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Coactivators for the Orphan Nuclear Receptor RORa

G. Brandon Atkins, Xiao Hu, Matthew G. Guenther, Christophe Rachez, Leonard P. Freedman, and Mitchell A. Lazar Division of Endocrinology (G.B.A., X.H., M.G.G., M.A.L.), Diabetes, and Metabolism Departments of Medicine and Genetics, and The Penn Diabetes Center University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104 Cell Biology Program (C.R., L.P.F.) Memorial Sloan-Kettering Cancer Center New York, New York 10021

A mutation in the nuclear orphan receptor RORa results in a severe impairment of cerebellar development by unknown mechanisms. We have shown previously that RORa contains a strong constitutive activation domain in its C terminus. We therefore searched for mammalian RORa coactivators using the minimal activation domain as bait in a two-hybrid screen. Several known and putative coactivators were isolated, including glucocorticoid receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205). These interactions were confirmed in vitro and require the intact activation domain of RORa although different requirements for interaction with GRIP-1 and PBP were detected. Even in the absence of exogenous ligand, RORa interacts with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors. Both PBP and GRIP-1 were shown to be present in these complexes. Thus we have identified several potential RORa coactivators that, in contrast to the interactions with hormone receptors, interact with RORa in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand. GRIP-1 functioned as a coactivator for the RORa both in yeast and in mammalian cells. Thus, GRIP-1 is the first proven coactivator for RORa. (Molecular Endocrinology 13: 1550–1557, 1999)

0888-8809/99/$3.00/0 Molecular Endocrinology Copyright © 1999 by The Endocrine Society

INTRODUCTION Members of the nuclear receptor superfamily are known to play roles in development, cellular growth, differentiation, and homeostasis (1). The nuclear orphan receptor RORa has been shown to play an important role in cerebellar development, as demonstrated with its mutation in naturally occurring staggerer mice (2), as well as in targeted knockout experiments (3). The exact mechanism by which RORa mutation leads to this defect is not known. RORa has also been implicated in the transcriptional regulation of the N-myc protooncogene (4) and in the apolipoprotein A-I gene (5), suggesting potential roles in neoplastic and metabolic processes. RORa is categorized as an orphan nuclear receptor because it is not known whether it binds a ligand or requires a ligand to activate gene transcription (6). In the case of nuclear receptors with known ligands, such as thyroid hormone receptor (TR) and retinoic acid receptor, the biological ligands are not found in yeast, rabbit reticulocyte lysate, or in most cultured mammalian cell lines. In these settings, the unliganded receptors interact with corepressor proteins N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) (7, 8). In the presence of ligand, these receptors undergo a conformational change, which leads to dissociation of the corepressors and recruitment of coactivators (9). Several putative nuclear receptor coactivator proteins have been identified, including SRC1/N-CoA-1, GRIP-1/TIF-2/N-CoA-2, PCIP/ACTR/ AIB1, RIP-140, TRIP-1, TIF-1, TRIP-230, PCAF, TRIP2/PBP/DRIP205/TRAP220, and CBP (reviewed in Ref. 7). Several studies have shown that these coactivator proteins are in multiprotein complexes that function, at least in part, by acetylating nucleosomal histones, thereby opening chromatin structure in a manner favorable for gene transcription (reviewed in Ref. 10). 1550

RORa Interactions with GRIP-1 and PBP

We previously showed that RORa contains a Cterminal activation domain that functions both in the context of the native protein and when fused to a heterologous DNA-binding domain (11). Here we describe a yeast two-hybrid screen using the C-terminal activation domain of RORa as bait that identified interactions between RORa and several known coactivators. Two of these, glucocorticoid receptor-interacting protein-1 (GRIP-1) (12, 13) [also known as hTIF-2 (14)]rsqb] and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP) (15) [also known as hDRIP205 (16), hTRAP220 (17), and RB18A (18)] were studied in detail. GRIP-1 and PBP interactions correlated with the ability of RORa polypeptides to function as activation domains and were demonstrated both in vitro and in vivo. In addition, endogenous GRIP-1 and PBP were among a complex of proteins that interact with RORa. The GRIP-1 and PBP interactions occurred with RORa in a variety of settings including yeast, bacterial extracts, rabbit reticulocyte lysates, and human-derived tissue culture cells, raising the possibility that RORa activation is truly constitutive. Furthermore, GRIP-1 was shown to function as a coactivator for RORa in yeast as well as in mammalian cells.

RESULTS The C Terminus of RORa Contains a Strong Activation Domain The C-terminal activation domain of RORa was fused to the DNA-binding domain of the Gal4 transcription factor and transiently transfected into JEG-3 human choriocarcinoma cells. Figure 1 confirms that the region of RORa containing amino acids 272–523 contains a strong activation domain. Moreover, amino acids 272–385 were required for this activation. All of the Gal4 fusion proteins were expressed at comparable levels (data not shown). These results indicate that in addition to the AF2 helix, which has been previously shown to be required for activation by RORa (11, 19), these more N-terminal amino acids may be required for interaction of RORa with cellular coactivators. Interaction of RORa Activation Domain with Several Putative Coactivators The C-terminal activation domain of RORa (amino acids 272–523) was used as bait to screen a 17-day mouse embryo library in yeast, where this activation domain is not functional (data not shown). In addition to double selection, interacting proteins were not considered further if they also interacted with the transcriptionally inactive RORa polypeptide (amino acids 385–523). Table 1 lists the cDNAs that fulfilled these criteria. Remarkably, several of the proteins that interacted with RORa activation domain in the absence of exogenous ligand were identical to proteins called

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Fig. 1. Transcriptional Activation Domains in RORa Expression vectors encoding the indicated proteins were transfected into JEG-3 cells, along with a reporter plasmid containing five Gal4-binding sites upstream of SV40-luciferase. The transcriptional activity in a representative experiment is shown. Similar results were obtained three to five times for each construct.

Table 1. Results of Yeast Two-Hybrid Screen with RORa Identity

TRIP-1 TIF-1 TRIP 230/TRIP-11 PBP/TRIP-2/DRIP 230/TRAP220/ RB18A GRIP-1/TIF-2

No. of Times Isolated

Interaction with RORa 272–523

Interaction with RORa 385–523

3 9 1 13

1 1 1 1

2 2 2 2

2

1

2

RORa amino acids 272–523 were used as bait in a screen of 2 million clones of a 17-day mouse embryo library in the absence of exogenous ligand. Shown in the table are the identities and frequency of isolation of clones that were reproducibly positive for interaction in yeast with RORa 272– 523 but not for interaction with RORa 385–523.

TRIPs that were previously shown by Moore and colleagues (20) to interact with thyroid hormone receptor in a ligand-dependent manner (20). These included TRIP-1, TRIP-2, and TRIP-11. TRIP-1 is a component of the 26S proteasome, whose physiological role in nuclear receptor function is unclear (21). Full length TRIP-11 was recently identified as a retinoblastoma protein- and TR-binding protein, called TRIP230 (22). Full-length TRIP-2 was cloned as a PBP (15), as well as a component of transcriptionally active complexes that interact with liganded vitamin D receptor (VDR) [DRIP205 (16) and liganded TR (TRAP220) (17)]. Other coactivators isolated in this screen were TIF-1 (23) and GRIP-1 (12, 13). In addition, one novel partial cDNA was isolated, but this has been difficult to characterize for technical reasons. In the remainder of this paper, we focus on GRIP-1 and PBP and putative coactiva-

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tors for RORa. GRIP-1 is representative of the p160/ SRC-1 family of nuclear receptor coactivators while PBP is a component of a large multiprotein complex including the mediator proteins that play a role in transcription by nuclear receptors as well as numerous other classes of transcriptional activatiors (reviewed in Ref. 24). RORa Interacts with GRIP-1 and PBP in Vitro The yeast in vivo interactions were next confirmed in vitro. The nuclear receptor interaction domain of GRIP-1 (amino acids 624-1121, containing three LXXLL sequences, also known as NR boxes) and PBP (amino acids 488–739) were expressed as glutathioneS-transferase (GST) fusion proteins in Escherichia coli. Pulldown experiments were carried out using in vitro translated nuclear receptors. Figure 2 demonstrates that RORa interacts with both GRIP-1 and PBP in vitro in the absence of any exogenous ligand. GRIP-1 and PBP interacted with RORa amino acids 272–523 but not with amino acids 385–523, correlating with the

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transactivation properties of these polypeptides and with the interactions observed in yeast. Differential Requirements for RORa Interaction with GRIP-1 and PBP We next studied the effects of mutations in helix 3, which is contained within amino acids 272–385, and helix 12 of RORa. The mutations used, V335R and LF510AA, are analogous to mutations in the GRIP-1 interaction surface of TR that have been shown to abrogate ligand-dependent TR activation and interaction with GRIP-1 (25). Both mutations effectively blocked transcriptional activation by RORa (Fig. 3A). The helix 12 mutant actually represses transcription, as has been previously described for a mutant in which RORa helix 12 is deleted (11). As expected from the analogous mutations in TR, both RORa mutants exhibited markedly reduced interactions with GRIP-1 (Fig. 3B). Consistent with the importance of 272–385 in RORa interaction with PBP, the helix 3 mutation interacted poorly with RORa. Interestingly, however, the helix 12 mutant of RORa, although not a transcriptional activator, retained the ability to interact with PBP. This is consistent with the observation that while VDR helix 12 is required for activation as well as for interaction with the DRIP complex, not all conserved residues in helix 12 are required for this interaction, which is dependent upon PBP (16). Endogenous GRIP-1 and PBP Interact with RORa

Fig. 2. Interactions between RORa and Nuclear Receptor Coactivators GRIP-1 and PBP A, Gal4-RORa fusion proteins were labeled during in vitro translation and assayed for interaction with GST and GSTGRIP-1 (amino acids 624-1121). Input shown is 20% of the amount used in each interaction incubation. B, The identical fusion proteins as in panel A were assayed for interaction with GST-PBP (amino acids 488–739).

Recently, liganded nuclear receptors have been shown to interact with endogenous protein complexes that can function as coactivators in cell-free transcription (16, 17, 26). We compared the abilities of RORa and VDR to interact with endogenous proteins in Namalwa cell nuclear extracts. Figure 4A shows that ligand-bound VDR interacts with a number of nuclear proteins, including proteins in the 230- and 160-kDa range. PBP has previously been shown to be present in this transcriptionally active multisubunit complex (16). By contrast, unliganded VDR does not interact with these proteins. Interestingly, in the absence of any exogenous ligand, RORa interacts with many proteins, some of which comigrate with those in the liganded VDR complexes (i.e. the DRIP coactivator complex). We next tested whether endogenous GRIP-1 and PBP were in these complexes. Western analysis shows that, indeed, both endogenous GRIP-1 and PBP were bound by RORa (Fig. 4B) and VDR (Fig. 4C). The RORa interactions occurred in the absence of exogenous ligand, whereas 1,25-(OH)2-vitamin D3 was required for interaction with VDR. The efficiencies of the interactions, assessed semiquantitatively by comparing the amounts of bound and input proteins, were similar for RORa and VDR, with GRIP-1 interacting perhaps more efficiently with liganded VDR than with RORa. It is important to note that although both PBP and GRIP-1 are present in the RORa pulldowns,

RORa Interactions with GRIP-1 and PBP

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Fig. 3. Differential Determinants of RORa Interaction with GRIP-1 and PBP A, Mutations in helix 3 and helix 12 of RORa abrogate activation. 293T cells were transfected with the indicated reporter constructs. B, Effects of helix 3 and helix 12 mutation upon interaction of Gal4-RORa(272–523) with GRIP-1 and PBP in vitro.

Fig. 4. RORa Interacts with Several Endogenous Nuclear Proteins, including GRIP-1 and PBP, in the Absence of Exogenous Ligand A, GST protein affinity binding assays with VDR-LBD and RORa. Namalwa nuclear extract (2 mg) was incubated with GST-RORa and GST-VDR LBD (amino acids 110–427), in the absence or presence of 1 mM 1,25-(OH)2D3. Interacting proteins were eluted from the GST-VDR LBD or GST RORa column by incubation with N-lauroyl sarkosine (Sarkosyl). The eluates were separated by SDS-PAGE and analyzed by silver nitrate staining. The approximate molecular mass of the common interacting proteins is shown at the right. B and C, Endogenous GRIP-1 and PBP from Namalwa cells interact with RORa (B) and VDR (C). Namalwa cell nuclear extract (2 mg) was incubated with GST (control), GST-RORa, and GST-VDR. Interacting proteins were resolved by SDS-PAGE and blotted using antibodies to GRIP-1 and PBP. Input shown is 10% of amount used in each interaction incubation.

it is not yet clear whether they are in the same or different complexes. In the case of the VDR, they appear to be in different complexes (26). GRIP-1 Is a Coactivator for RORa in Yeast We next sought to determine whether these RORainteracting proteins can actually function as coactivators for RORa. GRIP-1 has previously been shown to function as coactivator in yeast for multiple nuclear

receptors, including glucocorticoid receptor (12, 13) and TR (27). To date, such coactivator function has required cognate ligand. Figure 5 clearly demonstrates that full-length GRIP-1 (a generous gift of M. Stallcup) functioned as a strong coactivator for RORa, activating transcription 9-fold over control. Importantly, and consistent with interaction data, full-length GRIP-1 did not act as a coactivator for RORa amino acids 385– 523 (data not shown). In contrast, PBP did not function similarly as a coactivator in yeast, despite expression

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of the full-length protein similar to that achieved for GRIP-1 (data not shown). GRIP-1 Is a Coactivator for RORa in Mammalian Cells We next tested the ability of GRIP-1 to function as a coactivator for RORa in mammalian cells, utilizing

Fig. 5. GRIP-1 Functions as a Coactivator for RORa in Yeast Yeast was cotransformed with Gal4-RORa 272–523 and either full-length GRIP-1 or full-length PBP in the absence of exogenous ligand. b-gal activity was determined and plotted relative to the activity of Gal4-RORa 272–523 alone.

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293T cells. Unlike yeast, these cells already contain both PBP and GRIP-1, which interacted with RORa as shown in Fig. 6A. RORa interactions did not require any exogenous ligand, whereas TR interacted with both GRIP-1 and PBP in the presence of added T3 (Fig. 6A). Figure 6B shows that cotransfection of GRIP-1 potentiated transcriptional activation by RORa (272–523) by more than 2-fold. This modest level of transcriptional potentiation presumably reflects the basal abundance of GRIP-1, that contrasts dramatically with the situation in yeast. Figure 6C shows the incremental effect of transfection on overall GRIP-1 abundance. Transfection efficiency of 293T cells was determined to be 50–70%, indicating that the exogenous GRIP-1 was overexpressed by less than a log order. No stimulation of transcription by RORa (385– 523) was observed, again confirming the interaction results. In contrast to the ability of GRIP-1 to potentiate activation by RORa, PBP had little or no effect (data not shown) despite expression of PBP at levels that were increased in the transfected levels by approximately the same fold as was observed for transfected GRIP-1 (Fig. 6C). PBP actually inhibited RORa and TRa transcription in some experiments (data not shown). PBP also did not potentiate RORa transcription in other cell types, including JEG-3 cells. Since endogenous PBP is abundant in all of the mammalian cell lines tested, we cannot rule out the possibility that its basal expression is already in excess of what is required for maximal transcription activity. However,

Fig. 6. GRIP-1 Functions as a Coactivator for RORa in Mammalian Cells A, Endogenous GRIP-1 and PBP from 293T cells interact with RORa. 293T whole cell extract (3 mg) was incubated with GST (control), GST-RORa, and GST-TRa in the presence or absence of 1 mm T3. Interacting proteins were resolved by SDS-PAGE and blotted using antibodies to PBP and GRIP-1. Input shown is 4% of the total. B, 293T cells were transfected with the indicated Gal4-RORa expression plasmids and increasing concentration of GRIP-1 expression plasmid (20–320 ng), along with a reporter plasmid containing five GAL4-binding sites upstream of SV40 luciferase. The transcriptional activity in a representative experiment is shown. C, Both GRIP-1 and PBP are expressed above endogenous levels in transfected 293T cells. 293T cells were transfected with 320 ng of GRIP-1, PBP, or pCMX (control) as indicated. Harvested whole-cell extract (75 mg) was resolved by SDS-PAGE and blotted using antibodies to PBP and GRIP-1.

RORa Interactions with GRIP-1 and PBP

even when cotransfected with GRIP-1, PBP did not potentiate RORa activity (data not shown).

DISCUSSION The nuclear orphan receptor RORa has been shown to play important roles in brain development (2, 3), lipid metabolism (5), and oncogenesis (28). Here we show that RORa can interact with several known and putative coactivators, including GRIP-1 and PBP. This is the first demonstration that GRIP-1 can serve as a coactivator for RORa. We also demonstrate that a complex of proteins including GRIP-1 and PBP can associate with RORa. This observation further supports previous data from other laboratories showing that a complex of coactivator proteins may be involved in mediating nuclear receptor activation function (16, 17, 29). At the same time, differences in the composition of proteins that interact with RORa suggests that the formation of coactivation complexes may serve as another level of transcriptional regulation. The dramatic effects of GRIP-1 upon RORa activity in yeast contrasts with the modest potentiation observed in mammalian cells. The most likely explanation is the absence of endogenous coactivator in yeast and its relative abundance in mammalian cells. This could also explain the lack of potentiation by PBP in mammalian cells; in earlier studies PBP only weakly potentiated activation by PPAR (15) and had little or no effect on other receptors (16, 17). This point is unresolved, however, since while this paper was under review, Treuter et al. (30) reported that PBP contains an activation domain and potently coactivates TR in mammalian cells as well as in yeast. The present observation that full-length PBP does not function as coactivator for RORa in yeast suggests that PBP may not be a coactivator for RORa in the same sense as GRIP-1. Indeed, mutations in helix 12 that abolish activation and interaction with GRIP-1 did not alter PBP interaction. Interestingly, helix 12 is also dispensable for interaction of hepatocyte nuclear factor 4 (HNF4), another ligand-independent nuclear receptor, with the coactivator CBP (31). Unlike GRIP-1 or CBP, PBP does not possess intrinsic histone acetyltransferase activity (data not shown). Rather, the role of PBP is primarily to anchor a multisubunit complex to the nuclear receptor ligand-binding domain (LBD) (16, 17), thus serving as a bridging subunit of a much larger complex that has a transcriptional activation function (16, 17). The fact that exogenous ligand was not necessary to demonstrate coactivator interaction or activation in systems as varied as yeast, bacteria, rabbit reticulocyte, and human cells strongly suggests that RORa is a constitutively active nuclear receptor. The ability of RORa to interact with numerous other proteins that interact with TR only in the presence of endogenous ligand is a further argument that RORa exists in the

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liganded conformation in the absence of exogenous ligand. While this work was in progress, the constitutively active nuclear receptors HNF4 and constitutive androstane receptor b (CARb) were also shown to interact with coactivators in the absence of ligand (31–33). Interestingly, androstane ligands lead to dissociation of coactivators from CARb (33). This raises the possibility that, if there is a ligand for RORa, it might inhibit its activity. In this context it is noteworthy that deletion of the AF2 activation helix allows RORa to repress transcription and interact with corepressors (11), which is normally a characteristic of unliganded hormone receptors (7, 8).

MATERIALS AND METHODS Plasmids The Gal4 reporter used in Fig. 1 has been described previously (34). The Gal4 reporter construct used in Fig. 5 was kindly provided by M. Brown. pCMX-Gal4 and pCMX-Gal4 RORa 272–523 constructs have been described previously (11). pCMX-Gal4 RORa 385–523 was constructed from PCR products of pCMX-RORa using the primers 59-CCGGGGATCCAGTATGCC-39 and 59-CTCTGTAGGTAGTTTGTCC-39 and were cloned into the BamHI site of pCMX-Gal4. pSG5 GRIP-1 full length (FL) was kindly provided by M. Stallcup. pcDNA3 PBP will be described elsewhere (26). RORa 272–523 and 385–523 were subcloned into pGBT9 (CLONTECH Laboratories, Inc., Palo Alto, CA) from the corresponding Gal4 constructs. Helix 3 and Helix 12 mutations in RORa 272–523 were created by PCR. pGAD424 GRIP-1 was kindly provided by M. Stallcup. PBP was subcloned into pGAD424 by standard restriction/ligation procedures. A DNA fragment encoding PBP 488–739, the yeast two-hybrid clone, was subcloned into pGEX-2TK (Pharmacia Biotech, Piscataway, NJ) for preparing GST-fusion protein. A DNA fragment encoding GRIP-1 624-1121 was amplified from pSG5 GRIP-1 using the primers 59-CAGGGATCCGACAGGGCTGAGGGACACAG-39 and 59-GTTGGATCCGGACTCCTGACTTGAGAACT-39 and cloned into the BamHI site of pGEX-2TK. Plasmids for production of GST-VDR and GST-TR have been previously described (16, 35). The GSTRORa expression plasmid was constructed by BamHI and XhoI digestion of a PCR fragment amplified from pCMXRORa using the primers 59-CACGGATCCGAGTCAGCTCCGGCAGCCCCCGAC-39 and 59-GTCGGATCCCATCCGGTGTTTCTGTACTTC-39, which was ligated into the BamHI site of pGEX-2TK with the 39 XhoI and BamHI fragment of pCMX RORa. All constructs were confirmed by automated DNA sequencing. Yeast Two-Hybrid Screen Saccharomyces cerevisiae HF7c [MATa, ura3–52, his3–200, lys2–801, ade2–101, trp1–901, leu2–3, 112, gal4–542, gal80– 538, LYS2::Gal1-HIS3, URA3::(Gal4 17-mers)3-CYC1-lacZ] containing pGBT9 ROR 272–523 was transformed with a 17-day mouse embryo library in pGAD10 (CLONTECH Laboratories, Inc.) and plated on SD medium lacking tryptophan, leucine, and histidine (containing 5 mM 3-aminotriazole). His1 colonies exhibiting positive b-galactosidase activity in the filter lift assay (CLONTECH Laboratories, Inc.) were further characterized. To recover library plasmids, total yeast DNA was isolated, electroporated into E. coli HB101, and isolated on minimal media lacking leucine and containing ampicillin. Isolated plasmid was then cotransformed into HF7c yeast

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with pGBT9, pGBT9 ROR 272–523, or pGBT9 ROR 385–523 and those plasmids which only exhibited positive b-galactosidase activity with pGBT9 ROR 272–523 were further characterized. Yeast Liquid b-Galactosidase Assay The Y190 strain yeast [MATa, ura3–52, his3–200, lys2–801, ade2–101, trp1–901, leu2–3, 112, gal4D, gal80D, cychr2, LYS2::Gal1UAS-HIS3TATA-HIS3, URA3::Gal1UAS-Gal1TATA-lacZ] was cotransformed with pGBT9 ROR 272–523 or pGBT9 ROR 385–523 and pGAD10, pGAD424 GRIP-1, or pGAD424 PBP as indicated. Five independent colonies of each transformation were used to inoculate overnight cultures grown in SD medium lacking tryptophan and leucine. Yeast liquid b-galactosidase assays were carried out with each culture in triplicate, using the protocol provided by CLONTECH Laboratories, Inc. Protein Interaction Assays Using GST Fusion Proteins GST fusion proteins were expressed in BL21 cells induced with 0.5 mM isopropylthio-b-O-galactoside at 30 C. Proteins were isolated by cell lysis with lysozyme and detergent followed by sonication. GST beads (50 ml) containing the fusion protein were incubated at room temperature in GST binding buffer (50 mM KCl, 20 mM HEPES, pH 7.9, 2 mM EDTA, 0.1% NP-40, 10% glycerol, 0.5% nonfat dry milk, and 5 mM dithiothreitol). In vitro translated protein, using the Promega Corp. TNT T7 Quick kit with [35S]methionine, Namalwa nuclear extracts, or 293T whole-cell extracts were added to the beads as indicated. Binding was allowed to proceed for 1 h, and the beads were washed four times in the same buffer. The bound proteins were eluted by boiling in 30 ml of SDSPAGE loading buffer and resolved by electrophoresis. SDSPAGE gels performed with in vitro translated protein were dried and visualized by autoradiography. SDS-PAGE gels performed with cellular extract were silver stained or else the proteins were transferred to PVDF membrane for Western blot analysis. Immunoblotting Western blots were performed with 1:750 dilution of rabbit polyclonal antibody raised against GRIP-1 624-1121 or PBP 488–739 and a 1:5000 dilution of goat antirabbit IgG-peroxidase (Roche Molecular Biochemicals, Nutley, NJ) secondary as previously described (36). Nuclear and Whole-Cell Extract Preparation 293T whole-cell extracts were prepared by washing and harvesting cells in PBS, and subsequent incubation at 4 C for 20 min in lysis buffer or in GST binding buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin. Extracts were quantified using the Bio-Rad assay (Bio-Rad Laboratories, Inc., Richmond, CA). Namalwa B cells were cultured as previously described (16) and nuclear extracts were prepared as previously described (37). Cell Culture and Transfection 293T cells were maintained and transfected in DMEM high glucose with 10% FCS. JEG-3 cells were maintained and transfected in DMEM low glucose with 10% FCS. For JEG-3 cells, at 80% confluence, 60-mm dishes were transfected by the calcium phosphate precipitation method using 2 mg luciferase reporter, 0.5 mg b-galactosidase (B-gal) expression

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vector, and 2 mg receptor expression vector. For 293T cells, at 80% confluence, 24-well plates were transfected by the calcium phosphate precipitation method using 50 ng luciferase reporter, 50 ng b-galactosidase (b-gal) expression vector, and 15 ng receptor expression vector and additional coactivator expression vector as indicated. Equivalent amounts of empty expression vector (pCMX) were included in cells transfected with submaximal amounts of receptor or coactivator. Cells were lysed in Triton X-100 buffer, and b-gal and luciferase assays were carried out using standard protocols. The measured relative light units were normalized to b-gal activity, which served as an internal control for transfection efficiency. Fold activation was calculated as the activity of a given reporter after transfection with control expression vector divided by the activity of the same reporter in the presence of RORa expression vector. Figures show the results of representative experiments in which individual data points were assayed in duplicate and the range of the data is shown. Each experiment was repeated two to five times. The degree of activation from a given site was highly consistent from experiment to experiment.

Acknowledgments Received January 26, 1999. Revision received May 10, 1999. Accepted June 1, 1999. Address requests for reprints to: Mitchell A. Lazar, M.D., Ph.D., University of Pennsylvania School of Medicine, 611 CRB, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6149. E-mail: [email protected]. This work was supported by NIH Grant DK-45586 to M.A.L. G.B.A. was supported by a UNCF/Merck Graduate Science Research Dissertation Fellowship as well as by NIH Training Grant (GM-08216–12). Automated DNA sequencing was supported in part by the Molecular Biology Core of the Penn Center for Molecular Studies of Digestive Diseases (NIH Center Grant P30 DK-50306).

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RORa Interactions with GRIP-1 and PBP

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