T0070907, a Selective Ligand for Peroxisome Proliferator-activated Receptor (cid:1) , Functions as an Antagonist of Biochemical and Cellular Activities*

The nuclear hormone receptor peroxisome prolifera-tor-activated receptor (cid:1) (PPAR (cid:1) (NR1C3)) plays a cen-tral role in adipogenesis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. In a search for novel non-TZD ligands for PPAR (cid:1) , T0070907 was identified as a potent and selective PPAR (cid:1) antagonist. With an apparent binding affinity (concen-tration at 50% inhibition of [ 3 H]rosiglitazone binding or IC 50 ) of 1 n M , T0070907 covalently modifies PPAR (cid:1) on cysteine 313 in helix 3 of human PPAR (cid:1) 2. T0070907 blocked PPAR (cid:1) function in both cell-based reporter gene and adipocyte differentiation assays. Consistent with its role as an antagonist of PPAR (cid:1) , T0070907 blocked agonist-induced recruitment of coactivator-de-rived peptides to PPAR (cid:1) in a homogeneous time-re-solved fluorescence-based assay and promoted recruitment of the transcriptional corepressor NCoR to PPAR (cid:1) in both glutathione S -transferase pull-down assays and a PPAR (cid:1) /retinoid X receptor (RXR) (cid:2) -dependent gel on LGD1069-induced recruitment of coactivator of T0070907 on LGD1069-induced coactivator the PPAR (cid:1) /RXR (cid:2)

The nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥ (NR1C3)) plays a central role in adipogenesis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. In a search for novel non-TZD ligands for PPAR␥, T0070907 was identified as a potent and selective PPAR␥ antagonist. With an apparent binding affinity (concentration at 50% inhibition of [ 3 H]rosiglitazone binding or IC 50 ) of 1 nM, T0070907 covalently modifies PPAR␥ on cysteine 313 in helix 3 of human PPAR␥2. T0070907 blocked PPAR␥ function in both cell-based reporter gene and adipocyte differentiation assays. Consistent with its role as an antagonist of PPAR␥, T0070907 blocked agonist-induced recruitment of coactivator-derived peptides to PPAR␥ in a homogeneous time-resolved fluorescence-based assay and promoted recruitment of the transcriptional corepressor NCoR to PPAR␥ in both glutathione S-transferase pull-down assays and a PPAR␥/retinoid X receptor (RXR) ␣-dependent gel shift assay. Studies with mutant receptors suggest that T0070907 modulates the interaction of PPAR␥ with these cofactor proteins by affecting the conformation of helix 12 of the PPAR␥ ligand-binding domain. Interestingly, whereas the T0070907-induced NCoR recruitment to PPAR␥/RXR␣ heterodimer can be almost completely reversed by the simultaneous treatment with RXR␣ agonist LGD1069, T0070907 treatment has only modest effects on LGD1069-induced coactivator recruitment to the PPAR␥/RXR␣ heterodimer. These results suggest that the activity of PPAR␥ antagonists can be modulated by the availability and concentration of RXR agonists. T0070907 is a novel tool for the study of PPAR␥/RXR␣ heterodimer function.
Peroxisome proliferator-activated receptor ␥ (PPAR␥ 1 (NR1C3)) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcription factors (1,2). At least two PPAR␥ isoforms exist, ␥1 and ␥2, resulting from transcription from two different promoters upstream of the PPAR gene (3,4). PPAR␥2 possesses 30 additional amino acids at its amino terminus. PPAR␥1 is expressed broadly in many tissues, whereas PPAR␥2 is expressed predominantly in adipose tissue. Both "gain of function" and "loss of function" studies strongly support a critical role for PPAR␥ in adipocyte gene expression and differentiation (5).
Like other members of the NHR superfamily, PPAR␥ binds to a DNA-response element (PPAR-response element or PPRE) upstream of the coding regions of target genes and forms a heterodimeric complex with one of the three retinoid X receptor (RXR) proteins (1). Binding of ligands to PPAR␥ causes conformational changes in the receptor, in particular to ␣-helix 12 (H12), which is located at the carboxyl-terminal end of the protein and forms part of the transcriptional activation function (AF-2). When agonists bind to PPAR␥, H12 along with H3, H4, and H5 form a charge clamp and a hydrophobic pocket that allows the recruitment of coactivator protein complexes that are essential for transcriptional activation of PPAR␥ target genes (6). Although PPAR␥, in isolation, is capable of binding to transcriptional corepressor proteins NCoR and SMRT in the absence of ligand, PPAR␥ does not interact with these corepressors in the context of the RXR heterodimer nor does the PPAR␥/ RXR heterodimer repress transcription of PPAR␥ target genes, unlike heterodimers of RXR with thyroid hormone receptor or retinoic acid receptor (7). Two explanations for the difference in PPAR␥/corepressor interaction on and off DNA have been offered. The orientation of PPAR␥ and RXR on a PPRE could simply inhibit the binding of corepressor (8). Alternatively, PPAR␥ may be unable to stabilize a conformation of RXR that is permissive for corepressor interaction; unlike TR and retinoic acid receptor, PPAR␥ is unable to interact with H12 from RXR (9). Because other NHR antagonists stabilize the interaction of corepressors with their cognate receptors, PPAR␥ antagonists or inverse agonists would be useful tools to study PPAR␥/corepressor interaction. One way to test these hypotheses is to study the effects of a PPAR␥ antagonist or inverse agonist on corepressor binding. This avenue has not yet been explored.
Both natural and synthetic ligands have been reported for PPAR␥ (reviewed in Ref. 10). Naturally occurring compounds that have been reported to bind PPAR␥ include a number of fatty acids and eicosanoid derivatives such as 9-or 13-hydroxyoctadienoic acid and prostaglandin derivative 15-deoxy-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Here, we describe a novel, potent, and selective PPAR␥ ligand, T0070907. By using a variety of biochemical and cellbased assays, we demonstrate that T0070907 is a PPAR␥ antagonist. Our studies suggest that T0070907 modulates the interaction of PPAR␥ with cofactor proteins by affecting the conformation of helix 12 of the PPAR␥ ligand-binding domain (LBD). Finally, our studies reveal a functional asymmetry between the effects of PPAR␥ and RXR ligands on the activity of the permissive PPAR␥/RXR␣ heterodimer.
Mass Spectrometry-After incubation with 10 M T0070907 for 4 h at room temperature in 50 mM Tris, pH 7.9, 50 mM KCl, 1 mM EDTA, GST-PPAR␥ (12 g) was purified on an SDS-polyacrylamide gel. The excised gel fragment containing PPAR␥ was digested with trypsin at 37°C for 12 h without reduction and alkylation in 100 mM ammonium bicarbonate using an enzyme/substrate ratio of 1:50 (w/w). Analysis of covalent binding of T0070907 to PPAR␥ was performed with a Voyager-DE TM MALDI-TOF Mass Spectrometer (Perspective Biosystems, Framingham, MA) and an Esquire TM Nano-electrospray Tandem Mass Spectrometer (Bruker Daltonik, Billerica, MA).
The in-gel tryptic digest was desalted and concentrated prior to analysis by nano-ESI-MS/MS. The in-gel digests were extracted twice with 10 l of a 50% acetonitrile, 5% trifluoroacidic acid solution. All extracts were pooled and dried to 5 l with a SpeedVac. An additional 15 l of a 0.1% acidic acid solution was added, and a 10-l sample was loaded into a Ziptip TM (Millipore, Bedford, MA) with C 18 resin for desalting. After washing the column with 1% trifluoroacidic acid (in H 2 O), 5 l of 50% acetonitrile, 0.1% acidic acid was used to elute the sample into a nanospray needle. On the basis of the MALDI-TOF mass analysis results, the tandem mass spectrometric sequencing was only acquired on selected precursor ions (18).
Homogeneous Time-resolved Fluorescence (HTRF) Assay-HTRF assays were performed as described previously (19) with the following modifications. Reaction conditions were as follows: a 100-l reaction volume contained 50 mM Tris, pH 7.9, 50 mM KCl, 1 mM EDTA, 0.5 mM 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin, 800 ng/ml anti-GST-(Eu)K antibody (PerkinElmer Life Sciences), 1 ng/l GST-PPAR␥, 1.5 g/ml streptavidin conjugated with allophycocyanin (Streptavidin-APC, PerkinElmer Life Sciences), 200 nM biotin-peptide, and 5 l compound of interest in dimethyl sulfoxide (Me 2 SO) as indicated in the figure legends. GST-PPAR␥/anti-GST-(Eu)K (20 l) and biotin-peptide/ streptavidin (20 l) were incubated separately for 1 h at room temperature before being combined with the remaining components, and the complete mixture was incubated for an additional 1 h at room temperature. Reactions were carried out in 96-well plates (black polypropylene, Whatman), and fluorescence was measured on an LJL Analyst (LJL BioSystems, Sunnyvale, CA). Data were expressed as the ratio of the emission intensities at 665 and 620 nm multiplied by a factor of 1000.
Corepressor Recruitment Assay (Pull-down Assay)-Purified GST-PPAR␥ fusion protein (15 g) was incubated with 10 l of glutathione-Sepharose beads (50% slurry in GST binding buffer, Amersham Biosciences) in GST binding buffer (20 mM HEPES, pH 7.7, 100 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl 2 , 0.01% Nonidet P-40, 2 mM dithiothreitol, 10% glycerol) for 90 min at room temperature. After washing 5 times (5 min each wash) with 1 ml of binding buffer, the bead-bound GST-PPAR␥ protein was incubated with 7.5 l of [ 35 S]methionine-labeled in vitro translated hNCoR protein (TNT T7 Rabbit Reticulocyte Lysate Translation System, Promega Corp., Madison, WI) and the indicated ligand concentration in a final volume of 300 l at room temperature for 2 h. After washing with binding buffer as indicated above, the bound protein was eluted with 20 l of 2ϫ SDS buffer at 95°C, separated on a 10% SDS-PAGE, and analyzed by autoradiography.
Transient Transfection and 3T3L1 Differentiation Assay-Luciferase reporter assays were carried out following transient transfection of HEK293 cells using GenePORTER2 reagent (GTS Inc., San Diego, CA) according to the manufacturer's protocol. 3T3-L1 preadipocytes were cultured and induced to differentiate as described (24) with the following modifications. 3T3-L1 cells were grown to confluence in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and induced to differentiate with 0.25 M dexamethasone, 0.5 mM isobutylmethylxanthine, and 1 g/ml insulin. Medium was replaced 2 days post-induction (and every 2-3 days thereafter) with Dulbecco's modified Eagle's medium, 10% fetal bovine serum supplemented with 1 g/ml insulin.

T0070907 Is a Novel and Selective PPAR␥ Ligand-In a search
for novel non-TZD ligands for PPAR␥, T0070907 was identified to bind PPAR␥ with high affinity, capable of displacing [ 3 H]rosiglitazone with an apparent K i of 1 nM as shown in Fig. 1. Furthermore, T0070907 shows high selectivity among PPAR subtypes with a Ͼ800-fold preference for PPAR␥ over PPAR␣ and PPAR␦. In competition with the PPAR␣ and PPAR␦ co-ligand [ 3 H]GW2433 (21), T0070907 has an apparent K i of 0.85 M to PPAR␣ and 1.8 M to PPAR␦ (Fig. 1B).
T0070907 Is a Specific Potent PPAR␥ Antagonist in Tran-sient Transfection Assays-The effect of T0070907 on the transcriptional activity of PPAR␥ in a cell-based reporter gene assay was examined. HEK293 cells were transiently transfected with an expression construct that contained the PPAR␥ LBD fused to the Gal4-DNA binding domain, together with a luciferase reporter gene under the transcriptional control of the Gal4 upstream activating sequence (Gal4-UAS). As shown in Fig. 2A, rosiglitazone activated transcription up to 20-fold, whereas T0070907 has no effect (or perhaps even a slight inhibitory effect) on basal transcription. In addition, T0070907 is a potent inhibitor (IC 50 value in the nM range) of PPAR␥ transactivation in the presence of rosiglitazone ( Fig. 2A). This inhibition is not due to cytotoxicity as the concentration required to kill 50% of cells is greater than 10 M (data not shown). The specificity of T0070907 was also examined in cellbased reporter gene assays. HEK293 cells were transiently transfected with a GAL4-UAS reporter and expression constructs encoding the LBDs of PPAR␣, PPAR␦, the farnesoid X receptor, the liver X receptor ␣, the liver X receptor ␤, or pregnane X receptor fused to the Gal4-DNA binding domain. As shown in Fig. 2A, T0070907 at 1 M has no effect on the transcriptional activity of any other receptor besides PPAR␥. These results demonstrate that T0070907 is a PPAR␥-specific antagonist. T0070907 Blocks Hormone-mediated Differentiation of the Adipogenic Cell Line 3T3-L1-We next investigated whether T0070907 could block the induction of adipogenesis by various treatments of the adipogenic cell line 3T3-L1. As shown in Fig.  2B, the standard treatment of dexamethasone, 3-isobutyl-1methylxanthine, and insulin promoted lipid accumulation in 3T3-L1 cells. In contrast, lipid accumulation in these cells was completely inhibited when cells were treated with both 1 M T0070907 and the differentiation mixture. Similar inhibitory effects of T0070907 were observed when adipogenesis was induced by treatment with the PPAR␥ agonist, rosiglitazone (data not shown).
T0070907 Covalently Modifies PPAR␥ on Cys 313 -To under-stand the mechanism by which T0070907 antagonizes PPAR␥ function, its binding properties were first examined. That rosiglitazone was unable to displace T0070907 prebound to PPAR␥ suggested that the binding of T0070907 was irreversible (data not shown). To verify the covalent nature of the interaction between T0070907 and PPAR␥, and to identify the site of covalent attachment, we performed proteolytic mapping studies via mass spectrometry. The covalent binding of T0070907 (mass of 277.7 Da) to PPAR␥ would result in a mass change of the modified tryptic peptide(s) by 241.1 Da. By comparing the tryptic digests of PPAR␥ with and without T0070907 treatment, a candidate peptide containing the T0070907 attachment site (amino acids 272-279, IFQGCQFR, m/z 998.49 Da) was identified based on its mass shift to m/z 1239.56 (data not shown). The precise binding site on this peptide was determined with ESI-tandem mass spectrometry (Fig. 3A). The calculated dominant y-and b-ion fragments of this peptide are shown at the top of Fig. 3A, with the ions observed in the mass spectrum underlined. The mass difference between the y3-and y4-ions (m/z 344.1) identified Cys 313 as the site of modification by T0070907. In addition, several double-charged y-ions and small internal fragment ions obtained also confirmed this conclusion.
To confirm the importance of Cys 313 in T0070907 binding to PPAR␥, a mutant PPAR␥ was constructed in which Cys 313 was converted to a serine residue, and the corresponding recombinant GST-PPAR␥ LBD (C313S) fusion protein was expressed and purified. [ 3 H]T0070907 was first incubated with either wild (wt) type PPAR␥ or the C313S mutant as described, and the reaction mixtures were separated by SDS-PAGE. As shown in Fig. 3B, [ 3 H]T0070907 was only able to modify the wild type protein (upper panel, autoradiograph), although equal amounts of wild type and C313S mutant PPAR␥ were added to each reaction (lower panel, Coomassie staining). Thus, Cys 313 is necessary for the binding of T0070907 to PPAR␥.
The specificity of the covalent modification was examined in a whole-cell extract (WCE) made from HEK293 cells. Purified GST-PPAR␥ LBD fusion protein (Fig. 3C)  T0070907 Behaves as an Inverse Agonist of PPAR␥ LBD in Vitro-By using the homogeneous time-resolved fluorescence (HTRF) technology, we developed an assay to study the effects of PPAR␥ ligands on the interaction of PPAR␥ with fragments of coactivator or corepressor proteins. Reporter peptides of ϳ20 amino acids in length were synthesized from sequences derived from various coactivator and corepressor proteins (Table I) (25,26). The effects of various ligands on PPAR␥ binding to this collection of peptides are shown in Fig. 4A. The patterns that emerged from this peptide profiling have allowed us to distinguish between different functional classes of PPAR␥ ligands. First, known PPAR␥ agonists such as rosiglitazone, troglitazone, and the GSK compound GI262570 (27) showed very similar peptide profiles. Rosiglitazone and troglitazone, which both belong to the TZD chemical class, were more similar to each other than to tyrosine-based GI262570. GI262570 recruited additional peptides (peptides 11, 19, 23, and 27), suggesting perhaps that PPAR␥ adopted a slightly different conformation when bound to GI262570, compared with the conformation assumed by the receptor when bound to the TZD compounds. In contrast, the novel PPAR␥ ligand T0070907 shows a unique peptide profile (Fig. 4A), exclusively promoting recruitment of peptides derived from corepressor proteins NCoR and SMART (peptides 2 and 3, respectively). Furthermore, compared with the Me 2 SO control, T0070907 seems to suppress the basal interactions between PPAR␥ and coactivator-derived peptides.
In order to confirm these results, more extensive titration and competition experiments were carried out with two peptides derived from a representative coactivator and a representative corepressor (peptides 1 and 2). As shown in Fig. 4, B and C, rosiglitazone promoted the dose-dependent recruitment of peptide derived from coactivator DRIP205 to PPAR␥, while suppressing the interaction between PPAR␥ and a peptide derived from corepressor NCoR. In contrast, T0070907 suppressed the interaction between PPAR␥ and the coactivatorderived peptide in the absence of ligand, while promoting the recruitment of the NCoR-derived peptide to PPAR␥. T0070907 also effectively antagonized the effects of rosiglitazone in a dose-dependent manner.
To confirm independently these observations by using an alternative nonfluorescence-based format, the effects of T0070907 on PPAR␥/NCoR interactions were examined using a GST pull-down assay. As shown in Fig. 4D, rosiglitazone suppresses the interaction between the GST-PPAR␥LBD and NCoR in a dose-dependent fashion, with an IC 50 consistent with its binding affinity to PPAR␥. On the other hand, T0070907 promoted a dramatic increase in NCoR binding to GST-PPAR␥ consistent with the results observed in the HTRF assay.
Effects of T0070907 and LGD1069 on PPAR␥ and RXR␣ Heterodimer in GMSAs-By having shown that T0070907 strongly promotes recruitment of NCoR to the PPAR␥ LBD in both HTRF and pull-down assays, we next used a GMSA to examine whether this could also occur in the context of the PPAR␥/RXR␣ heterodimer. As shown in Fig. 5A, in vitro translated PPAR␥ and RXR␣ can bind simultaneously to a PPREcontaining DNA fragment derived from the promoter of acyl-CoA oxidase gene (lane 2). This shift in fragment mobility is absolutely dependent on the presence of both PPAR␥ and RXR␣ (data not shown), indicating the proper formation of a functional PPAR␥/RXR␣ heterodimer under these conditions. Whereas NCoR could not bind efficiently to the PPAR␥/RXR␣ heterodimer in the absence of ligand (compare lanes 2 and 3, and similar to Ref. 9), T0070907 was able to promote a significant increase in the recruitment of NCoR to the heterodimeric complex ( compare lanes 3 and 4).
To ensure that this increased NCoR recruitment was the result of T0070907 binding to PPAR␥ and to understand the nature of the PPAR␥ conformational changes associated with T0070907 binding, we next investigated the effects of deleting H12 from both receptors on the binding of NCoR to the het- erodimer. The deletion of PPAR␥ H12 (PPAR␥ ⌬H12) increased the basal interaction of NCoR with the heterodimer; however, T0070907 did not provide further enhancement of binding (Fig.   5A, lanes 5-7). In contrast, the PPAR␥ wt/RXR␣⌬H12 heterodimer responded to T0070907 and almost all PPAR␥ wt/ RXR␣ heterodimer could be super-shifted to form the PPAR␥/ RXR␣/NCoR complex in the presence of the antagonist (Fig. 5B,  lanes 2-4). Complexes containing H12 deletions in both PPAR␥ and RXR␣ interacted very efficiently with NCoR in the absence of ligand, but as in the case of the PPAR␥ wt/RXR ⌬H12 complex, T0070907 had almost no effect on NCoR recruitment (Fig. 5B, lanes 5-7).
The allosteric effects between PPAR␥ and RXR␣ were studied next by examining the effects of simultaneous treatments of RXR␣ agonist and PPAR␥ antagonist on the recruitment of coactivator and corepressor proteins to the heterodimer. Because the RXR␣ ⌬H12-containing heterodimers interacted much more strongly with NCoR than the wild type-containing heterodimers (Fig. 5B), we examined the effects of an RXR␣ agonist, LGD1069 (28), on T0070907-induced NCoR recruitment to PPAR␥ wt/RXR␣ ⌬H12 and PPAR␥ ⌬H12/RXR␣ ⌬H12 complexes. Strikingly, the addition of LGD1069 dramatically inhibited NCoR binding to both pairs of heterodimer complexes (Fig. 5C, lanes 5-10 and lanes 14 -19). Importantly, LGD1069 was not able to inhibit completely corepressor binding to the PPAR␥ ⌬H12/RXR␣ ⌬H12 complex as it was in the PPAR␥ wt/RXR⌬H12 complex. To ensure that the effect of LGD1069 on NCoR recruitment was not due to blocking T0070907 from binding to the PPAR␥/RXR␣ heterodimer, experiments were performed during which PPAR␥ was treated with T0070907 for an extended period prior to the addition of other components of the GMSA reaction mixture and LGD1069 to saturate all available binding sites on PPAR␥. No difference on the recruitment of NCoR to the PPAR␥/RXR␣ heterodimer was observed with or without preincubation with T0070907 (data not shown).
The effects of T0070907 binding to PPAR␥ on the ability of RXR␣ to interact with coactivators were also studied. In the presence of the RXR␣ agonist LGD1069, the coactivator protein SRC-1 can be recruited to the PPAR␥/RXR␣ heterodimer, as evidenced by the super-shifted complex observed in GMSAs (Fig. 4D, lanes 5-10). This super-shifted complex was not observed in the presence of rosiglitazone (BRL, lane 4) or in reactions containing an RXR H12 mutant (23) suggesting that SRC-1 was specifically recruited to the RXR␣ subunit. When added to the GMSA reaction mixture together with LGD1069, T0070907 seems to have a modest effect on formation of the PPAR␥/RXR␣/SRC-1 super-shifted complex induced by LGD1069 (compare lanes 5-10 and lanes 12-17). DISCUSSION We have identified a specific, high affinity PPAR␥ antagonist, T0070907, that blocks PPAR␥ activity in both biochemical and cell-based assays. T0070907 is highly selective for PPAR␥ over PPAR␣, PPAR␦, other NHRs, and proteins in an HEK293 WCE. Proteolytic mapping indicated that T0070907 irreversibly modifies PPAR␥ on Cys 313 in helix 3 of the LBD, a residue that is conserved in all three PPAR subtypes. This indicates that other residues in the binding pocket confer the specific binding of T0070907 to PPAR␥. Interestingly, this is also the site of covalent modification by L-764406, a PPAR␥ partial agonist that was described previously (12).
T0070907 functions as a PPAR␥ antagonist in cell-based assays. It effectively blocked TZD-induced transactivation by the GAL4-PPAR␥ LBD, as well as adipogenesis in 3T3-L1 cells treated with a differentiation mixture. Overall, these results further support the important role for PPAR␥ in fat cell differentiation. The antagonist properties of T0070907 were also demonstrated in a variety of in vitro biochemical assays using the PPAR␥ LBD. T0070907 suppressed agonist-induced interactions between the PPAR␥ LBD and coactivator-derived peptides and promoted recruitment of corepressor-derived peptide in HTRF assays (Fig. 4). The effect of T0070907 on assembly of corepressor NCoR/PPAR␥ complex was also observed in the pull-down assay (Fig. 4D).
Previous studies (29 -31) have suggested that corepressors bind to a hydrophobic groove on NHR LBDs formed by H3, H5, and H6. This binding site partially overlaps with that utilized by coactivators. NHR agonists disrupt the interaction between NHR LBDs and corepressors, and it is believed that the conformation of H12 stabilized by agonists partially occludes the corepressor binding site. In the unliganded state, H12 of NHR LBDs is thought to exist in multiple conformations, including the agonist-bound conformation. Consistent with this hypothesis, H12 is inhibitory for NCoR binding to most NHRs. Mutations and deletions of H12 from either PPAR␥ or RXR␣ significantly increase the recruitment of NCoR to the PPAR␥/RXR␣ heterodimer (9, 32) (Fig. 5). T0070907 can also promote NCoR recruitment to PPAR␥/RXR␣ heterodimer, but T0070907 can only promote recruitment of NCoR to complexes containing wt PPAR␥ but not to complexes containing PPAR␥ ⌬H12. These results suggest that the effect of T0070907 on the heterodimer is indeed mediated through PPAR␥ and that T0070907 induced NCoR recruitment requires H12. Indeed, T0070907 treatment or the deletion of H12 stabilize NCoR recruitment to comparable extents (Fig. 5) suggesting that T0070907 most likely acts on PPAR␥ by preventing H12 from adopting the agonist-bound conformation. The deletion of RXR␣ H12 domain has a synergistic effect with either T0070907 treatment or PPAR␥ ⌬H12 on the recruitment of NCoR to PPAR␥/RXR␣ heterodimer (Fig.  5B). Two NHR-binding motifs are present on NCoR protein (29 -31), the deletion of H12 from RXR␣ together with either T0070907 treatment or the deletion of H12 from PPAR␥ perhaps allows the cooperative binding of both motifs to PPAR␥/ RXR␣ heterodimer.
In order to dissect the contributions of the PPAR␥ and RXR␣  4. T0070907 is an inverse agonist of the GST-PPAR␥ LBD in vitro. A, HTRF peptide profiling of T0070907, rosiglitazone, troglitazone, and GI262570. All compounds were tested at 1 M, and peptides (x axis) were in numerical order as listed in Table I to NCoR recruitment to the heterodimer, the effects of simultaneous treatment with T0070907 and LGD1069 were determined. Notably, LGD1069 dramatically inhibited the T0070907-mediated increase in NCoR recruitment to the PPAR␥ wt/RXR␣ ⌬H12 and PPAR␥ ⌬H12/RXR␣ ⌬H12 heterodimers in a dose-dependent manner (Fig. 5C). Recent x-ray crystallographic studies of the apo-RXR␣ LBD (unliganded), the holo-RXR␣ LBD (agonist bound), and a PPAR␥/RXR␣ heterodimer (each bound to agonist) suggest possible molecular mechanisms for these effects. The rosiglitazone-bound PPAR␥/ 9-cis-retinoic acid-bound RXR heterodimer interface which is largely composed of residues from H10 and H11 of both receptors contains several important salt bridges. In particular, a salt bridge formed between the carboxylic acid of Tyr 477 from PPAR␥ H12 and Lys 431 from RXR␣ H10 stabilizes the positioning of H12 from PPAR␥ in the agonist-bound conformation (33). In addition, comparison of the apo-and holo-RXR␣ LBD structures reveal that ligand-binding triggers several large conformational changes. For example, H11, which partially fills the ligand binding pocket in the apo-RXR␣ structure, moves out of the binding pocket and rotates by ϳ180°around its own axis upon binding of 9-cis-retinoic acid, allowing H10 and H11 to form an almost continuous helix (34). Although the structure of a PPAR␥/apo-RXR␣ heterodimer has not yet been described, these structural results suggest that LGD1069 binding could lead to significant alterations in the PPAR␥/RXR␣ heterodimer interface. Given that Lys 431 is located near the site of the conformational changes involving H10 and H11, RXR agonists could also influence the stability of the Tyr 477 /Lys 431 salt bridge and hence the positioning of PPAR␥ H12. Thus, we suggest that LGD1069 binding inhibits binding of NCoR to the wild type heterodimer (with or without T0070907) by orienting PPAR␥ and RXR␣ such that binding of NCoR is disfavored and by stabilizing PPAR␥ H12 in the agonist-bound conformation. Consistent with this view, the effect of LGD1069 on T0070907induced recruitment of NCoR is more potent on the PPAR␥ wt/RXR␣ ⌬H12 heterodimer than on PPAR␥ ⌬H12/RXR␣ ⌬H12 heterodimer. In addition, a residual amount of NCoR remained on PPAR␥ ⌬H12/RXR␣ ⌬H12 heterodimer even at the highest LGD1069 concentrations (Fig. 5C), and LGD1069 was also ineffective in preventing NCoR binding to PPAR␥ ⌬H12/RXR␣ wt heterodimer complexes (data not shown).
The effect of T0070907 on LGD1069-induced recruitment of coactivator SRC-1 was also tested. Whereas the T0070907induced NCoR recruitment to PPAR␥/RXR␣ heterodimer can be almost completely reversed by the simultaneous treatment with RXR␣ agonist LGD1069, the effects of T0070907 on LGD1069-induced coactivator recruitment to the PPAR␥/RXR␣ heterodimer are more modest by comparison. These results suggest that RXR␣ agonists may have a greater influence on the conformation of the PPAR␥/RXR␣ heterodimer than do PPAR␥ antagonists, and more importantly, PPAR␥ antagonist activity could be modulated by the availability and concentration of RXR␣ agonist. The in vivo relevance of these effects is the focus of our current and future studies.