Differential Recruitment of Coactivator RIP140 byAh and Estrogen Receptors

The Ah receptor (AhR), a soluble cytosolic protein, mediates most of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related environmental contaminants. The mechanism of ligand-mediated AhR activation has been, in part, elucidated. The sequence of events following the binding of the AhR/AhR nuclear translocator protein (ARNT) heterodimer to dioxin response elements has yet to be completely understood. The role of coactivator, RIP140, in the modulation of transcriptional activity of AhR/ARNT heterodimer was examined. RIP140 enhanced TCDD-mediated, dioxin response element-driven reporter gene activity in three cell lines. Co-immunoprecipitation and co-localization assays revealed that RIP140 interacted with AhR, but not with ARNT, both in vitro and in cells. Mapping of the interaction sites revealed that RIP140 was recruited by the AhR transactivation domain via the Q-rich subdomain. The RIP140 domain that interacts with the AhR was mapped to a location between amino acid residues 154 and 350, which is distinct from those involved in estrogen receptor binding. The signature motif, LXXLL, which is responsible for binding of several coactivators to nuclear receptors, is not required for RIP140 binding to AhR. These results demonstrate that the AhR recruits coactivators that are capable of enhancing transcription and, thus, the AhR may compete with steroid receptors for a common coactivator pool. In addition, the data suggest that there are distinct motif(s) for the recruitment of RIP140 to AhR and possibly other non-steroid receptors/transcription factors.

The Ah receptor (AhR) 1 is a ligand-activated transcription factor that mediates most, if not all, of the biological effects of TCDD, an environmental contaminant (1). The AhR is also apparently involved in hepatic growth and development, based on the phenotype of AhR Ϫ/Ϫ mice (2), and may play a role in the Hepa 1c1c7 cell cycle (3). In addition, the AhR may also be involved in the development of the immune system, as indicated by decreased accumulation of lymphocytes in the spleen and lymph nodes in AhR Ϫ/Ϫ mice (4). The AhR exists in the cytosol of cells as a heterotetrameric 9 S complex (5-7) with two Hsp90 molecules and the X-associated protein 2 (8 -10). Upon ligand binding, the AhR undergoes a conformational change and translocates to the nucleus as a 9 S complex. ARNT (11), which localizes in the nucleus, heterodimerizes with the AhR after Hsp90 dissociation to yield a 6 S complex (12,13). This heterodimeric AhR/ARNT complex binds to DRE located upstream of the cyp1A1, cyp1A2, glutathione S-transferase Ya, and cyp1B1 genes, and modulates their transcription (14 -16). TCDD induces a myriad of species-specific tissue damage, including hyperkeratosis and chloracne of the skin in rabbits and humans, liver damage in rodents, and lymphoid involution and embryotoxicity in many species (for a review, see Ref. 1). In most cases the affected tissues include the epithelium, where TCDD acts to alter proliferation and differentiation. The apparent specificity of these TCDD-induced toxicities suggests that there may be tissue-specific factors that alter the regulation of the AhR-mediated response (17).
Several lines of evidence point to the role of the AhR/ARNT complex in the disruption of chromatin (18,19) and the direct recruitment of basal transcription factors (20,21). Mapping of the transactivation domain of ARNT revealed a 34-amino acid transactivation domain consisting of mostly hydrophobic and acidic amino acid residues. In contrast, the AhR has a complex C-terminal activation domain that has been subdivided into acidic, Q-rich, and P/S/T-rich subdomains (17,(22)(23)(24)(25). The subdomains are capable of activating transcription when expressed independently, but can act synergistically when expressed together as a chimeric protein (21). Sp1 binds to GC boxes in the promoter region of cyp1A1 and has been shown to interact with AhR and ARNT via the zinc finger domain of Sp1. Sp1, AhR, and ARNT were found to synergistically enhance both transcription in in vitro transcription assays and reporter gene activity in transient transfections (26).
Several proteins in the basal transcription machinery have been shown to interact with the ER, and may contribute to enhancement of transcriptional activity. However, these interactions are unaffected by ER agonists or antagonists (27), suggesting that other factors may be necessary for ligand-dependent transactivation as well as cell and tissue specificity. Several nuclear receptor accessory factors, also referred to as coactivators, may act as bridging factors between the enhancer-binding activator proteins and the basal transcription complex or chromatin remodeling factors. These include SRC-1 (28,29), CBP (30), RIP140 (27), transcriptional intermediary factor 1 (31), transcriptional intermediary factor 2 (32), GRIP1 (33), and ARA70 (34).
RIP140 is recruited by the ER and modulates the receptor's transcriptional activity in the presence of estrogen. This coactivator has also been shown to interact directly with the ER and, upon overexpression in cells, caused a 2-fold enhancement of transcriptional activity (27). In addition, RIP140 also enhanced transcription of ER and RAR in vivo in yeast (35). RIP140 was also found to interact with other nuclear receptors, including RAR, TR, and retinoid X receptor, albeit less stably than with ER. It has also been observed that, while the ER, TR␤, and RAR␤ interact with two distinct sites located in the N and C termini, the retinoid X receptor predominantly interacts through the N-terminal site of RIP140, suggesting a possible mechanism for differential regulation of transcription of cognate genes (36). A short motif (LXXLL), also referred to as a NR box or LXD, has been identified in the nuclear receptor-interacting domains of several coactivators, nine copies of which are found in RIP140 (37,38). Mutation of this motif completely abolished binding of SRC-1 to the ER-AF2 transactivation domain, but not to CBP. Mutant SRC-1a also failed to enhance ER-dependent reporter gene activity in transiently transfected HeLa cells. Subsequent studies have demonstrated that the NR boxes are necessary for several other coactivator-nuclear receptor interactions (37, 39 -48). This has been further characterized by crystallographic analyses of co-crystals of TR ligand binding domain (LBD)/GRIP1 and peroxisome proliferatoractivated receptor LBD/SRC-1 (39,44), clearly demonstrating the role of this motif.
The AhR has been shown to interact with various factors in the basal transcription complex, including TATA-binding protein and transcription factor IIF (49), but no coactivators have been shown to interact with and enhance AhR-mediated transcriptional activation. Although CBP has been shown to interact with ARNT, there is no direct evidence indicating its role in AhR-mediated transcriptional activity in vivo (50). In this paper, the role of RIP140 in modulating transcriptional activity of the AhR/ARNT heterodimer in a minimal DRE-driven reporter context was examined in co-transfection experiments. RIP140 overexpression resulted in enhanced reporter gene activity in HeLa, Hepa 1c1c7, and COS-1 cells. Interaction of RIP140 with the AhR was demonstrated in vitro and in cells using coimmunoprecipitation assays and GFP-tagged proteins. Functional domains necessary for RIP140/AhR interaction in both RIP140 and AhR were mapped and mutations in the LXXLL motifs did not significantly destabilize the AhR-RIP140 interaction. Thus, AhR may employ differential mechanisms of coactivator recruitment in the process of transactivation relative to steroid receptors.
Generation of GST Fusion Proteins-The cDNA sequences corresponding to residues 1-350 of RIP140 were PCR-amplified and cloned into the BamHI/EcoRI site of pGEX-2TK to generate pGEX-RIP 1-350. The mAhR cDNA sequences corresponding to amino acids 500 -861, containing the mAhR transactivation domain, were PCR-amplified and cloned into the BamHI site of pGEX-2TK (Amersham Pharmacia Biotech) to generate pGEX-mAhR/TAD. The nucleotide sequence of the transactivation domain insert was confirmed by nucleotide sequencing. The mARNT cDNA sequences corresponding to amino acids 419 -791, containing the mARNT transactivation domain, were PCR-amplified and cloned into the BamHI and EcoRI site of pGEX-2TK to generate pGEX-mARNT/TAD. The cDNA s encoding hAhR amino acid sequences 713-848, 500 -600, 598 -848, 713-848 500 -713, and 600 -713 were PCR-amplified and cloned into the BamHI site of pGEX-2TK to derive truncated GST fusions of hAhR/TAD. GST and GST fusion proteins were expressed in E. coli (BL-21) and purified using glutathione-agarose by standard techniques.
In Vitro Binding Assay for Interaction of RIP140 with AhR-mAhR/ FLAG, mARNT/FLAG (51) or other cDNA constructs were in vitro transcribed and translated separately using the TNT Coupled Reticulocyte Lysate system (Promega, Madison, WI) and mixed with in vitro translated [ 35 S]methionine-labeled RIP140 or its deletion mutants and incubated at 4°C for 1.5 h The complexes were immunoprecipitated by incubation with anti-FLAG M2 affinity gel in CSB buffer (150 mM NaCl or as indicated, 15 mM CHAPS, 18.75% glycerol (v/v), 0.125% Nonidet P-40 (v/v), 20 mM Tris (pH 8.0), and 2 mg/ml bovine serum albumin) for 2 h, followed by several washes with CSB buffer without bovine serum albumin. The immunoprecipitated complexes were eluted with 50 g of FLAG peptide in MENG buffer containing 150 mM NaCl plus 3 mg/ml soybean trypsin inhibitor. The elution was repeated, and eluted proteins were separated on an 8% polyacrylamide gel. The proteins were transferred onto Immobilon-P membrane and subjected to autoradiography or phosphoimage analysis (Bio-Rad). In certain experiments the gels were treated with Amplify (Amersham Pharmacia Biotech) and the radioisotope was detected by fluorography. The relative amount of radioactivity was quantitated using a phosphoimager (Bio-Rad).
For GST pull-down assays, GST, GST-ARNT/TAD or GST-AhR/TAD fusion proteins immobilized on 30 l of glutathione-agarose (Sigma) were incubated with [ 35 S]methionine-labeled in vitro translated RIP140 or its deletion derivatives in CSB buffer. Following a 1.5-h incubation at 4°C, the agarose was washed four times with CSB buffer without bovine serum albumin. The immobilized proteins were eluted with 2ϫ Tricine sample buffer, separated using SDS-PAGE, and analyzed by fluorography.
Ligand Dependence Assays-GST-RIP 1-350 and GST were prebound on 30 l of glutathione-agarose for 30 min at 4°C. The agarose was washed three times in CSB buffer and incubated with 5 l of 35 S-labeled in vitro translated AhR in the presence of 20 nM TCDD or Me 2 SO for 1.5 h at 4°C, followed by several washes with CSB buffer. The proteins were eluted and separated using an 8% polyacrylamide gel and visualized by fluorography.
Cell Culture and Transient Transfection Experiments-Cells were routinely cultured in ␣-modified Eagle's medium containing 10% fetal bovine serum (v/v). Cells were transfected with a total of 750 ng of DNA using LipofectAMINE reagent (Life Technologies, Inc.) in 24-well plates for reporter gene assays. The transfected DNA mixture included 100 ng each of reporter plasmid, pGUDLUC 6.1 and the ␤-galactosidase internal control plasmid-pCMV/LacZ (Invitrogen, San Diego, CA) along with variable amounts of control plasmid and RIP140 expression plasmid, pEF-RIP. Cells were treated with 10 nM TCDD or Me 2 SO 24 h after transfection, for a period of 12 h. Cells were harvested, and extracts were assayed for luciferase activity. Luciferase activity was normalized to the observed ␤-galactosidase activity. All transfections were performed in triplicate.
For the expression of GFP and its fusion proteins, COS-1 cells were seeded on 2-mm 2 coverslips in 60-mm dishes and transfected with a total of 400 ng of GFP or GFP fusion plasmid along with pEF-RIP or control plasmid. The cells were washed with phosphate-buffered saline and visualized using fluorescence microscopy 24 h after transfection.
Interaction of AhR and RIP140 in Vivo-COS-1 cells seeded in 100-mm culture dishes were co-transfected with 4.5 g of pBOS-GST-AhR or pBOS-GST-AhR418 in the presence of 4.5 g of pEF-RIP or empty vector. The cells were treated with 10 nM TCDD or Me 2 SO for a period of 12 h. The cells were harvested in buffer D containing 20 mM Hepes (pH 7.9), 420 mM NaCl, 0.2 mM EDTA, 20% glycerol (v/v), 1 mM dithiothreitol, 10 mM NaF, 1ϫ protease inhibitor mixture (Sigma), and whole cell extracts were made following the procedure outlined by Kobayashi et al. (50). About 400 g of whole cell extracts were incubated with 50 l of glutathione-agarose at 4°C for 3 h. The agarose was washed four times with buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.01% Nonidet P-40 (v/v) and subjected to 8% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membrane and analyzed by Western blotting by using an anti-RIP140 antibody (Affinity BioReagents, Golden, CO) and 125 I-labeled donkey anti-rabbit secondary antibody. To demonstrate actual expression of the GST proteins in each case, 10% of cell extracts used in actual GST pull-down assays were also separated using SDS-PAGE and transferred to a polyvinylidene difluoride membrane and proteins were visualized using an anti-GST antibody.
Peptide Block Assays-GST or the indicated GST fusion proteins were absorbed on to glutathione-agarose in the presence of appropriate ligands or Me 2 SO. The immobilized GST fusion protein was washed and incubated with peptides P1 or P2 (37) (PQAQQKSLLQQLLT and PQAQQKSLLQQAAT, respectively) at varying concentrations (0, 10, 25, 50, and 200 M) in the presence of the appropriate ligand or Me 2 SO in CSB buffer containing 0.2 M NaCl. Recombinant RIP 1-439 cDNAs were transcribed and translated in a TNT system in the presence of [ 35 S]methionine following manufacturer's instructions. The translation products were then added to the GST pull-down mixture, and this was incubated for 1.5 h at 4°C. Following several washes, the radiolabeled proteins were eluted from the beads using 2ϫ Tricine sample buffer and analyzed by SDS-PAGE, followed by fluorography.
Statistical Analysis-Experiments were done in triplicate and statistical analysis was performed using the standard t test in the Sig-maStat program (SSSP Inc., Chicago, IL). Sample comparisons with p Ͻ 0.05 were considered to be significantly different from each other.

Modulation of Transcription of AhR/ARNT Activated Reporter Genes by RIP140 -
In an effort to understand the mechanism of AhR/ARNT-mediated transcriptional regulation of target genes, we analyzed the potential effects of overexpression of a number of steroid receptor coactivators on the activity of a minimal DRE-driven luciferase reporter gene in transient co-transfection experiments. Preliminary data had suggested that RIP140 overexpression resulted in a marked enhancement of DRE-driven reporter gene activity (data not shown). Therefore, the role of RIP140 in this minimal promoter context was examined in three cell lines: Hepa 1clc7, HeLa, and COS-1 ( Fig.  1). COS-1 cells contain negligible amounts of endogenous AhR and were used to clearly delineate the role of RIP140 in AhRmediated transcription. COS-1 cells, transfected with increasing amounts of RIP140, resulted in a dose-dependent biphasic response only in the presence of TCDD and co-transfected AhR (Fig. 1A). In the absence of co-transfected RIP140, there was a 4.1-fold increase in TCDD-mediated reporter gene activity compared with Me 2 SO treatment. An additional 2-fold TCDD-dependent induction of DRE-driven reporter gene activity was seen in the presence of 10 ng of co-transfected RIP140 (Fig. 1A). When the amount of co-transfected RIP140 was increased to 50 -100 ng, TCDD-stimulated reporter gene activity was reduced. When RIP140 was overexpressed in the absence of cotransfected AhR, no RIP140-mediated increase in reporter gene activity was seen (Fig. 1A), clearly suggesting that the transactivation process involved and required the AhR. In the absence of TCDD, no increase in reporter gene activity was seen, further supporting the role of AhR in the transactivation process.
In order to determine whether the modulation of DRE-driven reporter activity was cell line-dependent, HeLa and Hepa 1c1c7 cells were transfected with increasing amounts of RIP140. In the absence of exogenous expression of RIP140 in HeLa cells TCDD caused a nearly 6-fold activation of reporter gene activity when compared with controls (Fig. 1B). An additional TCDDdependent 2.6-fold increase in reporter gene activity was observed when cells were transfected with 100 ng of RIP140 ( Fig.   FIG. 1

. Effect of RIP140 overexpression on AhR-mediated transactivation in COS-1, HeLa, and Hepa 1c1c7 cells. COS-1 cells (A), HeLa (B), and
Hepa 1c1c7 cells (C) were co-transfected with increasing amounts of RIP140 expression vector, pEF-RIP (0 -250 ng); 100 ng of DRE-driven luciferase gene-containing plasmid, pGUDLUC 6.1; internal control plasmid, pCMV/LacZ (100 ng); and empty plasmid. COS-1 cells were also co-transfected with 100 ng of pcDNA3/ ␤mAhR where indicated. Luciferase activity was measured following 12 h of induction with 10 nM TCDD or carrier solvent and normalized to ␤-galactosidase activity. Each transfection was performed in triplicate in 24-well plates. For statistical analysis, the values from the RIP140 transfected samples were compared with those of RIP140 non-transfected samples within each cell line. Asterisks indicate that statistically significant differences were observed (p Ͻ 0.05).
1B). In Hepa 1c1c7 cells without co-transfected RIP140, a 3.2fold activation of reporter activity was observed with TCDD treatment compared with controls. When co-transfected with 100 ng of RIP140, a 4.4-fold TCDD-dependent increase in reporter gene activity was seen (Fig. 1C). These results together suggest a potential coactivation role for RIP140 in the presence of exogenous or endogenous human or murine AhR, as well as a cell-line dependent level of coactivation. No significant increase in reporter gene activity was seen at any level of cotransfected RIP140 in HeLa and Hepa 1c1c7 cells in the absence of TCDD (Fig. 1, B and C, and data not shown). The RIP140-induced 4.4-fold increase in DRE-driven reporter activity observed in Hepa 1c1c7 cells is higher than the ligandmediated 2-fold induction obtained with ERE-driven reporter genes (52). The RIP140-induced increase in reporter gene activity in other cell lines is comparable to those obtained with ER.
In Vitro Interaction of AhR and RIP140 -The in vitro interaction between RIP140 and AhR or ARNT was examined in co-immunoprecipitation assays using in vitro transcribedtranslated FLAG-tagged AhR and ARNT and [ 35 S]methioninelabeled in vitro translated RIP140. While RIP140 was found to co-immunoprecipitate with AhR-FLAG (Fig. 2), no RIP140 was found to co-immunoprecipitate with ARNT-FLAG. This is consistent with earlier reports, which indicate that the AhR has a more complex transactivation domain (21), and is the dominant transactivation partner in the AhR/ARNT heterodimer (18). ARNT thus provides a negative control that further suggests that the AhR/RIP140 interaction is not a nonspecific proteinprotein interaction artifact. No binding of [ 35 S]methionine-labeled RIP140 was observed when it was incubated with the anti-FLAG M2 affinity gel alone in absence of in vitro translated AhR, indicating that there was no observed background binding to the affinity gel matrix or to the antibody (Fig. 2, control lane).
The AhR domain required for binding of RIP140 was mapped using deletion derivatives of AhR-FLAG. RIP140 co-immunoprecipitated with full-length AhR-FLAG, as well as with AhR-N⌬315 FLAG, the N-terminal deletion derivative of AhR-FLAG (Fig. 2), which contains the transactivation domain, but lacks the ligand-and DNA-binding domains. Curiously, more RIP140 was found to co-immunoprecipitate with the AhR N⌬315. The N-terminal deletion derivative may provide less steric hindrance to the binding of RIP140 in the absence of the ligandand DNA-binding domains of the AhR or it could induce a conformational change in this AhR mutant, which would facilitate the binding of RIP140. As expected, the C-terminal deletion of AhR (AhR⌬TAD-FLAG), lacking the entire transactivation domain, displayed greatly reduced binding to RIP140 (Fig.  2), suggesting that RIP140 interacts, directly or indirectly, with the transactivation domain. These data are consistent with results indicating that the AF2 transactivation domain of the estrogen and other nuclear receptors interacts with RIP140 (36,52). As expected, ARNT⌬TAD-FLAG, lacking the transactivation domain failed to interact with RIP140.
Interaction of RIP140 with AhR in Cells-In order to test for an interaction between AhR and RIP140 in cells, COS-1 cells were transiently transfected with pBOS-GST/AhR or pBOS-GST/AhR 1-418, which express GST fusion proteins of fulllength AhR, or a truncated AhR lacking the transactivation domain, respectively, along with RIP140, or a control plasmid, followed by treatment with TCDD or carrier solvent 24 h after transfection. Whole cell extracts from transfected cells were incubated with glutathione-agarose, and the proteins pulled down were analyzed by SDS-PAGE and protein blotting (Fig.  3B). Marginal binding of RIP140 to GST-AhR in the absence of TCDD was observed (Fig. 3B, lane 7). However, in the presence of TCDD, a 3-fold increase in co-immunoprecipitated RIP140 was detected (lane 8). RIP140, when transfected into COS-1 cells along with GST-AhR 1-418, lacking the transactivation domain, showed no binding (lane 5), suggesting the following conclusions: 1) the interaction was specific and not due to background binding to the resin or GST, and 2) RIP 140 interacts with the transactivation domain. As a control, COS-1 extracts transfected with only GST-AhR were used. In this case, no detectable RIP140 was found to interact with AhR (lane 6). Protein blot analysis revealed that equal amounts of RIP140 (lanes 1, 3, and 4) and GST fusion proteins (Fig. 3C) were expressed and loaded in each of these lanes.
To further show interaction of RIP140 in cells, GFP-tagged AhR/TAD (AhR/TAD-GFP), GFP-tagged ARNT/TAD (ARNT/ TAD-GFP) fusions, or GFP were expressed in COS-1 cells along with full-length RIP140. Since neither AhR/TAD or ARNT/TAD are known to possess nuclear localization signals, they would be expected to be localized mostly in the cytoplasm. Interaction with RIP140 would lead to the translocation of GFP-AhR/TAD. In contrast, GFP-ARNT/TAD should fail to be localize to the nucleus. As a control, we expressed only GFP in the presence or absence of RIP140. GFP (Fig. 3C) was found to be localized mostly in the cytoplasm and to some extent in the nucleus, which has also been observed by the manufacturer (CLON-TECH). The distribution remained the same in the presence of co-expressed RIP140 (Fig. 3D). AhR/TAD-GFP, when co-expressed in the presence of a control plasmid, was found to be distributed predominantly in the cytoplasm and, to a some extent, in the nucleus (Fig. 3E). However, when RIP140 was co-transfected, GFP-AhR/TAD was found to localize in discrete foci in the nucleus (Fig. 3, F and G). This shift in distribution of GFP-AhR/TAD, seen in other reports with GFP-GR (53, 54) and GFP-TR (55), suggests that it interacts with RIP140 in cells. This localization into discrete foci was also observed with GFPtagged mouse RIP140 (55). In contrast, the expression of GFP-ARNT/TAD showed no shift in the intracellular localization in the presence or absence of co-expressed RIP140 (Fig. 3, H and I). These results provide additional evidence for specific interaction of RIP140 with AhR and not with ARNT. Taken together, these results indicate that AhR interacts with RIP140 via the transactivation domain in cells.
Effect of Ligand on AhR-RIP140 Interaction-Preliminary results indicated that the binding of AhR to RIP140, in vitro, occurred even in the absence of apparent ligand. The fulllength AhR requires Hsp90 for proper folding, and hence a full-length AhR-GST fusion protein cannot be generated in bacteria. Instead, a GST fusion protein consisting 1-350 amino acid residues of RIP140 was generated and mixed with in vitro translated 35 S-labeled AhR and assayed for interaction in the presence or absence of TCDD. Significantly, considerable binding of AhR to RIP140 was observed even in the absence of an apparent ligand (Fig. 4A). GST-RIP 1-350 was able to bind AhR both in the presence and absence of TCDD, although an increase was observed in the presence of TCDD (Fig. 4A). However, some variation in the level of enhanced binding was observed from experiment to experiment. Overall, there appeared to be a rather marginal increase in ligand-dependent binding. Our results are consistent with the fact that in vitro reticulocyte lysate translated AhR and ARNT can successfully form a functional heterodimer capable of binding to DRE in the absence of an exogenous ligand in gel shift assays (26). 2 This is also consistent with the fact that RIP140 can bind efficiently to AhR without its LBD (Fig. 2). This is in contrast to the absolute ligand dependence of ER binding to RIP140 and that of several other nuclear/steroid receptors to other coactivators presenting possible differences in the mechanism of coactivator recruitment.
The relative binding affinities of AhR and ER to RIP140 were compared under increasing salt concentrations. The interaction of RIP140 and ER (in the presence of 1 M E2) was found to be stronger, with considerable binding at salt concentrations as high as 1.0 M NaCl (Fig. 4B). In contrast, the interaction of RIP140 with AhR was found to be less stable at concentrations higher than 0.5 M NaCl, which appears to be comparable to results observed with the retinoid acid receptor (Fig. 4B) (36). RIP140 bound AhR 30% and 35% more weakly compared with the ER, even at concentrations of 250 and 500 mM NaCl, respectively (Fig. 4B). These combined results are consistent with differential RIP140 recruitment mechanisms between the Ah and estrogen receptors.
Mapping of AhR Interacting Sites on RIP140 -We used a series of N-and C-terminal deletion derivatives of RIP140 to determine the AhR-interacting site(s) using co-immunoprecipitations with AhR/FLAG. Analysis of N-terminal deletion derivatives of RIP140 clearly indicated that AhR, unlike ER, interacted exclusively with the N-terminal region (Fig. 5A). While RIP 393-1158 clearly showed binding, RIP 301-1158 displayed weak binding to AhR. Analysis of the C-terminal deletion derivatives indicated that residues residues 1-439 were necessary and sufficient for AhR binding (Fig. 5A). This is in contrast to results from ER binding analysis, which indicated that ER bound to regions both in the N and C termini, suggesting a different mechanism of recruitment of RIP140 to AhR. Interestingly, these amino acid sequences include one of the acidic regions that were found in RIP140 (28,37). Further N-and C-terminal deletion constructs were generated and tested for binding to AhR using GST-mAhR/TAD in GST pull-down assays. Among the N-terminal deletion derivatives RIP 53-350, RIP 103-350 and RIP 154 -350 were able to bind GST-mAhR/ TAD (Fig. 5B), clearly indicating that the N-terminal 154 residues are not required for AhR binding. However, RIP 201-350 failed to bind to AhR (Fig. 5B). Binding of C-terminal derivative, RIP 154 -325 was reduced when compared with RIP 154 -350 (Fig. 5C), suggesting that, while amino acids 154 -325 may contain some of the residues involved in binding function, optimal binding requires amino acid residues 154 -350. As ex-2 M. B. Kumar, R. W. Tarpey, and G. H. Perdew, unpublished data.

FIG. 3. Interaction of RIP140 with AhR in cells.
A and B, evidence of AhR/RIP140 interaction in cells using GST pull-down assays. COS-1 cells were transfected with pBOS/GST-AhR or pBOS/GST-AhR 1-418 in the presence or absence of co-transfected RIP140. Thirty-six hours after transfection, 440 g of whole cell extracts were incubated with glutathione-agarose. After extensive washing, the proteins were eluted and subjected to SDS-PAGE and Western blotting. A, proteins on Western blot were detected by anti-RIP140 antibodies. B, one-tenth input of proteins were used in GST pull-down assays followed by elution, SDS-PAGE, and Western blotting. Proteins were detected by anti-GST antibodies. C-I, interaction in cells using GFP-tagged proteins. COS-1 cells were transfected with GFP or GFP fusion constructs and RIP140 or control expression vectors and visualized by microscopy (magnification, ϫ100) 24 h after transfection. C, GFP; D, GFP plus RIP140; E, GFP-AhR; F, GFP-AhR plus RIP140; G, single cell; H, GFP-ARNT; I, GFP-ARNT plus RIP140.
pected, RIP 154 -300 failed to bind to AhR (Fig. 5C). Control assays with GST alone showed no or minimal binding to any of the RIP140 derivatives. These results suggest that amino acid residues 154 -350 are involved in binding of RIP140 to AhR. These sequences are clearly distinct from those involved in the binding of ER to RIP 140. However, this region contains two LXXLL motifs, which may or may not play a role in binding of AhR to RIP140 (Fig. 5D). Analysis of this AhR-binding RIP140 sequence indicated that the secondary structure is predicted to be mostly composed of ␣-helices.
Role of LXXLL Motifs in AhR-RIP140 Interaction-RIP140 and some known coactivators possess several copies of the short sequence motif, LXXLL. This motif has been found to be necessary and sufficient for binding of SRC-1a (37,44,48), RIP140 (37,55), GRIP1 (40,46), and CBP (37), among others, to the liganded nuclear receptors. The RIP1-439 fragment contains three LXXLL motifs and is capable of binding to the ER transactivation domain (36,37). Two peptides, P-1, with amino acid sequence, PQAQQKSLLQQLLT, corresponding to the motif at the C terminus of SRC-1a, and P-2 with amino acid sequence, PQAQQKSLLQQAAT, containing mutations in the LXXLL motif, were tested for their ability to block the binding of RIP 1-439 to the AhR and ER in GST pull-down assays. Increasing concentrations of wild type (wt) peptide, P-1, were able to block the ability of liganded GST-ER-HBD to bind to RIP1-439 (Fig. 6A, upper right panel). In contrast, mutant peptide, P-2 was unable to compete with RIP1-439 for binding to GST-ER-HBD (lower right panel). However, P-1 failed to compete with RIP1-439 for binding to GST-mAhR/TAD (upper left panel), suggesting that the interaction of RIP140 and AhR may be mediated by residues distinct from those involved in the ER-RIP140 interaction. This also suggests the absence of a role for the signature motif, LXXLL in AhR-RIP140 interaction and the presence of other interaction motif(s). However, the possible involvement of the LXXLL motif in other interactions within a transcriptionally active AhR complex cannot be ruled out. The mutant peptide P-2, as expected, was unable to compete with the binding of RIP1-439 to GST-mAhR/TAD (lower right panel).
In order to further determine the lack of requirement for LXXLL motifs, the two LXXLL motifs in the RIP 154 -350 truncated protein were mutated at residues 188/9 and 270/1 to LXXAA to generate a mutant protein, RIP 154 -350mut 184/ 266. The wt and the mutant cDNAs were in vitro translated and assayed for in vitro interaction with GST-AhR/TAD in a GST pull-down assay. As seen earlier, RIP 154 -350wt was able to bind to GST-AhR/TAD, but not to GST alone (Fig. 6B). When RIP 154 -350mut 184/266 was assayed for binding to GST-AhR/ TAD, it was also able to interact with AhR (Fig. 6B), indicating that the binding function was not disrupted. This clearly suggests that the LXXLL motifs are not required for binding of RIP140 to AhR. In addition, GST fusions of RIP154 -350wt and RIP154 -350mut 184/266 were generated and tested for interaction with full-length in vitro translated AhR in the presence or absence of TCDD. AhR, as noted earlier, bound strongly to GST-RIP 154 -350wt in both the presence and absence of TCDD, demonstrating again the lack of a direct role of TCDD in the binding of RIP140 to AhR (Fig. 6C). AhR also bound to a mutant derivative of GST-RIP 154 -350wt in which the two LXXLL motifs were replaced by LXXAA (GST-RIP 154 -350mut 184/266), although to a lesser extent. A marginal decrease in binding in the absence of TCDD was observed. However, clearly the AhR was capable of interacting with the mutant RIP 154 -350. In addition, when the interaction was assayed in the presence of TCDD, the binding of mutant RIP 154 -350 to AhR was slightly greater with only an 18% decrease compared with that of RIP 154 -350wt. The marginal decrease in binding of the mutant proteins suggests that the replacement of the four leucine residues may not be completely innocuous. The mutations may induce changes in tertiary structure, which may affect binding even though they may not be directly involved in binding. Nevertheless, the analysis of the mutants and the peptide competition assays together indicate a lack of a required role of the LXXLL motifs for AhR-RIP140 interaction.
Interaction of RIP140 with Subdomains of AhR Transactivation Domain-The AhR has an unusually complex transactivation domain comprising acidic, Q-rich, and P/S/T-rich subdomains. In order to determine the subdomain(s) of the human AhR transactivation domain required for RIP140 interaction, several AhR/TAD deletions were made and fused to glutathione S-transferase. The human and murine AhR sequences are 70% homologous and, thus, GST fusion proteins of the transactivation domains of the two proteins were assayed for comparison of their ability to bind full-length RIP140 (Fig. 7). In addition, GST-Sp1, which has a Q-rich transactivation domain, and GST-VP16, which has an acidic transactivation domain, were assayed for interaction with RIP140. The binding of RIP140 to full-length murine and human transactivation domains were comparable, clearly demonstrating the conservancy of specific functions (lanes 2 and 3). The acidic subdomain comprising residues 500 -600 was unable to bind RIP140 (lane 7). However, the Q-rich subdomain alone was sufficient for RIP140 binding (lane 6). The Q-rich and the P/S/T-rich subdomains together showed slightly increased binding to RIP140 (lane 4). When the P/S/T-region was deleted the fusion protein showed decreased binding to RIP140 (lane 5). However, P/S/T-rich subdomain alone failed to recruit any RIP140 (lane 8). The deletion of the acidic subdomain made no substantial difference to RIP140 binding function (lane 4), which is consistent with the fact that the acidic subdomain alone was not able to bind RIP140 (lane 7). Interestingly, the Sp1 transactivation domain, which is predominantly Q-rich, failed to bind any RIP140 (lane 9), clearly suggesting specificity in interaction among different transactivating proteins with apparently similar transactivation domains (lane 10). GST-VP16 also failed to interact with RIP140 (lane 10).
An alignment of the transactivation domains of human AhR and Sp1 showed conservation of several glutamine residues and several flanking hydrophobic residues, which have been shown to be important for binding downstream factors. However, these residues do not seem to be highly conserved in AhR from other species, e.g. mice and rat. Interestingly, the human and murine AhRs share extensive homology in other regions of the Q-rich subdomains in certain hydrophobic and glutamine residues, which may be involved in binding coactivators like RIP140.

DISCUSSION
In order to determine whether there are tissue-specific coactivator recruitment events that are key determinants in the overall level of gene transcription mediated by the AhR, two important aspects of the assembly of a functional AhR transcriptional complex need to be addressed. The first is the examination of the type of coactivators that are recruited to the complex AhR transactivation domain. The second aspect of this process is a detailed delineation of the subdomain(s) of the AhR TAD, as well as coactivator domains, that are key to formation of the AhR/coactivator complex. This information can then serve as a basis for further studies examining the role of each TAD subdomain in mediating AhR activity through recruitment of specific classes of coactivators in a cell type-specific manner. An additional aspect of coactivator recruitment is the emergence of motifs required for coactivator binding to steroid receptors and whether these motifs also play a role in coactivator binding to other transcription factors, such as the AhR.
In this report, the effect of RIP140 co-expression on DREdriven reporter gene activity was examined in transient transfections. We tested the effect of RIP140 overexpression in a variety of cell lines: COS-1, which have negligible amounts of endogenous AhR (Fig. 1A), HeLa (Fig. 1B), and Hepa 1c1c7 (Fig. 1C) cells, which have significant endogenous levels of AhR and ARNT. Upon RIP140 ectopic expression, a TCDD-dependent 2-fold increase in DRE-driven reporter gene activity was seen in COS-1 cells. When AhR was not co-transfected, RIP140 did not enhance reporter gene activity, clearly suggesting that the effects of RIP140 are mediated through the AhR and that RIP140 cannot act alone in transactivating the reporter gene. A 4.4-and 2.6-fold increase in DRE-driven reporter gene activity were observed in Hepa 1c1c7 and HeLa cells, respectively. Although the co-transfection experiments indicate that RIP140 can increase minimal DRE-driven reporter activity to different levels in a cell line-dependent manner, the level of enhanced coactivation could potentially depend on the existing pool of RIP140 and other coactivators in each cell line. In addition, other factors such as cell-specific repressors and promoter contexts may have a significant influence on the level of induction observed. Nevertheless, determining the influence of RIP140 in different cell lines further strengthens the hypothesis that RIP140 is a potential coactivator of AhR-mediated transcrip- tional activity. The use of HeLa and Hepa 1c1c7 cells also demonstrates the potential role of RIP140 in human and mouse AhR-mediated transcriptional activity and, under conditions where the AhR, and perhaps other factors, are present in varying amounts. This RIP140-mediated increase in reporter gene activity is clearly similar to results obtained with the estrogen receptor-mediated ERE-driven reporter genes. Cavailles et al. (53) reported a 2-fold increase in ERE-driven reporter activity in a RIP140 dose-dependent manner in the presence of estrogen. Additionally, a similar biphasic response to increasing RIP140 doses in reporter co-transfection assays was observed. This is consistent with other reports of coactivators, including SRC-1 (56) and CBP (57). When an increased amount of RIP140 is co-transfected, it could potentially sequester key transcription factors, leading to a decrease in transcription (53). RIP140 itself does not bind to TATA-binding protein or TFIIB (52), but it could potentially interact with any of the other factors involved in assembling a transcription initiation complex. RIP140 may behave like a repressor when overexpressed by sequestering SRC-1 from transcription complexes (58), as suggested in the case of peroxisome proliferator-activated receptor. RIP140 has also been suggested to have an auto-repressor function (55).
RIP140 and other coactivators have been shown to interact with the transactivation domain of several steroid/nuclear receptors (36). Our results suggest that the AhR also recruits RIP140 via its transactivation domain (Fig. 2). It has been seen in the case of several steroid receptors that the binding of ligand to the LBD induces a conformational change such that the AF-2 amphipathic ␣-helix (helix 12) is aligned over the LBD, unlike the case of an unliganded receptor, where helix 12 protrudes away from the LBD. This realignment may lead to the formation of a new interaction surface for coactivators and this sequence of events may explain the need for ligands for effective binding of coactivators (59). Unlike the ER and other steroid receptors, where the LBD is in close proximity to the transactivation domain, these two domains are well separated in the AhR, which may explain the lack of enhancement of RIP140 binding in the presence of ligand in vitro. In contrast to several steroid receptors, which require the presence of a ligand to bind RIP140 (36), the AhR ligand, TCDD, had marginal effect on RIP140 binding in vitro. However, in cells, enhanced interaction with RIP140 was observed in the presence of TCDD. In the case of AhR, which possesses a separate and potentially unmasked transactivation domain that can readily interact with coactivators, the need for a ligand for physical interaction with coactivators, at least in part, may be unnecessary. Although the possible role of endogenous ligands or factors in the reticulocyte lysate cannot be completely ruled out, pull-down experiments with GST fusion proteins, containing only the transactivation domain of the AhR without its LBD, clearly indicate that RIP140 can bind AhR in the absence of ligand. It should be noted that, although RIP140 seems to interact with the AhR in the absence of TCDD, in vivo transcriptional enhancement by RIP140 requires TCDD, as seen with the lack of enhanced reporter gene activity in the absence of TCDD (Fig. 1, A-C). There is considerable documented evidence for the role of ligands triggering the nuclear translocation of cytosolic AhR (5,10). AhR in a tetrameric complex (5,10) is thought to bind ligand, translocate to the nucleus, and subsequently transform into a heterodimeric complex (5). The modular, well separated nature of AhR LBD and its transactivation domain suggest that, although TCDD may be required for translocation of AhR to the nucleus, TCDD may not be required for physical interaction between the AhR and certain coactivators. This may preclude the necessity of a ligand to induce a conformational change for protein-protein interaction to occur with the transactivation domain. In contrast, estradiol is required for interaction of RIP140 with the ER, even in the context of the GST fusion protein with AF2 alone, since the AF2 domain includes both the transactivation domain and the ligand binding domain of ER (36). The in vitro interaction of RIP140 and AhR/ARNT heterodimer was also examined using co-immunoprecipitation experiments. There was no observed increase in the co-immunoprecipitation of RIP140 with AhR/ ARNT heterodimer as compared with AhR alone (data not shown), further pointing to the dominant nature of the AhR transactivation domain. This points to the existence of a discrete class of receptors, which can apparently physically interact with specific coactivators or other transcription factors in the absence of an exogenous ligand, although a ligand is clearly required for other functions, like transactivation.
Mapping of the AhR-interacting sites on RIP140 revealed significant differences between the binding of AhR and ER to RIP140 (Fig. 5, A-C). While ER interacts with RIP140 via two sites on the N and C termini of RIP140 (36), it was found that AhR interacts predominantly with the N terminus, involving residues 154 -350 in RIP140. No interaction was seen with any of the C-terminal residues of RIP140, which indicates a significant difference in binding mechanism of RIP140 to the ER versus the AhR. The AhR interacting RIP140 fragment, RIP 154 -350, contains two LXXLL motifs, which are necessary for binding of several coactivators to ER and other nuclear receptors (36). However, peptide competition assays showed that a peptide, containing a wild type LXXLL motif, competed with RIP140 for binding to the transactivation domain of the ER, but failed to compete with RIP140 for binding to the AhR (Fig.  6A). The lack of a necessary role for LXXLL motifs in RIP140/ AhR interaction was further established when LXXLL mutants of RIP 154 -350, the minimal AhR interaction domain, were found to bind AhR similar to wt RIP 154 -350 (Fig. 6B). This suggests that a different motif(s) may be involved in binding of RIP140 to non-steroid receptors like the AhR, which suggests the existence of diverse mechanisms for recruiting limited coactivator pools to different receptors, or classes of receptors, depending on the signal. There are numerous reports of the requirement of the LXXLL motifs for interaction with nuclear receptors (37, 39 -48) and of differential requirements for these motifs in cases where more than one motif exists in response to different ligands and to different receptors (39). The AhR presents a unique case where the LXXLL motif is not required for interaction with RIP140. However, this may depend on the actual biological situation existing in the cell, in terms of signals, promoter contexts, and cell cycle status. It is possible that other receptors which harbor similar transactivation domains may display differential mechanisms of recruiting common coactivators also. The flanking sequences clearly play a vital role in differential recruitment to nuclear receptors and the affinities of their interaction. The requirement of a relatively long RIP140 sequence for stable AhR binding also is a distinct feature that must be further investigated.
Analysis of AhR transactivation domain deletions indicated that the Q-rich subdomain was necessary and sufficient for binding of RIP140, while the acidic and P/S/T-rich subdomains clearly appeared to be dispensable for RIP140 interaction (Fig.  7). The lack of a clear role for the P/S/T-subdomain in cyp1A1 activation has also been underscored in AhR transactivation in in vivo studies in other reports (24,60). However, reporter gene assays have suggested that the acidic subdomain (which also has some conserved glutamine residues) is required for optimal activation (24). However, the fact that the P/S/T and acidic subdomains do not interact with RIP140 does not rule out the requirement of these subdomains for interaction with other transcription factors, including basal transcription factors, repressors, and other coactivators. Indeed, while VP16 fused to the N terminus of AhR has been previously shown to induce cyp1A1 expression (60), suggesting that the VP16 activation domain could substitute for the AhR activation domain, we failed to detect any RIP140 binding to VP16. Sp1 fused to the N terminus of AhR was shown to be unable to induce cyp1A1 expression (60). Sp1 in the present study showed no binding to RIP140. It has been observed in reporter gene assays with a fusion protein of truncated AhR transactivation domains that, while individually the subdomains are incapable of substantial induction, a combination of two or more subdomains clearly leads to synergy in induction (21). It is possible that the different subdomains may be involved in recruiting different coactivators and/or basal transcription factors. This differential recruitment may also be dependent on cell, species, and tissue types along with promoter contexts.
Several coactivators have been isolated that modulate the transcriptional activity of steroid hormone receptors (28,32,33,52), but there is as yet no evidence for AhR-specific coactivators. Coactivators like ARA70 have been shown to be involved in regulating androgen receptor-mediated transcription of certain genes in specific tissues and thus, at least in part, help explain tissue-and cell line-specific phenotype(s) (34). AhR may differentially recruit coactivators relative to the ER and other steroid hormone receptors, depending on the signal impinging on the cell. There may also be other classes of coactivators, which may be uniquely recruited by transcription factors with transactivation domains similar to that of the AhR. Nevertheless, this report demonstrates that the AhR and steroid receptors share at least one common coactivator. However, whether these receptor systems compete for the existing coactivator pool will require additional investigation. Finally, determination of the type(s) of common or distinct motifs involved in the recruitment of various coactivators to AhR or AhR-like transcriptional enhancer proteins will aid in understanding possible differential coactivator recruitment mechanism(s) employed by various transcription factors and how these mechanisms may lead to cell-, species-, and tissue-specific activation of certain subsets of genes.