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Synergism Between ERα Transactivation Function 1 (AF-1) and AF-2 Mediated by Steroid Receptor Coactivator Protein-1: Requirement for the AF-1 α-Helical Core and for a Direct Interaction Between the N- and C-Terminal Domains

2001; Oxford University Press; Volume: 15; Issue: 11 Linguagem: Inglês

10.1210/mend.15.11.0727

1944-9917

Raphaël Métivier, Graziella Penot, Gilles Flouriot, Farzad Pakdel,

Receptor Mechanisms and Signaling

The transcriptional activity of ER␣ (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 ␣-helical structure within the AF-1 functional core, and by evaluating point mutants of human ER␣ (hER␣) within this region, we show that in transfection experiments this structure is required for synergism between SRC-1 and hER␣.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 hER␣.Glutathione-S-transferase pulldown assays demonstrate that the AF-1 core is able and sufficient for the hER␣ N-terminal region to interact with SRC-1.Interestingly, an enhancement of this recruitment in the presence of the hER␣ 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 hER␣, was an increase in the phosphorylation state of the N-terminal domain.Binding of 4-hydroxytamoxifen (OHT) to the hER␣ 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 hER␣ AF-1 was further substantiated by transfection experiments using the OHTresponsive human C3 promoter, which showed that OHT-induced hER␣ AF-1 activity was enhanced by SRC-1 and required the AF-1 ␣-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 ␣-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 ␣-helical core is essential for both E2-and OHT-induced ER␣ activity.(Molecular Endocrinology 15: 1953-1970, 2001) E STROGENS ARE POWERFUL regulators of both the developmental and functional aspects of reproductive functions (1).Estrogens are also involved in proliferative reproductive disorders, notably in breast and endometrium cancers (2-4).E2-regulated processes are predominantly transduced at the transcriptional level and are mediated by two related receptors, termed ER␣ and ER␤ (NR3A1 and NR3A2, respec-tively) (5).Both are ligand-inducible transcription factors (6, 7).ERs belong to the highly diverse superfamily of nuclear receptors (NRs), which are structurally organized into six independent domains, termed A to F, each of which encodes specific functions (8-10; Fig. 1A).The NR signature motif is the cysteine-rich, zinccoordinated structure of the C domain, which mediates binding to DNA (11-13).The C-terminal region, also termed the ligand-binding domain (LBD), is organized into 10-13 ␣-helices (14, 15).ERs have two major transcriptional regulatory functions, activation function 1 (AF-1) at the N terminus and AF-2 at the C terminus.AF-2 can be further subdivided into AF-2 core and AF-2a transactivating regions (16-19; Fig. 1A).These AFs exhibit distinct transactivation properties that depend on both cell and promoter contexts (20, 21).The full transcriptional activity of the ERs is