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J. Biol. Chem., Vol. 275, Issue 27, 20928-20934, July 7, 2000

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Functional Interactions between the Estrogen Receptor and DRIP205, a Subunit of the Heteromeric DRIP Coactivator Complex^*

Darya Burakov^§, Chi-Wai Wong^¶, Christophe Rachez, Boris J. Cheskis^¶, and Leonard P. Freedman

From the  Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Cornell University, Graduate School of Medical Sciences, New York, New York 10021 and ^¶ Wyeth Ayerst Research, Nuclear Receptors Department, Radnor, Pennsylvania 19087

Received for publication, March 10, 2000, and in revised form, April 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nuclear receptors regulate transcription in direct response to their cognate hormonal ligands. Ligand binding leads to the dissociation of corepressors and the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as potential chromatin remodelers through intrinsic histone modifying activities. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus. The DRIP complex is a 15-subunit complex required for nuclear receptor transcriptional activation in vitro. It is recruited to the receptor in response to ligand through specific interactions of one subunit, DRIP205. We present evidence that DRIP205 interacts with another member of the steroid receptor subfamily, estrogen receptor (ER). This interaction occurs in an agonist-stimulated fashion which in turn is inhibited by several ER antagonists. In vivo, a fragment of DRIP205 containing only its receptor interacting region acts to selectively inhibit ER's ability to activate transcription in response to estradiol. These observations suggest a key role for the DRIP coactivator complex in estrogen-ER signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nuclear receptors comprise a very large family of ligand-inducible transcription factors. The ligands for nuclear receptors include steroids, retinoids, vitamin D, thyroid hormone, prostanoids, and cholesterol metabolites, such as oxysterols and bile acids. Their combined effects are vast, influencing virtually every fundamental biological process, from development and homeostasis to proliferation and differentiation. Like other eukaryotic factors that regulate transcription, nuclear receptors bind selectively to DNA, primarily as dimers through two characteristic zinc finger modules and a dimerization region that directs self-interaction or hetero-partnering. Moreover, they possess identifiable transactivation functions (AFs),¹ which can independently confer activation potential to heterologous DNA-binding domains. Transactivation is mediated by both constitutive and inducible AFs (AF-1 and AF-2, respectively), the latter of which is conferred by its integral location within the ligand-binding domain (LBD).

A large number of nuclear receptor transcriptional cofactors have been isolated through interactions with receptors in yeast two-hybrid screens and from nuclear extracts in vitro, primarily using receptor LBDs as baits. One family of related proteins are collectively termed the p160 coactivators. They are represented by SRC-1/NCoA-1, TIF2/GRIP1/NCoA-2, and pCIP/ACTR/AIB1 (for review, see Refs. 1-3 and references therein). Besides sequence homology, p160 proteins share an ability to stimulate ligand-dependent transactivation by nuclear receptors in transient overexpression experiments. A distinctive structural feature of the p160 coactivators is the presence of multiple LXXLL signature motifs (also called LXDs, NR boxes, or NIDs), which comprise determinants for direct interactions with the nuclear receptor AF-2 (4, 5). A second shared feature of the p160 coactivators is an intrinsic histone acetyl transferase activity and the ability to interact with other histone acetyl transferase coactivators, such as CBP/p300 and pCAF (6-8). Other cofactors can interact with some nuclear receptors in the absence of ligand and confer transcriptional repression, most notably N-CoR and SMRT through the recruitment of histone deacetylases (HDACs). These corepressors have been found as part of multiple complexes containing various HDACs through indirect interactions with corepressors bridged by mSin3a or through direct corepressor-HDAC interactions (9, 10) (11-13). Remarkably, recent data indicate that corepressors and coactivators use very similar determinants (i.e. the LXXLL motif) to recognize and interact with receptor LBDs (14-16). Ultimately, it is the absence, presence, or nature of the ligand that influences whether or not a corepressor or coactivator is bound to essentially the same site within the LBD. Functionally, these factors act in concert to modify histone tails, presumably to destabilize or stabilize chromatin (recently reviewed in Ref. 17).

In addition to actions at the level of chromatin, other, distinct nuclear receptor coactivators also appear to act directly at promoters on key components of the transcriptional apparatus. A recently discovered multisubunit complex that was found to interact with the vitamin D receptor (VDR) (18, 19) and thyroid hormone receptor (TR) (20) probably function, at least in part, at the level of direct recruitment. This complex, alternatively called DRIP or TRAP, binds to the nuclear receptor LBD AF-2 in response to ligand through a single subunit (DRIP205/TRAP220 (19, 21), also cloned as PBP (22)). However, this single subunit anchors an additional 13-15 proteins comprising the DRIP/TRAP complex, thereby conferring hormone-dependent recruitment of what appears to be a preformed complex. Other activators unrelated to steroid/nuclear receptors, such as VP16, p65 subunit of NF-B, and SREBP-1a, also recruit this complex (called ARC; Ref. 23), and many of the DRIP-TRAP-ARC subunits are also present in three similar, if not identical, SRB-associated complexes, NAT, SMCC, and mammalian SRB/mediator, the latter targeted by adenovirus E1A (24-26). At least seven DRIP/TRAP/ARC subunits are homologous to proteins described as components of Srb/mediator, a complex that associates with RNA polymerase II (Pol II) through its large subunit's carboxylterminal repeat domain (reviewed in Ref. 27). This suggests that the DRIP/TRAP/ARC coactivator complex, perhaps through mediator components, functions in part by targeting polymerase II holoenzyme to promoters. In fact, these related complexes are required for transcription by these same activators, as demonstrated utilizing purified components in in vitro transcription assays.

The potent effect of the DRIP complex on transcriptional activation in vitro and the generality of the activators it cooperates with suggests that it is central to the process of transcription. While preliminary data indicate that the DRIP complex interacts with several nuclear receptors, in addition to VDR and TR, such as retinoic acid receptor and peroxisome proliferator-activated receptor- (18, 21),² its role as a ligand-dependent coactivator of steroid receptors, including estrogen receptor (ER), has not yet been clearly elucidated. We recently demonstrated that the glucocorticoid receptor (GR) LBD interacts with DRIP205 in a dexamethasone-dependent manner (28). In addition, the N-terminal transactivation function of GR, AF-1, interacts selectively with a distinct DRIP subunit, DRIP150. These results suggest that two separate activation functions might serve to bridge a multisubunit complex like DRIP through multiple interactions and perhaps offer a possible explanation for the transcriptional synergism between these two transactivation domains of GR in response to hormone.

In this work, we present evidence that DRIP205 interacts with another member of the steroid receptor subfamily, ER. This interaction occurs in an agonist-stimulated fashion, which in turn is inhibited by several ER antagonists. In transient transfection assays, a fragment of DRIP205 containing only its receptor-interacting region acts to selectively inhibit ER's ability to activate transcription in response to estradiol. These observations suggest a key role for the DRIP coactivator complex in estrogen-ER signaling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Equipment and Reagents-- The BIAcore 2000 system, sensor chips CM5 (certified), Tween 20, amine coupling, and GST capture kits were obtained from BIAcore Inc. For BIAcore experiments, purified recombinant human estrogen receptors  and  were obtained from PanVera Corp. The buffer used for all experiments was 50 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.2 mM EDTA, 0.05% Tween 20, 1 mM DTT. 17--Estradiol and 4-(OH) tamoxifen were obtained from Sigma. Raloxifene was synthesized by Wyeth-Ayerst Medicinal Chemistry group. ICI-182,780 was provided by Zeneca Pharmaceuticals.

Plasmids-- A region encoding amino acids 613-773 of SRC1 was polymerase chain reaction-amplified using primers designed for the specific sites. The amplified product was subcloned into pGEX-5X-3 using unique SmaI/XhoI sites. GST-ER-LBD (aa 312-595), GST-DRIP205 (aa 527-774), and well as DRIP205 mutant derivatives (GST-NR1, GST-NR2, GST-M1, and GST-M2) are all described elsewhere (29-31). BL21 cells were transformed with all GST expression plasmids for bacterial overexpression. pcDNA3-hER (aa 1-595) was obtained from M. Garabedian (New York University Medical School). pcDNA3-hER-N (aa 1-251) and pcDNA3-hER-C (aa 185-595) were generated through polymerase chain reaction amplification and ligated into the pcDNA3 vector.

Protein Purification-- BL-21 cells expressing GST-DRIP205, GST-DRIP205 mutants, GST-ER derivatives, or GST-SRC1 were grown at 37 °C until A₆₀₀ reached 0.4. Subsequently, the temperature was lowered to 24 °C and incubation was continued until cells reached A₆₀₀ = 0.6. Protein expression was induced by the addition of 1 mM isopropyl-1-thio--D-galactopyranoside (final concentration). Cells were allowed to grow at 24 °C for an additional 3 h, harvested by centrifugation at 5000 rpm in a Sorval SS-34 rotor for 20 min, and washed with phosphate-buffered saline buffer, containing 1 mM dithiothreitol, 1 mM EDTA, and protease inhibitor mixture (Sigma). Cells were sonicated, and the homogenate was centrifuged for 1.5 h at 45,000 rpm in a T45 rotor (Beckman). The supernatant was applied to a glutathione-agarose column (Pierce). Bound GST-fused proteins were eluted with a gradient of reduced glutathione from 5 to 50 mM in 20 mM sodium phosphate buffer, pH 7.5, 2 mM dithiothreitol, 1 mM EDTA. Eluted proteins were concentrated with ammonium sulfate (final concentration of 70%) and further purified by gel filtration on a Superdex-200 column.

Preparation of the Sensor Chip-- Protein immobilization on a BIAcore sensor chip surface was carried out as described previously (31). Briefly, anti-GST-antibody was immobilized using an amine coupling kit, as described by the manufacturer. 6000-8000 RU of protein was typically bound. Purified GST-DRIP205 or GST-SRC1 was then immobilized using antibody-antigen interactions. Surfaces with approximately 600-800 RU of immobilized protein were used for the interaction analysis.

SPR Binding Assay and Data Analysis-- Each binding cycle was performed with a constant flow of buffer at 10 µl/min. Samples of ER were injected across the surface via a sample loop. Once the injection plug had passed the surface, the formed complex was washed with buffer for an additional 1000 s. Following each injection, the surface was regenerated with one 10-µl injection of 0.05% SDS solution. To remove immobilized GST coactivator, a 10-µl injection of 10 mM glycine, pH 2.0, was used. All experiments were performed at 25 °C. Data were collected at 1 Hz and analyzed using the BIAEvaluation program 3.0 (BIAcore, Inc.) on a Compaq PC. This program uses a global fitting analysis method for the determination of rate and affinity constants of macromolecular interactions. Refractive index differences for the ERs at different protein/ligand concentrations were adjusted using the Sigma Plot 5.0 program.

GST Pull-down Assays-- GST-fusion proteins (20 µg), ER-LBD (aa 312-595), DRIP 205 (aa 527-774) or GST alone, immobilized on glutathione-Sepharose beads were preincubated in binding buffer (20 mM Tris, pH 7.9, 170 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM dithiothreitol, and 4 mg/ml bovine serum albumin) for 30 min at 4 °C. In vitro translated [³⁵S]methionine-labeled (Promega TNT reticulocyte lysate system) human ER (aa 1-595), human ER-N (aa 1-251), or human ER-C (aa 185-595) were incubated with the immobilized fusion proteins for 1 h at 4 °C. The beads were washed four times in the same buffer without bovine serum albumin, resuspended in 2× SDS sample buffer, and boiled for 3 min; the associated proteins were resolved by SDS-PAGE and visualized by autoradiography.

Immunoprecipitation and Immunoblotting-- The U2OS-ER cell line was a kind gift from M. J. Garabedian, I. Rogatsky, and J. Trowbridge (New York University Medical Center). Cells were cultured with or without estradiol in the medium (10⁶ M) for 24 h. Cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 mM EDTA, 50 mM NaF), in the presence of protease inhibitors (15 mM phenylmethylsulfonyl fluoride, 40 µg/ml aprotenin, 20 µg/ml leupeptin, 1 mM benzamidine). The extracts were cleared by centrifugation for 5 min at 14,000 rpm. For ER immunoprecipitation, 20 µg of SRA1010 anti-ER IgG antibody (StressGen) was used per 1 mg of protein extract. The same amount of normal rabbit serum was used as a control. The immunoprecipitation from hormone-treated cells was performed in the presence of 10⁴ M estradiol. The extracts were incubated for 2 h with the antiserum before adding a 1:1 mix of protein A and Gamma-bind G-Sepharose beads (Amersham Pharmacia Biotech) for another 1 h. The beads were washed four times with the buffer described above, washed three times with 50 mM Tris, pH 7.5, and then boiled in SDS sample buffer. The immunoprecipitated proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. DRIP 205 was detected by Western blot using anti-PBP polyclonal serum that was kindly provided by Dr. M. Lazar (University of Pennsylvania Medical School). Antibody was used at a 1:750 dilution.

Mammalian Cell Culture and Transient Transfections-- U2OS-ER cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% minimal essential medium nonessential amino acids solution (Life Technologies, Inc.), and 500 µg/ml G418 to maintain ER expression. Cells were seeded on 35-mm dishes at a density of 2 × 10⁵ in Dulbecco's modified Eagle's medium phenol red-free medium supplemented with 10% stripped fetal bovine serum and 1% nonessential amino acids. Cells were transfected by the calcium phosphate method with 15 µg of total DNA including 2 µg of ERE-TK-Luc, 2 µg of CMV--Gal, and various amounts of pcDNA3-DRIP205 box. 12 h following transfection, cells were washed with Tris-buffered saline, and fresh medium with or without estradiol was added. Cells were harvested 24 h later, and luciferase activity was quantified using a luminometer. -Galactosidase activity of the cell lysates was determined and used to normalize luciferase activity.

Mammalian Two-hybrid Assay-- 2 × 10⁴ COS-7 cells per well were seeded in Dulbecco's modified Eagle's medium without phenol red (BioWhitter) supplemented with 10% charcoal-stripped fetal calf serum. Cells were transfected with LipofectAMINE Reagent (Life Technologies, Inc.) according to the manufacturer's protocol. 100 ng of pGL-Luciferase reporter, 20 ng of pCMV -galactosidase control, 100 ng of pMGal4-DBD fused in frame to DRIP205-(527-774), and 100 ng of pVP16 fused to ER or ER LBD were co-transfected per well for 4 h. Cells were incubated in appropriate hormone-supplemented Dulbecco's modified Eagle's medium without phenol red medium with 10% charcoal-stripped serum for 36 h and then harvested for luciferase and -galactosidase assays. The relative level of luciferase activity was normalized to the -galactosidase activity.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ER Interacts with the DRIP Coactivator Complex-- We and others previously examined a subset of steroid/nuclear receptors for their ability to interact with the DRIP complex in the presence of their cognate ligands. In these experiments, very weak or no interaction with ER was detected (18, 21), raising the possibility that this receptor does not interact with or coactivate through the DRIP complex. However, binding conditions used in these GST pull-down assays included relatively high amounts of the detergent deoxycholate that apparently inhibited estradiol binding to ER. Using revised conditions that included significantly lower amounts of detergent, we reanalyzed the ability of ER to interact with individual DRIP subunits. By expressing individual cDNAs of seven of the subunits in a coupled transcription/translation system, we observed that ER-LBD interacted with three DRIP subunits, DRIP240, -205, and -77. However, estradiol-induced interaction occurred only with DRIP205 (Fig. 1A). This is the same subunit previously identified as interacting directly with VDR (19, 29) and TR (called TRAP220; Ref. 21) and found in a yeast two-hybrid screen as a peroxisome proliferator-activated receptor--interacting murine protein called PBP (22). DRIP77 and DRIP240 also associated with ER, albeit more weakly, and this interaction was not significantly enhanced by estradiol. The ligand-enhanced ER interaction with DRIP205 was observed regardless of which protein was used as bait (Fig. 1B). Moreover, under the identical binding conditions described above, we found that ER also bound DRIP205, in a strongly estradiol-dependent manner (Fig. 1B). The DRIP205 interaction with both ER isoforms was also detected in cells using a mammalian two-hybrid assay (Fig. 1C). DRIP205 therefore does not have a binding preference for one of the two known isoforms of ER.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   A single DRIP subunit, DRIP205, binds directly to ER in a ligand-dependent manner. A, individually in vitro translated, full-length 35S-labeled DRIP subunits, as indicated, were used together with bacterially expressed and affinity-purified GST-ERLBD or GST in each pull-down assay. B, both ER isoforms interact with DRIP205. GST pull-down assays were performed with in vitro translated, 35S-labeled ER or ER and a fragment of DRIP205 containing its nuclear receptor-interacting domains (residues 527-970) fused to GST (left panels), or in vitro translated, full-length 35S-labeled DRIP205 and full-length, bacterially expressed GST-ER or ER (right panels). 10% of the input for each translated protein is shown. All pull-downs were carried out in the absence () and presence (+) of 1 × 106 M 17-- estradiol. C, mammalian two-hybrid interaction. COS-7 cells were transfected with a Gal4DBD plasmid fused to the receptor interaction region of DRIP205 and a second plasmid encoding the VP16 transactivation domain fused to ER or  LBDs. -Fold induction in the presence of 106 M 17--estradiol was calculated with the level of relative luciferase activity in the absence of hormone set to 1. D, ER interaction with DRIP205 in vivo is ligand-dependent. ER was expressed stably in U2OS-ER cells and immunoprecipitated from cells untreated or treated with 1 × 106 M 17--estradiol (E2). The precipitate was separated by SDS-PAGE, blotted, and probed with an anti-DRIP205 antibody. NRS represents a parallel immunoprecipitation using a normal rabbit serum control.

In order to determine if DRIP205's interaction with ER anchors the entire multisubunit DRIP complex to the receptor, as it does with so-called class II nuclear receptors such as VDR, TR, and retinoic acid receptor, we initially attempted to view the complex bound to immobilized ER-LBD or full-length ER bound to glutathione beads. Under these conditions, we failed to detect the DRIP complex bound to liganded ER (data not shown). However, when endogenous ER was immunoprecipitated from human osteosarcoma U2OS cells overexpressing ER with an anti-ER antibody and immunoblotted against antibodies directed to DRIP subunits, we observed estradiol-dependent co-precipitation of DRIP205 (Fig. 1D) but not other DRIP subunits, such as DRIP 130 or DRIP 150 (data not shown). These results are consistent with either DRIP205 bound on its own to ER or as part of an as yet to be determined subcomplex of DRIP.

Quantitative Analysis of DRIP205 with ER and ER-- To examine more carefully aspects of DRIP205 binding with ER, we determined binding constants for the interaction in the presence of both agonist and antagonists, using surface plasmon resonance. Bacterially expressed and purified GST-DRIP205 was immobilized on the surface of a BIAcore sensor chip as described under "Materials and Methods." Fig. 2A demonstrates overlaid sensograms of the injections of 30 µl of ER liganded with 17-estradiol, at concentrations ranging from 4.4 to 280 nM. A saturable response was detected for ER liganded with estradiol, indicating that this DRIP derivative specifically interacted with purified ER. Essentially identical results were generated with ER (data not shown). Fig. 2B presents a graph of the amount of ER bound to DRIP205 at the end of each injection versus a logarithm of ER concentration. The sensograms generated were analyzed using the BIAEvaluation 3.0 program, as described previously (30), and values for apparent dissociation, association, and affinity rate constants were determined (Table I). Data analysis revealed that the ER-DRIP205 interaction in the presence of estradiol cannot be described using a simple monomolecular model. Rather, the data indicate that fast initial ER binding leads to the formation of an unstable ER-DRIP205 complex (k_off1 = 1.8e² s¹) that slowly undergoes conformational changes, resulting in a significantly more stable complex. (k_off2 = 2.11e⁴ s¹). As summarized in Table I, the apparent affinity rate constant for the ER-17--estradiol-DRIP205 interaction was K_D = 64 nM; the affinity of the ER-17--estradiol-DRIP205 interaction was found to be very similar (K_D = 72 nM).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Analysis of DRIP 205/ER binding by surface plasmon resonance. A, overlaid injections of 30 µl of ER at the protein concentrations of 4.4, 8.75, 17.5, 35, 70, and 140 nM over a chip surface containing immobilized GST-DRIP205 (680 RU). Prior to injection, ER was preincubated with 1 µM (final concentration) 17-estradiol at room temperature for 2 h. B, graph of the amount of ER, bound to DRIP205 at the end of each injection, versus logarithm of ER concentration.

View this table: [in this window] [in a new window]   Table I Affinities of DRIP205 to ER and ER in response to agonist or antagonists Dissociation constants were generated by surface plasmon resonance, as described under "Materials and Methods" and shown in Figs. 2 and 3. E2, 17--estradiol; 4HT, 4-(OH)-tamoxifen; Ral, raloxifene; ICI, ICI-182,780.

The effects of ER antagonists on DRIP205/ER binding were also analyzed by BIAcore. Fig. 3A presents overlaid sensograms of injections of unliganded ER, and ER liganded with 17-estradiol, 4-(OH) tamoxifen, raloxifene, and ICI-182,780 (all at 10⁶ M) run over immobilized DRIP205. While binding of agonist enhanced the affinity of ER interaction with DRIP205 compared with unliganded receptor, the pure antagonist ICI-182,780, and partial agonists 4-(OH)-tamoxifen and raloxifene all inhibited this interaction. Similar results were obtained for ER (data not shown). The data indicate that binding of 17-estradiol significantly accelerated formation of the ER-DRIP205 complex, where k_on1 was enhanced more than 3-fold compared with unliganded ER and more than 10-fold relative to ER liganded with ICI-182,780. Affinity and kinetic rates of DRIP205 interactions with both ER and ER in the presence of these antagonists are summarized in Fig. 3B.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effects of agonist and antagonists on ER/DRIP205 binding. A, overlaid sensograms of 30 µl injections of unliganded ER and ER liganded with 4-(OH)-tamoxifen, raloxifene, and ICI-182,780 over immobilized GST-DRIP205 (680 RU). Prior to injection, ER (75 nM) was incubated with the indicated ligands at 1 µM for 1 h at room temperature. B, apparent affinity rates of ER/DRIP205 binding. Rates were obtained by global fitting analysis. Experimental data fit into a model that describes ER-DRIP205 binding and conformational changes.

Surface plasmon resonance was used to directly compare the affinities of ER with DRIP205 and SRC-1, a p160 nuclear receptor coactivator. Serial injections of ER or ER liganded with 17-estradiol were run over flow cells immobilized with equivalent amounts of bacterially expressed GST-DRIP205 or GST-SRC1. Data analysis indicated that ER interacted with both DRIP205 and SRC1 with similar affinities, although somewhat higher for SRC1 (Table I). Essentially identical results were observed with ER (data not shown).

DRIP205 Binds Selectively to ER-AF-2 through One of Two NR Boxes-- Ligand-dependent interactions of most receptor coactivators, including DRIP205, have been mapped to the AF-2 domain within the receptor's ligand binding domain (18, 29). However, several recent reports indicate that p160 coactivators, such as SRC-1, also can interact with subregions of the functionally defined AF-1 (32-34), found within the N terminus of all steroid as well as some nuclear receptors. To determine if DRIP205 could also bind to the ER N terminus, we carried out in vitro pull-down assays with full-length ER and N- and C-terminal truncations (Fig. 4A). As shown in Fig. 4B, no binding was detected between DRIP 205 and the ER N terminus that contains the AF-1 region (lanes 5-7). Under the same binding conditions, the C-terminal half of ER and also full-length ER interacted with DRIP205 in an estradiol-stimulated or -dependent fashion (lanes 1-4 and 8-11). The LBD and ligand dependence of DRIP205 binding to ER suggested that this interaction is reminiscent of how DRIP205 as well as other coactivators interact with nuclear receptors, namely through key interactions with residues in the AF-2 domain that form part of a charged clamp that accommodates the coactivator within a hydrophobic cleft in the LBD. To test the involvement of the AF-2 in ER-DRIP205 binding, we asked if DRIP205 bound ER in the context of two key mutations in the AF-2 that both abolish p160 interaction and ER-mediated transactivation (4). As is clear from Fig. 4C, the presence of mutations at ER residues 539 and 540 completely abolished the ability of the receptor to interact with DRIP205, indicating that this ligand-dependent interaction is dependent on the structural integrity of the AF-2.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   DRIP205 associates exclusively with the ER-LBD in an AF-2-dependent manner primarily through contributions of NR box 1. A, schematic representation of full-length ER, ER-N (amino acids 1-251), and ER-C (amino acids 185-595). B, DRIP205 interacts with the ER-LBD only. ER, ER-N, or ER-C were in vitro transcribed and translated in the presence of [35S]methionine (lanes 1, 5, and 8; 10% input) and incubated with Sepharose beads containing bound GST-DRIP205, in the presence or absence of 17--estradiol. GST beads were used as a control for nonspecific binding. Bound ER, ER-N, or ER-C was resolved by SDS-PAGE and visualized by autoradiography. C, ER/DRIP205 interaction is AF-2-dependent. GST pull-downs of intact ER or ER containing AF-2 Leu to Ala mutations at residues 539 and 540 were carried out as described for B. D, relative contributions of NR boxes. GST pull-down assays using in vitro translated ER (lanes 1-11) or VDR (lanes 12-22) in the absence or presence of 106 M 17--estradiol or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), respectively, together with 2 µg of GST-DRIP205-(527-970) (wt; lanes 2 and 3 and lanes 13 and 14), GST-DRIP205NR1 (residues 604-774; lanes 4 and 5 and lanes 15 and 16), or GST-DRIP205NR2 (527-604; lanes 6 and 7 and lanes 17 and 18) are shown. Mutants 1 and 2 (lanes 8-11) correspond to GST-DRIP205-(527-970) containing point mutations in either NR1 or NR2 boxes, respectively, that change each LXXLL motif to LXXAA. GST proteins used in the experiments depicted in D were quantitated by visualization on Coomassie-stained SDS-PAGE.

DRIP205 contains two closely positioned consensus LXXLL nuclear receptor interaction motifs we have termed NR1 and NR2, and we have established that NR2 is required for DRIP complex interactions with VDR through DRIP205 (29). Similar results have been reported with TRAP220 and TR (21). These interactions occur with the AF-2 in a way presumably analogous to that previously described for p160 coactivators (2). To examine the contributions of NR1 and NR2 to DRIP205 binding to ER, we utilized both deletion and missense mutants in each NR box and tested these mutants for their estradiol-dependent interactions with ER, comparing this to the mutants' interactions with VDR. The missense mutations were alterations in which the last two Leu residues in each LXXLL box were changed to Ala. Whereas NR2 is critical and NR1 is dispensable to DRIP205's interaction with VDR (Fig. 4D, lanes 17 and 18 and lanes 21 and 22 versus lanes 15 and 16 and lanes 19 and 20), remarkably, the opposite appears to be the case for ER, where NR1 is the decisive interacting motif and NR2 is not required (lanes 4 and 5 and lanes 8 and 9 versus lanes 6 and 7 and lanes 10 and 11). Identical results were observed by assaying interactions using the BIAcore (data not shown). Since we have observed NR2's predominance with VDR and a number of other nuclear receptors, these results suggest that homodimer-binding steroid receptors such as ER employ a distinct mode of interaction with DRIP205, where the N-terminal LXXLL motif makes a decisive interaction with the receptor.

DRIP205 Enhances ER Transactivation in Vivo-- Many coactivators share an ability to stimulate ligand-dependent transactivation of steroid/nuclear receptors when overexpressed in transient transfection experiments. We have tested this characteristic for DRIP205 in the context of several nuclear receptors, including ER (Ref. 18 and data not shown). While we often observe modest coactivation, we believe that transient overexpression of a single subunit that derives from a multisubunit complex is not necessarily the best way to assess its functional relevance. Instead, we previously generated a small fragment of DRIP205 containing only its nuclear receptor-interacting region (Fig. 5A) (29). When cotransfected with a vitamin D-responsive reporter, this fragment, called the 205 box, selectively attenuated VDR-mediated transactivation, but not VP16 of E1A-stimulated activation (29). When tested in the osteosarcoma cell line U2OS-ER, overexpression of the 205 box resulted in a dose-dependent inhibition of ER/estradiol-dependent transactivation from an ERE-regulated reporter (Fig. 5B). This dominant-negative effect was selective for ER, since it did not result in the inhibition of VP16-mediated transactivation from a GAL4-regulated reporter (Fig. 5C). Note that although the DRIP/ARC complex can also interact with VP16 and potentiate its transactivation (23), it does not do so through direct contacts mediated by DRIP205 but rather through a different subunit DRIP(ARC)77/TRAP80 (25). Thus, endogenous DRIP205 (and perhaps the entire DRIP complex) is required for estradiol-dependent transactivation by ER and utilizes the same or similar ER-interacting determinants (i.e. the LXXLL motifs) as the p160 coactivators.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   A 190-amino acid DRIP205 fragment, containing both NR boxes (205-Box) selectively inhibits ER transactivation. A, schematic diagram of 205-Box, a fragment of DRIP205 (residues 527-714) containing two nuclear receptor-interacting LXXLL motifs (NR1 and NR2). B, U2OS cells stably overexpressing ER were transfected with a luciferase reporter plasmid containing a multimerized ERE, together with increasing amounts (in ng) of DRIP205-Box expression vector, in the absence (gray bars) or presence (black bars) of 10 6 M 17--estradiol (E2). Luciferase activity was normalized relative to -galactosidase activity. C, the 205-Box has no effect on transactivation by VP16. Transient transfection assays were performed as in B except that cells were transfected with a luciferase reporter plasmid containing a multimerized Gal4-UAS enhancer, together with increasing amounts (in ng) of 205-Box and Gal4-VP16 (50 ng) expression vectors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Coactivators appear to play central roles in mediating steroid/nuclear receptor transactivation. Among several ligand-dependent cofactors that associate with the receptors, the DRIP complex, co-discovered as TRAP and ARC, has been shown to directly and potently coactivate ligand-dependent transcription of nuclear receptors on naked and chromatin-assembled templates in vitro. Although originally described as a TR- and VDR-interacting complex, DRIP/TRAP/ARC shares several subunits with the mammalian SRB/mediator complex and is capable of enhancing the transcriptional activation functions of several classes of activator proteins unrelated to nuclear receptors.

Despite the apparent generality of the DRIP complex, its putative role in steroid receptor signaling has not been well developed. Two subunits of the DRIP complex, DRIP205 and DRIP150, interact with the AF-2 and AF-1 domains of GR, respectively (28), suggesting that different activation surfaces specify distinct DRIP subunits. The relatively well conserved AF-2 domain among members of the steroid/nuclear receptor superfamily predicts that DRIP205 would be a common subunit interaction with the AF-2 and one that would be directly regulated by ligand binding. We have shown in this work that DRIP205 does indeed interact selectively with the ER LBD in a ligand-dependent manner that is enhanced by agonist and inhibited by antagonists such as tamoxifen and raloxifene. A fragment of DRIP205 containing only the receptor-interacting region can inhibit ER-mediated activation from a responsive reporter, indicating that this dominant negative mutant competes part or all of the DRIP coactivator complex required for transcriptional enhancement at a target promoter. Interestingly, DRIP205 has been found to be overexpressed in 50% of breast tumors examined, albeit in a relatively small sample size (35). This observation coupled with DRIP205's association with ER suggests its possible role in tumorigenesis, although both the requirement of the DRIP complex in ER transactivation and its involvement in breast cancer are strictly correlative at this point and remain to be established.

Indeed, it should be pointed out that we failed to observe other DRIP subunits recruited to ER. In the case of several nuclear receptors, it is through the ligand and AF-2-dependent interaction with DRIP205 that the entire 15-subunit DRIP complex is recruited, and it is this complex, rather than a single DRIP subunit alone, that constitutes the functionally active DRIP species. Why we cannot detect other DRIP subunits associated with DRIP205 when the latter is recruited to ER is unclear. The biochemical conditions we utilized, although similar to what we used originally to isolate and purify the complex with VDR, may simply not be optimal in the context of ER. Alternatively, DRIP may be composed of several distinct subcomplexes, differentially recruited to steroid/nuclear receptors by DRIP205. The subfamily distinction of steroid versus nuclear receptors might also distinguish between such subcomplexes. Finally, the so-called class I steroid receptors, defined in part by their association with heat shock proteins, may require additional factors, perhaps cytosolic, for proper DRIP complex association that are missing from our nuclear extract preparations.

We have previously reported that the same ER ligands examined here have similar potentiating or inhibiting effects on the binding of SRC3 (ACTR/AIB-1) to ER (36). Moreover, the DRIP and p160 classes of coactivators share very similar determinants for binding to steroid and nuclear receptors (i.e. the AF-2 domain in the receptor and the LXXLL motifs in the coactivator). This raises the question of how the binding of these coactivators is regulated vis-a-vis each other, if, in fact, they are at all. We and others have previously proposed a sequential model whereby the CBP/p160 complex modification of histones leads to chromatin remodeling, which in turn renders the preinitiation complex accessible to recruitment by the DRIP complex (17, 21). A second possible scenario is a combinatorial type of mechanism, whereby both complexes are recruited separately by two bound receptor heterodimers on the same promoter, functioning in concert to activate. Very recent results of Chen and co-workers (37) demonstrated that acetylation of SRC3/ACTR by p300/CBP actually disrupts the former's interaction with ER; this could conceivably allow the DRIP complex to bind ER as a second step in the activation process.

ER ligands that in some cell types exhibit agonist activity, such as 4-(OH)-tamoxifen and raloxifene, inhibit ER/DRIP205 interaction in vitro. Similar results have also been observed for ER and ER interactions with SRC1, SRC3, GRIP1, and CBP.³ The fact that a compound that consistently inhibits ER-coactivator interactions can in some circumstances stimulate ER-mediated transactivation suggests that unidentified ER-interacting factors might mediate the activity of these compounds in modulating ER function, perhaps through additional activation domains in the ER (i.e. AF-1). It will be interesting to determine whether or not additional DRIP subunits interact with ER-AF-1, as is the case with DRIP150 and GR (28), and whether or not in the context of full-length ER partial agonists have differential effects on this interaction in vivo.

	ACKNOWLEDGEMENTS

We thank B. Komm for critical comments on the manuscript, M. Garabedian and M. Parker for ER constructs and valuable discussions, I. Rogatsky and J. Trowbridge for the U2OS-ER cell line, and M. A. Lazar for anti-PBP antibody.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK45460 (to L. P. F.).

^§ Supported by the Abraham J. and Phyllis Katz Foundation.

To whom correspondence should be addressed: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, Box 470, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2976; Fax: 212-717-3298; l-freedman@ski.mskcc.org.

Published, JBC Papers in Press, April 17, 2000, DOI 10.1074/jbc.M002013200

² W. Yang, C. Rachez, and L. P. Freedman, unpublished data.

³ C. W. Wong, B. Komm, and B. J. C., unpublished results.

	ABBREVIATIONS

The abbreviations used are: AF, transactivation function; LBD, ligand-binding domain; VDR, vitamin D receptor; TR, thyroid hormone receptor; ER, estrogen receptor; GR, glucocorticoid receptor; GST, glutathione S-transferase; aa, amino acids; PAGE, polyacrylamide gel electrophoresis; RU, resonance units.

	REFERENCES

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