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Endocrinology Vol. 145, No. 4 1527-1538
Copyright © 2004 by The Endocrine Society

Tyrosine Agonists Reverse the Molecular Defects Associated with Dominant-Negative Mutations in Human Peroxisome Proliferator-Activated Receptor {gamma}

Maura Agostini, Mark Gurnell, David B. Savage, Emily M. Wood, Aaron G. Smith, Odelia Rajanayagam, Keith T. Garnes, Sidney H. Levinson, H. Eric Xu, John W. R. Schwabe, Timothy M. Willson, Stephen O’Rahilly and V. Krishna Chatterjee

Departments of Medicine (M.A., M.G., D.B.S., E.M.W., A.G.S., O.R., S.O., K.C.) and Clinical Biochemistry (D.B.S., S.O.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; GlaxoSmithKline (K.T.G., S.H.L.), Isotope Chemistry, Upper Merion, Pennsylvania 19406; GlaxoSmithKline (H.E.X., T.M.W.), Nuclear Receptor Discovery Research, Research Triangle Park, North Carolina 27709; and Medical Research Council Laboratory of Molecular Biology (J.W.R.S.), Addenbrooke’s Hospital, Cambridge CB2 2QH, United Kingdom

Address all correspondence and requests for reprints to: V. K. Chatterjee, Department of Medicine, University of Cambridge, Level 5, Box 157, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom. E-mail: kkc1{at}mole.bio.cam.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Loss-of-function mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) are associated with a novel syndrome characterized by partial lipodystrophy and severe insulin resistance. Here we have further characterized the properties of natural dominant-negative PPAR{gamma} mutants (P467L, V290M) and evaluated the efficacy of putative natural ligands and synthetic thiazolidinedione (TZD) or tyrosine-based (TA) receptor agonists in rescuing mutant receptor function. A range of natural ligands failed to activate the PPAR{gamma} mutants and their transcriptional responses to TZDs (e.g. pioglitazone, rosiglitazone) were markedly attenuated, whereas TAs (e.g. farglitazar) corrected defects in ligand binding and coactivator recruitment by the PPAR{gamma} mutants, restoring transcriptional function comparable with wild-type receptor. Transcriptional silencing via recruitment of corepressor contributes to dominant-negative inhibition of wild type by the P467L and V290M mutants and the introduction of an artificial mutation (L318A) disrupting corepressor interaction abrogated their dominant-negative activity. More complete ligand-dependent corepressor release and reversal of dominant-negative inhibition was achieved with TA than TZD agonists. Modeling suggests a structural basis for these observations: both mutations destabilize helix 12 to favor receptor-corepressor interaction; conversely, farglitazar makes more extensive contacts than rosiglitazone within the ligand-binding pocket, to stabilize helix 12, facilitating corepressor release and transcriptional activation. Farglitazar was a more potent inducer of PPAR{gamma} target gene (aP2) expression in peripheral blood mononuclear cells with the P467L mutation. Having shown that rosiglitazone is of variable and limited efficacy in these subjects, we suggest that TAs may represent a more rational therapeutic approach.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR {gamma} (PPAR{gamma}), a member of the nuclear receptor superfamily, was first characterized as a transcriptional regulator of adipocyte-specific gene expression (1) and preadipocyte differentiation (2). A number of unsaturated fatty acids (arachidonic, linoleic, {gamma}-linolenic, eicosapentaenoic) activate PPAR{gamma} and may represent endogenous ligands for the receptor in this context (3, 4). Eicosanoid derivatives of fatty acids can act as endogenous PPAR{gamma} activators in other biological processes: in the macrophage, hydroxyoctadecadienoic acid (HODE) and hydroxyeicosatetraenoic acid (HETE), the 15-lipooxygenase products of arachidonic and linoleic acids, inhibit the production of inflammatory cytokines (5) and promote the uptake and catabolism of oxidized low-density lipoprotein (6); 15-deoxy {Delta}12, 14 prostaglandin J2 (15d-PGJ2), a terminal metabolite of prostaglandin D2, which binds PPAR{gamma} and promotes adipocyte differentiation, has been most widely studied as a putative naturally occurring ligand (7, 8).

The thiazolidinediones (TZDs) were synthesized as potentially hypolipidemic derivatives of clofibrate but then developed as antidiabetic agents because of their unexpected insulin sensitizing action in vivo. TZDs are high-affinity PPAR{gamma} ligands (9), with the rank order of their binding affinities mirroring antihyperglycemic activity, suggesting a role for this receptor in mediating their antidiabetic action. In keeping with this, we have previously described two different mutations (P467L, V290M) in the ligand-binding domain (LBD) of human PPAR{gamma} (10) in two families, with affected subjects exhibiting severe insulin resistance and early-onset type 2 diabetes mellitus (T2DM), together with other features of the human metabolic syndrome (e.g. dyslipidemia [low high-density lipoprotein cholesterol, high triglycerides], hypertension). Consonant with a central role for PPAR{gamma} in adipogenesis, these individuals also exhibit a stereotyped pattern of partial lipodystrophy (11), a feature that has also been observed in other reported cases with receptor mutations (12, 13).

In addition to being functionally impaired, the P467L and V290M mutant receptors inhibit wild-type (WT) PPAR{gamma} action in a dominant-negative manner, consistent with heterozygosity for mutant PPAR{gamma} in affected subjects and dominant inheritance of the disorder in one family (10). The syndrome of resistance to thyroid hormone (RTH), a disorder characterized by elevated circulating thyroid hormones with tissue refractoriness to thyroid hormone action, is associated with similar dominant-negative mutations in the human thyroid hormone ß-receptor (TRß) (14). Here functional studies have shown that higher concentrations of ligand can overcome dominant-negative inhibition by many TRß mutants in vitro (15) and that the administration of supraphysiological doses of thyroid hormone can restore target tissue responsiveness in vivo (16). By analogy, we reasoned that the administration of a PPAR{gamma} agonist to enhance mutant receptor function and reverse dominant-negative activity might represent a rational approach to the treatment of the severe metabolic disturbance observed in our affected subjects. Three TZD PPAR{gamma} agonists have been developed for clinical use: troglitazone, the first insulin-sensitizing antidiabetic agent to be licensed, was later withdrawn due to unpredictable and potentially fatal hepatotoxicity; however, the newer agents, pioglitazone and rosiglitazone, offer comparable efficacy and appear to be devoid of this side effect (17). Clinical studies with rosiglitazone in two subjects harboring the P467L and V290M PPAR{gamma} mutations have demonstrated variable efficacy in ameliorating the insulin resistance and metabolic phenotype (11), suggesting a role for more potent receptor agonists. Recently high-affinity tyrosine-based PPAR{gamma} agonists, with potent glucose-lowering activity in vivo (18) and proven antidiabetic efficacy in patients with T2DM (19), have been developed. The lead compound, farglitazar (GI262570), is currently being evaluated in human clinical trials.

Here we report more detailed functional characterization of the previously reported dominant-negative natural PPAR{gamma} mutants. Consonant with the severe clinical phenotype, an array of putative endogenous natural ligands were unable to activate mutant PPAR{gamma}. The mutant receptors exhibited markedly impaired transcriptional responses with TZDs, but in contrast, tyrosine-based receptor agonists (TAs) corrected defects in ligand-binding, corepressor release, and coactivator recruitment, permitting transcriptional activation comparable with WT receptor. In comparison with the TZD rosiglitazone, the TA farglitazar completely reversed dominant-negative inhibition by both mutant receptors in vitro and activated PPAR{gamma} target gene (adipocyte P2) expression in P467L mutant peripheral blood mononuclear cells (PBMCs) more effectively. Crystallographic modeling suggests a structural basis for these observations: both mutations in PPAR{gamma} destabilize helix 12 (20), and, as in the recently elucidated PPAR{alpha}/silencing mediator of retinoid and thyroid receptors (SMRT) structure (21), this may facilitate corepressor interaction; conversely, unlike rosiglitazone, the synthetic ligand farglitazar is able to make additional contacts within the receptor ligand-binding pocket, thereby providing additional stability to helix 12, which mediates transactivation. Tyrosine-based PPAR{gamma} agonists, rather than TZDs, may therefore represent a more rational approach to restoring mutant receptor function in vivo, thereby ameliorating insulin resistance in our patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs
Full-length human PPAR{gamma}1 cDNA was cloned by RT-PCR from total human preadipocyte RNA and introduced into the pcDNA3 expression vector (Invitrogen, Groningen, The Netherlands). The P467L and V290M natural mutants and L318A artificial mutant were generated by site-directed mutagenesis of the WT receptor template as previously described (10). DNA sequences encoding residues 173–477 of the WT and mutant PPAR{gamma}1 LBDs were cloned into pGEX4T (Amersham Pharmacia Biotech, Buckinghamshire, UK) and AASV (22) to yield glutathione-S-transferase (GST)-PPAR{gamma} and VP16-PPAR{gamma} LBD fusions, respectively. Gal4-SMRT consists of the 468 C-terminal amino acids of SMRT-fused in-frame to the Gal4 DNA-binding domain in pCMX (23). Gal4-ID1 (amino acids 2302–2352), Gal4-ID2 (amino acids 2131–2201), and Gal4-ID1 + 2 (amino acids 2131–2352) contain one or more of the nuclear receptor interaction domains of SMRT as reported previously (24). PPARETKLUC (7) and UASTKLUC (22) have been described previously.

Protein-protein interaction assays
Bacterially expressed GST fusion proteins were prepared according to standard protocols (10). After purification, proteins bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech) in binding buffer [40 mM HEPES (pH 7.8), 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 2 mM dithiothreitol, 4 mg/ml BSA] were mixed with 5 µl of 35S-labeled in vitro-translated cAMP response element-binding protein (CBP) together with ligand or vehicle and incubated at 4 C for 2 h. After washing with NETN buffer [20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40], bound CBP was determined by SDS-PAGE. Comparable loading of the GST-PPAR{gamma} LBD fusion proteins was confirmed with Coomassie staining before autoradiography. The assay shown is representative of three separate experiments with similar results.

Ligand-binding assays
[3H]-farglitazar was synthesized as follows: Crabtree’s catalyst (25) (3 mg, 200 mol %) was added to a solution of farglitazar (1.0 mg) in methylene chloride (1.0 ml). The mixture was subjected to three freeze-pump-thaw cycles on a steel manifold before introduction of 1.96 Ci tritium gas. The reaction mixture was allowed to warm to room temperature and vigorously stirred for 18 h. After workup and exchange of labile tritium, 129 mCi crude [3H]-farglitazar was obtained at 50% radiochemical purity by HPLC. A 25.8-mCi portion of the crude product was purified by HPLC (Zorbax SB C18, 5 µm, 4.6 x 250 mm, 70:30:0.1 acetonitrile/water/trifluoroacetic acid at 1.0 ml/min, UV detection at 240 nm). The desired product fraction was collected, concentrated in vacuo, frozen, and lyophilized under vacuum to give a pale yellow solid. The solid was dissolved in 5 ml of absolute ethanol to provide 6.05 mCi [3H]-farglitazar (1.21 mCi/ml; 41 Ci/mmol) at 97.6% radiochemical purity by HPLC: 1H-NMR (CDCl3, 400 MHz), 8.84 (m, 1H), 7.94 (m, 2H), 7.58 (m, 2H), 7.51 (m, 1H), 7.46 (m. 1H), 7.43 (m, 2H), 7.38 (m, 2H), 7.34 (m, 1H), 7.21 (m, 2H), 6.80 (m, 2H), 6.69 (d, J = 8.3 Hz, 1H), 6.61 (ddd, J = 7.6, 7.6, 0.9 Hz, 1H), 4.38 (m, 1H), 4.134 (t, J = 6.6 Hz, 2H), 3.25 (dd, J = 13.9, 5.7 Hz, 1H), 3.14 (dd, J = 13.9, 7.1 Hz, 1H), 2.94 (t, J = 6.6 Hz, 2H), 2.33 (s, 3H). 3H-NMR (CDCl3, 426 MHz) 8.03 (dm, J = 1.2 Hz). Hormone-binding assays were performed using bacterially expressed GST-PPAR{gamma} LBD fusion proteins and the PPAR{gamma} ligands [3H]-rosiglitazone (9) and [3H]-farglitazar in a modification of a previously described filter binding assay (26). Filters were preincubated with BSA (1%) and Tween (1%) to reduce nonspecific binding with the [3H]-farglitazar compound. Again, addition of comparable amounts of PPAR{gamma} LBD fusion proteins was confirmed through Coomassie staining of aliquots subjected to SDS-PAGE. Results denote the mean ± SEM of experiments performed on three separate occasions.

Transfection assays
Calcium phosphate-mediated transfection was performed in 24-well plates of 293EBNA cells. Each well was cotransfected with 50–100 ng of receptor expression vector, 500 ng of reporter construct, 100 ng of the internal control plasmid Bos-ß-gal, and, where indicated, 50–100 ng of additional construct. Cells were harvested and assayed as described previously (15). Results represent the mean ± SEM of at least three independent experiments, each performed in triplicate.

aP2 assays in PBMCs
Blood was obtained from the index case harboring the P467L PPAR{gamma} mutation (10) and PBMCs were isolated by ficoll gradient centrifugation, washed in PBS, and cultured in RPMI 1640 (Sigma-Aldrich, Dorset, UK) with 1% charcoal-stripped fetal bovine serum in 6-well plates with 3 x 106 cells/well. After exposure to either rosiglitazone or farglitazar for 24 h, RNA was isolated from cells using a commercial kit (Qiagen, West Sussex, UK) and reverse transcribed to generate first-strand cDNA. This was serially diluted and analyzed by quantitative PCR as described previously (27). Results shown are the mean of two independent experiments in the individual carrying the P467L mutation (a deterioration in her clinical condition precluded venesection for a third determination).

Statistical analyses
All results are expressed as mean ± SEM; where appropriate, comparisons between values were made using the Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The transcriptional activities of WT receptor and PPAR{gamma} mutants were assayed by cotransfection of receptor expression vectors together with a reporter gene (PPARETKLUC) containing three copies of the PPARE from the acyltransferase-coenzyme A oxidase gene linked to the thymidine kinase promoter and luciferase, in the absence or presence of an array of putative natural ligands (Fig. 1Go). Western blotting of cell extracts after transfection of WT PPAR{gamma} or P467L and V290M mutants confirmed that their expression levels were equivalent in these assays (data not shown). As has been previously described, WT PPAR{gamma} exhibited some constitutive basal transcriptional activity (28) but showed a transcriptional response to unsaturated fatty acids (linoleic acid, arachidonic acid, {gamma}-linolenic acid), 15d-PGJ2, and eicosanoids (13-HODE, 15-HETE), which ranged from 50% to 80% of that obtained with a synthetic PPAR{gamma} agonist rosiglitazone (1 µM). In contrast, the P467L and V290M mutants were completely unresponsive to all the natural ligands tested, despite their partial response to the synthetic receptor agonist.



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  		FIG. 1. A panel of putative endogenous ligands fail to transactivate mutant PPAR{gamma}. Twenty-four-well plates of 293EBNA cells were transfected with 500 ng of PPARETKLUC reporter gene, 100 ng of Bos-ß-gal control plasmid, and 100 ng of receptor expression vector as shown. Transcriptional activity in response to a variety of endogenous ligands is shown. Results are expressed as a percentage of the maximal WT observed response.

 
To evaluate the potential therapeutic role of synthetic PPAR{gamma} agonists, we next examined the function of PPAR{gamma} mutants with each of the TZDs, including rosiglitazone, which is the most potent receptor agonist in this class that is licensed for clinical use. In comparison with WT PPAR{gamma}, the P467L and V290M mutant receptors were virtually unresponsive to both troglitazone (Fig. 2CGo) and pioglitazone (Fig. 2DGo), achieving only 40–50% of WT receptor activity at the highest concentration (10 µM) of ligand. Similarly, only 1–10 µM rosiglitazone elicited partial transcriptional responses (50–75% of WT) from the mutant receptors (Fig. 2EGo). Replacement of the 2,4-thiazolidinedione head group (Fig. 2AGo) with tyrosine-based substituents has led to the development of a series of high-affinity PPAR{gamma} agonists (Fig. 2BGo). In marked contrast to the TZDs, these compounds showed greater activity with PPAR{gamma} mutants. GW1929 (Fig. 2FGo) and GW7845 (Fig. 2GGo) induced significant transcriptional activation by both P467L and V290M mutant receptors even at low concentrations (10–100 nM) of ligand, enabling both mutants to achieve maximal responses comparable with WT receptor. Farglitazar, which is being developed for clinical use, showed the greatest activity, with the PPAR{gamma} mutants achieving greater than 75% of WT receptor activity at 10 nM concentration of ligand (Fig. 2HGo). Importantly, such greater potency of tyrosine agonists, compared with thiazolidinediones, was more evident with PPAR{gamma} mutants than WT receptor. Thus, whereas farglitazar was 100 times more potent than rosiglitazone with WT PPAR{gamma} [WT activation with 100 nM rosiglitazone (Fig. 2EGo) vs. 1 nM farglitazar (Fig. 2HGo)], the tyrosine agonist was up to 1000 times more potent than rosiglitazone with the PPAR{gamma} mutants [P467L and V290M activation with 10,000 nM rosiglitazone (Fig. 2EGo) vs. 10 nM farglitazar (Fig. 2HGo)].



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  		FIG. 2. A and B, Synthetic PPAR{gamma} agonists. Comparison of the chemical structures of rosiglitazone (thiazolidinedione, A) and farglitazar (tyrosine agonist, B). C–H, Tyrosine-based but not thiazolidinedione receptor agonists restore the transcriptional activity of P467L and V290M PPAR{gamma} mutants. Twenty-four-well plates of 293EBNA cells were transfected as outlined in Fig. 1Go. Transcriptional activity in response to ligand is shown for troglitazone (C) pioglitazone (D) rosiglitazone (E) GW1929 (F) GW7845 (G) and farglitazar (H). Results are expressed as a percentage of the WT maximum. The gray circle in H denotes the transcriptional response of WT PPAR{gamma} to 100 nM rosiglitazone, indicating that it is of the same magnitude as the receptor response to farglitazar.

 
We have shown previously that the impaired transcriptional function of the P467L PPAR{gamma} mutant reflects a combination of defects in binding to ligand and recruitment of coactivator (10). We therefore compared these properties of the P467L and V290M receptor mutants with TZD vs. tyrosine-based PPAR{gamma} agonists. In ligand-binding assays with bacterially expressed WT or mutant GST-PPAR{gamma} LBD fusion proteins and [3H]-rosiglitazone or [3H]-farglitazar, neither mutant receptor exhibited detectable specific binding to the radiolabeled TZD, whereas both mutant proteins showed significant specific binding to the TA (Fig. 3AGo). In a protein-protein interaction assay, both rosiglitazone and farglitazar mediated strong recruitment of the 35S-labeled coactivator CBP to WT receptor. However, the P467L and V290M mutants showed negligible coactivator binding even at high concentrations (10 µM) of TZD, whereas a lower concentration (1 µM) of TA promoted recruitment of CBP (Fig. 3BGo). Some members of the nuclear receptor family [e.g. TR and retinoic acid receptor (RAR)] are able to silence basal gene transcription through ligand-independent interaction with specific corepressor proteins such as nuclear receptor corepressor (NCoR) (29) and SMRT (23), with ligand-binding promoting corepressor dissociation. We therefore examined the effects of the P467L and V290M mutant receptors on basal gene transcription and their interaction with corepressor. In comparison with cells transfected with empty expression vector, WT PPAR{gamma} activated basal reporter gene activity (~5-fold); in striking contrast, both PPAR{gamma} mutants not only lacked such activation but also significantly repressed basal gene transcription (pcDNA3 = 1.0; P467L = 0.44; V290M = 0.53) (Fig. 4AGo), suggesting that they might interact aberrantly with corepressors in vivo. Several studies have identified domains (ID1 and ID2) within NCoR and SMRT that mediate interaction with nuclear receptors (24, 30, 31).



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  		FIG. 3. A, Binding of thiazolidinedione (3H-rosiglitazone) and tyrosine agonist (3H-farglitazar) radioligands to GST-PPAR{gamma} LBD chimaeras. Bacterially expressed GST-PPAR{gamma} LBD fusion proteins were incubated with radioligand as indicated in the absence or presence of 10 µM cold competing ligand (rosiglitazone or farglitazar, respectively). Inset, Coomassie-stained gel of proteins used in ligand-binding assays confirming comparable expression of WT and mutant receptors, with GST present in slight excess. B, Coactivator recruitment to mutant PPAR{gamma} is greater with TA (farglitazar) than thiazolidinedione (rosiglitazone). WT and mutant GST-PPAR{gamma} LBD fusion proteins (quantitated as in A) were tested for interaction with 35S-labeled in vitro-translated CBP in the presence of increasing concentrations of ligand (rosiglitazone or farglitazar). Control assays were performed with GST alone. Histograms below each panel quantify the amount of CBP bound. An asterisk (*) denotes the band corresponding to full-length CBP.

 


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  		FIG. 4. PPAR{gamma} mutants repress basal transcription and are recruited to the ID1 domain of the corepressor SMRT. A, Unlike their WT counterpart, both the P467L and V290M mutants silence basal gene transcription. 293EBNA cells were transfected with 500 ng reporter gene (PPARETKLUC), 100 ng Bos-ß-gal (internal control), and 100 ng of receptor construct (empty vector, WT, P467L, or V290M). B, WT and mutant PPAR{gamma} interact with the ID1 domain of SMRT. 293EBNA cells were transfected with 500 ng of the reporter construct UASTKLUC, 100 ng of the internal control Bos-ß-gal, 50 ng of expression vectors encoding the Gal4 DNA-binding domain (Gal4) alone or fused to the ID1, ID2, or ID1 + 2 domains of SMRT, and 50 ng of expression vector encoding VP16 alone or VP16 fused to the LBD of WT PPAR{gamma} (WT), P467L PPAR{gamma} (P467L), or V290M PPAR{gamma} (V290M). C, Farglitazar is more effective than rosiglitazone in promoting corepressor dissociation from mutant PPAR{gamma}. 293EBNA cells were transfected as in B and treated with vehicle (dimethylsulfoxide, DMSO), rosiglitazone, or farglitazar. **, P < 0.001.

 
To study the interaction between PPAR{gamma} mutants and corepressor, mammalian two-hybrid assays were performed, with cotransfection of fusions consisting of the ID1 + 2, ID1, or ID2 domains of SMRT linked to the DNA-binding domain of Gal4, together with VP16 linked to WT, P467L, or V290M PPAR{gamma} LBDs. In the absence of ligand, WT receptor and both PPAR{gamma} mutants were recruited comparably with Gal4-ID1 + 2, and additional experiments with individual ID domain fusions indicated that this interaction was mediated through the ID1 region (Fig. 4BGo). Next, we examined the effect of thiazolidinedione vs. tyrosine-based PPAR{gamma} agonists on receptor-corepressor interaction. With the addition of increasing concentrations (100–1000 nM) of TZD ligand (rosiglitazone), both mutant receptors exhibited significantly attenuated and incomplete dissociation from a Gal4-ID1 corepressor fusion when compared with the WT receptor (Fig. 4CGo). However, the addition of TA (farglitazar) induced progressive and nearly complete dissociation of both mutant receptors from Gal4-ID1 in a manner comparable with WT PPAR{gamma} (Fig. 4CGo).

Our previous studies indicated that inhibition of WT receptor function by the P467L and V290M PPAR{gamma} mutants is a likely mechanism for impaired receptor action in vivo (10). We therefore compared the relative efficacy of both natural and synthetic agonists in ameliorating such dominant-negative inhibition by PPAR{gamma} mutants. Cells transfected with WT receptor plus an equal amount of either P467L or V290M PPAR{gamma} mutants were studied with increasing concentrations of natural (15d-PGJ2) or synthetic ligands (rosiglitazone or farglitazar). In keeping with their transcriptional activities with each ligand when tested alone, the P467L and V290M mutants exhibited significant dominant-negative inhibition (30–35%) of WT receptor function even at maximal concentrations of 15d-PGJ2 (Fig. 5Go). Moreover, both mutants exerted strong dominant-negative activity at low (10 nM) concentrations of TZD, and such inhibition was retained at higher (1 µM) levels of ligand with the V290M mutant (Fig. 5Go). In contrast, low (10 nM) or high (1 µM) concentrations of farglitazar completely reversed dominant-negative inhibition by the PPAR{gamma} mutants (Fig. 5Go).



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  		FIG. 5. TA (farglitazar) reverses dominant-negative inhibition by PPAR{gamma} mutants more fully than natural ligand (15d-PGJ2) or thiazolidinedione (rosiglitazone). 293EBNA cells were transfected with 100 ng of WT receptor plus an equal amount of either WT or mutant (P467L; V290M) expression vector (with the same reporter gene and internal control constructs as described in Fig. 1Go) in the presence of increasing concentrations of ligand. The transcriptional responses mediated by either 100 or 200 ng of WT receptor were virtually identical (data not shown). Results are expressed as a percentage of the WT maximum response. **, P < 0.001; ns, not significant.

 
Failure of ligand-dependent corepressor release has been shown to mediate dominant-negative inhibition by natural TRß mutants in RTH (32). We therefore sought to determine whether corepressor interaction is important for dominant-negative activity of the natural PPAR{gamma} mutants. The crystalstructure of a PPAR{alpha}-SMRT complex has recently been elucidated (21), and residues in PPAR{alpha} that mediate binding to a polypeptide from SMRT are highly conserved in PPAR{gamma} (see Fig. 8BGo). One of these conserved residues in PPAR{gamma} (Leu 318) was mutated to alanine on either WT or P467L mutant PPAR{gamma} backgrounds, with comparison of their transcriptional properties in the absence of ligand. The L318A receptor mutant showed comparable constitutive activity to WT PPAR{gamma}; however, the P467L/L318A double mutation exhibited attenuated repression of basal transcription when compared with the P467L mutant (Fig. 6AGo: pcDNA3 = 1.0; P467L = 0.48; P467L/L318A = 0.85). Consistent with this, in a mammalian two-hybrid assay, the L318A mutation abolished interaction of the P467L mutant with the ID1 domain of SMRT corepressor (Fig. 6AGo, inset). Moreover, in comparison with the P467L mutation alone, the P467L/L318A double mutant exhibited almost negligible dominant-negative inhibition of WT PPAR{gamma} activity (Fig. 6BGo).



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  		FIG. 8. A, Crystallographic modeling demonstrating how the TA (farglitazar) may preferentially stabilize helix 12 in mutant PPAR{gamma}. Superimposition of PPAR{gamma} structures bound to either tyrosine (farglitazar) or thiazolidinedione (rosiglitazone) agonists showing part of the cavities (gray mesh) containing either ligand. On the helical backbone (green), the side chains (pink) of residues (P467, V290), mutated in our patients with severe insulin resistance, are depicted. Both mutations are predicted to disrupt the orientation of helix 12 as described previously (10 ), thereby perturbing known important interactions of this helix with ligand [rosiglitazone (yellow) or farglitazar (red)] and coactivator. B, An alignment of amino acid sequences corresponding to the corepressor interaction interface in PPAR{alpha} in the three receptor subtypes. Residues in PPAR{alpha} mediating contact with the SMRT motif are highlighted (*), and boxes denote complete conservation of 13 of 14 of these amino acids between the receptors. L318 in PPAR{gamma} is denoted in bold. C, A molecular model showing interface between SMRT (white) and PPAR{gamma} (green) with the V290M mutation (purple). The key SMRT residues that form the interface (I+4, I+5, L+1, L+9) are numbered as reported in the PPAR{alpha}/SMRT crystal structure (21 ).

 


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  		FIG. 6. Introduction of the L318A mutation attenuates both transcriptional repression and dominant-negative activity of P467L through abolition of its interaction with corepressor. A, Basal transcriptional repression by the P467L natural mutant is reversed, but constitutive activity of WT PPAR{gamma} is not affected by the addition of an L318A mutation. Inset, Interaction of P467L with the ID1 domain of SMRT is abolished after introduction of the L318A mutation. 293EBNA cells were transfected and results analyzed as in Fig. 4Go, A and B. B, Introduction of the L318A mutation significantly attenuates the dominant-negative activity of P467L. 293EBNA cells were transfected as in Fig. 5Go and treated with vehicle (dimethylsulfoxide, DMSO) or rosiglitazone. **, P < 0.001.

 
The adipocyte P2 (aP2) gene, a well-validated PPAR{gamma} target gene, has previously been shown to be expressed and regulated by PPAR{gamma} ligands in PBMCs (33). To determine whether the differences in mutant PPAR{gamma} responses to synthetic agonists observed in vitro might correlate with ligand-dependent responses in cells from our affected subjects, we examined the ability of both rosiglitazone and farglitazar to induce aP2 expression in cultured PBMCs taken from the index case harboring the P467L mutation (10). Rosiglitazone induced aP2 expression in patient PBMCs in a dose-dependent manner, but with farglitazar the dose-response curve of the target gene activation was significantly left shifted (Fig. 7Go). The magnitude of maximal aP2 target gene induction inresponse to either ligand was similar. The results suggest that the tyrosine agonist is a more potent activator of PPAR{gamma}-mediated transcription than its thiazolidinedione counterpart in primary cells from an affected subject.



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  		FIG. 7. The TA (farglitazar) enhances target gene (aP2) expression in P467L mutant receptor containing PBMCs more effectively than thiazolidinedione (rosiglitazone). After 24 h exposure to increasing concentrations of rosiglitazone or farglitazar, aP2 gene expression in PBMCs was quantitated by RT-PCR. The results are expressed as a percentage of the maximum observed response. The SEM was less than 10% and has been omitted for clarity.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously described two different heterozygous, loss-of-function mutations (P467L, V290M) in the LBD of human PPAR{gamma}. Affected individuals exhibited marked hyperinsulinemia and the skin lesion acanthosis nigricans, signifying severe insulin resistance; importantly, subjects had developed complications secondary to insulin resistance, including characteristic dyslipidemia (elevated triglycerides, low high-density lipoprotein cholesterol), ovarian dysfunction, and T2DM; they also showed early-onset hypertension unrelated to diabetic comorbidity (10). Whereas both receptor mutants were markedly functionally impaired and dominant-negative inhibitors of wild-type receptor action, they retained some transcriptional activity at the highest concentrations of ligand (10). We therefore reasoned that if either higher levels of endogenous natural ligands or synthetic receptor agonists could overcome the functional defect and dominant-negative inhibition by PPAR{gamma} mutants in vitro, they might be useful to treat the severe clinical phenotype when administered in vivo.

Despite being able to activate transcription via WT PPAR{gamma}, even micromolar concentrations of putative endogenous ligands, including omega-3 ({gamma}-linolenic) and omega-6 (linoleic, arachidonic) polyunsaturated fatty acids, eicosanoids (13-HODE, 15-HETE) and 15d-PGJ2, were unable to induce transcriptional activity from the mutant receptors (Fig. 1Go). Furthermore, high levels of 15d-PGJ2 were unable to reverse significant dominant-negative inhibition of WT receptor function by the P467L and V290M PPAR{gamma} mutants (Fig. 5Go). Such unresponsiveness of mutant receptors to endogenous ligands correlates with recent clinical findings of partial lipodystrophy in adults and significant insulin resistance, even in two young children aged 4 and 7 yr with the P467L mutation (11), which underscore the severity of the clinical phenotype. In addition, such unresponsiveness in vitro suggests that raising levels of endogenous PPAR{gamma} ligands in affected subjects is unlikely to be a successful therapeutic approach.

With thiazolidinedione PPAR{gamma} agonists, both the lower-affinity (WT PPAR{gamma} EC50 = 500 nM) agents, troglitazone and pioglitazone, and the more potent (WT PPAR{gamma} EC50 = 43 nM) rosiglitazone, induced significant transcriptional activity with the P467L and V290M mutants only at 10- or 1-µM concentrations of ligand, respectively (Fig. 2Go, C–E). A novel class of synthetic PPAR{gamma} ligands (GW1929, GW7845, and farglitazar), where N-tyrosine moieties have been substituted for the 2,4-thiazolidinedione head group, have been developed (34) and are known to be higher-affinity (EC50 = 0.3–6 nM) agonists for WT PPAR{gamma}. In marked contrast to TZDs, the TAs proved capable of rescuing mutant PPAR{gamma} function, even at low concentrations of ligand (1–10 nM), eliciting a maximal transcriptional response comparable with WT receptor (Fig. 2Go, F–H). Furthermore, the greater potency of tyrosine vs. thiazolidinedione agonist is more marked with the PPAR{gamma} mutants than WT receptor, indicating that this class of ligand acts specifically to restore mutant receptor function.

Further comparisons of rosiglitazone vs. farglitazar indicated that the ability of the TA to correct deficits in ligand binding, coactivator recruitment and corepressor displacement mediated its enhancement of mutant receptor function (Figs. 3Go and 4Go). To elucidate the molecular basis for the observed differences between the two classes of PPAR{gamma} ligand, we examined the crystal structures of the PPAR{gamma}/retinoid X receptor-{alpha} heterodimer (35) complexed with either rosiglitazone or farglitazar. In keeping with other nuclear receptors, an amphipathic {alpha}-helix (H12) at the receptor carboxyterminus mediates important interactions with both ligand and coactivator (steroid receptor coactivator-1) (36): in both crystal structures, Tyr473 makes contact with ligand, forming hydrogen bonds with either the 2,4-thiazolidinedione head group of rosiglitazone or the carboxylate head group of farglitazar; the side chain of Leu468 from the opposite side of H12 contributes to a hydrophobic cleft on the receptor surface, which accommodates the coactivator peptide, whereas Glu471 acts in concert with Lys301 to form a charge clamp that stabilizes interaction with coactivator. Pro467 forms the amino-terminal boundary of helix 12 and Val290 (within helix 3) packs against H12. We have previously demonstrated, using fluorescence anisotropy, that mutation of either residue disrupts the position and orientation of helix 12, thereby compromising interactions with both ligand and coactivator (20). Inspection of the TZD vs. TA-bound PPAR{gamma} structures reveals that farglitazar occupies more (~40% vs. 25%) of the ligand-binding pocket with a 5-methyl-2-phenyloxazole tail and benzophenone head group, making additional hydrophobic interactions in the cavity, which probably account for its increased PPAR{gamma}-binding affinity, compared with rosiglitazone (35) (Fig. 8AGo).

Unlike a subset of nuclear receptors (including TR and RAR), which are capable of repressing basal transcription in the absence of ligand through recruitment of corepressor proteins such as NCoR (29) and SMRT (23), WT PPAR{gamma} exhibits constitutive transcriptional activity (Fig. 4AGo) (28). Whether such activity represents receptor activation by endogenous PPAR{gamma} ligands or is an intrinsic property of unliganded PPAR{gamma}, with H12 being in an active conformation in the apo-receptor crystal structure (36), remains unclear. In contrast, both the P467L and V290M PPAR{gamma} mutants not only lacked such constitutive activity but also acted as potent transcriptional repressors in the absence of exogenous ligand (Fig. 4AGo). These properties are similar to those of artificial dominant-negative human [L468A/E471A (37)] and murine [L466A (38)] PPAR{gamma} mutants described previously. However, in a two-hybrid assay, both WT and natural PPAR{gamma} mutants interacted with corepressor (Fig. 4BGo). To reconcile these apparently discordant observations, we suggest that corepressor is greatly overexpressed relative to endogenous coactivators in the two-hybrid assay, probably promoting its interaction with WT PPAR{gamma} in a manner that is not relevant to its normal action in cells containing more physiological levels of each cofactor type. Evidence in favor of this notion is provided by our observation that the introduction of a mutation (L318A), which disrupts corepressor interaction with both WT PPAR{gamma} and the P467L mutant, has no discernible effect on the constitutive transcriptional activity of WT receptor, whereas it reverses transcriptional silencing and dominant-negative inhibition by the P467L mutant (Fig. 6Go, A and B).

The ability to silence basal gene transcription is also a characteristic of dominant-negative inhibition by mutant nuclear receptors in other disorders, e.g. TRß mutants in RTH (32), the promyelocytic leukemia-RAR fusion protein in acute promyelocytic leukemia (39), and the oncogene v-erbA (40). Furthermore, some TRß mutants in RTH have been shown to interact aberrantly with corepressor, exhibiting failure to dissociate fully with ligand (41, 42) and corepressor interaction with PLZF-RAR fusions in acute promyelocytic leukemia is refractory to retinoic acid treatment (39, 43, 44). In this context, both PPAR{gamma} mutants exhibited delayed and incomplete corepressor release in the presence of saturating levels (1 µM) of rosiglitazone (Fig. 4CGo), whereas a moderate concentration (100 nM) of farglitazar promoted near normal dissociation of corepressor (Fig. 4CGo). Furthermore, such failure of natural PPAR{gamma} mutants to release corepressor fully with TZD is analogous to the properties of the artificial helix 12 PPAR{gamma} mutants (L468A/E471A; L466A) described previously (37, 38).

Recently the crystal structure of a ternary complex consisting of the PPAR{alpha} LBD bound to an antagonist and a polypeptide motif from the corepressor SMRT has been solved (21). Notable features of this structure include displacement of helix 12 such that it adopts a different position, compared with its active conformation in the agonist-bound structure, and docking of a SMRT motif in a hydrophobic groove formed by helices 3, 4, and 5 of the receptor. The LBDs of PPAR{gamma} and PPAR{alpha} are similar (~71% homology) and an alignment of residues in helix 3 from the receptors (Fig. 8BGo) indicates striking homology, with 13 of 14 amino acids mediating PPAR{alpha}-SMRT interaction being identical in PPAR{gamma}. These observations permit crystallographic modeling to provide insights into how the natural PPAR{gamma} mutations (P467L, V290M) facilitate interaction with corepressor. Both mutations destabilize helix 12, preventing it from adopting the agonist-bound conformation (20). By analogy with the altered conformation of helix 12 in the antagonist-bound PPAR{alpha}/SMRT structure, we suggest that such displacement of H12 in the natural PPAR{gamma} mutants favors corepressor recruitment. In addition, with the V290M mutation, an additional factor may stabilize corepressor binding. A crystallographic model of PPAR{gamma} complexed with SMRT (Fig. 8CGo) shows that the side chain of V290 is in contact with an isoleucine residue (I + 4) of the SMRT motif. However, the interaction is relatively weak due to the distance (~4 Å) between the isoleucine and valine residues and the fact that these hydrophobic side chains are partially solvent exposed. In contrast, when residue 290 is substituted by methionine, its extended side chain has improved van der Waals contacts, predicting stabilization of corepressor interaction.

Whereas both PPAR{gamma} mutants inhibited WT receptor function significantly at lower (10 nM) concentrations of rosiglitazone (Fig. 5Go), the same concentration of farglitazar fully relieved dominant-negative inhibition by both mutant receptors (Fig. 5Go). To determine whether differential responses of the mutant receptors to the two ligands in vitro might translate into differences in clinical efficacy in vivo, we compared the ability of both rosiglitazone and farglitazar to induce PPAR{gamma} target gene (aP2) expression in PBMCs from one patient with the P467L receptor mutation. As anticipated, even at low concentrations (1–10 nM), farglitazar evoked a greater target gene response from mutant PBMCs than was observed with rosiglitazone, indicating greater efficacy of the tyrosine agonist vs. its TZD counterpart (Fig. 7Go). Although peak plasma drug levels after oral administration of farglitazar (5 mg) are slightly lower (300 nM) (45) than after 8 mg (1 µM) of rosiglitazone (46), our studies indicate that they still exceed concentrations required to restore the function and abrogate dominant-negative activity of mutant receptors in vitro. Accordingly, the tyrosine-based PPAR{gamma} agonist may have greater potential efficacy in vivo, and future clinical studies will determine whether it does represent a more rational therapeutic approach to treating the severe insulin resistance in our affected patients.


   Acknowledgments
 
We thank Dr. Xue-Ming Shen for HPLC radiochemical purity analysis, Ms. Tong Ni for radioactive concentration analysis, and Dr. Alan Freyer for NMR assignments. We also acknowledge the secretarial expertise of Mrs. T. D. Wallman.


   Footnotes
 
This work was supported by the Wellcome Trust (to V.K.C., S.O., and M.G.), a European Union network grant (to J.S. and V.K.C.), and the Raymond and Beverly Sackler Foundation (to M.G.).

M.A. and M.G. contributed equally to this work.

Abbreviations: aP2, Adipocyte P2; CBP, CREB (cAMP response element binding protein) binding protein; 15d-PGJ2, 15-deoxy {Delta}12, 14 prostaglandin J2; GST, glutathione-S-transferase; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; LBD, ligand-binding domain; NCoR, nuclear receptor corepressor; PBMC, peripheral blood mononuclear cell; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; RAR, retinoic acid receptor; RTH, resistance to thyroid hormone; SMRT, silencing mediator of retinoid and thyroid receptors; TA, tyrosine-based receptor agonist; T2DM, type 2 diabetes mellitus; TRß, thyroid hormone ß-receptor; TZD, thiazolidinedione; WT, wild-type.

Received September 23, 2003.

Accepted for publication November 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR{gamma}2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8:1224–1234[Abstract/Free Full Text]
  2. Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblasts by PPAR{gamma}2, a lipid-activated transcription factor. Cell 79:1147–1156[CrossRef][Medline]
  3. Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand P, Wahli W, Willson TM, Lenhard JM, Lehmann JM 1997 Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors {alpha} and {gamma}. Proc Natl Acad Sci USA 94:4318–4323[Abstract/Free Full Text]
  4. Desvergne B, Wahli W 1999 Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20:649–688[Abstract/Free Full Text]
  5. Huang JT, Welch JS, Ricote M, Binder CJ, Willson TM, Kelly C, Witztum JL, Funk CD, Conrad D, Glass CK 1999 Interleukin-4-dependent production of PPAR-{gamma} ligands in macrophages by 12/15-lipoxygenase. Nature 400:378–382[CrossRef][Medline]
  6. Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM 1998 Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR{gamma}. Cell 93:229–240[CrossRef][Medline]
  7. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-Deoxy-{Delta} 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR{gamma}. Cell 83:803–812[CrossRef][Medline]
  8. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM 1995 A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor {gamma} and promotes adipocyte differentiation. Cell 83:813–819[CrossRef][Medline]
  9. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J Biol Chem 270:12953–12956[Abstract/Free Full Text]
  10. Barroso I, Gurnell M, Crowley VEF, Agostini M, Schwabe JW, Soos MA, Maslen GLI, Williams TDM, Lewis H, Schafer AJ, Chatterjee VKK, O’Rahilly S 1999 Dominant-negative mutations in human PPAR{gamma} are associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 402:880–883[Medline]
  11. Savage DB, Tan GD, Acerini CL, Jebb SA, Agostini M, Gurnell M, Williams R, Umpleby AM, Thomas EL, Bell JD, Dixon AK, Cinti S, Vidal-Puig A, Karpe F, Chatterjee VKK, O’Rahilly S 2003 Clinical and pathophysiological features of a metabolic syndrome resulting from mutations in the nuclear receptor PPAR{gamma}. Diabetes 52:910–917[Abstract/Free Full Text]
  12. Agarwal AK, Garg A 2002 A novel heterozygous mutation in peroxisome proliferator-activated receptor-{gamma} gene in a patient with familial partial lipodystrophy. J Clin Endocrinol Metab 87:408–411[Abstract/Free Full Text]
  13. Hegele RA, Cao H, Frankowski C, Mathews ST, Leff T 2002 PPARG F388L, a transactivation-deficient mutant, in familial partial lipodystrophy. Diabetes 51:3586–3590[Abstract/Free Full Text]
  14. Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of resistance to thyroid hormone. Endocr Rev 14:348–399[CrossRef][Medline]
  15. Collingwood TN, Adams M, Tone Y, Chatterjee VKK 1994 Spectrum of transcriptional dimerization and dominant negative properties of twenty different mutant thyroid hormone ß receptors in thyroid hormone resistance syndrome. Mol Endocrinol 8:1262–1277[Abstract]
  16. Hayashi Y, Weiss RE, Sarne DH, Yen PM, Sunthornthepvarakul T, Marcocci C, Chin WW, Refetoff S 1995 Do clinical manifestations of resistance to thyroid hormone correlate with the functional alteration of the corresponding mutant thyroid hormone ß receptors? J Clin Endocrinal Metab 80:3246–3256[Abstract]
  17. Krentz AJ, Bailey CJ, Melander A 2000 Thiazolidinediones for type 2 diabetes. New agents reduce insulin resistance but need long term clinical trials. BMJ 321:252–253[Free Full Text]
  18. Brown KK, Henke BR, Blanchard SG, Cobb JE, Mook R, Kaldor I, Kliewer SA, Lehmann JM, Lenhard JM, Harrington WW, Novak PJ, Faison W, Binz JG, Hashim MA, Oliver WO, Brown HR, Parks DJ, Plunket KD, Tong WQ, Menius JA, Adkison K, Noble SA, Willson TM 1999 A novel N-aryl tyrosine activator of peroxisome proliferator-activated receptor-{gamma} reverses the diabetic phenotype of the Zucker diabetic fatty rat. Diabetes 48:1415–1424[Abstract]
  19. Fiedorek FT, Wilson GG, Frith L, Patel J, Abou-Donia M 2000 Monotherapy with G1262570, a tyrosine-based non-thiazolidinedione PPAR{gamma} agonist, improves metabolic control in type 2 diabetes mellitus patients. Diabetes 49(Suppl 1):157-OR (Abstract)
  20. Kallenberger BC, Love JD, Chatterjee VK, Schwabe JW 2003 A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease. Nat Struct Biol 10:136–140[CrossRef][Medline]
  21. Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT, Parks DJ, Moore JT, Kliewer SA, Willson TM, Stimmel JB 2002 Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR{alpha}. Nature 415:813–817[Medline]
  22. Tone Y, Collingwood TN, Adams M, Chatterjee VKK 1994 Functional analysis of a transactivation domain in the thyroid hormone ß receptor. J Biol Chem 269:31157–31161[Abstract/Free Full Text]
  23. Chen JD, Evans RM 1995 A transcriptional corepressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  24. Nagy L, Kao H-Y, Love JD, Li C, Banayo E, Gooch JT, Chatterjee VKK, Evans RM, Schwabe JWR 1999 Mechanism of co-repressor binding and release from nuclear hormone receptors. Genes Dev 13:3209–3216[Abstract/Free Full Text]
  25. Shu AYL, Saunders D, Levinson SH, Landvatter SW, Mahoney A, Senderoff SG, Mack JF, Heys JR 1999 Direct tritium labeling of multifunctional compounds using organoiridium catalysis. J Labelled Comp Radiopharm 42: 797–807
  26. Adams M, Matthews CH, Collingwood TN, Tone Y, Beck Peccoz P, K CVK 1994 Genetic analysis of twenty-nine kindreds with generalised and pituitary resistance to thyroid hormone. J Clin Invest 94:506–515
  27. Savage DB, Sewter CP, Klenk ES, Segal DG, Vidal-Puig A, Considine RV, O’Rahilly S 2001 Resistin/Fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-{gamma} action in humans. Diabetes 50:2199–2202[Abstract/Free Full Text]
  28. Zamir I, Zhang J, Lazar MA 1997 Stoichiometric and steric principles governing repression by nuclear hormone receptors. Genes Dev 11:835–846[Abstract/Free Full Text]
  29. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CJ, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor corepressor. Nature 377:397–404[CrossRef][Medline]
  30. Hu X, Lazar MA 1999 The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402:93–96[CrossRef][Medline]
  31. Perissi V, Staszewski LM, McInerney EM, Kurokawa R, Krones A, Rose DW, Lambert MH, Milburn MV, Glass CK, Rosenfeld MG 1999 Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev 13:3198–3208[Abstract/Free Full Text]
  32. Yoh SM, Chatterjee VKK, Privalsky ML 1997 Thyroid hormone resistance syndrome manifests as an aberrant interaction between mutant T3 receptors and transcriptional corepressors. Mol Endocrinol 11:470–480[Abstract/Free Full Text]
  33. Pelton PD, Zhou L, Demarest KT, Burris TP 1999 PPAR{gamma} activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes. Biochem Biophys Res Commun 261:456–458[CrossRef][Medline]
  34. Henke BR, Blanchard SG, Brackeen MF, Brown KK, Cobb JE, Collins JL, Harrington WWJ, Hashim MA, Hull-Ryde EA, Kaldor I, Kliewer SA, Lake DH, Leesnitzer LM, Lehmann JM, Lenhard JM, Orband-Miller LA, Miller JF, Mook RAJ, Noble SA, Oliver WJ, Parks DJ, Plunket KD, Szewczyk JR, Willson TM 1998 N-(2-Benzoylphenyl)-L-tyrosine PPAR{gamma} agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents. J Med Chem 41:5020–5036[CrossRef][Medline]
  35. Gampe J, RT, Montana VG, Lambert MH, Miller AB, Bledsoe RK, Milburn MV, Kliewer SA, Willson TM, Xu HE 2000 Asymmetry in the PPAR{gamma}/RXR{alpha} crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. Mol Cell 5:545–555[CrossRef][Medline]
  36. Nolte RT, Wisely BG, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and coactivator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  37. Gurnell M, Wentworth JM, Agostini M, Adams M, Collingwood TN, Provenzano C, Browne PO, Rajanayagam O, Burris TP, Schwabe JW, Lazar MA, Chatterjee VKK 2000 A dominant negative peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) mutant is a constitutive repressor and inhibits PPAR{gamma}-mediated adipogenesis. J Biol Chem 275:5754–5759[Abstract/Free Full Text]
  38. Park Y, Freedman BD, Lee EJ, Park S, Jameson JL 2003 A dominant negative PPAR{gamma} mutant shows altered cofactor recruitment and inhibits adipogenesis in 3T3–L1 cells. Diabetologia 46:365–377[Medline]
  39. Lin JR, Nagy L, Satoshi I, Shao W, Miller W, Evans RM 1998 Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391:811–814[CrossRef][Medline]
  40. Damm K, Thompson CC, Evans RM 1989 Protein encoded by v-erbA functions as a thyroid-hormone receptor antagonist. Nature 339:593–597[CrossRef][Medline]
  41. Clifton-Bligh RJ, de Zegher F, Wagner RL, Collingwood TN, Francois I, van Helvoirt M, Chatterjee VKK 1998 A novel TRß mutation (R383H) in resistance to thyroid hormone predominantly impairs corepressor release and negative transcriptional regulation. Mol Endocrinol 12:609–621[Abstract/Free Full Text]
  42. Safer JD, Cohen RN, Hollenberg AN, Wondisford FE 1998 Defective release of corepressor by hinge mutants of the thyroid hormone receptor found in patients with resistance to thyroid hormone. J Biol Chem 273:30175–30182[Abstract/Free Full Text]
  43. Grignani F, De Matteis S, Nervi C, Tomassoni L, Gelmetti V, Cioce M, Fanelli M, Ruthardt M, Ferrara FF, Zamir I, Seiser C, Lazar MA, Minucci S, Pelicci PG 1998 Fusion proteins of the retinoic acid receptor-{alpha} recruit histone deacetylase in promyelocytic leukaemia. Nature 391:815–818[CrossRef][Medline]
  44. He L-Z, Guidez F, Tribioli C, Peruzzi D, Ruthardt M, Zelent A, Pandolfi PP 1998 Distinct interactions of PML-RAR{alpha} and PLZF-RAR{alpha} with co-repressors determine differential responses to RA in APL. Nat Genet 18:126–135[CrossRef][Medline]
  45. Sorbera LA, Leeson PA, Martin L, Castaner J 2001 Farglitazar. Antidiabetic PPAR{gamma} agonist. Drugs of the Future 26:354–363[CrossRef]
  46. Cox PJ, Ryan DA, Hollis FJ, Harris AM, Miller AK, Vousden M, Cowley H 2000 Absorption, disposition, and metabolism of rosiglitazone, a potent thiazolidinedione insulin sensitizer, in humans. Drug Metab Dispos 28:772–780[Abstract/Free Full Text]



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