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

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. 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. These AFs exhibit distinct transactivation properties that depend on both cell and promoter contexts (20, 21). The full transcriptional activity of the ERs is