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

Mol Endocrinol. 2001 Nov;15(11):1953-70. doi: 10.1210/mend.15.11.0727.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cells, Cultured
  • Coenzyme A / metabolism
  • Cricetinae
  • DEAD-box RNA Helicases
  • Estradiol / analogs & derivatives*
  • Estradiol / metabolism
  • Estradiol / pharmacology
  • Estrogen Antagonists / metabolism
  • Estrogen Antagonists / pharmacology
  • Estrogen Receptor alpha
  • Histone Acetyltransferases
  • Humans
  • Ligands
  • Molecular Sequence Data
  • Mutation
  • Nuclear Receptor Coactivator 1
  • Phosphorylation
  • Polyunsaturated Alkamides
  • Protein Conformation
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • RNA Helicases / genetics
  • RNA Helicases / metabolism
  • Rabbits
  • Receptors, Estrogen / chemistry*
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism*
  • Tamoxifen / analogs & derivatives*
  • Tamoxifen / metabolism
  • Trans-Activators / chemistry
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*


  • Estrogen Antagonists
  • Estrogen Receptor alpha
  • Ligands
  • Polyunsaturated Alkamides
  • Receptors, Estrogen
  • Trans-Activators
  • Transcription Factors
  • Tamoxifen
  • afimoxifene
  • Estradiol
  • ICI 164384
  • Histone Acetyltransferases
  • NCOA1 protein, human
  • Nuclear Receptor Coactivator 1
  • Protein Kinases
  • Ddx5 protein, human
  • DEAD-box RNA Helicases
  • RNA Helicases
  • Coenzyme A