Identification of Nuclear Receptor Corepressor as a Peroxisome Proliferator-activated Receptor alpha  Interacting Protein*

Paul DowellDagger §, Jane E. Ishmael§, Dorina Avram§, Valerie J. Peterson§, Daniel J. NevrivyDagger §, and Mark LeidDagger §parallel

From the Dagger  Program in Molecular and Cellular Biology and § Laboratory of Molecular Pharmacology, College of Pharmacy, Oregon State University, Corvallis, Oregon 97331

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor alpha  (PPARalpha ), and PPARalpha ligands suppressed this interaction. In contrast to the interaction of PPARalpha with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARalpha ligand binding domain. NCoR was found to suppress PPARalpha -dependent transcriptional activation in the context of a PPARalpha ·retinoid X receptor alpha  (RXRalpha ) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARalpha ·RXRalpha heterodimeric complexes in mammalian cells. NCoR appears to influence PPARalpha signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Members of the steroid/thyroid hormone receptor superfamily function by binding to specific DNA response elements within the regulatory regions of target genes and modulating expression of these genes at the transcriptional level (1-3). Regulation of target gene expression mediated by nuclear receptors may occur in response to activation of the receptors by ligands (4) or by phosphorylation (5) or by a combination of both events (5).

The mammalian PPAR1 family is composed of at least three genetically and pharmacologically distinct subtypes, PPARalpha , -gamma , and -beta /delta (also referred to as NUCI; reviewed in Refs. 6 and 7). The primary physiological roles for the alpha  and gamma  subtypes of PPAR appear to be regulation of lipid metabolism and adipogenesis, respectively, and both subtypes have been implicated in modulating inflammatory responses (8-12). A physiological role for PPARbeta /delta has not been elucidated but this receptor subtype is expressed ubiquitously in the mouse, possibly suggesting a more general function (13).

Retinoid X receptors (RXRs) serve as obligate heterodimeric partners for all PPAR subtypes, and the PPAR-RXR heterodimeric complex binds most efficiently to degenerate direct repeats of the hexameric nucleotide sequence, AGGTCA, separated by 1 base pair (DR1; Refs. 14-17). PPAR/RXR heterodimeric binding sites, known as peroxisome proliferator response elements (PPREs), have been identified within the regulatory regions of several genes that encode proteins implicated in lipid metabolism (8, 9, 14, 16, 18) and adipocyte function (19, 20).

PPARalpha was initially identified in a search for novel superfamily members and was shown to be activated by a group of compounds known to elicit proliferation of hepatic peroxisomes in rodents (21). Structurally diverse peroxisome proliferators include phthalate ester plasticizers (di(-2-ethylhexyl)-pthalate), herbicides (2,4,5-trichlorophenoxyacetic acid), and several fibric acid anti-hyperlipidemic agents (WY-14, 643, clofibric acid, gemfibrozil; Ref. 22). Additional compounds have since been shown to activate PPARalpha , including the fatty acid, arachidonic acid (23), and the corresponding synthetic analog 5,8,11,14-eicosotetraynoic acid (ETYA; Ref. 24), the leukotriene D4 antagonist, LY-171883 (25), and the arachidonic acid derivative, leukotriene B4, which has been proposed to be an endogenous ligand for PPARalpha (12).

In addition to activating expression of target genes upon binding cognate ligands, receptors for all-trans retinoic acid (RAR) and thyroid hormone (TR) actively repress genes in the absence of ligand (26, 27). The related corepressor proteins, NCoR and silencing mediator of retinoid and thyroid hormone receptors (SMRT), have been demonstrated to interact with and mediate the repression functions of unliganded RAR and TR (28, 29). NCoR and SMRT are associated with a multiprotein corepressor complex that minimally contains Sin3a and the histone deacetylase, HDAC1/Rpd3 (30, 31). Ligand binding by RAR and TR promotes dissociation of the receptor-corepressor complex (28, 29) and subsequent interaction of the receptor with one or more coactivator proteins that possess intrinsic histone acetyltransferase activity (32-35). Several nuclear receptor-associated coactivator proteins that exhibit histone acetyltransferase activity have been identified including p300/CREB binding protein (CBP; Refs. 35 and 36), steroid receptor coactivator-1 (SRC-1; Ref. 37), and p300/CBP-interacting protein (p/CIP; Ref. 34). These findings, when considered together with previous observations that hyperacetylated histones are associated with actively transcribed chromatin (reviewed in Ref. 38), offer a molecular explanation for ligand-dependent transcriptional modulation by nuclear receptors. Thus, ligand binding may serve as a molecular switch between transcriptional repression and activation by promoting exchange of a receptor-associated deacetylase-containing corepressor complex with that of a histone acetyltransferase-containing coactivator complex.

PPAR-dependent transcriptional activation of many genes is well documented, and direct, ligand-enhanced interactions between PPARs and the coactivators, p300/CBP (39), SRC-1 (39-41), PPAR-binding protein (PBP; Ref. 42), and PGC-1 (43) are thought to play a role in such activation. In contrast, PPAR-mediated transcriptional repression of target genes, as observed for RAR and TR (see above), is relatively unexplored or at best controversial. PPARgamma has been shown to interact in solution with the corepressors, NCoR and SMRT, but weakly if at all when bound to DNA, possibly suggesting that neither of these corepressors mediate putative PPAR-dependent gene repression (44). However, Lavinsky and co-workers (45) demonstrated SMRT-dependent gene repression mediated by a phosphorylated form of PPARgamma . Such findings illustrate the need for a more complete mechanistic understanding of potential PPAR-dependent repression of gene expression. We report here the isolation of NCoR from a yeast two-hybrid screen using PPARalpha as a bait. We describe results from studies in yeast, in mammalian cells, and in vitro that were conducted to characterize PPARalpha -NCoR interactions and to examine the influence of PPAR ligands upon such interactions.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids and Receptor Constructs-- Plasmids encoding the receptors described below were used either directly or as templates for polymerase chain reaction to assemble all constructs described herein using standard techniques. All plasmids were kind gifts from the following individuals: mouse PPARalpha (21) from Drs. S. Green and J. D. Tugwood (Zeneca, Macclesfield, United Kingdom); mouse RXRalpha (RXRalpha ; Ref. 46) and human RARgamma (RARgamma ; Ref. 47) from Drs. P. Kastner, A. Krust and P. Chambon (Institute de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France); human p300 (48) from Dr. D. Livingston (Dana Farber Cancer Institute, Boston, MA), NCoR (29) from Dr. T. Heinzel (German Cancer Research Center, Heidelberg, Germany), SMRT (28) from Dr. R. Evans (Salk Institute, La Jolla, CA). The integrity of all constructs was verified by restriction digest and/or sequence analysis.

The parental bait vector for the yeast two-hybrid screen (pBTM16) and the yeast reporter strain L40 (Refs. 49 and 50, respectively) were kind gifts from Drs. R. Losson and P. Chambon (Institute de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France). The yeast expression vectors encoding p300 amino acids 39-221 and the following amino acids of the indicated receptors have been described previously (39): PPARalpha (91-468 of mPPARalpha ), PPARalpha D/E (166-468 of mPPARalpha ), RXRalpha (132-467 of mRXRalpha ), and RARgamma (90-454 of hRARgamma ). PPARalpha Delta 448 (91-447 of mPPARalpha ), PPARalpha Delta 425 (amino acids 91-424 of mPPARalpha ), PPARalpha E (282-468 of mPPARalpha ), and identical fragments of all receptors and mutants thereof listed above were subcloned in pTL1 (46) and have been described previously (39, 51). PPARalpha Delta 288 (amino acids 91-287 of mPPARalpha ), PPARalpha Delta 227 (amino acids 91-226 of mPPARalpha ), PPARalpha Delta 202 (amino acids 91-201 of mPPARalpha ), and PPARalpha Delta 180 (amino acids 91-179 of mPPARalpha ) were constructed by polymerase chain reaction amplification of the indicated receptor regions with primers containing appropriate restriction sites for insertion into pTL1. Receptor proteins and derivatives thereof were expressed by in vitro transcription/translation for use in GST pulldown experiments as described previously (39, 51). Note that all PPARalpha constructs encode receptors truncated in the amino-terminal (Delta AB).

GST/p300 has been described previously as GST/p300 (39-221; Ref. 39). GST-NCoR (Delta ID II) and GST-NCoR, encoding GST fusions with NCoR amino acids 2218-2381 and 2110-2453, respectively, were prepared by polymerase chain reaction amplification with primers containing BamHI and EcoRI sites using the original pACT2/NCoR plasmid isolated in the yeast two-hybrid screen as a template. The resulting fragments were digested and subcloned into BamHI/EcoRI-digested pGEX2T (Amersham Pharmacia Biotech).

A (PPRE)3-tk-CAT reporter construct was prepared by insertion of a multimerized acyl-CoA oxidase PPRE (8) into the XbaI site of pBL2CAT2 (52). Additional information concerning any of the above constructs described herein can be obtained upon request.

Yeast Two-hybrid Screening-- The yeast two-hybrid screen was conducted as described (39) except that a mouse brain cDNA library inserted into the GAL4 activation domain-encoded yeast expression vector, pACT2 (CLONTECH), was used in place of the mouse embryo library and approximately 2 × 106 yeast transformants were screened. Plasmid DNA from positive clones was then isolated, and the resulting cDNAs were re-tested for interaction with PPAR baits, bait DBDs alone, and non-PPAR-related baits. cDNA clones, which exhibited a specific interaction with the PPAR baits, were then sequenced using the standard dideoxynucleotide chain termination method.

beta -Galactosidase Assays and Data Analysis-- beta -Galactosidase assays were conducted as described previously (39). All titration data were analyzed using an iterative, curve-fitting routine (GraphPad Prism) and the four-parameter logistic equation. Yeast beta -galactosidase results were analyzed using a two-tailed Student's t test.

Protein-Protein Interaction Assay-- GST pulldown experiments were conducted as described previously (39), and ligand-dependent assays were carried out by inclusion of 1 µM 9-cis-RA, 1 mM clofibrate, 100 µM troglitazone, 100 µM LY-171883, 100 µM ETYA, 100 µM WY-14,643 or vehicle in binding buffers except where otherwise noted. GST and GST fusion proteins were produced as described previously (39).

Mammalian Cell Transfection Experiments-- Human embryonic kidney 293 (HEK293) cells were maintained and transiently transfected as described previously (53). Dose-response curves were fit using GraphPad Prism software as described above.

Chemicals and Reagents-- WY-14,643 and clofibrate were purchased from Chemsyn Science Laboratories (Lenexa, KS) and Sigma, respectively. LY-171883 and ETYA were obtained from Biomol (Plymouth Meeting, PA). Troglitazone was kindly supplied by Dr. S. Kliewer (Glaxo Wellcome). All radioisotopes were purchased from NEN Life Science Products.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A previously described yeast two-hybrid system (49) was used to isolate a carboxyl-terminal NCoR (29) fragment as a PPARalpha -interacting protein from an oligo(dT)-primed, mouse brain cDNA library (NCoR amino acids 2110-2453, Fig. 1). Previous studies have identified two domains, ID I and ID II, that mediate interactions between NCoR and nuclear receptors (29, 54). The NCoR fragment isolated in this screen encompasses the last 33 amino acids of ID II and the entirety of ID I (Fig. 1).


View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1.   Isolation of NCoR as a PPARalpha -interacting protein. Carboxyl-terminal NCoR amino acid sequence. The encoded fragment isolated in the screen is indicated by bold, underlined text (amino acids Lys2110-Asp2453 of NCoR; Ref. 29). Previously identified nuclear receptor interaction domains, ID I (Asp2239-Met2300) and ID II (Arg2062-Ser2142), are delineated by boxed amino acids (see Ref. 54). Note that amino acids Lys2110-Asp2453 of NCoR are identical to Lys319-Asp662 of the originally reported RXR-interacting protein (RIP) 13 isolate (55). For clarity we have referred to our clone throughout the text as NCoR amino acids 2110-2453.

PPARalpha and interaction domains of the coactivators, p300 and SRC-1, exhibit strong ligand-independent association when examined in a yeast two-hybrid system (39). When examined in vitro, however, these interactions are strictly ligand-dependent, suggesting that yeast may contain endogenous PPARalpha activating ligands (39). The isolation of NCoR in a yeast two-hybrid screen was therefore unexpected as we (39) had hypothesized that PPARalpha existed in a liganded state in yeast that may not facilitate receptor interactions with potential corepressor proteins as has been observed for other liganded nuclear receptors (28, 29). Toward the goal of understanding the influence of NCoR on PPARalpha signaling mechanisms, we chose to conduct a more thorough analysis of the interaction between these two proteins.

WY-14,643 Inhibits PPARalpha -NCoR Interaction in a Dose-dependent Manner-- The yeast two-hybrid system was used to compare NCoR interactions with PPARalpha , RARgamma , and RXRalpha baits. PPARalpha interacted specifically and robustly with amino acids 2110-2453 of NCoR, and the PPARalpha ligand, WY-14,643, potently suppressed this interaction, while the retinoid receptor ligand, 9-cis-RA, had no influence (Fig. 2A). RARgamma also interacted with NCoR and 9-cis-RA abolished this interaction (Fig. 2A). In contrast, a relatively weak interaction was observed between RXRalpha and NCoR, which was enhanced by 9-cis-RA (Fig. 2A, see below).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2.   Interactions between NCoR and PPARalpha , RARgamma , and RXRalpha . A, interactions between NCoR and nuclear receptors in yeast. LexA DBD/receptor fusions were coexpressed with a GAL4 AD/NCoR (amino acids 2110-2453) fusion (GAD/NCoR) and examined for the ability to activate an integrated LexA-responsive lacZ reporter in the yeast strain L40 (49). Ligand-dependent interactions were examined in the presence of 100 µM WY-14,643, 1 µM 9-cis-RA, or vehicle where indicated. At least three independent transformants were assayed for each experiment and the results shown represent the mean ± S.E. for each determination. No reporter activity was detected when the GAD/NCoR fusion was coexpressed with either LexA DBD alone or LexA DBD fused to an unrelated protein (data not shown). Statistical significance at the 99% (p < 0.01) confidence level is indicated by ** symbols for ligand versus vehicle treatment. B, in vitro protein-protein interactions between NCoR and nuclear receptors. GST alone or GST fused to NCoR amino acids 2110-2453 (GST/NCoR) were bound to glutathione-Sepharose and used as an affinity matrix to examine interactions with in vitro translated [35S]methionine-labeled PPARalpha , RARgamma , and RXRalpha in the absence and presence of ligand (100 µM WY-14,643 for PPARalpha or 1 µM 9-cis-RA for RARgamma and RXRalpha as indicated). Input lanes (IN) represent 25% of receptor preparations used in binding reactions for the indicated samples. Note that the signals present in lanes 7-9 were obtained by increased exposure time relative to those in all other lanes. All receptors preparations expressed in yeast or translated in vitro were truncated in the amino-terminal (Delta AB) region (see "Materials and Methods"). Shown in both A and B are representative experiments that were carried out three (RARgamma -NCoR and RXRalpha -NCoR) to eight (PPARalpha -NCoR) times.

To confirm the observed interactions in yeast, in vitro protein-protein interaction assays were conducted. A GST/NCoR fusion protein containing amino acids 2110-2453 of NCoR was examined for the ability to interact with radioactively labeled PPARalpha , RARgamma , and RXRalpha prepared by in vitro translation. PPARalpha interacted strongly with GST/NCoR in vitro, but not GST alone, in the absence of ligand (Fig. 2B, lanes 2 and 10), and the presence of WY-14,643 did not significantly affect this interaction (Fig. 2B, lane 3). Similarly, RARgamma interacted with GST/NCoR in vitro, and this interaction was not significantly affected by the presence of 9-cis-RA (Fig. 2B, lanes 5 and 6). As observed in yeast, RXRalpha interacted weakly with GST/NCoR and 9-cis-RA appeared to stimulate this interaction modestly (3.4-fold; Fig. 2B, lanes 8 and 9). A weak, ligand-enhanced interaction between RXR and the related corepressor, SMRT, has also been observed by Chen and co-workers (28). In addition, Seol and collaborators (55) observed an approximately 3-fold increased interaction between NCoR/RIP13 (see Fig. 1 legend) and RXR in the presence of 9-cis-RA.

To probe the ligand dependence of PPARalpha -NCoR interactions further, several additional PPAR ligands were examined using the yeast two-hybrid system as described above. Clofibrate, LY-171883, and ETYA, as well as the PPARgamma -specific ligand, troglitazone, did not significantly affect PPARalpha -NCoR interactions in yeast (Fig. 3A). The only ligand examined in yeast that efficiently promoted dissociation of PPARalpha and NCoR was WY-14,643, and this effect was dose-dependent with an apparent IC50 of 134 nM (Fig. 3B).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Influence of PPAR ligands on NCoR-PPARalpha interactions. A, ligand-dependent dissociation of NCoR and PPARalpha in yeast. PPARalpha interaction with NCoR was examined in the presence of 1 µM 9-cis-RA, 100 µM troglitazone, 1 mM clofibrate, 100 µM LY-171883, 100 µM ETYA, 100 µM WY-14,643 or vehicle where indicated. Statistical significance at the 99% (p < 0.01) confidence level is indicated by ** symbols for ligand versus vehicle treatment. Each determination represents the mean ± S.E. of three independent experiments. B, WY-14,643 dose-response curve for dissociation of NCoR-PPARalpha in yeast. PPARalpha interaction with NCoR was examined in the presence of increasing concentrations of WY-14,643 (1-10,000 nM in one log unit increments) or vehicle. Each point represents the mean ± S.E. of four independent experiments. The theoretical curve shown was obtained by fitting the data using an iterative, curve-fitting routine (GraphPad Prism), which yielded an IC50 of 134 nM.

NCoR and p300 Require Distinct PPARalpha Regions for Interaction-- To determine which regions of PPARalpha are required for association with NCoR, we examined GST/NCoR interactions with several carboxyl- and amino-terminal PPARalpha truncation mutants in vitro using standard GST pulldown methodology (Fig. 4A). The results of these in vitro studies are depicted schematically in Fig. 4A. As shown above, PPARalpha interacted strongly with both GST/NCoR and GST/p300 in vitro (Fig. 4B, lanes 4-7). The former interaction was only modestly inhibited by ligand, while the latter interaction was strictly ligand-dependent. In contrast, PPARalpha Delta 448, which lacks 21 carboxyl-terminal amino acids, efficiently interacted with GST/NCoR but ligand-dependent interaction between this truncation mutant and GST/p300 was abolished (Fig. 4B, lanes 11-14). The hinge/LBD region of PPARalpha , PPARalpha D/E, interacted in vitro with GST/NCoR and GST/p300 in a manner similar to that of PPARalpha (Fig. 4B, lanes 18-21), although the WY-14,643-induced dissociation of receptor·GST/NCoR complexes was more apparent (lanes 18-19). In contrast, PPARalpha E, which lacks residues contained within the hinge region of PPARalpha (amino acids 166-281), did not interact with either GST/NCoR or GST/p300 (Fig. 4B, lanes 25-28). These results demonstrate that both NCoR and p300 require the hinge region of the receptor for efficient interaction, while only the latter requires an intact PPARalpha LBD. Therefore, the regions of PPARalpha required for interaction with NCoR and p300 are partially overlapping but largely distinct.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 4.   Interactions between NCoR and truncation mutants of PPARalpha . A, schematic representation of PPARalpha truncation mutants and summary of NCoR and p300 binding activity as determined using in vitro, GST pulldown experiments shown in B and C. PPARalpha A/B, C, D, and E domains are as defined in Ref. 21. Stippled boxes (PPARalpha amino acids Lys196-Ala225) represent the putative CoR box present in TR and RAR (29). Note that PPARalpha refers to an amino-terminally truncated receptor lacking the A/B domain (see "Materials and Methods"). ND, not determined. B, in vitro protein-protein interactions between PPARalpha truncation mutants and both GST/NCoR (2110-2453) and GST/p300 (39-221). These GST pulldown experiments were carried out as described in Fig. 2B and ligand concentrations were the same as those indicated in Fig. 3A. C, in vitro protein-protein interactions between PPARalpha carboxyl-terminal truncation mutants and GST/NCoR (2110-2453). GST pulldown assays were conducted as described in B. Representative experiments that were replicated three to five times are shown in B and C.

The carboxyl-terminal PPARalpha truncation mutants, PPARalpha Delta 425, PPARalpha Delta 288, PPARalpha Delta 227, and PPARalpha Delta 202, all interacted efficiently with GST/NCoR (Fig. 4C, lanes 3, 6, 9, and 12). PPARalpha Delta 180, which lacks the entire LBD, also interacted with GST/NCoR (Fig. 4C, lane 15). PPARalpha amino acids 166-179 within the hinge region are common to all receptor proteins observed to interact with GST/NCoR, suggesting that these residues may play a role in PPARalpha interactions with NCoR. However, this isolated region of the receptor did not interact with NCoR either in yeast or in vitro under a variety of conditions (data not shown), suggesting that this region of PPARalpha is necessary but not sufficient to mediate interaction with NCoR.

NCoR ID II Is Not Necessary for PPARalpha -NCoR Interactions-- Protein-protein interaction studies carried out with a GST/NCoR fusion protein lacking the entire ID II, GST/NCoR (Delta ID II), demonstrated that the ID II region of NCoR was not required for interaction with PPARalpha (Fig. 5). As observed for interactions between PPARalpha and GST/NCoR (amino acids 2110-2453, see Fig. 2B, lanes 2 and 3), WY-14,643 did not significantly affect receptor-NCoR interactions in vitro (Fig. 5, lanes 4 and 5).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5.   ID II of NCoR is not required for interaction with PPARalpha . In vitro protein-protein interactions between PPARalpha and amino acids encompassing ID I, but not ID II, of NCoR (amino acids 2218-2381, see Fig. 1). Assays were carried out as described in the legend to Fig. 2B with GST or GST/NCoR (Delta ID II). Shown is a representative experiment that was replicated three times.

PPARalpha Ligands Promote Both PPARalpha -NCoR Dissociation and PPARalpha -p300 Association-- The yeast two-hybrid system was used to compare the influence of several PPAR ligands on receptor interactions with the corepressor, NCoR (amino acids 2110-2453), and the coactivator, p300 (amino acids 39-221). PPARalpha D/E was used as the receptor component in this series of experiments because we previously observed a readily detectable WY-14,643-enhanced interaction in yeast between the D/E region of PPARalpha and p300 amino acids 39-221 (39). As observed for yeast two-hybrid analyses using PPARalpha (see Fig. 3), WY-14,643, but not troglitazone, clofibrate, LY-171883, or ETYA, significantly repressed the interaction between PPARalpha D/E and NCoR (Fig. 6A). Similarly, troglitazone, clofibrate and LY-171883 had no significant influence on the strong ligand-independent PPARalpha D/E interaction with p300, while both ETYA and WY-14,643 modestly, but significantly, enhanced this interaction (Fig. 6A). Thus, while both ETYA and WY-14,643 promoted p300-PPARalpha interactions in yeast and in vitro, only WY-14,643 was observed to induce dissociation of NCoR-PPARalpha complexes, and this was only significantly apparent in yeast.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6.   Influence of ligands on NCoR and p300 interactions with PPARalpha . A, interactions between PPARalpha D/E and both NCoR (amino acids 2110-2453) and p300 (amino acids 39-21) in yeast. Assays were conducted as described in Fig. 3A. Each determination represents the mean ± S.E. of three independent experiments. Statistical significance at the 95% (p < 0.05) and 99% (p < 0.01) confidence levels are indicated by * and ** symbols, respectively, for ligand versus vehicle treatment. B, in vitro protein-protein interactions between PPARalpha D/E and both GST/NCoR (2110-2453) and GST/p300 (39-221). Assays were carried out as described in the legend to Fig. 2B, and ligand concentrations were the same as those indicated in Fig. 3A. C, in vitro protein-protein interactions between PPARalpha and both GST/NCoR and GST/p300. Assays were carried out as described above (Fig. 6B). Shown in B and C are representative GST pulldown experiments that were replicated four to six times.

To confirm the observed interactions in yeast, in vitro protein-protein interaction assays were conducted as described above. GST/NCoR (amino acids 2110-2453) and GST/p300 (amino acids 39-221) were examined for interaction with both PPARalpha D/E and PPARalpha in the absence and presence of several, above-mentioned PPAR ligands. None of the ligands tested significantly affected the in vitro interaction of NCoR with either PPARalpha D/E (Fig. 6B) or PPARalpha (Fig. 6C). In contrast, in vitro association between GST/p300 and both PPARalpha D/E (Fig. 6B) and PPARalpha (Fig. 6C) was observed only in the presence of ETYA or WY-14,643 (lanes 18 and 19, respectively).

NCoR Represses PPARalpha /RXRalpha -mediated Transcriptional Activation from a PPRE in HEK293 Cells-- Cotransfection experiments in HEK293 cells were conducted to determine the physiological significance of the strong interaction between PPARalpha and NCoR observed in yeast and in vitro. The reporter construct used for these studies, (PPRE)3-tk-CAT, exhibited low basal or ligand-stimulated activity in the absence of cotransfected receptor (Fig. 7, lanes 1-6). However, cotransfection of PPARalpha and RXRalpha resulted in a strong, constitutive activity of this reporter that was only minimally stimulated by PPARalpha (WY-14,643 and ETYA) or RXRalpha (9-cRA) agonists or both types of ligands together (Fig. 7, lanes 7-12). Cotransfection of full-length NCoR (2 µg) dramatically reduced constitutive, PPARalpha ·RXRalpha -dependent transcriptional activation (compare lanes 7 and 13 of Fig. 7), and this repression was reversed by treating the cells with either PPARalpha agonists alone or in combination with 9-cis-RA (lanes 13-18). Cotransfection of a SMRT expression vector (28) similarly repressed PPARalpha ·RXRalpha -dependent transcriptional activation (data not shown). NCoR-dependent repression of transcriptional activation mediated by PPARalpha ·RXRalpha was also reversed by 9-cis-RA alone (Fig. 7, lane 15), suggesting that RXRalpha may be competent to bind ligand and/or activate transcription in the context of a PPARalpha ·RXRalpha heterodimeric complex bound to the acyl-CoA oxidase PPRE (8). The potencies with which WY-14,643, 9-cis-RA, and ETYA activated PPARalpha ·RXRalpha in the presence of cotransfected NCoR were determined in a series of concentration-response experiments using HEK293 cells. WY-14,643 (EC50 = 288 ± 81 nM; n = 5, Fig. 8A) and ETYA (EC50 = 189 ± 108 nM, n = 5; Fig. 8C) were roughly equipotent, while 9-cis-RA activated this reporter construct with an EC50 = 40 ± 8 nM (n = 3, Fig. 8B). These EC50 values are in general agreement with previous estimates of the affinity of PPARalpha for WY-14,643 (21, 51) as well as that of RXRalpha for 9-cis-RA (56). These findings demonstrate that NCoR interacts with and represses transcriptional activation mediated by PPARalpha ·RXRalpha heterodimeric complexes bound to a PPRE in mammalian cells and this repression is reversed by agonists of either receptor. From these results, we conclude that the cellular interaction between NCoR and PPARalpha and/or RXRalpha is likely to be of physiological relevance and may influence tissue responsiveness to both peroxisome proliferators and retinoic acids.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of NCoR on transcriptional activation mediated by PPARalpha ·RXRalpha heterodimeric complexes in HEK293 cells. HEK293 cells were cotransfected with 2 µg of the (PPRE)3-tk-CAT reporter plasmid and, where indicated, expression vectors for full-length NCoR (2 µg) and PPARalpha /RXRalpha (0.5 µg of each). Cells were treated with the indicated concentrations of WY-14,643, 9-cis-RA (9cRA), or ETYA for 24 h. Extracts were normalized for transfection efficiency by cotransfection with a beta -galactosidase expression vector (pCMV-Sport-beta gal; Life Technologies, Inc.), and the chloramphenicol acetyltransferase (CAT) activity was determined in the presence [14C]chloramphenicol and acetyl-CoA using standard methodology. Acetylated and unacetylated [14C]chloramphenicol were separated by thin layer chromatography and visualized by autoradiography. Shown is a representative experiment that was replicated four times.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 8.   Concentration-response curves for activation of PPARalpha ·RXRalpha heterodimeric complexes by WY-14,643, 9-cis-RA, and ETYA in HEK293 cells. HEK293 cells were cotransfected with 2 µg of the (PPRE)3-tk-CAT reporter plasmid and expression vectors for NCoR (2 µg) and PPARalpha /RXRalpha (0.5 µg of each). Cells were treated with increasing concentrations of WY-14,643 (A), 9-cis-RA (B), or ETYA (C) for 24 h, and CAT activity was determined as described in the legend of Fig. 7. The curves shown for each agonist were obtained using an iterative, curve-fitting routine (GraphPad Prism). Shown is a representative CAT experiment that was replicated three (9-cis-RA (9cRA)) or five (WY-14,643 and ETYA) times. The following EC50 values represent the mean ± S.E. of these multiple determinations: WY-14,643, 288 ± 81 nM; 9-cis-RA, 40 ± 8 nM; ETYA, 189 ± 108 nM.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated that PPARalpha interacts strongly with NCoR, and the PPARalpha ligand, WY-14,643, inhibits this interaction in yeast and in mammalian cells. The PPARalpha ligand, ETYA, also inhibits PPARalpha -NCoR interaction in mammalian cells but fails to influence this interaction in yeast. Neither ligand significantly affected NCoR-PPARalpha interactions in vitro under the conditions examined in these studies. ETYA does, however, promote interaction of the receptor with the coactivator, p300, in yeast, suggesting that the strain of yeast used for these studies is permeable to ETYA. Thus, the simplest explanation for these results is that ETYA may be metabolized in yeast to compounds which, while capable of promoting p300·PPARalpha complex formation, are incapable of inducing dissociation of NCoR·PPARalpha complexes. It is also possible that ETYA may elicit production of an endogenous yeast compound that promotes PPARalpha -p300 interaction but fails to influence PPARalpha -NCoR complexes. Furthermore, we cannot rule out the possibility that a combination of these two possibilities may be responsible for our observations in yeast using ETYA. Given that treatment of transfected HEK293 cells with ETYA activates a PPRE-containing reporter construct, the anomalous results that we obtained in yeast with this compound do not appear to have direct applicability to function of PPARalpha in mammalian cells.

We previously hypothesized the existence of putative, endogenous agonists in yeast that may disfavor PPARalpha -corepressor interactions (39). However, if putative, endogenous ligands contribute to the constitutive PPARalpha -coactivator interactions observed in yeast (39), the presence of these agonists clearly does not preclude PPARalpha interaction with NCoR. In this case, endogenous yeast agonists may function in a manner similar to that of ETYA or metabolic derivatives thereof which promote PPARalpha -p300 association but do not inhibit PPARalpha -NCoR interaction. These hypotheses may provide an explanation for how NCoR was unexpectedely isolated as a PPARalpha -interacting protein in our yeast two-hybrid screen.

Previously we demonstrated that the coactivators, p300 and SRC-1, require 21 carboxyl-terminal residues of the PPARalpha LBD for interaction with PPARalpha (39). By analogy with other nuclear receptors, this region of PPARalpha is predicted to contain the putative core of the ligand dependent-transcriptional activation function (57). We demonstrate herein that the PPARalpha truncation mutant, PPARalpha Delta 448, which lacks the 21 carboxyl-terminal amino acids encompassing the putative AF-2 core, interacts with NCoR but not p300. Therefore, the corepressor, NCoR, and the coactivators, p300 and SRC-1, appear to interact with the receptor in mechanistically distinct manners that utilize different regions of PPARalpha as protein-protein interaction surfaces. However, simultaneous interaction between the receptor and both NCoR and p300/SRC-1 are unlikely, because both types of interaction require common amino acid residues within the hinge region of the receptor.

Deletion of the hinge region of PPARalpha (amino acids 166-281) abolished NCoR-PPARalpha interaction, and amino acids 166-179 within the amino-terminal portion of the PPARalpha hinge region were common to all receptor fragments that exhibited interaction with NCoR. The PPARalpha mutant, PPARalpha Delta 180 (amino acids 91-179), which interacted efficiently with NCoR, lacks the entirety of both the putative CoR box (29) and the ligand binding domain. These results suggest that PPARalpha amino acids 166-179 may mediate interactions with NCoR. However, extensive analyses in yeast and in vitro have failed to demonstrate that PPARalpha 166-179 are sufficient to mediate interaction with NCoR (data not shown), possibly indicating that additional contacts are required for efficient interaction. Nonetheless, our results suggest that PPARalpha likely contains a NCoR interaction surface that is clearly not contained within the LBD of the receptor and, thus, may be distinct from that present in either RAR or TR (28, 29).

Zamir and collaborators (44) have shown that PPARgamma can interact with the corepressors, SMRT and NCoR, in solution, but weakly if at all when bound to DNA. Similarly, we were unable to demonstrate the formation of a DNA bound PPARalpha ·RXRalpha ·NCoR complex in vitro (data not shown). However, in contrast to the findings of Zamir and colleagues (44) who observed no corepressor-dependent PPARgamma -mediated repression, our transient transfection studies clearly demonstrate a functional interaction between a PPRE-bound, PPARalpha ·RXRalpha complex and NCoR. Such discrepant results could simply be a result of either differing PPAR subtypes (alpha  versus gamma ) or cell lines (HEK 293 versus 293T) or a combination of these two possibilities. The inability to observe a DNA-bound PPARalpha ·RXRalpha ·NCoR complex in vitro may be due to an inherent instability of such complexes, and indeed, other groups have reported that cross-linking reagents are required to stabilize similar complexes in vitro (58). NCoR clearly associates with and represses the transcriptional activity of PPRE-bound, PPARalpha ·RXRalpha heterodimeric complexes in HEK293 (Fig. 7). However, we cannot exclude the possibility that additional cellular factor(s) present in HEK293 cells, but lacking in vitro, are required for the assembly of a DNA-bound, PPARalpha ·RXRalpha ·NCoR complex.

Finally, it is conceivable that interaction of NCoR or SMRT with either PPARalpha or PPARalpha ·RXRalpha complexes may influence other signaling pathways by titration of limiting amounts of these corepressors. This form of receptor cross-talk may serve to relieve transcriptional repression mediated by other nuclear receptors, such as RAR, TR, or RevErb, that utilize common corepressors. Results presented herein raise the possibility that PPAR interactions with corepressors in solution or on DNA may play a prominent role in regulating PPAR-dependent transcriptional regulation of target genes.

    ACKNOWLEDGEMENTS

We thank P. Chambon, R. Losson, P. Kastner, T. Lufkin, J. D. Tugwood, S. Green, and D. Livingston for plasmid constructs and reagents; S. Kliewer and Glaxo Wellcome for generously providing troglitazone; Drs. W. Wahli and B. Desvergne for useful discussion and suggestions; and J. Webster for expert technical assistance.

    FOOTNOTES

* This work was supported by American Heart Association Grant 9640219N and National Institute of Environmental Health Science Grant ES00040.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a predoctoral fellowship from the American Foundation for Pharmaceutical Education. Current address: The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.

parallel Established Investigator of the American Heart Association. To whom correspondence should be addressed: Laboratory of Molecular Pharmacology, College of Pharmacy, Oregon State University, Corvallis, OR 97331. Tel.: 541-737-5809; Fax: 541-737-3999; E-mail: mark.leid{at}orst.edu.

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; DR1, direct repeat separated by one nucleotide; ETYA, 5,8,11,14-eicosatetraynoic acid; RAR, retinoic acid receptor; TR, thyroid hormone receptor; NCoR, nuclear receptor corepressor; SMRT, silencing mediator of retinoid and thyroid hormone receptors; CBP, CREB-binding protein; SRC-1, steroid receptor coactivator-1; p/CIP, p300/CBP-interacting protein; mPPARalpha , mouse PPARalpha ; ID, interaction domain; 9-cis-RA, 9-cis-retinoic acid; RIP13, RXR-interacting protein 13; GAD, GAL4 activation domain; PPRE, peroxisome proliferator response element; GST, glutathione S-transferase; CAT, chloramphenicol acetyltransferase; LBD, ligand binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. O'Malley, B. W., and Tsai, M.-J. (1992) Biol. Reprod. 46, 163-167[Abstract]
  2. Beato, M., Herrlich, P., and Schutz, G. (1995) Cell 83, 851-857[Medline] [Order article via Infotrieve]
  3. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Shutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835-839[Medline] [Order article via Infotrieve]
  4. Moras, D., and Gronemeyer, H. (1998) Curr. Opin. Cell. Biol. 10, 384-391[CrossRef][Medline] [Order article via Infotrieve]
  5. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract]
  6. Green, S. (1995) Mutat. Res. 333, 101-109[CrossRef][Medline] [Order article via Infotrieve]
  7. Schoonjans, K., Staels, B., and Auwerx, J. (1996) Biochim. Biophys. Acta 1302, 93-109[Medline] [Order article via Infotrieve]
  8. Tugwood, J. D., Issemann, I., Anderson, R. G., Bundell, K. R., McPheat, W. L., and Green, S. (1992) EMBO J. 11, 433-439[Abstract]
  9. Bardot, O., Aldridge, T. C., Latruffe, N., and Green, S. (1993) Biochem. Biophys. Res. Commun. 192, 37-45[CrossRef][Medline] [Order article via Infotrieve]
  10. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156[Medline] [Order article via Infotrieve]
  11. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K. (1998) Nature 391, 79-82[CrossRef][Medline] [Order article via Infotrieve]
  12. Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43[CrossRef][Medline] [Order article via Infotrieve]
  13. Braissant, O., Foufelle, F., Scotto, C., Dauca, M., and Wahli, W. (1996) Endocrinology 137, 354-366[Abstract]
  14. Varanasi, U., Chu, R., Huang, Q., Catellon, R., Yeldani, A. V., and Reddy, J. K. (1996) J. Biol. Chem. 271, 2147-2155[Abstract/Free Full Text]
  15. Keller, H., Dreyer, C., Medin, J., Mahfoudi, A., Ozato, K., and Wahli, W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2160-2164[Abstract]
  16. Issemann, I., Prince, R. A., Tugwood, J. D., and Green, S. (1993) Biochimie (Paris). 75, 251-256[CrossRef][Medline] [Order article via Infotrieve]
  17. Gearing, K. L., Gottlicher, M., Teboul, M., Widmark, E., and Gustafsson, J.-A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1440-1444[Abstract]
  18. Lemberger, T., Desvergne, B., and Wahli, W. (1996) Annu. Rev. Cell Dev. Biol. 12, 335-363[CrossRef][Medline] [Order article via Infotrieve]
  19. Tontonoz, P., Graves, R. A., Budavari, A. I., Erdjument-Bromage, H., Lui, M., Hu, E., Tempst, P., and Spiegelman, B. M. (1994) Nucleic Acids Res. 22, 5628-5634[Abstract]
  20. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes Dev. 8, 1224-1234[Abstract]
  21. Issemann, I., and Green, S. (1990) Nature 347, 645-650[CrossRef][Medline] [Order article via Infotrieve]
  22. Lake, B. G. (1995) Annu. Rev. Pharmacol. Toxicol. 35, 483-507[CrossRef][Medline] [Order article via Infotrieve]
  23. Gottlicher, M., Widmark, E., Li, Q., and Gustafsson, J.-A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4653-4657[Abstract]
  24. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G., and Wahli, W. (1992) Cell 68, 879-887[Medline] [Order article via Infotrieve]
  25. Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7355-7359[Abstract]
  26. Damm, K., Thompson, C. C., and Evans, R. M. (1989) Nature 339, 593-597[CrossRef][Medline] [Order article via Infotrieve]
  27. Graupner, G., Wills, K. N., Tzukerman, M., Zhang, X. K., and Pfahl, M. (1989) Nature 340, 653-656[CrossRef][Medline] [Order article via Infotrieve]
  28. Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457[CrossRef][Medline] [Order article via Infotrieve]
  29. Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., Glass, C. K., and Rosenfeld, M. G. (1995) Nature 377, 397-403[CrossRef][Medline] [Order article via Infotrieve]
  30. Heinzel, T., Lavinsky, R. M., Mullen, T. M., Soderstrom, M., Laherty, C. D., Torchia, J., Yang, W. M., Brard, G., Ngo, S. D., Davie, J. R., Seto, E., Eisenman, R. N., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 43-48[CrossRef][Medline] [Order article via Infotrieve]
  31. Nagy, L., Kao, H. Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997) Cell 89, 373-380[Medline] [Order article via Infotrieve]
  32. Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract]
  33. Kamei, Y., Xu, L., Heinzel, T., Torchia, J., Kurokawa, R., Gloss, B., Lin, S. C., Heyman, R. A., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1996) Cell 85, 403-414[Medline] [Order article via Infotrieve]
  34. Torchia, J., Rose, D. W., Inostroza, J., Kamei, Y., Westin, S., Glass, C. K., and Rosenfeld, M. G. (1997) Nature 387, 677-684[CrossRef][Medline] [Order article via Infotrieve]
  35. Bannister, A. J., and Kouzarides, T. (1996) Nature 384, 641-643[CrossRef][Medline] [Order article via Infotrieve]
  36. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H., and Nakatani, Y. (1996) Cell 87, 953-956[Medline] [Order article via Infotrieve]
  37. Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J., Mizzen, C. A., McKenna, N. J., Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1997) Nature 389, 194-198[CrossRef][Medline] [Order article via Infotrieve]
  38. Struhl, K. (1998) Genes Dev. 12, 599-606[Free Full Text]
  39. Dowell, P., Ishmael, J. E., Avram, D., Peterson, V. J., Nevrivy, D. J., and Leid, M. (1997) J. Biol. Chem. 272, 33435-33445[Abstract/Free Full Text]
  40. Zhu, Y., Qi, C., Calandra, C., Rao, M. S., and Reddy, J. K. (1996) Gene Expr. 6, 185-195[Medline] [Order article via Infotrieve]
  41. Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa, R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., and Milburn, M. V. (1998) Nature 395, 137-143[CrossRef][Medline] [Order article via Infotrieve]
  42. Zhu, Y., Qi, C., Jain, S., Rao, M. S., and Reddy, J. K. (1997) J. Biol. Chem. 272, 25500-25506[Abstract/Free Full Text]
  43. Puigserver, P., Wu, Z., Park, C. W., Graves, R., Wright, M., and Spiegelman, B. M. (1998) Cell 92, 829-839[Medline] [Order article via Infotrieve]
  44. Zamir, I., Zhang, J., and Lazar, M. A. (1997) Genes Dev. 11, 835-846[Abstract]
  45. Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen, T. M., Schiff, R., Del-Rio, A. L., Ricote, M., Ngo, S., Gemsch, J., Hilsenbeck, S. G., Osborne, C. K., Glass, C. K., Rosenfeld, M. G., and Rose, D. W. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2920-2925[Abstract/Free Full Text]
  46. Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J.-Y., Staub, A., Garnier, J.-M., Mader, S., and Chambon, P. (1992) Cell 68, 377-395[Medline] [Order article via Infotrieve]
  47. Krust, A., Kastner, P., Petkovich, M., Zelent, A., and Chambon, P. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5310-5314[Abstract]
  48. Eckner, R., Ewen, M. E., Newsome, D., Gerdes, M., DeCaprio, J. A., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 869-884[Abstract]
  49. Le Douarin, B., Pierrat, B., vom Baur, E., Chambon, P., and Losson, R. (1995) Nucleic Acids Res. 23, 876-878[Medline] [Order article via Infotrieve]
  50. Pierrat, B., Heery, D., Lemoine, Y., and Losson, R. (1992) Gene (Amst.) 119, 237-245[CrossRef][Medline] [Order article via Infotrieve]
  51. Dowell, P., Peterson, V. J., Zabriskie, T. M., and Leid, M. (1997) J. Biol. Chem. 272, 2013-2020[Abstract/Free Full Text]
  52. Luckow, B., and Schutz, G. (1987) Nucleic Acids Res. 15, 5490[Medline] [Order article via Infotrieve]
  53. Avram, D., Ishmael, J. E., Nevrivy, D. J., Peterson, V. J., Lee, S.-H., Dowell, P., and Leid, M. (1999) J. Biol. Chem. 274, 14331-14336[Abstract/Free Full Text]
  54. Seol, W., Mahon, M. J., Lee, Y. K., and Moore, D. D. (1996) Mol. Endocrinol. 10, 1646-1655[Abstract]
  55. Seol, W., Choi, H. S., and Moore, D. D. (1995) Mol. Endocrinol. 9, 72-85[Abstract]
  56. Leid, M. (1994) J. Biol. Chem. 269, 14175-14181[Abstract/Free Full Text]
  57. Wurtz, J. M., Bourguet, W., Renaud, J. P., Vivat, V., Chambon, P., Moras, D., and Gronemeyer, H. (1996) Nat. Struct. Biol. 3, 87-94[Medline] [Order article via Infotrieve]
  58. Schulman, I. G., Shao, G., and Heyman, R. A. (1998) Mol. Cell. Biol. 18, 3483-3494[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.