1) RN - EC 2.7.- (Protein Kinases) RN - EC 3.6.1.- (Ddx5 protein, human) RN - EC 3.6.4.13 (DEAD-box RNA Helicases) RN - EC 3.6.4.13 (RNA Helicases) RN - SAA04E81UX (Coenzyme A) SB - IM MH - Amino Acid Sequence MH - Animals MH - Cells, Cultured MH - Coenzyme A/metabolism MH - Cricetinae MH - DEAD-box RNA Helicases MH - Estradiol/*analogs & derivatives/metabolism/pharmacology MH - Estrogen Antagonists/metabolism/pharmacology MH - Estrogen Receptor alpha MH - Histone Acetyltransferases MH - Humans MH - Ligands MH - Molecular Sequence Data MH - Mutation MH - Nuclear Receptor Coactivator 1 MH - Phosphorylation MH - Polyunsaturated Alkamides MH - Protein Conformation MH - Protein Kinases/genetics/metabolism MH - Protein Structure, Tertiary MH - RNA Helicases/genetics/metabolism MH - Rabbits MH - Receptors, Estrogen/*chemistry/genetics/*metabolism MH - Tamoxifen/*analogs & derivatives/metabolism MH - Trans-Activators/chemistry/genetics/*metabolism MH - Transcription Factors/genetics/*metabolism EDAT- 2001/10/30 10:00 MHDA- 2002/03/22 10:01 CRDT- 2001/10/30 10:00 PHST- 2001/10/30 10:00 [pubmed] PHST- 2002/03/22 10:01 [medline] PHST- 2001/10/30 10:00 [entrez] AID - 10.1210/mend.15.11.0727 [doi] PST - ppublish SO - Mol Endocrinol. 2001 Nov;15(11):1953-70. doi: 10.1210/mend.15.11.0727.