Synergism Between ER{alpha} 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

Raphaël Métivier1, Graziella Penot, Gilles Flouriot and Farzad Pakdel

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

Address all correspondence and requests for reprints to: Farzad Pakdel, Equipe d’Endocrinologie Moléculaire de la Reproduction, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6026, Université de Rennes I, Rennes Cedex, France. E-mail: farzad.pakdel{at}univ-rennes1.fr


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcriptional activity of ER{alpha} (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 ER{alpha} (hER{alpha}) within this region, we show that in transfection experiments this structure is required for synergism between SRC-1 and hER{alpha}. 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{alpha}. Glutathione-S-transferase pulldown assays demonstrate that the AF-1 core is able and sufficient for the hER{alpha} N-terminal region to interact with SRC-1. Interestingly, an enhancement of this recruitment in the presence of the hER{alpha} 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{alpha}, was an increase in the phosphorylation state of the N-terminal domain. Binding of 4-hydroxytamoxifen (OHT) to the hER{alpha} 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{alpha} AF-1 was further substantiated by transfection experiments using the OHTresponsive human C3 promoter, which showed that OHT-induced hER{alpha} 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 ER{alpha} activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGENS 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, 3, 4). E2-regulated processes are predominantly transduced at the transcriptional level and are mediated by two related receptors, termed ER{alpha} and ERß (NR3A1 and NR3A2, respectively) (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, 9, 10 ; Fig. 1AGo). The NR signature motif is the cysteine-rich, zinc-coordinated structure of the C domain, which mediates binding to DNA (11, 12, 13). The C-terminal region, also termed the ligand-binding domain (LBD), is organized into 10–13 {alpha}-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, 17, 18, 19 ; Fig. 1AGo). These AFs exhibit distinct transactivation properties that depend on both cell and promoter contexts (20, 21). The full transcriptional activity of the ERs is thought to proceed through a synergism between these AFs (11, 17, 18, 19, 20, 21).



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Figure 1. Scheme of hER{alpha} and SRC-1 Structural Determinants Involved in Transcriptional Activation

A, hER{alpha} sequence segmentation revealed modular properties to the six domains (A–F) of this protein. Numbers indicate the amino acids that delineate these domains. This figure is based on previously published work (11 19 31 44 ). hER{alpha} has two AFs located at the N-terminal (AF-1) and C-terminal (AF-2) parts of the protein. As shown, the AF-2 helical core resides within helix 12 of the E domain. The hER{alpha} N-terminal region contains two internal domains that repress AF-1, termed IR boxes 1 and 2. IR box 1, corresponding to the A domain, was determined to function through a direct interaction with the DF domains of hER{alpha}. This interaction is relieved in the presence of agonists. AF-1 was shown to possess three independent activation boxes (A boxes 1–3). N-terminal serine residues that can be phosphorylated are indicated. The AF-1 {alpha}-helical core resides within A box 1, and its sequence, are shown, with critical hydrophobic amino acids indicated in boldface. Regions of the protein determined to be involved with p160 CoA (such as SRC-1) recruitment are illustrated, and the critical residues (amino acids 38–64 in AF-1 and helices 11 and 12 in AF-2) are indicated by thick lines. B, Scheme of the SRC-1/NCoA1 protein, based on previously published work (26 31 32 ). The regions of SRC-1 involved in its interaction with NR AF-1 and AF-2, as well as with the CBP/p300 integrator protein, are shown. The domains of SRC-1 required for histone acetylase activity (HAT) are also shown. Asterisks indicate NR boxes (LxxLL), and "Q-rich" indicates a glutamine-rich region involved in physical contact with NR AF-1. The basic helix-loop-helix domain (bHLH/PAS) is involved in the transcriptional activation property of SRC-1. Numbers indicate the limits of each domain.

 
Structural and functional studies have shown that AF-2-mediated activation requires ligand-induced conformational changes within the C-terminal LBD (14, 22). This structural remodeling causes ERs to interact with components of multiprotein coactivator (CoA) complexes through their LxxLL NR boxes (23). A hydrophobic cleft on the surface of the ER LBD, involving helices 3–5 and the AF-2 core located in helix 12 (24, 25), interacts with the LxxLL motif. Two classes of CoA complex are recruited either subsequently or combinatorially, the CBP/p300, steroid receptor coactivator protein-1 (SRC-1), transcriptional intermediary factor 2 (TIF-2) class, which promotes the nucleosomal remodeling required for transcriptional activation, and the SMCC/TRAP/DRIP/ARC class, which forms a direct bridge to the transcriptional machinery apparatus (26, 27, 28). Whereas the molecular mechanisms underlying transactivation through the AF-2 of ER are beginning to be well understood, much less is known about how AF-1 achieves transactivation. Only two related specific ER{alpha} AF-1 CoAs have been identified to date, the p68 and p72 RNA helicases (29, 30). However, recent evidence indicates that some CoAs that are known to mediate AF-2 activity could also interact with the N-terminal region of ERs to mediate AF-1 activity (Fig. 1AGo). Indeed, both ER{alpha} and ERß are able to recruit SRC-1 or TIF-2 through their N-terminal regions (31, 32, 33). Interactions involved in this process seem not to require the CoA NR boxes, in contrast to CoA recruitment by AF-2 (Refs. 31 and 32 ; Fig. 1BGo). Moreover, in the case of ERß, SRC-1 targeting is modulated by phosphorylation on N-terminal serines that are involved in AF-1 regulation (33). Thus, members of the CBP/p300, SRC-1, TIF-2 CoA subclass are able to interact with both the N- and C-terminal regions of ERs. This led several groups to propose the concept of AF-bridging proteins, allowing for transcriptional synergism between the N- and C-terminal AFs (31, 32, 33, 34, 35, 36, 37). For some NRs, such as the ARs and PRs (NR3C4 and NR3C3, respectively), AF-bridging CoA recruitment might be facilitated by the existence of direct contacts established between the N- and C-terminal domains of these receptors (38, 39, 40, 41, 42, 43).

We previously identified a conserved potential {alpha}helix at the beginning of the B domain of ER{alpha} as the AF-1 functional core (44 ; Fig. 1AGo). This putative {alpha}-helix was required for 100% and for 30–50% of the AF-1 activity detected for the rainbow trout (rt) and human (h) ER{alpha}, respectively. The present study was undertaken to determine if this {alpha}-helix could participate in the synergism between both of the ER{alpha} AFs. Our data demonstrate that this structure is necessary for ER{alpha} total activity and that it is directly involved in recruiting SRC-1 but not in recruiting the p68 RNA helicase. Moreover, we show that the E2-induced functional synergism between both ER{alpha} AFs, which occurs in cells sensitive to both AFs, relies on a direct physical interaction between the N- and C-terminal regions that does not require the AF-1 core {alpha}-helix. This process has two consequences: first, phosphorylation of the N-terminal region can take place, and second, cooperative recruitment of the SRC-1 CoA by the AF-1 helical core and AF-2 can occur. Induction of AF-1 activity by the selective agonist 4-hydroxytamoxifen (OHT) was also found to require both the interaction of SRC-1 with the hER{alpha} AF-1 helical core and the C-terminal-mediated activation of AF-1.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hER{alpha} AF-1 {alpha}-Helical Core Is Required for SRC-1-Mediated hER{alpha} Transcriptional Activity
NR CoAs may be divided into two classes: those that are specific for one AF (such as AF-1) and those that make a physical bridge between the AFs. The p68 RNA helicase (p68) belongs to the first class (29). Alternatively, members of the p160 subfamily, such as SRC-1 and CBP/p300, are thought to mediate a synergistic process between ER{alpha} AF-1 and AF-2 (31, 37). The identification of a potential {alpha}-helix in the ER{alpha} AF-1 core (Ref. 44 and Fig. 1Go) led us to evaluate if this structure could be involved in synergism between AF-1 and AF-2. Transient transfection experiments were carried out by expressing hER{alpha}, hER{alpha} C to F domains [the hER 46-kDa isoform (45)], or hER{alpha} point mutants in which the AF-1 helical core region is rendered functionally inactive [hER{alpha} L39P or hER{alpha} Y43P, because the mutation of the L39 or Y43 residue to a proline destroys the {alpha}-helical structure (44)] together with SRC-1 or p68. Estrogen-responsive element-thymidine kinase-luciferase (ERE-TK-Luc) was used as an E2-responsive reporter gene. Transfections were performed in three cell lines exhibiting differential sensitivity to ER{alpha} AFs: HeLa cells, which are predominantly sensitive to ER{alpha} AF-2; HepG2 cells, which are almost entirely sensitive to AF-1; and CHO-K1 cells, in which ER{alpha} transactivation is mediated by both the AF-1 and AF-2 regions (18, 20, 44, 46). This experimental design was used to discriminate between processes caused by either AF acting on its own and those caused by the synergism between AF-1 and AF-2. Corresponding histograms are shown in Fig. 2Go.



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Figure 2. The AF-1 {alpha}-Helical Core Is Required for the Synergism Between hER{alpha} AF-1 and AF-2

HeLa (A), HepG2 (B), or CHO-K1 (C) cells were transfected with ERE-TK-Luc reporter and an internal control for transfection, pCH110. These cell lines exhibit differential sensitivity to the two hER{alpha} AFs, which is indicated on the left. The hER{alpha} wild type and the N-terminally truncated receptor hER{alpha} CF or point mutants (hER{alpha} L39P and hER{alpha} Y43P) were transiently expressed in these three cell lines, alone or in combination with SRC-1 or p68 RNA helicase. After 16 h, cells were treated for 36 h with EtOH or 10 nM E2. Cells were harvested, and the activities of both reporters were assessed. Luciferase activities were divided by the LacZ internal reporter activity, and the results are expressed as the percentage of the reporter fold induction in the presence of E2. A 100% value was ascribed to the transcriptional activity of hER{alpha} wild-type alone. Values shown in the histograms are means ± SEM from four independent experiments.

 
In HeLa cells, as anticipated, hER{alpha}-induced transactivation was enhanced 2-fold by SRC-1 but not by the specific AF-1 CoA p68. This was also observed with the hER{alpha} CF mutant and both hER{alpha} L39P and hER{alpha} Y43P mutants, although they lack either the whole N-terminal region or the AF-1 {alpha}-helix (Fig. 2AGo). These data reflect and confirm the AF-2-sensitive context of HeLa cells. Conversely, hER{alpha} transactivation ability was increased approximately 2.5-fold by both SRC-1 and p68 RNA helicase in HepG2 cells (Table 1Go). As expected in these cells, deletion of the N-terminal region (hER{alpha} CF) suppressed almost all hER{alpha} transactivation, and coexpression of p68 or SRC-1 with this mutant did not increase hER{alpha} transactivation (Fig. 2BGo). Similarly, replacement of the L39 or Y43 residue with proline gave rise to a 2-fold weaker induction of the reporter gene compared with that of wild-type hER{alpha}. The transactivation ability of these two hER{alpha} point mutants, however, was still enhanced by p68 but not by SRC-1. These results indicate that p68 coactivation does not require the presence of a functional AF-1 {alpha}-helical core structure. However, SRC-1-mediated enhancement of hER{alpha} activity absolutely requires this structure in an AF-1-sensitive cell context. Moreover, the strongest induction of hER{alpha} activity by SRC-1 was observed in CHO-K1 cells, in which both hER{alpha} AF-1 and AF-2 are functional. Indeed, as shown in Fig. 2CGo and Table 1Go, hER{alpha} transactivation was increased 5.6-fold by SRC-1, whereas an enhancement of 2.4-fold was obtained with the AF-1-specific CoA p68 RNA helicase. Deletion of the hER{alpha} A and B domains (mutant hER{alpha} CF) reduced by 2-fold the hER{alpha} activity and totally abolished p68 coactivation. SRC-1 functioned as a CoA for this mutant hER{alpha} CF, but resulted in only 2-fold induction. Finally, destruction of the AF-1 {alpha}-helical core structure in hER{alpha} (hER{alpha} L39P and hER{alpha} Y43P) did not reduce the p68 effect, although it decreased the SRC-1 enhancement of ER{alpha} activity 2-fold (Fig. 2CGo). These data clearly demonstrate that the AF-1 {alpha}-helical core is absolutely required for SRC-1 to mediate ER{alpha} AF-1 transcriptional activity in cells sensitive to AF-1 as well as in cells in which synergism occurs between AF-1 and AF-2.


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Table 1. Fold Enhancement of E2-Liganded hER{alpha} Activity by Two CoAs on the ERE-TK-Luc Reporter

 
The AF-1 {alpha}-Helical Core Physically Interacts with SRC-1 but Not with p68 RNA Helicase
To evaluate if SRC-1-mediated hER{alpha} AF-1 activity results from a physical interaction between the AF-1 {alpha}-helical core region and SRC-1, glutathione-S-transferase (GST) pull-down assays were performed. The hER{alpha} AF-1 {alpha}-helical core or a point mutant version that possesses no intrinsic transactivation ability, as assessed in yeast by fusion to the Gal4DBD (Fig. 3AGo), was fused to GST. The hER{alpha} AB domains or B domain alone, containing point mutations within the AF-1 {alpha}-helix core or at residues S104, S106, S118, and S167, known to be target sites for phosphorylation events (47, 48, 49, 50), were also fused to the GST. These fusion proteins were correctly expressed, as indicated by Coomassie blue staining (Fig. 3BGo) of SDS-PAGE gels containing the purified fusion proteins. Representative interactions of these GST fusion proteins with the SRC-1 and p68 CoAs are shown in Fig. 3Go, C–F. As expected (51), SRC-1 interacted with the C-terminal region of hER{alpha} in an E2-dependent manner, validating the pull-down conditions (Fig. 3CGo). Likewise, consistent with previous studies (28, 29), SRC-1 and p68 were able to directly interact with the hER{alpha} B domain; moreover, the presence of the A domain did not affect these interactions (Fig. 3Go, C and D). Whereas point mutations of the phosphorylation target serine residues had no effect on the interaction with SRC-1, substitution of S118 by alanine reduced contact with p68 (Fig. 3EGo). Strikingly, point mutations within the {alpha}-helical core of hER{alpha} AF-1 in the B domain markedly diminished (8-fold) the interaction of the B domain with SRC-1 but not with p68 (hER{alpha} B L39P and hER{alpha} B Y43P; Fig. 3EGo).



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Figure 3. The AF-1 {alpha}-Helical Core Interacts with SRC-1 but Not with p68 RNA Helicase

A, Gal4DBD alone or fused with either wild-type sequence for the {alpha}-helix or sequence harboring point mutations in the {alpha}-helix (to proline) were expressed in Y190 yeast cells in which the LacZ gene is under the control of three Gal4-responsive elements. After auxotrophy and growth selection in appropriate conditions, yeast cells were cultured in liquid medium, and the resulting ß-galactosidase activity was quantified and expressed as means ± SD from three independent experiments. B, Sequence encoding the AF-1 {alpha}-helical core was fused to the GST with additional alanine residues to detect the fusion protein. Wild-type hER{alpha} AB or B domains, as well as point-mutated B domains [Leu-39 to Pro (L39P), Tyr-43 to Pro (Y43P), Ser-167 to Ala (S167A), Ser-118 to Ala (S118A), and Ser-104 and Ser-106 to Ala (S104, 106A)], were fused to GST. Expression of these proteins was confirmed by Coomassie blue staining, shown on the right. GST pulldown assays were performed using 250 ng of GST fusion proteins with the AB, B, or DF domains of hER{alpha} and 2 µl of either [35S]methionine-labeled SRC-1 (C) or p68 (D) in the presence of EtOH vehicle or E2 where indicated. E, The influence of some point mutations within the hER{alpha} B domain on its interaction with SRC-1 or p68 was determined by GST pulldown assays, performed as in C, using GST B domain fusion proteins harboring the point mutations indicated. F, To determine the direct association of the AF-1 {alpha}-helical core with p160 or p68, GST fusion proteins with either the corresponding wild-type sequence or the point mutant were used in classic pulldown assays. Input lanes (I) represent 25% of the proteins used in the assay, and control procedures were performed using 250 ng of GST.

 
To directly assess the implication of this {alpha}-helical structure in the physical interaction between SRC-1 and the hER{alpha} N-terminal domain, GST pull-down experiments were performed with the wild-type or mutant AF-1 {alpha}-helix core region (Fig. 3BGo). Although p68 did not interact with these GST fusion proteins (Fig. 3F, bottom), SRC-1 was specifically retained on beads coupled to the wild-type form of the AF-1 {alpha}-helical core region (Fig. 3FGo, top). A point mutation in this sequence, which abolished both structure and transactivation property (43 ; Fig. 3AGo), also totally inhibited the SRC-1 interaction with the GST fusion proteins. Therefore, these data demonstrate that the hER{alpha} AF-1 core region spatial organization is required for SRC-1 interaction.

SRC-1 Recruitment Is Enhanced When Both hER{alpha} N- and C-Terminal Domains Are Present
The SRC-1 protein was shown to mediate synergism between AF-1 and AF-2 in cells sensitive to both hER{alpha} AFs, such as CHO-K1 cells (Fig. 2CGo). Therefore, we determined whether this synergism could be correlated with an increased recruitment of SRC-1 in the presence of both the hER{alpha} N- and C-terminal domains. Competition experiments in GST pull-down assays were performed using as bait either hER{alpha} AB (or B) (Fig. 4AGo) or DF (Fig. 4BGo) domains fused to GST. As anticipated, complexes were formed between hER{alpha} AB (or B) or DF fusion proteins with labeled SRC-1, and these were competed by increasing amounts of AD or liganded CF regions, respectively (Fig. 4Go, A and B, left). In the same way, full-length hER{alpha} was able to decrease N- and C-terminal interactions with SRC-1 in the presence of estrogens (Fig. 4Go, A and B, middle). In contrast, increasing the amounts of hER{alpha} regions complementary to those fused to GST enhanced the recruitment of SRC-1 (Fig. 4Go, A and B, right). Similar experiments performed with p68 RNA helicase showed that recruitment of this CoA by the N-terminal region of hER{alpha} was not affected by the presence of the hER{alpha} C-terminal domain (data not shown). Therefore, the presence of both N- and C-terminal domains of hER{alpha} results in a specific increase in SRC-1 CoA recruitment, which might explain hER{alpha} AF-1/AF-2 synergism obtained in cells in which both AFs are functional (Fig. 2Go and Table 1Go).



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Figure 4. The hER{alpha} N and C Termini Cooperatively Recruit SRC-1

The binding of 1 µl of in vitro labeled SRC-1 to 250 ng of GST fusion proteins in either the hER{alpha} AB or B (A) or DF (B) domains was competed by increasing amounts of in vitro produced proteins as indicated at the top of each gel (0.5- to 4-fold). Equal amounts of each competitor were determined by labeling an aliquot of the rabbit reticulocyte lysate and then estimating the molar concentration by taking account of their specific radioisotopic activity. The hER{alpha} full-length or hER{alpha} CF domains were used after incubation with EtOH (-E2) or 50 µM E2 (+E2). Input lanes are 25% of proteins used in the assays, whereas control lanes represent assays performed with 250 ng of GST.

 
Evidence for an Agonist-Enhanced and CoA-Independent Direct Physical Association Between hER{alpha} N- and C-Terminal Regions
At least two mechanisms might lead to the enhancement of SRC-1 recruitment in the presence of both the N- and C-terminal regions of ER{alpha}. First, SRC-1 protein complexed with one ER{alpha} AF domain might result in a higher affinity for the other AF domain. Second, additional interactions involving surfaces different from those involved in the initial recruitment of SRC-1 may also lead to an increase in SRC-1 binding. Such additional contacts might imply a direct interaction between the N- and C-terminal regions of ER{alpha}. To determine whether such physical contacts between these modules of hER{alpha} could occur, in vivo experiments using yeast two-hybrid assays were performed. However, a positive result in these assays could reflect either the existence of a direct interaction or the occurrence of a ternary complex composed of hER{alpha} B and C-terminal domains bridged by a third protein such as a CoA. Therefore, it was critical to ensure that any functional interactions detected between ER{alpha} N- and C-terminal regions were not caused by the establishment of a CoA-mediated bridge between the AF domains. We decided to use mutated or deleted ER{alpha} N-terminal regions that are unable to transactivate ER element reporter constructs in yeast and thus incapable of interacting with CoAs.

As illustrated in Fig. 5AGo, the AF-1 {alpha}-helical core is not the only AF-1 CoA contacting region within hER{alpha} (30, 44), because hER{alpha} {Delta}1–44 and hER{alpha} {Delta}1–38 L39P receptors exhibit residual AF-1 activity. However, deletion of this structure within the rtER{alpha} abolishes AF-1 transactivation in yeast (Fig. 5BGo). For this reason, the yeast two-hybrid approach was used with rtER{alpha}/Gal4AD (activation domain) fusion proteins. Moreover, the fact that the rtER{alpha} AF-1 {alpha}-helical core is able to replace the hER{alpha} core (44) legitimizes this comparative approach. Fusions of the rtER{alpha} DF and rtER{alpha} B domains either with or without the AF-1 helical core (B and B{Delta}8–17) were generated (Fig. 5CGo). Y190 yeast cells were transformed with different combinations of these constructs and, after auxotrophy selection, treated for 4 h with ethanol (EtOH) as control, 10-8 M E2, or 10-5 M of the anti-estrogens OHT and ICI164,384 (ICI). Interestingly, the results indicated that a functional interaction between the N- and C-terminal regions of rtER{alpha} occurred whether or not the B domain contained the AF-1 helical region (Fig. 5CGo). Moreover, this functional association was enhanced 5-fold in the presence of full or partial agonists (E2 or OHT, respectively), but not in the presence of ICI164,384. Furthermore, we observed the functional dimerization of two rtER{alpha} C-terminal domains, which was strictly ligand dependent, as anticipated (7, 52). These two-hybrid data indicate that the ER{alpha} B domain is able to interact with the ER{alpha} C-terminal region. This is most likely to occur without a CoA bridge, because an ER{alpha} B domain devoid of CoA interaction surface could contact the C-terminal domain. Thus, the association detected in the yeast two-hybrid experiments between the N- and C-terminal regions of ER{alpha} is probably the result of physical contact between the two regions.



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Figure 5. The Functional Interaction Between ER{alpha} N- and C-Terminal Regions Does Not Result from a Bridging Property of SRC-1

Yeast strain BJ2168 was transformed by the pLG{Delta}178/3EREc vector together with plasmids encoding hER{alpha} AD, hER{alpha} CF, hER{alpha}{Delta}1–37, hER{alpha}{Delta}1–44, or hER{alpha}{Delta}1–37L39P (A) or rtERS, rtER CF, rtER BD, or rtER{Delta}8–17 (B). Reporter activity was assessed as in Fig. 2Go after 4 h of incubation with either EtOH as vehicle control or 10 nM E2. Results shown are means ± SD from at least four independent experiments. C, To assess for an association between the N- and C-terminal domains of rtER, two-hybrid experiments were conducted using the Gal4DBD and Gal4AD fusion proteins alone or in combination, as illustrated at the left. After Y190 yeast clone selection for growth and auxotrophy, yeast cells were cultured in liquid medium and treated for 4 h with EtOH, 10 nM E2, or 10 µM OHT or ICI164,384. Specific ß-galactosidase activity was quantified and is expressed as means ± SD from at least four independent experiments.

 
The hER{alpha} C-Terminal Region Physically Interacts with the B Domain and Induces Its Hyperphosphorylation
To test the possible occurrence of a direct association between the N- and C-terminal regions, GST pull-down assays were performed using the A, B, or AB domain as bait. We have previously defined a physical interaction between the A domain and the C-terminal region of ER{alpha} that was disrupted in the presence of ligands (44). Therefore, we wished to discriminate between interactions mediated by the B domain and those involving the A domain. As illustrated in Fig. 6AGo, the hER{alpha} B domain fused to the GST was able to interact directly with the hER{alpha} C-terminal (DF) region under pull-down conditions. This interaction occurred in a ligand-dependent manner, reproducing previous yeast two-hybrid results (Refs. 34, 36 , and 37 and our unpublished results). E2 and OHT but not ICI164,384 were able to promote induction of this interaction, although a weak but significant physical contact was also detected in the absence of ligand. Furthermore, results obtained with the A domain fused to the GST were in agreement with those previously published (44). Finally, compared with the A or B domain alone, the combined AB domains had better interaction with the hER{alpha} C-terminal region. Already significant in the absence of ligand, this association was enhanced 3- to 4-fold in the presence of ligands, although ICI164,384 actively repressed this hER{alpha} N- and C-terminal interaction. These data provide evidence that in the full-length ER{alpha} context, the interactions of the A and B domains with ER{alpha} C-terminal regions occur in an integrated manner. However, the AF-1 {alpha}-helical core region was not involved in these interactions (Fig. 6BGo). Indeed, point mutations within the AF-1 helical structure did not abrogate the hER{alpha} N- and C-terminal interaction, and a GST fusion protein that included only the AF-1 {alpha}-helix was unable to retain the C-terminal region of hER{alpha} on the beads (Fig. 6CGo).



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Figure 6. Evidence That a Direct Interaction Between hER{alpha} N- and C-Terminal Domains Induces N-Terminal Region Hyperphosphorylation

A, GST pull-down experiments were performed using 250 ng of GST fusion proteins with the hER{alpha} A, B, or AB domains. Beads coated with these proteins were incubated for 3 h with 2 µl of in vitro produced and labeled hER{alpha} DF domain treated with EtOH as control, 50 µM E2, or 500 µM OHT or ICI164,384 (ICI). B and C, Each GST fusion protein indicated (250 ng) was assessed for their interaction with 2 µl of labeled hER{alpha} DF domain either free or containing 50 µM E2. Input lanes represent 15% of the proteins used in the assay, and the control lanes represent assays performed with GST. D, hER{alpha} AB or hER{alpha} B wild-type or point mutant domains (250 ng) fused to GST were first used to pull down in vitro translated hER{alpha} DF before being phosphorylated by endogenous kinases from 10 µg of CHO-K1 whole cell extract (WCE) in the presence of 10 µCi of {gamma}-32P. Reactions were performed 30 min at 30 C, after which the beads were washed. Proteins were eluted by boiling in SDS-PAGE sample buffer and analyzed by SDS-PAGE. To control for equivalent loading, gels were stained with Coomassie blue before drying and autoradiography.

 
To date, most if not all of the NRs studied have been found to be phosphoproteins (53, 54). In addition to providing a ligand-independent activation of NRs through protein kinase-mediated pathways, phosphorylation is a key event that could modulate DNA binding or CoA recruitment during ligand activation (53, 54). As with other NRs, ERs are phosphoproteins whose phosphorylation state is enhanced on binding ligand (53, 54). One way to induce hER{alpha} N-terminal phosphorylation on ligand binding to the C-terminal LBD could be through intramolecular signaling between these two regions, as for PPAR (55). Therefore, to unequivocally illustrate the occurrence and crucial importance of this interaction in hER{alpha} functions, we determined whether the hER{alpha} N- and C-terminal interaction had further functional importance than the recruitment of SRC-1. We developed a new in vitro phosphorylation assay in which protein complexes from GST pull-down experiments were incubated with whole-cell extracts. The cell extract was used to provide appropriate kinase activity(ies). As illustrated on Fig. 6DGo, rabbit reticulocyte lysate had no effect on the phosphorylation status of a GST/hER{alpha} AB, GST/hER{alpha} B, or GST/hER{alpha} BL39P fusion protein. However, the ligand-dependent interaction of the hER{alpha} C-terminal region with these N-terminal domains increased the incorporation of 32P. Because this also occurred with GST/hER{alpha} BL39P, this increase in phosphorylation reflects the physical interaction between the hER{alpha} N- and C-terminal regions.

Ligand Determinants in the Direct ER{alpha} N- and C-Terminal Domain Physical Interaction and Links to SRC-1-Mediated Synergism
Throughout our experiments, OHT was able to mimic E2 action in the interaction between the N and C termini of hER{alpha} in two ways: it promoted dissociation of the A domain from the C-terminal region and enhanced the interaction of the B domain with the C terminus. These results, in conjunction with OHT being a partial hER{alpha} agonist whose activity depends on hER{alpha} AF-1 (19, 20), led us to assess the role of the interaction between the hER{alpha} N and C termini in OHT agonism. We first performed transient transfection experiments with the human complement C3 promoter region (-307 to +58), a well characterized OHTresponsive promoter (56, 57). As shown in the response curves (Fig. 7AGo), the maximum hER{alpha} activity under our transfection conditions was obtained with high doses of OHT (i.e. 5 x 10-6 M). Control experiments using the ERE-TK-Luc reporter (Fig. 7BGo) demonstrate that 5 x 10-6 M OHT alone cannot induce hER{alpha} activity on this reporter construct, indicating that the human C3 activation by OHT-liganded hER{alpha} is specific. Moreover, this dose was the most efficient one competing for E2-induced hER{alpha} activity (Fig. 7BGo). Therefore, the involvement of SRC-1 and the N- and C-terminal interaction in AF-1 activation by OHT was determined using these conditions and was evaluated in HeLa, HepG2, and CHO-K1 cell lines (Fig. 7Go, C, D, and E, respectively). Transient transfection experiments were carried out using constructs expressing hER{alpha}, hER{alpha} CF, or the hER{alpha} L39P and hER{alpha} Y43P point mutants, together with SRC-1 or p68, which was used as a control. In HeLa cells, hER{alpha} and all mutants used were able to induce the human C3 (-307/+58)-Luc reporter only when liganded to E2, demonstrating that OHT is unable to activate AF-2 (20, 46). Furthermore, in the presence of E2, only SRC-1 and not the specific AF-1 CoA p68 was able to coactivate hER{alpha} transcriptional activity, albeit to a lesser extent than on the ERE-TK-Luc reporter (1.5-fold vs. 2-fold; Fig. 7CGo and Tables 1Go and 2Go).



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Figure 7. Partial Agonism of OHT Requires That the hER{alpha} AF-1 Helical Core Region Activity Is Enhanced by SRC-1

To examine OHT agonism, transient transfection experiments were carried out using the human complement C3 promoter (-307/+58) controlling luciferase gene expression. A, HepG2 cells were transfected with this reporter, an internal control for transfection (pCH110), and vector directing expression of the hER{alpha} protein. After 16 h of transfection, cells were treated for 36 h with EtOH or with increasing concentrations of either E2 or OHT. Normalized luciferase activities are indicated as means ± SEM from three independent experiments. B, To control for OHT antagonism under our transfection conditions, HepG2 cells were transiently transfected using the ERE-TK-Luc reporter, pCH110, and pSG5/hER{alpha}. After 16 h of contact with the precipitate, cells were treated with EtOH or with increasing concentrations of E2 (closed circles) or OHT (open circles) or simultaneously with 10-8 M E2 and increasing concentrations of OHT (open triangles). Transcription was assessed and expressed from the results of three independent experiments. C, D, and E, The three cell lines HeLa, HepG2, and CHO-K1, which have differential sensitivity to the different hER{alpha} AFs, as indicated at the left, were transfected with pCH110 and the human C3 (-307/+58)-Luc reporter and different combinations of the expression vectors, as indicated at the bottom. Cells were treated with EtOH, 10-8 M E2, or 5 x 10-6 M OHT. Luciferase activities were normalized by LacZ internal reporter activity, and the results are expressed as the percentage of the reporter fold induction in the presence of E2. A value of 100% was ascribed to the transcriptional activity of hER{alpha} wild- type alone. Values shown in the histograms are means ± SEM from three independent experiments.

 

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Table 2. Fold Enhancement of hER{alpha} Activity by Two CoAs on the Human C3 (-307/+58)-Luc Reporter

 
In HepG2 cells, both SRC-1 and the p68 RNA helicase were able to coactivate E2-liganded hER{alpha} activity on the human C3 (-307/+58)-Luc reporter by 2-fold (Fig. 7DGo and Table 2Go). In the presence of OHT, SRC-1 but not p68 was able to coactivate wild-type hER{alpha} protein activity, again by 2-fold. This indicates that p68 is not involved in AF-1-mediated activity when hER{alpha} is complexed with this ligand. SRC-1-mediated coactivation of OHT-liganded hER{alpha} on this reporter construct depends on AF-1, because neither SRC-1 nor p68 was able to induce transactivation activity from hER{alpha} CF protein in the presence of OHT. Consistent with the data presented in Fig. 1Go, SRC-1 was unable to increase the activity of the hER{alpha} L39P and hER{alpha} Y43P point mutant proteins in HepG2 cells, illustrating the absolute requirement of the AF-1 helical motif in transactivation by E2-liganded, hER{alpha}-mediated coactivation by SRC-1. This is a similar pattern of coactivation to that obtained with the ERE-TK-Luc reporter, but we demonstrate, for the first time, that the AF-1 helical core is also required for AF-1 activation by OHT, because SRC-1 was unable to enhance the activity of the two point mutant proteins when they were liganded to OHT. As previously seen, CHO-K1 cells have a cell context sensitive to both hER{alpha} AFs. However, the pattern of activation obtained in these cells was similar to that observed in HepG2 cells. This demonstrates the predominance of AF-1-mediated transactivation in hER{alpha} activation of the human C3 promoter. From all of these results, we conclude that the hER{alpha} AF-1 helical core region is required for the agonistic activities of OHT, which mostly rely on AF-1 (20). Moreover, this structure is necessary for SRC-1 to enhance hER{alpha} activity when it is bound to this molecule.

The hER{alpha} C-terminal region, when associated with OHT, was shown to exhibit an altered conformation that could not recruit p160 CoAs (14, 58 ; Fig. 8AGo). Because these transfection results could also reflect altered SRC-1 recruitment by the C-terminal region, we performed, as described previously, GST pull-down assays with increasing amounts of either hER{alpha} full-length or hER{alpha} CF domains in the presence of OHT or ICI164,384. Interestingly, increasing amounts of OHT-liganded hER{alpha} resulted in a decrease in the amount of SRC-1 retained by the GST/hER{alpha} AB fusion protein (Fig. 8BGo, top). However, increasing the amount of hER{alpha} CF protein, either unliganded (-OHT) or liganded to either of the two anti-estrogens (+OHT, +ICI), did not affect the interaction of SRC-1 with the hER{alpha} AB domain (Fig. 8BGo, bottom). These results are likely to indicate that the spatial organization of the OHT-liganded hER{alpha} C-terminal region is unable to support a recruitment of SRC-1 (Fig. 8AGo) and is thus also unable to cooperatively recruit this CoA through interaction with the hER{alpha} N- and C-terminal regions. More importantly, these experiments demonstrate that within hER{alpha}, OHT is able to induce a functional conformation that favors SRC-1 recruitment, because full-length hER{alpha} is able to compete for this process. Furthermore, the fact that the isolated hER{alpha} C-terminal region is unable to reproduce this decrease in SRC-1 interaction with the hER{alpha} AB domain demonstrates that this is caused by competition for SRC-1 recruitment to the same hER{alpha} region (i.e. the B domain). Thus, OHT binding in the context of hER{alpha} mimics E2 action by inducing an interaction between the B domain and the C-terminal regions, which in turn allows the B domain to recruit SRC-1.



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Figure 8. Assessment of the Involvement of the hER{alpha} N- and C-Terminal Interaction in OHT Partial Agonism

A, GST pulldown assays were performed using 250 ng of GST/hER{alpha} DF fusion protein with 2 µl of [35S]methionine-labeled SRC-1 in the presence of EtOH, 50 µM E2, or 500 µM OHT or ICI164,384 (ICI) as indicated. B, Binding of 1 µl of in vitro labeled SRC-1 to 250 ng of GST/hER{alpha} B protein was competed by increasing amounts of in vitro produced hER{alpha} or hER{alpha} CF, as indicated at the top of each gel (0.5- to 4-fold) after treatment with EtOH or 500 µM OHT or ICI164,384. Input lanes are 25% of proteins used in the assays, whereas control lanes represent assays performed with 250 of GST.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We recently identified the ER{alpha} AF-1 core as a short segment that has been conserved during evolution, likely organized as an {alpha}-helix and that is positioned within the beginning of the B domain (amino acids 39–45 in hER{alpha}) (Ref. 44 and Fig. 1AGo). Helical structures are involved in many protein-protein contacts, including those established between NR AF-2 regions and their coregulators (23, 24, 25). In this study, we wished to characterize the role of the AF-1 {alpha}-helical structure in hER{alpha} function, particularly in the recruitment of p160 subfamily CoA proteins such as SRC-1. A previous report had shown that the major surface of the hER{alpha} N-terminal region interacting with p160 lies between amino acids 38 and 64, thereby overlapping the AF-1 {alpha}-helical sequence (31 ; Fig. 1AGo). Transfection experiments in cell lines that exhibit differential responses to hER{alpha} AF-1 and AF-2 revealed that this structure was absolutely required for an enhancement of hER{alpha} AF-1 activity by SRC-1. Moreover, in cells responsive to both hER{alpha} AF-1 and AF-2, such as CHO-K1 cells, we demonstrate that this AF-1 structure is essential for synergism between hER{alpha} AFs. Furthermore, this structure was found to recruit SRC-1 and to be responsible for approximately 70% in the interaction between SRC-1 and the hER{alpha} N-terminal region. The length and sequences of NR N-terminal regions are highly divergent among individual AF-1 structures depending on the receptor. Moreover, despite data describing secondary structures within the N-terminal region of NRs, there may not be a general rule for all of the superfamily members, because SF-1 and VDR possess very short AB domains (59). In the case of GR, three {alpha}-helices reside within its {tau}1 core region (60), allowing interactions with some TATA box protein- associated factors (TAFIIs) and other general transcription factors (61). AR also contains a structural unit within its AF-1a that is organized around a ß-turn and an {alpha}-helix (62), as does the short AF-1 of HNF-4, which also has an {alpha}-helix (63). These structures could contact general transcription factors (64, 65). However, this report provides the first example to our knowledge of an interaction between an AF-1 structure and a p160 CoA protein.

SRC-1, and more generally p160 members, use different regions to interact with the N- and C-terminal regions of ER{alpha}. EF domains make contact through the NR boxes, whereas AB domains use the glutamine-rich region (Refs. 31 and 32 and Fig. 1BGo). This could signify that the interaction between the hER{alpha} AF-1 helical core and SRC-1 could require both structural and charge components. Interestingly, the proposed AF-1 {alpha}-helix contains positively charged residues. However, disrupting the structure through proline substitution abolished the interaction with SRC-1. This likely reflects a critical role for the structure of the hER{alpha} AF-1 core. In contrast to SRC-1, p68 RNA helicase is not involved in a synergistic process between hER{alpha} AFs. This is likely to occur, because p68 is not recruited to the hER{alpha} N terminus through the hER{alpha} AF-1 helical structure but through a region encompassing the phosphorylated S118 residue (29). In consequence, we observed that the recruitment of SRC-1 and p68 by hER{alpha} N-terminal region is not mutually exclusive (data not shown), indicating that these two proteins may be recruited concomitantly.

hER{alpha}, like other NRs, exhibits modular properties. Functions have been ascribed to individual domains, but some data are now consistent with a more flexible organization, because some functional aspects require the entire receptor. Indeed, binding to DNA results in allosteric control of NR function (66) and was recently shown to affect N-terminal region structures in both PR-A and GR (67, 68) or the overall ER{alpha} helical content (69). Such interrelations between different domains of NR were considered possible between the N- and C-terminal regions of ER. Indeed, in the absence of ligand, AF-1 ligand-independent activity is repressed by C-terminal domains in full-length protein (6, 19, 70). The synergism between hER{alpha} AFs was also ascribed to a functional interrelation between these two regions of hER{alpha}, and it was proposed that this was mediated through CoA recruitment (36, 37). However, it was not clear if hER{alpha} N- and C-terminal domains were able to interact directly with each other. Indeed, a bridging CoA, able to interact with both regions, was hypothesized to be necessary for an indirect interaction between hER{alpha} N and C termini. However, pull-down competition assays unexpectedly indicated that SRC-1 interaction with one region was enhanced by the other. This observation did not reflect a bridging property of SRC-1, because in this case either no changes in the amounts of retained SRC-1 or a reduction in these amounts attributable to competition for labeled SRC-1 should have occurred. This enhancement in SRC-1 recruitment provided evidence for a third interaction surface. We then went on to prove, using two different approaches, that a direct interaction between the hER{alpha} N- and C-terminal regions occurs and that this does not require the AF-1 {alpha}-helical core. This is similar to situations already described for other NRs such as the PR and the AR (38, 39, 40, 41, 42, 43). Our data provide evidence for a functional importance of this direct interaction between the N- and C-terminal regions of hER{alpha} at two levels. First, a cooperative recruitment of SRC-1 can occur, and second, hyperphosphorylation of the receptor N-terminal region can occur. This could be a general rule for other members of the NR superfamily, because N-terminal domain phosphorylation of some NRs was shown to be modulated by their C-terminal regions (54, 55, 71).

Our results are in sharp contrast with those of two recent reports, which failed to detect any direct interaction between the hER{alpha} N and C termini. Consequently, bridging properties were ascribed to NCoA-2 and CBP/p300 (34, 35). These conflicting results may arise from different conditions used in pull-down assays, particularly because Kobayashi et al. (35) were unable to find a recruitment of p160 proteins to hER{alpha} or hERß N-terminal domains, which has been demonstrated by several other groups (31, 32, 33). These two reports were also compromised by results obtained in a mammalian two-hybrid system, which is affected by the endogenous expression of NCoA-2 and CBP/p300 proteins. Finding a direct interaction between the N- and C-terminal domains would resolve and simplify these indirect observations. We propose that the physical interaction between isolated N- and C-terminal domains in the presence of ligand occurs and results in an enhanced recruitment of endogenous CoAs such as p160 or CBP/p300. The resulting transcriptional activation using isolated hER{alpha} N- and C-terminal domains in combination, as previously reported (36), reflects AF-1/AF-2 synergism. In the study by Benecke et al. (34), however, this transcriptional activity was not observed, perhaps because they used a less sensitive system. Because a significant promoter activation required increasing amounts of p160 to be detected, these authors suggested that the p160s acted as a bridging protein, because detectable synergism between the hER{alpha} N and C termini was observed only when they were present. In the report by Kobayashi et al. (35), transcriptional activity attributable to interaction between the N and C termini was observed under basal conditions, reproducing previous results (36, 37). We propose that, in this case, the p160 coactivation of hER{alpha} was maximal, as a result of the saturation of transcription factors involved in hER{alpha} recruitment of the transcription machinery. This may be attributable to the CBP/p300 integrator, which is limiting for transcriptional activation in cells (72). Increasing the amounts of available CBP/p300 by transfection would relieve this process and allow greater stimulation of the reporter gene construct. In this case, CBP/p300 was considered as a bridging factor between the hER{alpha} N- and C-terminal regions mediating synergism between AF-1 and AF-2. Finally, our proposals imply that physical interaction between the B and C-terminal domains could involve different regions than those determined for the hER{alpha} B domain interrelations with CBP/p300 and p160s.

We also show that, in addition to interacting with the B domain, the hER{alpha} C-terminal region interacts with the A domain, and this physical contact is reduced in the presence of agonists (44). A complex network of interrelations occurs between hER{alpha} N- and C-terminal domains, because this interaction is also responsible in part for the inhibition of the hER{alpha} AF-1 in the absence of ligand. Two contrasting effects can occur after the interaction of the AB domain with the C-terminal part of hER{alpha}, a suppression of activity mediated by the A domain that occurs in absence of ligand, and the ligand-induced synergism between AF-1 and AF-2 caused by the B domain. These two mechanisms appear to be independent of each other because the A domain has no influence on the cooperative recruitment of SRC-1 by the B domain and the C-terminal region. This indicates that precise molecular determinants discriminate between the influences of two adjacent regions. The binding of hER{alpha} agonists is central in this process, because they are able to switch the negative effect of a direct interaction (A domain/C-terminal region) to an interaction between the B domain and the C-terminal region that results in transactivation. Thus, it is interesting that a proline-rich stretch or a glycine/alanine-rich sequence is present at the end of all known ER{alpha} A domains. These sequences may provide a hinge region between the A and B domains and result in the structural involvement of this region in the agonist-mediated activation of hER{alpha}. Structural changes in this hinge region geometry may cause a reorientation of hER{alpha} AF-1 with AF-2, resulting in SRC-1-mediated synergism in the presence of ligand.

One of the most interesting features of AF-1 is its involvement in OHT agonism, because AF-1 seems to be the only hER{alpha} AF to be activated when hER{alpha} binds OHT (19, 20). This is supported by our observation of a strong correlation between OHT agonism and the ability of this compound to mimic E2 action in the interaction between the hER{alpha} N- and C-terminal regions. Both E2 and OHT binding inhibit the interaction of the C-terminal region with the A domain, thereby promoting an interaction between C-terminal domains and the B domain. We thus hypothesized that OHT-mediated activation of AF-1 might involve an interaction between SRC-1 and the N- and C-terminal regions of hER{alpha}. Using the human C3 promoter (-307/+58 region) as an OHT-responsive promoter (56, 57), we show that the interaction of SRC-1 with the AF-1 helical core is required for an enhancement of hER{alpha} activity in the presence of OHT. This highlights the importance of this structure in AF-1 function and in the induction of transactivation by agonists and also the involvement of SRC-1/N-terminal/C-terminal interrelations in OHT agonism. Correspondingly, within the full-length receptor, AF-1 is a ligand-induced transactivation domain. Binding of E2 or OHT to the LBD results in an activation of the "far" N-terminal AF-1, likely as a result of a direct interaction between these regions. OHT was unable to promote a coactivation of hER{alpha} activity by the p68 RNA helicase, indicating that the B domain conformation and/or the S118 phosphorylation (29) is inadequate for such a process within OHT-complexed hER{alpha}. In vitro pull-down experiments demonstrated that OHT is able to induce a specific conformation within full-length hER{alpha}, allowing AF-1 to function through the recruitment of SRC-1. Conversely, in the presence of OHT, there was no cooperative recruitment of SRC-1 by the hER{alpha} B and C-terminal domains, resulting in a 2-fold enhancement of AF-1 activity by this CoA in cells responsive to AF-1. Therefore, it seems that although the C-terminal domain interacts with the B domain in the presence of OHT, this physical contact does not provide a conformation that can mediate a synergistic effect of SRC-1 by both hER{alpha} AFs, reflecting the inability of SRC-1 to interact with the C-terminal region when hER{alpha} is complexed with OHT.

Our data show that E2- and OHT-bound hER{alpha} are able to recruit SRC-1 mostly through the AF-1 helical core. This implies that the different conformations induced by E2 and OHT result in the activation of AF-1 but that OHT acts solely on AF-1. This demonstrates that the activation of AF-1 by partial or full agonists relies on common structural features within the known crystal structures of the hER{alpha} C-terminal region when complexed with E2 or OHT. During the course of these experiments, we unexpectedly found that OHT was able to induce hER{alpha} CF activity to 35% of the full-length protein activity in cell types sensitive to AF-1, such as CHO-K1, HepG2, or yeast (data not shown). However, this transactivation ability could not be related to the AF-2 core, which is unable to generate cofactor-binding surfaces upon binding OHT (58). However, the AF-2a subdomain was reported to function in AF-1-sensitive cells (18). Therefore, we suggest that OHT is able to induce this AF-2a function in such cells. This extends the potential agonist properties of OHT, which is considered a strict antagonist of the C-terminal functions of hER{alpha} (73).

We provide evidence of a recruitment of SRC-1 to hER{alpha} that depends on the AF-1 helical core. Moreover, SRC-1 is cooperatively recruited to full-length hER{alpha} through AF-1 and AF-2, and this is caused by a direct association of the B domain with the C-terminal region of hER{alpha}. A precise understanding of the stoichiometry of the hER{alpha}/SRC-1 complex is now required. It was recently shown that one SRC-1-derived protein is complexed with each hER{alpha} dimer (74). As a consequence, the SRC-1 monomer interacting with the hER{alpha} C-terminal dimer could also interact with the hER{alpha} B domain, or the B domain could recruit another SRC-1 monomer. Other CoAs, such as CBP/p300, which could be recruited to an hER{alpha}/p160 subfamily CoA complex, were found to interact directly with both hER{alpha} AFs, but to another sequence/structure within the N-terminal domain. This leads to an emerging concept in which multiple combinatorial protein-protein interactions are required for full NR activity (26). Other proteins, however, cannot be excluded, such as the DRIP/TRAP complexes that may bridge the N- and C-terminal domains. Indeed, one component (DRIP150) of these Mediator-like complexes was shown to be recruited by GR {tau}1, presumably through its potential structures (75). Other CoA complexes not yet identified may be required to fulfill hER{alpha} AF-1 function and to dictate the cell specificity of hER{alpha} function. Indeed, although considerable advances have been gained in understanding the mechanisms underlying hER{alpha} AF-2 function by isolating many CoA complexes, no clear scheme is emerging to describe the differential sensitivity of different cell types to AF-1 or AF-2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Receptor Constructions and Reporter Plasmids
Construction of the pSG5/hER{alpha} L39P, pSG5/hER{alpha} Y43P, pSG5/hER{alpha} AD, YEPrtERS, YEPrtER CF, YEPrtER{Delta}8–17, YEPhER{alpha} CF, YEPhER{alpha}{Delta}1–37, and YEPhER{alpha}{Delta}1–37L39P vectors, the yeast reporter pLG{Delta}178/3EREc construct, and the plasmids encoding the GST/hA, hER{alpha}35–47/Gal4DBD, hER{alpha}35–47L39P/Gal4DBD, and hER{alpha}35–47Y43P/Gal4DBD fusion proteins have been described (44, 76). The diverse Gal4DBD and Gal4AD fusion proteins were obtained by subcloning the corresponding inserts in either pAS2-1 or pACT-2 vectors (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively. The GST/hER{alpha} DF protein was expressed using the pGEX2TK/hER{alpha}282–595 plasmid, a gift from Dr. B.S. Katzenellenbogen (77). GST/hER{alpha} AB, GST/hER{alpha} B, GST/hER{alpha} B L39P, GST/hER{alpha} B Y43P, GST/hER{alpha} B S118A, GST/hER{alpha} B S167A, and GST/hER{alpha} B S104A,S106A were expressed using pGEX2T (Pharmacia, Little Chalfont, Buckinghamshire, UK) constructs containing the corresponding PCR inserts. The serine point mutant used as a PCR matrix [hER{alpha} S118A (HE457)] was a gift from Prof. P. Chambon (78), whereas the hER{alpha} S167A and hER{alpha} S104A,S106A were kindly provided by Dr. B. S. Katzenellenbogen (47). The GST fusion proteins with the hER{alpha} AF-1 {alpha}-helical core sequence were obtained by subcloning double-stranded oligonucleotides within the SmaI-EcoRI sites of the pGEX2T vector. All of these fusions were verified by sequencing. For transient expression in mammalian cells and in vitro expression in rabbit reticulocyte lysate, we used the pSG5/hER{alpha} and pSG5/hER{alpha} p46 (hER{alpha} CF) vectors and the ERE-TK-Luc reporter gene, which were gifts from Prof. F. Gannon (45). The human C3 (-307/+58)-Luc reporter was obtained by nested PCR on human genomic DNA (CLONTECH Laboratories, Inc.) using 5'-AACTGGGGATGAGGTCCAAGACATC-3' and 5'-TCCATGGTGCTGGGACAGTGCAGGG-3' as forward and reverse primers, respectively. These oligonucleotides were designed as referred to the human complement C3 promoter sequence (Ref. 79 ; accession no. X62904). The second round of amplification was performed with internal primers possessing a SmaI or BglII restriction site in 5' and 3', respectively. This SmaI-BglII insert was inserted within the corresponding sites of the pGL2-Basic vector (Promega Corp., Madison, WI). Plasmids encoding the SRC-1 and p68 RNA helicase CoAs were gifts from Dr. M. J. Tsai (51) and Dr. S. Kato (29), respectively.

In Vitro Translation in Rabbit Reticulocyte Lysate
Proteins were in vitro synthesized using the T7 RNA polymerase in the rabbit reticulocyte-coupled transcription/translation kit (Promega Corp.), as recommended by the manufacturer. Labeled protein expression was monitored by estimating the relative amounts of protein on SDS-PAGE gels. To study the ligand effects, lysates were submitted to EtOH, 50 µM E2 (Sigma, St. Quentin-Fallavier, France), 500 µM OHT or ICI164,384 (from Dr. A. Wakeling, ICI Pharmaceuticals, Macclesfield, Cheshire, UK) treatments for 1 h in TEG buffer (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 50 mM NaCl, 10% glycerol, 5 mM MgCl2, and 1 mM dithiothreitol).

Host Strains
The bacteria strain used for subcloning and protein expression was Escherichia coli DH5{alpha} [supE44{Delta}lacU169 (Ø80 lacZ{Delta}M15) hsdR17 Rec A1 end A1 gyrA96 thi-1 relA1]. The yeast strains used in this study were Y190 (CLONTECH Laboratories, Inc.; MAT{alpha}, ura 3-52, his 3-200, ade 2-201, lys 2-801, trp 1-901, leu 2-3, 112, gal 4{Delta}, gal 80{Delta}, cyhr 2, LYS2::GAL1UAS-HIS3TATA-HIS3, URA3::GAL1UAS-GAL1TATA- LacZ) and BJ2168 (Yeast Genetic Stock Center, Berkeley, CA; a leu2 trp1 ura3-52 prb 1-1122 pep4-3 prc1-407 gal2) for the transactivation assay. Yeast cells were transformed using a modified lithium acetate method (76). Selection of stable transformants and liquid ß-galactosidase assays were performed as previously described (44, 76).

Bacterial Production of GST Fusion Proteins and GST Pull-Down Assay
After 4 h of expression with 0.1 mM isopropyl ß-D-thiogalactoside, E. coli DH5{alpha} cells were harvested by centrifugation, resuspended in NETN buffer [100 mM NaCl, 20 mM Tris, pH 8, 1 mM EDTA, 0.5% Nonidet, 1 mM phenylmethylsulfonyl fluoride (PMSF), and the protease inhibitors leupeptin, pepstatin, and aprotinin at a final concentration of 10 µg/ml], and then lysed by sonication. Lysates were clarified by centrifugation and immediately placed in contact with a 50% suspension of glutathione-agarose beads (Sigma) in NETN buffer. Incubation was performed overnight under rotation. Washed fusion proteins bound to the beads were resuspended in binding buffer (50 mM NaCl, 50 mM Tris, pH 8, 0.02% BSA, 0.02% Tween-20, 1 mM PMSF, and 10 µg/ml of the proteases inhibitors). For quantification, Bradford assay was performed, together with analysis by SDS-PAGE, allowing an evaluation of the stability of the fusion protein and the amounts of protein recovered. GST fusion proteins (250 ng) bound to the beads were incubated for 3 h with equal amounts of [35S]methionine-labeled proteins (near 0.5–2 µl of the hormone- or EtOH-treated lysates) in the presence or absence of 50 µM E2 and 500 µM OHT or ICI164,384 in a total volume of 300 µl of binding buffer. Beads were collected by centrifugation and washed five times in 400 µl of washing buffer 300 (WB300; 50 mM Tris, pH 8, 300 mM NaCl, 1 mM PMSF, 0.05% Tween-20, and 10 µg/ml of the protease inhibitors). Washed beads were denatured at 100 C for 5 min, and the supernatant was subsequently submitted to SDS-PAGE. 35S-Labeled proteins were visualized after autoradiography using Kodak (Rochester, NY) BioMax films. All experiments were performed at least three times.

Cell Culture, Transient Transfection Experiments, and Whole-Cell Extracts
CHO-K1 and HeLa cells were routinely maintained in DMEM-F12 (Sigma) supplemented with 5% FCS (Sigma), whereas HepG2 cells were grown in DMEM/5% FCS. All of the cell lines were cultured at 37 C and 5% CO2, and all media contained 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml Amphotericin (Sigma). One day before transfection, cells were dispatched in 6-well plates in the case of HeLa and HepG2 cell lines and 24-well plates for CHO-K1 cells. The three cell lines were transfected at 60–70% confluence with a classic calcium phosphate/DNA precipitation protocol after replacing the media 1 h before transfection with DMEM-F12/4% charcoal/dextran-treated (desteroided) FCS. Transfection in 6-well plates was performed using 3 µg of total DNA per well and containing 250 ng of expression vector, 500 ng of ERE-TK-Luc or human C3 (-307/+58)-Luc reporter plasmids, and 1.5 µg of the internal control pCH110. For CHO-K1 cells, 1 µg of total DNA per well was used, containing 25 ng of expression vector, 50 ng of ERE-TK-Luc or human C3 (-307/+58)-Luc reporter, and 150 ng of pCH110. In all cases, the total amount of DNA was maintained constant by adding Bluescript plasmid. Cells were kept in contact with the precipitate overnight at 37 C with 2% CO2, washed once with PBS, and replaced in fresh DMEM-F12/8% desteroided FCS medium supplemented with EtOH, or increasing concentrations of E2 or OHT, or a mix of 10-8 M E2 and increasing concentrations of OHT. After 36 h of transient expression, cells were harvested and 10% of the cellular extract was used to measure luciferase activity. Half of the remaining extract was used for the ß-galactosidase assay. Luciferase activities were normalized for transfection efficiency with the ß-galactosidase activity and expressed as the E2 fold induction vs. the activity obtained with the promoter alone. A value of 100% was ascribed to the hER{alpha} activity. All transfections were performed in triplicate at least three times. Whole-cell extracts from untransfected cells were obtained by harvesting confluent cells, and lysis in whole-cell extract buffer (20 mM HEPES, pH 7.9, 400 mM KCl, 2 mM dithiothreitol, and 20% glycerol) was obtained by three thawing/freezing cycles performed at -80 and 37 C. After lysis, soluble proteins were recovered in the supernatant by centrifugation.

In Vitro Phosphorylation
Two hundred fifty nanograms of purified bacterially expressed GST/hER{alpha} AB or B (wild type or point mutant) fusion proteins linked to glutathione-agarose beads were incubated for 3 h with 1.5 µl of in vitro labeled and E2- or EtOH-treated hER{alpha} DF. Beads were then washed four times in pull-down WB300 before being resuspended in binding buffer and submitted to the phosphorylation reaction. This was performed for 30 min at 30 C with 10 µCi of [{gamma}-32P]ATP in phosphorylation buffer [40 mM Tris-HCl, pH 7.4, 50 mM MgCl2, 5 mM dithiothreitol, and 250 µg ATP plus phosphatase inhibitors (1 µM okadaic acid and 200 µM sodium orthovanadate)] with 10 µg of whole-cell extract. The total volume of whole-cell extract buffer was maintained constant, and the final reaction volume was 50 µl. Beads were then washed two times in WB300, resuspended in 15 µl of SDS-PAGE loading buffer, boiled for 5 min, and centrifuged, and the supernatant was fractionated on SDS-PAGE gels. After Coomassie blue staining and drying, gels were submitted to autoradiography for 4–16 h with Kodak BioMax films. Experiments were carried out at least two times.


    ACKNOWLEDGMENTS
 
We are grateful to the Prof. P. Chambon, Dr. B. S. Katzenellenbogen, Dr. M. J. Tsai, Dr. S. Kato, and Prof. J. Camonis for their kind gifts of vectors and inserts. We gratefully acknowledge Dr. G. Reid and Prof. F. Gannon for assistance in the writing of the manuscript. We also thank F. Gay for her helpful advice on GST pull-down experiments. We are grateful to the Fondation Langlois, the Association pour la Recherche contre le Cancer, and the Ligue Contre le Cancer for funding and technical support.


    FOOTNOTES
 
This work was supported by the Centre National de la Recherche Scientifique, by the Ministère de l’Education et de la Recherche, and by grants from the Association pour la Recherche contre le Cancer (to R.M.), and by funds from the Region de Bretagne (to G.P.).

1 Current address: EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Back

Abbreviations: AF, Activation function; CBP, CEBP binding protein; CEBP, cAMP response element binding protein; CoA, coactivator; ERE-TK-Luc, estrogen-responsive element-thymidine kinase-luciferase; EtOH, ethanol; GST, glutathione-S-transferase; hER{alpha}, human ER{alpha}; LBD, ligand-binding domain; NR, nuclear receptor; OHT, 4-hydroxytamoxifen; PMSF, phenylmethylsulfonyl fluoride; rtER{alpha}, rainbow trout ER{alpha}; SRC-1, steroid receptor coactivator protein-1; TAFIIs, TATA box binding protein-associated factors; TIF-2, transcriptional intermediary factor 2; WB300, washing buffer 300.

Received for publication January 22, 2001. Accepted for publication August 1, 2001.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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