The Steroid Receptor Coactivator-1 Contains Multiple Receptor Interacting and Activation Domains That Cooperatively Enhance the Activation Function 1 (AF1) and AF2 Domains of Steroid Receptors*

Sergio A. Onate, Viroj BoonyaratanakornkitDagger , Thomas E. Spencer, Sophia Y. Tsai, Ming-Jer Tsai, Dean P. EdwardsDagger , and Bert W. O'Malley§

From the Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 and the Dagger  Department of Pathology and Molecular Biology Program, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Steroid receptors are ligand-inducible transcription factors, and their association with steroid receptor coactivators (SRCs) upon binding to DNA is necessary for them to achieve full transcriptional potential. To understand the mechanism of SRC-1 action, its ability to interact and enhance the transcriptional activity of steroid receptors was analyzed. First, we show that SRC-1 is a modular coactivator that possesses intrinsic transcriptional activity when tethered to DNA and that it harbors two distinct activation domains, AD1 and AD2, needed for the maximum coactivation function of steroid receptors. We also demonstrate that SRC-1 interacts with both the amino-terminal A/B or AF1-containing domain and the carboxyl-terminal D/E or AF2-containing domain of the steroid receptors. These interactions are carried out by multiple regions of SRC-1, and they are relevant for transactivation. In addition to the inherent histone acetyltransferase activity of SRC-1, the presence of multiple receptor-coactivator interaction sites in SRC-1 and its ability to interact with components of the basic transcriptional machinery appears to be, at least in part, the mechanism by which the individual activation functions of the steroid receptors act cooperatively to achieve full transcriptional activity.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Steroid receptors belong to a superfamily of transcription factors that regulate hormone-responsive genes and thereby cellular growth and differentiation. In the absence of hormone, the receptor is maintained in an inactive or repressive state by association with heat shock proteins and/or corepressors. Activation of the aporeceptor by ligand binding involves structural and functional changes in the receptor molecule that promote release from the inactive or repressive state to bind specific DNA hormone response elements. In addition, the ligand-bound receptor promotes the recruitment of coactivators to the receptor-DNA complex and thus entitles the receptor to achieve its full transactivation capacity (for review, see Refs. 1-4).

Formation of the preinitiation complex at the promoter involves numerous general transcription factors that recruit RNA polymerase II binding to DNA and initiation of transcription (5). Steroid receptors activate target genes by stabilizing this preinitiation complex through direct interactions with components of the transcription machinery, including TFIIB, TATA-binding protein (TBP), TFIID, and TFIIF (6-16). However, the mechanism by which receptors activate transcription is more complex. The squelching observed between receptor family members and between their various AFs1 suggests that limiting intracellular coactivators are also needed for mediating receptor function (17, 18). Furthermore, the synergism observed between the two transactivation functions (AF1 and AF2) of a single receptor suggest that the proper assembly of the individual activation functions of the steroid receptor is required to render the steroid receptor-DNA complex transcriptionally productive. Today, several receptor coactivators have been identified and characterized. Sequence comparisons indicate that they belong to a family of proteins termed steroid receptor coactivators (SRCs) and include SRC-1 (19) (or NCoA-1 (20, 21)); SRC-2, (TIF2 (22), GRIP1 (23), or NCoA2 (21)); and SRC-3 (ACTR (24), RAC3 (25), AIB1 (26), p/CIP (21), or TRAM (27)). In addition, other protein factors that interact with the receptor in a ligand-dependent manner have been identified. These include p160 and ERAP140 (28), RIP160 and RIP 140 (29), TRIP1/SUG1 (30, 31), TRIP230 (32), TIF1 (33), ARA70 (34) and CBP/p300 (20, 35, 36). SRC-1, SRC-2, SRC-3, ARA70, TRIP230, and CBP are capable of enhancing receptor transcription activity in intact cells. The finding that SRC-1 interacts in vitro with general transcription factors, such as TFIIB and TBP (27), as well as with other coactivators, such as CBP/p300 (20, 37) and p/CIP (21), suggests that SRC-1 plays an important role in bridging the interactions of receptor activation functions with the basal transcription machinery to stimulate transactivation. The recent finding that members of the SRC-1 family and coactivator proteins CBP and P/CAF possess intrinsic histone acetyltransferase activity suggests that activated receptors may also recruit these cofactors to remodel chromatin structure for better accessibility of the transcription machinery to DNA (24, 38-41). The precise mechanism by which steroid receptor coactivators modulate transactivation function remains to be determined.

To better understand the role of coactivators in receptor action, we studied the effect of SRC-1 and SRC-2/TIF2 on the transcriptional activity of several steroid receptors. We show that SRC-1 is a modular coactivator harboring intrinsic independent activation functions required for coactivation in intact cells. We also show that SRC-1 is able to interact with both the amino terminus A/B-C (AF1) and the carboxyl terminus C-D/E (AF2) regions of several steroid receptors. In addition, the cooperativity observed between AF1 and AF2, regions of the steroid receptors, appears to be assisted by SRC-1 and SRC-2/TIF2. We proposed that coactivators provide, in part a mechanism by which the independent AFs of the steroid receptors communicate within a transcription complex on target DNA elements.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmids-- The mammalian expression vectors for human progesterone receptor (PR) and estrogen receptor (ER) (19), rat glucocorticoid receptor (GR) (42), the A/B-C and C-D/E domains for ER and GR (42), the ER-responsive (ERE-TATA-luciferase), PR- and GR-responsive (PRE-TATA-luciferase), and UAS-TATA-luciferase target gene constructs (40, 43) have been described previously. The A/B-C (aa 1-638) and C-D/E (aa 538-933) domains for human PR and the SRC-1 and TIF2 (isolated from pSG5-TIF2 (22) as a BglII fragment) were cloned into the pABDelta Gal and pCR3.1 (Invitrogen) mammalian expression vectors, respectively. The Gal147-SRC-1 and VP16-SRC-1 expression plasmid encoding the Gal4 DNA binding domain (aa 1-147) and the VP16 activation domain (aa 411-487) (both of which fused to the SRC-1 regions, shown by amino acid numbers in the figure legends of Figs. 1, 2, 4, and 5) were constructed according to standard procedures as described previously (40, 43).

Cell Culture and Transfections-- HeLa cells were maintained and transfected as described previously (43). The DNA mixture, as indicated in the figure legends of Figs. 1, 2, and 5-7, was mixed with transfection reagent, Lipofectin (Life Technologies, Inc.) or Superfect (Qiagen), and used to transfect 3 × 105 cell in six-well plates. Five percent charcoal-treated fetal calf serum medium containing hormone or vehicle alone was then added, and the cells were incubated at 37 °C for 30 h. Luciferase assays were carried as described previously (43), and the luciferase activity was normalized to protein content, in µg, as assessed by Bradford assay.

Construction and Expression of Recombinant Baculovirus Vectors for Human PR and PR Domains-- Recombinant baculovirus vectors expressing full-length human PRA and PRB, and PR domains were constructed by insertion of human PR sequences into the baculovirus transfer plasmid pBlueBacHis (Invitrogen). PR coding sequences were inserted in-frame with the six-histidine aa and the enterokinase cleavage site and include human PR sequences for PRB (aa 1-933), PRA (aa 165-933), and the C-D/E (aa 538-933), D/E (aa 634-933), and E (aa 688-933) PR domains; amino-terminal to the B receptor isoform, it included the A/B (aa 1-535) and A isoform A/B (aa 165-535) domains plus the DNA binding domain for the B (A/B-C, aa 1-688) and A (aa 165-688) domain. PR-polypeptide expression in infected Sf9 cells with the corresponding recombinant virus were confirmed by Western blot of cell lysates using a panel of region-specific mAbs to human PR. For expression of PR and PR domains bound to ligand, 200 nM R5020 was added to the Sf9 cultures for the last 6-8 h of infection.

In Vitro Synthesis of SRC-1 and Different Sequence Regions of SRC-1-- SRC-1 was synthesized in vitro by a coupled transcription/translation assay using the TNT kit (Promega) in the presence of [35S]methionine. Additional sequence regions of SRC-1 were cloned in frame with an ATG translation start site in the pT7B vector.

Production of Monoclonal Antibodies to SRC-1 and Immunoprecipitation-- Mouse monoclonal antibody (IgG1, clone 1135/H4) was produced against SRC-1 sequences 477-947 fused to glutathione S-transferase and expressed and purified from yeast on a glutathione-Sepharose column. Immunization of mice and construction of hybridomas were performed by methods previously described (44). For immunoprecipitation, protein A-Sepharose beads were prebound with rabbit antimouse IgG bridging antibody, followed by 1135/H4 monoclonal antibody. Unfractionated reticulocyte lysates containing [35S]SRC-1 polypeptides (20 µl) were incubated with protein A-Sepharose beads (100 µl) 4 °C for 3 h. Resins were washed with TEDG (10 mM Tris-base, pH 7.4, 1 mM EDTA, and 10% glycerol containing 100 mM NaCl) and eluted with 2% SDS sample buffer, and eluates were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.

Immobilized Metal-Ion Affinity Pull-down Assays to Detect PR-SRC-1 Interactions-- Unfractionated reticulocyte lysates containing [35S]SRC-1 polypeptides were incubated with polyhistidine-tagged PR peptides for 30 min at 4 °C in 20 mM Tris-Cl, pH 8.0, 10% glycerol, 100 mM NaCl, and 15 mM imidazole. Samples were added to a 100-µl suspension (1:1 v/v) of Talon metal ion affinity resins (CLONTECH) and incubated for 1 h at 4 °C. Resins were then washed with the binding buffer, and bound proteins were eluted with SDS-sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. As controls for nonspecific binding to Talon resins, [35S]SRC-1 peptides were incubated either with blank Talon resins or with unrelated polyhistidine-tagged proteins, including the polyhistidine-tagged DNA binding domain of PR, which does not physically or functionally interact with SRC-1. To quantitate SRC-1 binding to His-tagged PR, the SRC-1 bands of dried SDS-PAGE gels were directly scanned for 35S radioactivity with a series 400 Molecular Dynamics PhosphorImager. Specific association with PR was determined by subtracting any [35S]SRC-1 binding to control Talon from the total binding obtained in the presence of His-tagged PR polypeptides.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

SRC-1 Contains Two Distinct Activation Domains Important for Coactivation Function-- SRC-1 is a coactivator for steroid receptor family members and contains several structural/functional domains (Fig. 1A). The ability of the carboxyl terminus to act as a dominant negative inhibitor on receptor transactivation suggested that the N terminus of SRC-1 is important for coactivation (19). To determine whether SRC-1 contains an intrinsic activation domain, we fused SRC-1 to the Gal4 DNA binding domain and determined its ability to stimulate a luciferase-driven reporter plasmid containing four Gal4 binding sites. As shown in Fig. 1B, Gal4-SRC-1 activates reporter activity in a dose-dependent manner. To further define the boundary of the activation domain, deletion mutants of SRC-1 were generated. Two regions in SRC-1 were able to stimulate transcription in mammalian cells: aa 1-93, hereafter termed AD1, and aa 840-948, or AD2 (Fig. 1C). Similarly, AD1 and AD2 stimulate transcription of the galactosidase gene reporter plasmid containing four Gal4 binding sites when tested in yeast (not shown). The carboxyl terminus region of SRC-1 (aa 948-1441) also exhibits intrinsic activation activity, although to a lesser extent. To determine the relevance of these activation domains in the coactivation function of SRC-1, deletion of these domains was performed in the context of the full-length molecule, and the ability of these mutated proteins to coactivate the steroid receptor activity was determined in transient transfection assays. As shown in Fig. 1D, SRC-1 stimulates PR transcription activity by ~25-fold. Deletion of either AD1 (Delta AD1) or AD2 (Delta AD2) from SRC-1 reduced the ability of SRC-1 to enhance the receptor transcription activity by ~50%. These data indicate that these two activation domains are necessary for optimal coactivation function of SRC-1 and that they function independently from each other.


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Fig. 1.   SRC-1 contains intrinsic transcription activation domains. A, schematic representation of SRC-1 functional domains. Depicted are the activation domains AD1 and AD2, the basic helix-loop-helix and Per-Arnt-Sim domain (bHLH/PAS), the serine- and threonine-rich (S/T) and glutamine-rich (Q) regions, the histone acetyltransferase activity domain (HAT), and the SRC-1(.8) fragment that can serve as dominant negative inhibitor of receptor function. SRB1 and SRB2 correspond to the steroid receptor binding regions 1 and 2. B, SRC-1 activates gene expression when tethered to DNA. The Gal4 DNA binding domain alone (Gal147) or fused to SRC-1 (in various amounts: 0.1, 0.5, and 2 µg) were expressed in HeLa cells along with 2 µg of a reporter containing four binding sites for Gal4 (17-mer-TATA-luciferase) and their activity compared with the activity of reporter alone (control). The relative luciferase activity represents the average of three transfections normalized to protein content. C, specific regions in SRC-1 activate transcription. The region of SRC-1 indicated at the left (amino acid numbers) was fused to Gal147 and tested for its ability to transactivate the 17-mer-TATA-luciferase reporter in HeLa cells using 1 µg of Gal4-DNA binding domain-SRC-1 fusion expression plasmid determined as above. D, SRC-1 activation domains are required for PR coactivation. HeLa cells were transfected with 0.5 µg of PRE-TATA-luciferase reporter along with 0.3 µg of PR mammalian expression vector in the absence (-) or presence (+) of 20 nM R5020. The effect of SRC-1 and its deletion mutants Delta AD1 (deletion of aa 1-93) and Delta AD2 (deletion of aa 840-948) on PR transcriptional activity were tested by adding 1.5 µg of SRC-1 mammalian expression vectors, and their relative luciferase activity was determined as above.

Defining the Receptor Interaction Domain of SRC-1-- It has been shown previously that the carboxyl-terminal region of SRC-1 interacts with the E region of the PR in a hormone-dependent manner and that mutations in the conserved AF2 motif significantly decrease these interactions (19, 27). Other reports indicated that full-length SRC-1 is able to interact with receptor, also, in a ligand-independent manner and with receptor mutated in the AF2 region (27, 45, 46). These data suggest the presence of multiple receptor-coactivator interacting domains. To map the relevant regions of SRC-1 involved in receptor-coactivator interactions, a modified version of the mammalian two-hybrid system was used. The human PR A/B-C (containing AF1) and C-D/E (containing AF2) regions was used as bait, and SRC-1 fused to the VP16 activation domain was employed as a target (Fig. 2A). Interaction was assessed by the ability to transactivate a PRE-TATA-luciferase reporter. As shown in Fig. 2B, a fusion protein of SRC-1, aa 1-1441, interacts with the PR C-D/E region. The major interaction is observed with the carboxyl terminus of SRC-1, aa 1139-1441. The middle region of SRC-1, aa 361-1139, also interacts with the C-D/E region of receptor. The most amino-terminal region of SRC-1, aa 1-399, containing the basic helix-loop-helix and Per-Arnt-Sim (PAS) conserved domains, fails to interact with the C-D/E region of the receptor (Fig. 2B). Surprisingly, SRC-1 also interacted with the A/B-C region of PR (Fig. 2C). In contrast to interaction with the C-D/E region, the aa 361-1139 region of SRC-1 exhibited the strongest receptor interaction activity, and the carboxyl-terminal fragment did not bind, which is the opposite of that observed when using the PR ligand binding domain. In addition, the SRC-1, aa 1-399, exhibits moderate interaction activity with the A/B-C region of PR (Fig. 2C).


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Fig. 2.   SRC-1 interacts with PR by multiple regions. Protein-protein interactions between receptor and coactivator were analyzed by the mammalian two-hybrid system. The SRC-1 regions indicated in A were fused to VP16 activation domain (aa 411-490) of the pABVP16 target mammalian expression vector. The receptor domains A/B-C and C-D/E were used as bait, and the interactions were assessed by their ability to transactivate the PRE-TATA-luciferase reporter plasmid. HeLa cells were transfected with 0.3 µg of DNAs, including the A/B-C (B) and C-D/E (C) regions of PR, along with the VP16-SRC-1 fusion protein and PRE-TATA-luciferase reporter. The relative luciferase activity was determined as in Fig. 1 and represents the average of three independent experiments.

In an attempt to define the boundaries of SRC-1 interaction within the A/B-C region of PR, several deletion mutations in SRC-1 were used. As shown in Fig. 2C, subfragments of SRC-1 361-1139, aa 361-633, 633-782, and 782-1139, were able to interact with A/B-C region of PR, although to a lesser extent as compared with the full-length SRC-1.

Defining The SRC-1 Interaction Domains of PR by in Vitro Pull-down Assays-- We also examined the ability of SRC-1 to interact with subdomains of PR in vitro. Full-length human PRB and PRA and various subdomains of PR were expressed as polyhistidine-tagged proteins in baculovirus (Fig. 3A). Protein-protein interactions between SRC-1 and PR were measured as an association of [35S]SRC-1 with His-tagged PR polypeptides immobilized to metal ion affinity resins. Specifically bound [35S]SRC-1 was detected by SDS-PAGE and autoradiography, and the extent of the interaction was quantitated by phosphor-image analysis. Western blot with an mAb to the polyhistidine tag was employed to ensure that the assay input of PR polypeptides and the amount of immobilized His-tagged PR were equal (not shown). As shown by the autoradiograph of a representative experiment in Fig. 3B (top panel) and from quantitative analysis of multiple independent experiments (Fig. 3B, bottom panel), SRC-1 consistently interacted weakly with the A/B, A/B-C, and E regions, moderately with the D/E and C-D/E regions, and strongly with PRA and PRB. Efficient interaction with the ligand binding domain required the presence of hinge sequences. There was no significant difference between SRC-1 interactions with the A and B isoforms of PR; the presence of the C region did not affect SRC-1 binding to either the A/B or D/E regions of PR (Fig. 3B). The results in Fig. 3 were obtained using amino-terminally truncated SRC-1 (381-1441); identical results were obtained with full-length SRC-1 (not shown). It was also of interest that the extent of SRC-1 interaction with full-length PR was 3-4-fold greater than the sum of the interactions obtained with individual A/B-C and C-D/E regions of PR, suggesting a synergistic interaction of SRC-1 with multiple regions within the full-length receptor. In Fig. 3B, PR constructs that contained the ligand binding domain were bound by the hormone agonist R5020. To determine whether SRC-1 interactions with these PR polypeptide are hormone-dependent, experiments were also done in the presence and absence of the progesterone agonist R5020. PR-polypeptides that contain the C-D/E region of progesterone receptor failed to interact in the absence of R5020 (Fig. 3C). The presence of hormone strongly enhanced SRC-1 interactions with the B, C-D/E, and D/E regions of PR (Fig. 3C).


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Fig. 3.   SRC-1 interactions in vitro with full-length PR and domains of PR. A, schematic representation of polyhistidine-tagged full-length human PR (A and B isoforms) and PR domains C-D/E, D/E, E, A/B-C, and A/B (both PR isoforms) expressed in the baculovirus system. His6x, six sequential histidine residues. B, interaction of SRC-1 with PR polypeptides. Unfractionated reticulocyte lysates containing [35S]SRC-1 (aa 381-1441) were incubated for 30 min on ice with whole cell extracts of Sf9 insect cells containing equal amounts of polyhistidine-tagged PR polypeptides shown in A above. Full-length PR and PR polypeptides containing the E domain were bound to the progestin R5020. Samples were bound to 100 µl of metal ion affinity resin (Talon), the resins were washed, and proteins were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE and autoradiography (top panel). Lane 1 is 10% of input [35S]SRC-1, lane 2 is the background binding of [35S]SRC-1 on Talon resin in the absence of His-tagged PR (Control), and lanes 3-11 are the binding of [35S]SRC-1 in the presence of His-tagged PR polypeptides indicated. Quantitation of [35S]SRC-1 binding to PR polypeptides from multiple experiments (bottom panel) as determined by phosphorimage scanning of SDS-PAGE gels. Binding of [35S]SRC-1 to PRB was set as 100% control, and binding to all other His-tagged PR polypeptides regions was calculated as a percentage of the control. Values are the averages of three independent experiments ± S.E. C, hormone-dependent interaction of SRC-1 with PR polypeptides. [35S]SRC-1 was incubated with equal amounts of His-tagged PRB, C-D/E, D/E, and PRB A/B-C in the presence and absence of R5020. Samples were bound to Talon resins and analyzed by SDS-PAGE and autoradiography (top panel) and phosphorimage analysis as in B above. R5020 was added to Sf9 cells at 200 nM for the last 6-8 h of infection.

Determination of in Vitro Receptor Binding Sites within SRC-1-- To map the regions of SRC-1 that are required for interaction with PR in vitro, the amino-terminal (aa 1-399), middle (aa 361-1139) and carboxyl-terminal (aa 1139-1441) regions of SRC-1 were synthesized in vitro (see Fig. 2A) in the presence of [35S]methionine and compared with full-length SRC-1 for binding to His-tagged full-length PRB immobilized on metal ion affinity resins. As shown in Fig. 4A, each of the in vitro synthesized sequences of SRC-1 exhibited the predicted size (in kDa), and as expected, the 1135/H4 mAb immunoprecipitated only the full-length SRC-1 and the SRC-1 aa 361-1139 polypeptide that harbors the antibody epitope (Fig. 4B). To quantitate the interaction of these SRC-1 polypeptides with PR, [35S]SRC-1 polypeptides bound to immobilized His-tagged PR were submitted to SDS-PAGE and phosphorimage analysis. Because each of the SRC-1 polypeptides contains different numbers of methionine aa, the phosphorimage units were corrected for the number of methionines. As shown in Fig. 4B, the middle region of SRC-1 (aa 361-1139) exhibited the highest level of binding to PRB, comparable to that observed with SRC-1. The carboxyl-terminal region of SRC-1, aa 1139-1441, exhibited a moderate level of interaction with PRB, whereas the amino terminus of SRC-1, aa 1-399, gave a weak but detectable binding to PRB. With all sequence regions of SRC-1, the interaction with full-length PRB was hormone-dependent (Fig. 4B). Because SRC-1, aa 361-1139, exhibited the strongest interaction with PRB, we further mapped this region for receptor interacting sequences. As shown in Fig. 4C, subregions in SRC-1 corresponding to aa 634-782 and 361-633 were able to bind to PRB. In contrast, the SRC-1 782-1139 polypeptide did not bind to PR-B (Fig. 4C). When these observations are taken together, it can be seen that SRC-1 interacts with more than one region of PR and that there are multiple PR binding sites within SRC-1.


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Fig. 4.   Interaction of SRC-1 domains with PR in vitro. A, full-length SRC-1 (aa 1-1441) and regions of SRC-1 (aa 1-399, 361-1139, and 1139-1441) were synthesized in vitro in the presence of [35S]methionine and analyzed by SDS-PAGE and autoradiography (lanes 1-4). Samples were also immunoprecipitated with an unrelated control antibody (-) or with the 1135 mAb to SRC-1 (+), and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography (lanes 5-12). B, the SRC-1 domains synthesized above were incubated with His-tagged PRB, either unliganded (-) or bound to R5020 (+). Because each of the expressed SRC-1 domains contains a different number of methionines, the phosphorimage units have been corrected for these differences. The data were plotted as relative phosphorimage units from a single experiment that is representative of three independent experiments (number of methionine residues for SRC-1 domains: 42 for SRC-1:1-1441, 10 for SRC-1:1-399, 17 for SRC-1:361-1139, and 15 for SRC-1:1139-1441). C, subregions of the SRC-1 middle domain, SRC-1:361-1139, were synthesized in vitro in the presence of [35S]methionine, including SRC-1:361-633, SRC-1:634-782, and SRC-1:782-1139, and bound to His-tagged PRB (liganded to R5020); data were analyzed by phosphorimage as in B. The number of methionine residues in SRC-1 domains was 9 for SRC-1:361-633, 1 for SRC-1:634-782, and 7 for SRC-1:782-1139.

The SRC-1 Regions That Bind Receptor Can Independently Coactivate the Steroid Receptor AFs-- SRC-1 is a modular coactivator possessing two major receptor interacting regions and two separable transcriptional activation domains. To determine whether these interacting regions are in part relevant for SRC-1 function in intact cells, we analyzed their coactivation function on the individual AFs of the steroid receptors. As shown in Fig. 5, SRC-1 is able to enhance both the A/B-C and the C-D/E regions of PR containing AF1 and AF2, respectively. The region in SRC-1 (aa 1-1139) primarily responsible for interaction with the amino terminus of PR A/B and containing the two activation domains, AD1 and AD2, was as efficient as full-length SRC-1 in coactivation of receptor AF1. Coactivation of AF2 by this SRC-1 region was also observed, although to a lesser extent as compared with full-length SRC-1. This result is consistent with the weaker ability of SRC-1, aa 1-1139, to interact with the AF2 region of receptor. The first 1-361 aa of SRC-1 weakly interacts with the AF1-containing region of PR and exhibits a minimal coactivation effect on receptor transactivation (not shown). Thus, specific regions in SRC-1 can independently coactivate either of the two AFs of PR when expressed in intact mammalian cells.


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Fig. 5.   Subregions of SRC-1 can coactivate steroid receptors ex vivo. HeLa cells were transiently transfected with 0.3 µg of PR A/B-C (left panel) or C-D/E (right panel) along with PRE-TATA-luciferase reporter plasmid and 1.5 µg of SRC-1 or the region in SRC-1 containing the SRB1, aa 1-1139. The relative luciferase activity is the average of three independent experiments.

SRC-1 and Its Related Family Member TIF2 Facilitate AF1 and AF2 Interactions for Efficient Receptor Transactivation-- In an attempt to identify functional differences between coactivators, SRC-1 and TIF2 were tested for their ability to coactivate different steroid receptors and their AFs when expressed separately and simultaneously in intact cells. As shown in Fig. 6A, both SRC-1 and TIF2 were able to enhance transcription activity of PR to a similar extent. When SRC-1 and TIF2 were expressed simultaneously, they exhibited an additive effect on PR-driven gene expression (not shown). Furthermore, both SRC-1 and TIF2 were able to enhance the individual AF1 and AF2 for PR in a dose-dependent manner (Fig. 6B). SRC-1 and TIF2 also mediate transcriptional enhancement by the AF1 and AF2 of ER and GR in a similar manner (Fig. 6, C and D, respectively).


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Fig. 6.   SRC-1 and TIF2 coactivate steroid receptors to a similar extent. A, HeLa cells were transfected with 0.3 µg of PR or PRE-TATA-luciferase reporter in the absence (-) or presence (+) of 20 mM R5020 and 1.5 µg of SRC-1 or TIF2 expression vector under the same promoter and enhancer to regulate their expression. B-D, cells were transfected with PR (B), ER (C), or GR (D) A/B-C (right panels) or PR C-D/E (left panels) domains along with PRE-TATA-luciferase reporter and increasing amounts of SRC-1 (black-square) or TIF2 (square ), given in µg, and the relative reporter activity was determined as in A. Cells transfected with the C-D/E region of receptors were treated with R5020 for PR, E2 for ER, and dexamethasone for GR 20 nM final concentration.

SRC-1 appears to promote functional interactions between AF1 and AF2 by an unknown mechanism (47, 48). Because SRC-1 and TIF2 interact and coactivate the transcription activity of the individual AFs, we analyzed their effects on the AFs of PR when expressed simultaneously inside the cell. As shown in Fig. 7, coexpression of either SRC-1 or TIF2 with simultaneously coexpressed AF1 and AF2 of PR resulted in a more efficient enhancement of the reporter transcription activity (2-3-fold), as compared with the AFs expressed separately. As reported previously (47), SRC-1 and TIF2 appear to stabilize or promote the interactions between AF1 and AF2 domains of the steroid receptors via multiple interactions, resulting in a virtual synergism of the transcription activity for both AFs.


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Fig. 7.   SRC-1 and TIF2 enhance the transcriptional activity of AF1 and AF2 of PR in a cooperative manner. Cells were transfected with 0.3 µg of A/B-C (square ), C-D/E (), or both A/B-C and C-D/E (black-square) regions of PR in the absence (control) or presence of 1.2 µg of SRC-1 or TIF2, along with 0.5 µg of PRE-TATA luciferase reporter, and the relative luciferase activity was determined as in Fig. 1. Cells transfected with PR or the C-D/E region were treated with 20 nM R5020 (final concentration).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Considerable understanding of the mechanism by which nuclear receptors activate gene expression has been gained with the cloning and characterization of several steroid receptor coactivators. Modulation of receptor activity by coactivators is a complex multistep process and appears to involve enzymatic remodeling at the chromatin level for access of receptor to DNA (12, 24, 38-41), as well as interactions with components of the RNA polymerase II transcriptional machinery at the promoter of hormone-responsive genes (6-16). Consistent with the predicted properties of a coactivator, we show here that SRC-1 activates transcription when tethered to DNA by fusion with a heterologous DNA binding domain. Deletion of either AD1 or AD2 decreases the coactivation function of SRC-1 in intact cells (Fig. 1). Thus, the data highlight the relevance of SRC-1 and its domains in coactivation and steroid receptor function. AD1 includes the basic helix-loop-helix motif, which is highly conserved among the SRC-1 family members (20-26). This may explain, at least in part, the intrinsic transcription activity observed in some SRCs family members, including TIF2/GRIP1 (22, 23) and RAC3 (25). In view of the role of basic helix-loop-helix domains in dimerization and DNA binding for several activators (49), the intrinsic transcription activity of AD1 may be the result of the direct interaction(s) with general transcription factors and/or the recruitment of intermediary factors capable of dimerization and consequent stabilization of the preinitiation complex. In contrast, sequence comparison of AD2 failed to reveal homology to any protein canonical domain, including SRC-1 family members. Therefore, mapping of the activation domain and elucidation of the downstream interacting protein(s) for the SRCs are critical for understanding the mechanism by which SRC-1 modulates steroid receptor function.

At least two AF domains have been identified for the steroid/nuclear hormone receptors: a poorly characterized AF1 located at the amino-terminal A/B region and the ligand-inducible AF2 located at the D/E region of the steroid receptors. The AF2 domain contains an amphiphatic alpha -helix (helix 12) that is highly conserved between the steroid receptor superfamily members (50). Here, we have shown that SRC-1 is capable of interacting with both the A/B and D/E regions via multiple receptor interaction sites. The original receptor interacting region for SRC-1 was found at the carboxyl terminus (19). Our present data indicate that this region in SRC-1 (hereafter termed SRB2) is largely responsible for interaction with the ligand binding D/E domain of the receptor; it does not interact with the A/B region of steroid (Fig. 2). Hence, the carboxyl-terminal region in SRC-1, aa 1139-1441, is proposed to provide specificity for ligand-bound AF2.

Consistent with previous findings, the middle region of SRC-1, aa 361-1139, also interacts with the D/E region of the receptor in a ligand-dependent manner (Fig. 2). Three highly conserved hydrophobic LXXLL motifs within aa 570-833 in SRC-1 family members and other nuclear receptor interacting proteins have been shown to be relevant for the interactions with ER, retinoic acid receptor, and TR ligand binding domains (45, 51). A unique observation of this study is that the SRC-1 region from aa 361 to aa 1139, hereafter termed SRB1, also interacts with the ligand-independent AF1 in the A/B region of PR (Fig. 2). Interactions of SRB1 with the D/E region were observed to have about <FR><NU>1</NU><DE>5</DE></FR> the efficiency observed with the A/B region of PR. In present experiments, we observe that several subregions of SRC-1 are involved in the interactions with the A/B region of PR in intact cells (Fig. 4). Thus, aa 633-772, containing a cluster of three LXXLL motifs, and aa 361-633 bind to the N terminus and full-length PR. However, the region comprising aa 782-1139 fails to interact with PR in vitro, but is active in a mammalian two-hybrid interaction assay (Fig. 2), suggesting that this region in SRC-1 requires additional cellular components for interaction with the A/B region of PR in intact cells. The interaction of SRB1 with the AF1- and AF2-containing regions of the receptor is not limited to PR. Other receptors, including ER, GR, and AR, exhibited similar patterns of interaction in intact cells. Therefore, there are multiple interaction sites in the SRC-1 and in the receptor molecule that are important for receptor-coactivator function.

Additional variants for SRC-1 containing distinct amino-terminal and carboxyl-terminal sequences, reflecting alternative initiation and splicing events, have been proposed (20). To test the hypothesis that truncated forms of SRC-1 may be of physiological relevance, deletion mutants for SRC-1 were tested for their ability to coactivate PR and PR domains. We observed that a carboxyl-terminally truncated form of SRC-1, lacking the ligand-dependent steroid receptor interacting region, was as efficient as the wild type SRC-1 to coactivate PR (Fig. 5). Therefore, one might speculate that different isoforms of SRC-1 could be relevant for ligand-independent activation of target gene expression. Because steroid receptors are phosphoproteins and the major phosphorylation sites are at the amino-terminal A/B domain (52, 53), the present data raise the interesting possibility that phosphorylation of steroid receptors may modulate SRC-1 interactions. It will be of interest to determine whether ligand-dependent and/or independent phosphorylation of the A/B region of steroid receptors is directly involved in receptor-coactivator interactions and transactivation.

Previous reports indicate that the interactions and coactivation of steroid receptors by SRC-1 and SRC-2 (TIF2/GRIP1) are specific for AF2-mediated transactivation of the steroid receptors (22, 45, 51, 54). The failure of coactivators to enhance the transcription activity of the A/B region has been ascribed to the ability of AF1 to activate transcription by a mechanism dependent on coactivator(s), presumably different from those described for AF2-interacting coactivators (54). However, we observed here that there are functional interactions of SRC-1 and SRC-2/TIF2 with the AF1 and AF2 domains for several steroid receptors (Fig. 6). These differences support the hypothesis that additional accessory proteins, which appear to be absent in the yeast systems previously employed for analysis, are relevant for AF1 interactions and coactivation in mammalian cells. We observed that SRC-1 can directly interact with the receptor more efficiently when both AF1 and AF2 are present, as compared with the interactions with the individual AFs (Fig. 3). Similarly, SRC-1 coactivates more efficiently when individual AF1 and AF2 of the steroid receptors were expressed simultaneously in intact cells (Fig. 7). Although the mechanism by which the AFs act in a synergistic manner remains unclear, the assembly of their functional domains likely depends on coactivators. Taken together, the existence of multiple coactivators and receptor-coactivator interaction sites may contribute to a mechanism by which steroid receptors achieve their specificity and the diversity of target gene expression.

    ACKNOWLEDGEMENTS

We thank Dr. Pierre Chambon for the expression plasmid TIF2, the University of Colorado Cancer Center Tissue Culture Core Facility for baculovirus production of PR polypeptides, and Lori Sterman and Kurt Christensen for technical assistance in production of MAbs to SRC-1.

    FOOTNOTES

* This work was supported by National Institute of Health grants (to B. W. O. and M.-J. T.) and Public Health Services Grant NCI CA 46938 (to D. P. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Cell Biology, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-6205; Fax: 713-790-1275.

1 The abbreviations used are: AF, activation function; SRC, steroid receptor coactivator; aa, amino acid(s); mAb, monoclonal antibody; PR, progesterone receptor; ER, estrogen receptor; PAGE, polyacrylamide gel electrophoresis; TBP, TATA-binding protein; GR, glucocorticoid receptor.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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