Ligand-induced Peroxisome Proliferator-activated Receptor alpha  Conformational Change*

(Received for publication, July 2, 1996, and in revised form, October 4, 1996)

Paul Dowell Dagger §, Valerie J. Peterson Dagger , T. Mark Zabriskie Dagger and Mark Leid Dagger §par

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
Note Added in Proof
REFERENCES


ABSTRACT

Structurally diverse peroxisome proliferators and related compounds that have been demonstrated to induce the ligand-dependent transcriptional activation function of mouse peroxisome proliferator-activated receptor alpha  (mPPARalpha ) in transfection experiments were tested for the ability to induce conformational changes within mPPARalpha in vitro. WY-14,643, 5,8,11,14-eicosatetraynoic acid, LY-171883, and clofibric acid all directly induced mPPARalpha conformational changes as evidenced by a differential protease sensitivity assay. Carboxyl-terminal truncation mutagenesis of mPPARalpha differentially affected the ability of these ligands to induce conformational changes suggesting that PPAR ligands may make distinct contacts with the receptor. Direct interaction of peroxisome proliferators and related compounds with, and the resulting conformational alteration(s) in, mPPARalpha may facilitate interaction of the receptor with transcriptional intermediary factors and/or the general transcription machinery and, thus, may underlie the molecular basis of ligand-dependent transcriptional activation mediated by mPPARalpha .


INTRODUCTION

Peroxisome proliferator-activated receptors (PPARs)1 are members of a large family of ligand-inducible transcription factors that includes receptors for retinoids, vitamin D, and thyroid and steroid hormones (1-5). The mammalian PPAR family is composed of at least three genetically and pharmacologically distinct subtypes, PPARalpha , -gamma , and -delta (reviewed in Ref. 6). Murine PPAR alpha  (mPPARalpha ) was originally isolated from a mouse liver cDNA library by Issemann and Green (7) who demonstrated that the receptor was activated in transfection experiments by a group of compounds known to induce peroxisome proliferation in rodents. A number of structurally diverse compounds have subsequently been demonstrated to activate PPARalpha in transient transfection experiments. Particularly noteworthy among these compounds are: 1) lipids such as arachidonic acid (8-11) and its synthetic analog 5,8,11,14-eicosatetraynoic acid (ETYA, Refs. 9, 11-13), 8-[S]-hydroxyeicosatetraenoic acid (14), a lipoxygenase metabolite of arachidonic acid, and linoleic acid (8-11, 14, 15); 2) fibric acid anti-hyperlipidemic drugs (WY-14,643, clofibric acid, gemfibrozil, ciprofibric acid; Refs. 10, 16, 17) that represent a class of therapeutic agents useful in the treatment of hypertriglyceridemia (18); and 3) a leukotriene D4 antagonist, LY-171883 (19). Many of these compounds, together with phthalate ester plasticizers (di(-2-ethylhexyl)-phthalate) and herbicides (2,4,5-trichlorophenoxyacetic acid), are known collectively as peroxisome proliferators (reviewed in Ref. 20). While chemically distinct, most of these compounds have been demonstrated to induce proliferation of peroxisomes leading to hepatic hyperplasia and hepatocarcinogenesis in many species (20). Peroxisome proliferator-induced alteration of hepatocyte phenotype is believed to result from activation of PPARalpha and subsequent modulation of gene expression downstream of this nuclear receptor (reviewed in Refs. 6, 20; see below). The central role of PPARalpha in xenobiotic-induced peroxisomal proliferation was recently demonstrated by the absence of hepatomegaly and peroxisome proliferation in mice null for expression of this gene (21).

PPARs modulate expression of target genes by binding to response elements comprised of a degenerate direct repeat of the hexameric nucleotide sequence, TGACCT, separated by one base pair (DR1). PPAR has been shown to bind cognate response elements with high affinity only in the context of a heterodimeric complex with the retinoid X receptor (RXR, Refs. 11, 17, 22-24). PPAR·RXR heterodimeric complexes appear to be responsive to both PPAR activators and 9-cis-retinoic acid, the endogenous ligand for RXR (11, 17, 22-24).

PPAR response elements (PPREs) have been identified in the 5' regions of several mammalian genes coding for proteins involved in lipid metabolism such as acyl-CoA oxidase (17, 25), bifunctional enzyme (26, 27), malic enzyme (16), liver fatty acid binding protein (28), 3-hydroxy-3-methylglutaryl-CoA synthase (15), and cytochrome P450 fatty acid omega -hydroxylase (29). Such findings indicate a prominent regulatory role for the PPAR receptor family in lipid metabolism and homeostasis. In addition, overexpression of PPARalpha and -gamma in cultured fibroblasts and subsequent exposure to PPAR ligands has been shown to confer adipogenicity (30, 31), further illustrating the central regulatory role of PPAR family members in lipid homeostasis.

In contrast to many other receptors in the retinoid/thyroid hormone receptor superfamily, functional domains of PPARs and critical amino acid residues within such putative domains have not been extensively characterized. Two previous studies with PPARalpha have identified: 1) a Glu282 right-arrow Gly point mutation in mPPARalpha that ameliorates transcriptional responses to WY-14,643 and ETYA (13), and 2) a Leu433 right-arrow Arg point mutation in human PPARalpha (hPPARalpha ) that abolishes heterodimerization with RXR (32). The present studies were undertaken to identify mPPARalpha carboxyl-terminal receptor regions that are important for both ligand responsiveness and heterodimerization with mRXRalpha and to determine if structurally diverse PPAR ligands induce similar conformational changes within mPPARalpha . To our knowledge, these studies provide the first direct biochemical evidence demonstrating that peroxisome proliferators induce conformational changes within mPPARalpha . Ligand-induced stabilization of particular mPPARalpha conformational states likely underlies the molecular basis for the ability of these compounds to activate the receptor and to modulate expression of mPPARalpha target genes including those implicated in peroxisome proliferation.


MATERIALS AND METHODS

Plasmids and Receptor Constructs

Full-length mPPARalpha (7) was kindly provided by Drs. S. Green and J. Tugwood (Macclesfield, UK) and was used as a template for the polymerase chain reaction during construction of all PPAR mutants described herein. Full-length mouse RXRalpha (mRXRalpha , Ref. 33) and pGEX-cs (34) were kind gifts from Drs. Ph. Kastner and P. Chambon (Strasbourg) and Dr. W. Dougherty (Oregon State University), respectively.

A mPPARalpha amino-terminal truncation mutant was constructed by polymerase chain reaction using a 5' primer (ML023) that introduced an EcoRI site, favorable Kozak sequence, and an initiator methionine fused to Asp91 of mPPARalpha and a 3' primer that introduced a BamHI site 3' of the mPPARalpha natural stop codon. The resulting fragment was appropriately digested and subcloned into the eukaryotic expression vector, pTL1 (33), yielding PPARDelta AB. PPARDelta AB is transcribed/translated in vitro at least 10-fold more efficiently than full-length receptor and exhibits DNA binding and heterodimerization activities that are indistinguishable from full-length receptor (data not shown). The carboxyl-terminal truncation mutants, PPARDelta AB/Delta 448 and PPARDelta AB/Delta 425 (Fig. 1A), were prepared by polymerase chain reaction using ML023 as the 5' primer and a 3' primer that introduced stop codons at positions 448 and 425, respectively, preceding a BamHI site. Both of the resulting fragments were appropriately digested and subcloned into pTL1 as described above. PPARDelta AB, PPARDelta AB/Delta 448, and PPARDelta AB/Delta 425 were transcribed/translated in vitro with equal efficiencies (data not shown).


Fig. 1. mPPARalpha carboxyl-terminal truncation mutants and PPAR ligands. A, schematic representation of hRARgamma , hRXRalpha , and mPPARalpha carboxyl-terminal regions. H9-H12 represent alpha -helical regions within the LBD of hRARgamma (38) and hRXRalpha (37). The carboxyl-terminal mPPARalpha residues of PPARDelta AB/Delta 448 and PPARDelta AB/Delta 425 (Ile447 and Pro424, respectively) are indicated by arrows. The linear alignment of mPPARalpha LBD with that of hRARgamma and hRXRalpha was adapted from Wurtz et al. (39). B, structures of the PPAR activators used in this study.
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GST-mRXRalpha was prepared by polymerase chain reaction amplification of full-length mRXRalpha using a 5' primer that introduced a HincII site immediately upstream of the natural initiator methionine and a 3' primer that introduced an EcoRI site 3' of the natural stop codon of mRXRalpha . The resulting fragment was appropriately digested and subcloned into a EheI/EcoRI-digested GST fusion vector (pGEX-cs).

In Vitro Transcription/Translation

Proteins were prepared by in vitro transcription/translation using rabbit reticulocyte lysate as described previously (3, 33). Translation reactions were carried out in the presence of [35S]methionine for production of radioactively labeled proteins used in DPSAs and GST-pull down experiments, whereas receptor proteins used in electrophoretic mobility shift assays were translated in the presence of unlabeled methionine. Unprogrammed lysates were generated identically using equal amounts of linearized pTL1 in place of receptor-coding templates.

Electrophoretic Mobility Shift Assays (EMSA)

Two probes were used in these studies as follows. DR1, 5'-cgag<UNL>TGACCT</UNL>c<UNL>TGACCC</UNL>ctaccctcga-3'; ACO-PPRE, 5'-cttcgcgaacg<UNL>TGACCT</UNL>t<UNL>TGTCCT</UNL>ccccttttgctcgatc-3'. One strand of each probe is shown for clarity, and the directly repeated motifs are indicated in uppercase letters and by underlining. Both DR1 (35, 36) and ACO-PPRE (25) probes have been described previously.

Receptor proteins (10 and 20 fmol of PPARDelta AB and mRXRalpha , respectively) were preincubated on ice for 15 min prior to addition of a mix containing ~50,000 cpm of Klenow end-filled DR1 or ACO-PPRE probes. Components of the probe mix were (in mM) HEPES-NaOH, pH 7.5, 10; EDTA, 1; dithiothreitol, 1; and NaCl, 150. The mix was supplemented with 10% glycerol, 1 µg/µl bovine serum albumin, and poly[d(I·C)] (2 µg/tube). The amount of lysate in each binding reaction was held constant by addition of unprogrammed reticulocyte lysate. Samples were loaded on a 5% polyacrylamide gel, electrophoresed, and gels were dried and subjected to autoradiography as described previously (33, 36).

Bacterial Expression and Purification of GST and GST-mRXRalpha Fusion Proteins

GST-mRXRalpha expression in the DH5alpha F' strain of Escherichia coli was induced by addition of isopropyl beta -D-thiogalactopyranoside (1 mM final concentration) to the growth media and cultured for an additional 2 h. Bacterial extracts were prepared using standard methods. The fusion protein was purified on a glutathione-Sepharose 4B column as per the manufacturer's (Pharmacia Biotech Inc.) recommendations.

GST Pull-down Experiments

Glutathione-Sepharose 4B (Pharmacia) was washed extensively in phosphate-buffered saline (PBS) and resuspended in a volume of PBS sufficient to generate a 50% slurry. This slurry (1 ml) was mixed with 2 volumes of PBS (2 ml) containing either no protein, purified GST, or purified GST-mRXRalpha (proteins were at a concentration of ~1 mg/ml) and incubated with rotation at 4 °C overnight. The resin slurry was gently centrifuged, washed 5 times in 2 volumes of PBS (2 ml) to remove all unbound protein, and finally resuspended in 1 volume of binding buffer (same as EMSA buffer but without poly[d(I·C)]). GST pull-down experiments were conducted using 20 µl (~100 fmol) of in vitro translated 35S-PPARDelta AB, 35S-PPARDelta AB/Delta 448, or 35S-PPARDelta AB/Delta 425, and 40 µl of GST bound, GST-mRXRalpha bound, or unbound glutathione-Sepharose slurries. After an overnight incubation at 4 °C with continuous rotation, samples were gently centrifuged and washed 10 times using 250 µl of binding buffer. After the final wash the resin was resuspended in 30 µl of 2 × loading buffer (125 mM Tris-HCl, pH 6.8; 4% (w/v) SDS; 1.4 M beta -mercaptoethanol; 25% (v/v) glycerol; 0.1% (w/v) bromphenol blue) of which one-half was electrophoresed on 12.5% denaturing gels and processed as described previously (33, 36). Some GST pull-down experiments were conducted as described above but with the addition of unlabeled, annealed oligonucleotides corresponding to ACO and DR1 PPREs to all incubations and wash buffers at a final concentration of 5 fmol/µl.

Differential Protease Sensitivity Assays

Two µl (~10 fmol) of in vitro translated 35S-PPARDelta AB, 35S-PPARDelta AB/Delta 448, or 35S-PPARDelta AB/Delta 425 were preincubated in 7 µl of binding buffer (as described under "GST Pull-down Experiments") containing either WY-14,643 (pirinixic acid), ETYA, LY-171883 (5-[4'-(4"-acetyl-3"-hydroxy-2"-propylphenoxy)butyl]tetrazole), clofibrate (2-(4-chlorophenoxy)-2-methylpropanoic acid ethyl ester), clofibric acid (2-(4-chlorophenoxy)-2-methylpropanoic acid), or an equal volume of vehicle for 30 min at 22 °C. The final concentration of vehicle did not exceed 0.15% (v/v) in any experiment conducted. Stock solutions of all ligands were prepared on the day of the experiment in dimethyl sulfoxide (WY-14,643, and LY-171883) or ethanol (ETYA, clofibric acid, and clofibrate). DPSAs were initiated by addition of 1 µl of 10 × stock solution of chymotrypsin in water and were allowed to proceed for 20 min at 22 °C. Reactions were terminated by addition of 1 volume of 2 × loading buffer (as described under "GST Pull-Down Experiments"). Electrophoresis, autoradiography, and densitometric quantification were carried out as described previously (33, 36). DPSAs conducted to determine the effect of heterodimerization with mRXRalpha were carried out essentially as described above except that [35S]methionine-labeled PPAR preparations were incubated with a 2-fold molar excess of in vitro translated mRXRalpha or unprogrammed lysate for 10 min at 22 °C prior to addition of ligand.

Chemicals and Reagents

WY-14,643 was purchased from Chemsyn Science Labs (Lenexa, KS). LY-171883 and ETYA were obtained from BIOMOL (Plymouth Meeting, PA). Clofibric acid, clofibrate, and chymotrypsin were purchased from Sigma.


RESULTS

Carboxyl-terminal truncation mutants of PPARDelta AB were constructed to define regions of the receptor required for interaction with RXR and to determine if diverse ligands require distinct mPPARalpha structural features. Based on the crystal structures of RXRalpha (37, 38) and retinoic acid receptor gamma  (RARgamma , Ref. 38) LBDs and the predicted structural similarity of these receptors to mPPARalpha (39 and data not shown) two PPARDelta AB carboxyl-terminal truncation mutants were prepared as follows: 1) PPARDelta AB/Delta 448 that lacks a portion of putative helix H11 and all of helix H12, and 2) PPARDelta AB/Delta 425 that lacks putative helices H10-H12 (see Fig. 1A). Because both carboxyl-terminal truncation mutants lack the core of the putative ligand-dependent transcriptional activation function (AF-2, Ref. 39), neither would be expected to activate transcription in a ligand-dependent manner.

mPPARalpha Carboxyl-terminal Truncation Mutants Define PPAR·RXR Heterodimerization Interface

EMSAs were conducted to compare the ability of PPARDelta AB, PPARDelta AB/Delta 448, and PPARDelta AB/Delta 425 to bind two degenerate DR1 probes: a DR1 retinoid responsive element described previously (35) and a peroxisome proliferator-activated response element (PPRE) identified in the promoter region of the rat acyl-CoA oxidase gene (ACO-PPRE) that confers peroxisome proliferator inducibility on this gene (25). While none of the PPAR receptors bound either probe alone (Fig. 2, lanes 3, 5, 7, 11, 13, and 15), addition of in vitro translated mRXRalpha resulted in mRXRalpha ·PPARDelta AB (Fig. 2, lanes 4 and 12) and mRXRalpha ·PPARDelta AB/Delta 448 (Fig. 2, lanes 6 and 14) heterodimeric complex formation on both probes. PPARDelta AB/Delta 425 did not interact with mRXRalpha on either probe (Fig. 2, lanes 8 and 16). RXR homodimeric complexes have previously been demonstrated to bind DR1 response elements (36, 40-43), and indeed such complexes are observed in our binding assays on the DR1 but not the ACO-PPRE probe (Fig. 2, compare lanes 2 and 10). The efficiency of mRXRalpha ·PPARDelta AB/Delta 448 complex formation on both probes was reduced approximately 2-fold relative to that of mRXRalpha ·PPARDelta AB, suggesting that mPPARalpha residues 448-468 contribute to the stability of the heterodimeric complex but are not absolutely required for complex formation and DNA binding. However, truncation of an additional 23 mPPARalpha carboxyl-terminal residues (amino acids 425-468; PPARDelta AB/Delta 425) abolished the ability of the receptor to interact with mRXRalpha on either probe (Fig. 2, lanes 8 and 16).


Fig. 2. PPAR·RXR heterodimeric complex formation on a DR1 and ACO-PPRE. The filled and open arrows indicate positions of RXR homodimeric complexes and PPAR·RXR heterodimeric complexes, respectively. Lanes 1 and 9 represent nonspecific binding activity present in unprogrammed reticulocyte lysate (not indicated by an arrow). Experiments and gel processing were carried out as described under "Materials and Methods."
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Protein-protein interaction experiments were carried out to investigate the ability of PPARalpha carboxyl-terminal truncation mutants to interact with RXR independently of DNA binding. GST-mRXRalpha fusion protein, immobilized on glutathione-Sepharose, was used in standard GST pull-down experiments for this purpose. In vitro translated 35S-PPARDelta AB and 35S-PPARDelta AB/Delta 448 both interacted with GST-mRXRalpha (Fig. 3, lanes 4 and 5) while an interaction between 35S-PPARDelta AB/Delta 425 and GST-mRXRalpha was not detected (Fig. 3, lane 6). The efficiency of 35S-PPARDelta AB/Delta 448 interaction with GST-mRXRalpha was reduced approximately 2-fold relative to that of 35S-PPARDelta AB with GST-mRXRalpha in agreement with DNA binding experiments described above. No interactions between any of the PPAR receptor proteins and an immobilized GST protein (Fig. 3, lanes 7-9) or glutathione-Sepharose alone were observed (data not shown). Additionally, results from experiments conducted in the presence of unlabeled response elements, identical to those used in DNA binding assays (see above), were indistinguishable from those described above (data not shown).


Fig. 3. PPAR interactions with an immobilized GST-mRXRalpha fusion protein. GST-mRXRalpha (lanes 4-6) or GST (lanes 7-9) immobilized on glutathione-Sepharose beads was incubated with 35S-PPARDelta AB, 35S-PPARDelta AB/Delta 448, and 35S-PPARDelta AB/Delta 425 (~100 fmol) and extensively washed as described under "Materials and Methods." The beads were resuspended in 30 µl of 2 × SDS sample buffer and boiled, and 15 µl were loaded on a 12.5% SDS-polyacrylamide gel. Input lanes represent ~10 fmol of 35S-PPARDelta AB, 35S-PPARDelta AB/Delta 448, and 35S-PPARDelta AB/Delta 425 (lanes 1-3, respectively). Electrophoresis and gel processing were carried out as described under "Materials and Methods." The positions of Bio-Rad prestained low molecular mass standards are indicated.
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The Sensitivity of mPPARalpha to Chymotryptic Digestion Is Altered by Interaction with Ligands That Activate the Receptor

We have adapted a differential protease sensitivity assay (DPSA, Ref. 36) for use with 35S-PPARDelta AB to address the possibility that peroxisome proliferators and related compounds (see Fig. 1B) interact directly with and alter the protease sensitivity of the receptor. Digestion of 35S-PPARDelta AB with increasing concentrations of chymotrypsin in the presence of 100 µM LY-171883, ETYA, or WY-14,643 (Fig. 4A, lanes 11-13, 14-16, and 17-19, respectively) resulted in the appearance of protease-resistant fragments of approximately 33, 31, and 27 kDa, referred to hereafter as PF33, PF31, and PF27, respectively. Clofibric acid and clofibrate, when examined at concentrations of 100 µM, resulted in very weak signals (data not shown); therefore, these PPAR ligands were examined at concentrations of 1 mM. While clofibric acid clearly induced formation of PF33, PF31, and PF27 (Fig. 4A, lanes 5-7), clofibrate only weakly induced formation of these proteolytic fragments (Fig. 4A, lanes 8-10). The glucocorticoid receptor ligand, dexamethasone, had no effect on the proteolytic sensitivity of 35S-PPARDelta AB at concentrations up to 1 mM (data not shown). Moreover, none of the mPPARalpha activators examined affected the protease sensitivity of other nuclear receptors such as mRXRalpha (data not shown), indicative of the specificity of these observations. These results suggest that mPPARalpha undergoes a ligand-induced conformational change upon interaction with compounds previously demonstrated to activate the receptor in transient transfection experiments (7, 13, 19, 44). Chymotrypsin-resistant fragments induced by clofibric acid, clofibrate, LY-171883, ETYA, and WY-14,643 appear to be indistinguishable suggesting that a similar change within mPPARalpha may be induced by all five PPAR activators. The following rank order of efficacy of the five PPAR activators for induction of PFs within PPARDelta AB, at concentrations of 100 µM, was determined using quantitative densitometric scanning of autoradiographs from DPSAs (as described previously in Ref. 36; data not shown): WY-14,643 >>  ETYA > LY-171883 >>  clofibric acid > clofibrate.


Fig. 4. Ligand-induced mPPARalpha conformational change. A, 35S-PPARDelta AB subjected to DPSA. 35S-PPARDelta AB (~10 fmol) was preincubated for 30 min at room temperature with either vehicle (lanes 1-4), 1 mM clofibric acid (CFA, lanes 5-7), 1 mM clofibrate (CLO, lanes 8-10), 100 µM LY-171883 (lanes 11-13), 100 µM ETYA (lanes 14-16), or 100 µM WY-14,643 (lanes 17-19) before addition of chymotrypsin (final concentrations of 75, 150, and 300 µg/ml, respectively, in lanes 2-4, 5-7, 8-10, 11-13, 14-16) or water (lane 1). Proteolytic digestions were carried out at room temperature for 20 min, after which time samples were denatured and electrophoresed on a 12.5% SDS-polyacrylamide gel. Gels were processed as described under "Materials and Methods." B, 35S-PPARDelta AB/Delta 448 subjected to DPSA. Preincubations, electrophoresis, and gel processing were carried out as described in A. Final concentrations of chymotrypsin were 20, 50, and 100 µg/ml, respectively, in lanes 2-4, 5-7, 8-10, 11-13, 14-16. C, 35S-PPARDelta AB/Delta 425 subjected to DPSA. Preincubations, protease concentrations, electrophoresis, and gel processing were carried out as described in B. Arrows throughout the figure indicate positions of proteolytic fragments and migration of Bio-Rad prestained low molecular mass standards. Note that unproteolyzed receptor preparations incubated with vehicle alone (lane 1) were indistinguishable from those incubated with all ligands tested (data not shown). Clofibric acid and clofibrate are abbreviated as CFA and CLO, respectively.
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DPSAs were carried out using truncation mutants 35S-PPARDelta AB/Delta 448 and 35S-PPARDelta AB/Delta 425 (see Fig. 1A) toward the goal of determining if distinct receptor regions are required for responsiveness to structurally diverse PPAR ligands. A clear differential proteolytic pattern was observed with 35S-PPARDelta AB/Delta 448 in the presence of 1 mM clofibric acid, 100 µM LY-171883, and 100 µM WY-14,643 (Fig. 4B, lanes 5-7, 11-13, and 17-19, respectively) and a weaker but detectable differential proteolytic pattern was observed with 1 mM clofibrate (Fig. 4B, lanes 8-10). Ligand-induced alterations in the protease sensitivity of 35S-PPARDelta AB and 35S-PPARDelta AB/Delta 448 appeared to be qualitatively indistinguishable for all ligands examined except that the proteolytic fragments derived from 35S-PPARDelta AB/Delta 448 were of smaller mass reflecting the truncation of 21 carboxyl-terminal amino acids (see arrows in Fig. 4B; termed PF33Delta 448, PF31Delta 448, and PF27Delta 448). However, in contrast to 35S-PPARDelta AB, the protease sensitivity of 35S-PPARDelta AB/Delta 448 was only weakly affected by ETYA (Fig. 4B, lanes 14-16). The following rank order of the five PPAR activators for induction of PFs within PPARDelta AB/Delta 448, at concentrations of 100 µM, was determined using quantitative densitometric scanning (data not shown): WY-14,643 >>  LY-171883 >>  clofibric acid > ETYA = clofibrate.

These results suggest that the most carboxyl-terminal 21 mPPARalpha residues (448-468 corresponding to all of putative helix 12 and a portion of helix 11; see Fig. 1A) are important for mPPARalpha responsiveness to ETYA. With the possible exception of WY-14,643, 35S-PPARDelta AB/Delta 425 did not exhibit a differential proteolytic pattern in the presence of any PPAR ligands examined (Fig. 4C) suggesting that the extreme carboxyl-terminal mPPARalpha amino acids may be required for responsiveness to many PPAR ligands (see below).

Induction of Proteolytic Fragments Is Dependent on Ligand Concentration

DPSAs were conducted using 35S-PPARDelta AB at a constant chymotrypsin concentration and increasing concentrations of PPAR ligands (WY-14, 643, ETYA, LY-171883, CFA; see Fig. 1B) to determine the dependence of PF33, PF31, and PF27 on ligand concentration. Induction of all proteolytic fragments from 35S-PPARDelta AB was clearly ligand-dependent in all cases (Fig. 5A-D), and the relative potencies with which these compounds induced 35S-PPARDelta AB conformational change in vitro was generally consistent with previously reported transcriptional activation studies (Refs. 7, 13, 19; see "Discussion").


Fig. 5. Dose-response relationships for PPAR ligands with PPARDelta AB. A, 35S-PPARDelta AB subjected to DPSA in the presence of increasing concentrations of WY-14,643. 35S-PPARDelta AB (~10 fmol) was preincubated for 30 min at room temperature with either vehicle (lanes 1-2) or increasing concentrations of WY-14,643 (1-100,000 nM in log units, lanes 3-8). Water (lane 1) or chymotrypsin, at a final concentration of 150 µg/ml (lanes 2-9), was added and the reaction allowed to proceed for 20 min at room temperature. Electrophoresis and gel processing were as described in Fig. 4. B, 35S-PPARDelta AB subjected to DPSA in the presence of increasing concentrations of ETYA. Experiments were conducted as in A. C, 35S-PPARDelta AB subjected to DPSA in the presence of increasing concentrations of LY-171883. Experiments were conducted as in A. D, 35S-PPARDelta AB subjected to DPSA in the presence of increasing concentrations of clofibric acid (CFA). Experiments were conducted as in A; the additional lane 10 represents a final CFA concentration of 1 mM. The positions of proteolytic fragments and Bio-Rad prestained low molecular mass standards are indicated.
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PPAR Activator-induced Alteration in Chymotryptic Sensitivity of mPPARalpha Is Not Altered by Heterodimerization with mRXRalpha

Because PPARalpha has been demonstrated to heterodimerize with RXR (10, 11, 22, 23; see Figs. 2 and 3), DPSAs were conducted to examine the effects of heterodimerization with mRXRalpha on the induction of 35S-PPARDelta AB proteolytic fragments by WY-14,643. DPSAs, in the presence of unprogrammed lysate or in vitro translated mRXRalpha , were carried out at a constant protease concentration and increasing concentrations of WY-14,643. Interaction with mRXRalpha did not alter the protease sensitivity of unliganded 35S-PPARDelta AB (Fig. 6A, compare lanes 2 and 6) or 35S-PPARDelta AB/Delta 448 (Fig. 6B, compare lanes 2 and 6). In addition, the concentration dependence of WY-14,643 on the induction of proteolytic fragments derived from either receptor did not differ noticeably in the presence of mRXRalpha (compare lanes 2-5 with lanes 6-9 of Fig. 6A and B, respectively). Similar results were observed for both 35S-PPARDelta AB and 35S-PPARDelta AB/Delta 448 when using the PPAR ligands clofibric acid, clofibrate, LY-171883, and ETYA (data not shown). Moreover, the rank order of efficacy of the five compounds tested for induction of PFs within PPARDelta AB and PPARDelta AB/Delta 448 did not differ from that stated above (data not shown). Therefore, heterodimerization with mRXRalpha does not appear to influence, positively or negatively, the capacity of mPPARalpha to bind PPAR activators and undergo ligand-induced conformational changes. 35S-PPARDelta AB/Delta 425 was not examined in these experiments due the inability of this receptor mutant to interact with mRXRalpha (Figs. 2 and 3) or bind ligand (Fig. 4C).


Fig. 6. Effect of heterodimerization with RXR on ligand-induced PPAR conformational change. A, 35S-PPARDelta AB subjected to DPSA in the presence of vehicle (lanes 2 and 6) or increasing concentrations of WY-14,643 (0.1, 100, and 100,000 nM; lanes 3-5 and 7-9) in the absence (lanes 2-5) and presence of RXR (lanes 6-9). Lane 1 contains undigested 35S-PPARDelta AB. Experiments were conducted as described in Fig. 5A and under "Materials and Methods." B, 35S-PPARDelta AB/Delta 448 subjected to DPSA in the presence of vehicle (lanes 2 and 6) or increasing concentrations of WY-14,643 (0.1, 100, and 100,000 nM; lanes 3-5 and 7-9) in the absence (lanes 2-5) or presence (lanes 6-9) of RXR. Lane 1 contains undigested 35S-PPARDelta AB/Delta 448. Experiments were conducted as described in A except the final concentration of chymotrypsin was 50 µg/ml. Note that in A and B the amount of lysate was kept constant in all samples by addition of unprogrammed reticulocyte lysate. The positions of proteolytic fragments and Bio-Rad prestained low molecular mass standards are indicated.
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DISCUSSION

Our results suggest that the extreme carboxyl-terminal amino acids of mPPARalpha are required for formation of PPAR·RXR heterodimeric complexes both in solution and bound to DR1 and ACO-PPRE probes. This finding is in agreement with a previous study that characterized a hPPARalpha point mutation (Leu433 right-arrow Arg corresponding to the same residue in mPPARalpha ) which abolished heterodimerization with RXR (32) thus illustrating a critical role for this region (which is deleted in PPARDelta AB/Delta 425). A putative leucine zipper-like heptad repeat, located between residues 426-433 of mouse, human, and rat PPARalpha , has been postulated to mediate heterodimerization with RXR (32). Truncation of mPPARalpha to amino acid 447 (PPARDelta AB/Delta 448) gave rise to a receptor protein that was capable of interacting with RXR and binding to DR1 and ACO-PPRE probes, albeit at a 2-fold decreased efficiency as compared with a receptor protein with an intact carboxyl terminus (PPARDelta AB). Considered together, these results suggest that the mPPARalpha dimerization interface contains at least Leu433, which is 100% conserved across all PPAR subtypes (32, data not shown), and extends through at least Ile447. In addition to heterodimerizing with RXR, PPARs have been reported to interact with thyroid hormone receptor (Ref. 45), and more recently, Miyata et al. (46) reported that mPPARalpha interacts with a third member of the nuclear receptor superfamily, the orphan receptor LXRalpha . Therefore, it appears that there may be physiologically relevant cross-talk between PPARs and signaling pathways mediated by other nuclear receptors. It will be of interest to determine if other nuclear receptors interact with PPARs through distinct or common heterodimeric protein interfaces and if these protein-protein interactions and/or the functional capacities of the involved receptors are allosterically regulated by DNA binding as previously demonstrated for other nuclear receptors (47, 48).

PPARs, like other receptor proteins within the nuclear receptor superfamily, exhibit a conserved subdivision of receptor regions referred to as A/B, C, D, and E/F (49, reviewed in Refs. 2-5). Experiments conducted with various chimeric receptor proteins composed of putative PPARalpha ligand binding domains (LBDs) fused to heterologous DNA binding domains from estrogen (7, 50) and glucocorticoid (8) receptors, bacterial tetracycline repressor (14), and GAL4 (44, 51, 52) have demonstrated the requirement for a large portion of the carboxyl terminus of PPARs (D and E/F regions as defined in Ref. 7) for ligand-responsive transcriptional activation.

PPAR activating ligands constitute a chemically diverse group of compounds in which the most obvious common structural elements are an acidic group (free carboxyl group, a metabolically labile derivative thereof, or a bioisostere such as a tetrazole or sulfonamide moiety) and a pi  electron-rich region (aromatic ring or series of alkenes or alkynes) (53). When considering the structural diversity exhibited by these compounds, it seems possible that the molecular determinants of mPPARalpha interaction with each ligand or class of ligands may be distinct. Indeed, our results indicate that distinct mPPARalpha regions are required for responsiveness to different PPAR activators. While PPARDelta AB is responsive to WY-14,643, ETYA, LY-171883, clofibric acid, and clofibrate, as detected by DPSAs, deletion of mPPARalpha residues 448-468 (PPARDelta AB/Delta 448) severely compromises responsiveness to ETYA but not other PPAR ligands. The distal carboxyl-terminal amino acids of mPPARalpha that are deleted in PPARDelta AB/Delta 448 correspond to part (H12) of the region that has been proposed to stabilize ligand-receptor interactions with hRARgamma by functioning as a "lid" on the ligand binding cavity (38, 39). The greatly reduced efficacy with which ETYA induced PPARDelta AB/Delta 448 conformational change relative to that of PPARDelta AB suggests that the hydrophobicity of putative H12 may play a critical role in the stabilization of ETYA binding, perhaps by stabilizing an extended conformation of this compound. Truncation of mPPARalpha residues 425-468 (PPARDelta AB/Delta 425) gave rise to a receptor protein which was slightly responsive to WY-14,643 but unresponsive to all other PPAR ligands examined. In addition to deletion of putative helix H12, PPARDelta AB/Delta 425 also lacks putative helices H10 and H11, encompassing a region that has been proposed to form one side of the nuclear receptor ligand binding pocket (39), which may explain the inactivity of this mutant in DPSAs. However, we cannot presently rule out the possibility that the inactivity of PPARDelta AB/Delta 425 is due to improper protein folding and/or detrimental structural distortions outside the deleted region. Nonetheless, it is clear that mPPARalpha residues 448-468 are important for ligand binding and/or conformational change induced by ETYA while being dispensable for responsiveness to other PPAR ligands supporting the hypothesis that distinct mPPARalpha receptor regions may be required for interaction with structurally dissimilar PPAR activators.

Recently, several synthetic antidiabetic thiazolidinediones (44, 51, 52) and 15-deoxy-Delta 12,14-prostaglandin J2 (44, 52) have been shown to bind directly to the ligand binding domain of the mouse PPARgamma . Direct binding of any compounds to PPARalpha subtypes, however, has not been demonstrated. Issemann et al. (7) specifically report the lack of [3H]nafenopin binding by mPPARalpha which may be due to both a low affinity of nafenopin for mPPARalpha and a large amount of endogenous binding activity in the cell lines tested (7). In the current study, DPSA methodology was adapted to facilitate detection of ligand interactions with mPPARalpha . DPSAs have been demonstrated to be a useful method for detection of ligand-induced conformational change within the nuclear receptor superfamily (36, 54-64). The differential sensitivity of liganded and unliganded receptors is likely related to ligand-induced stabilization of one or more receptor conformations that exhibit sensitivity to proteolytic digestion different from that of unliganded receptor. Although DPSAs yield data that are somewhat less amenable to quantitative analyses than radioligand binding experiments, an advantage of the former is the ability to detect low affinity ligand-receptor interactions. All mPPARalpha activators tested induced mPPARalpha conformational change in vitro, albeit it with varying potencies. However, the relative potencies of these compounds with regard to induction of PPARDelta AB conformational change in vitro is generally consistent with previously reported transcriptional activation studies utilizing mPPARalpha : WY-14,643 >>  ETYA > LY-171883 >>  clofibric acid (7, 13, 19). ETYA, a potent activator of Xenopus PPARalpha (11), was a substantially weaker ligand than WY-14,643 in the in vitro studies employing PPARDelta AB described herein (Fig. 5). Hsu and co-workers (13) also reported that ETYA was approximately 10-fold weaker than WY-14,643 as an activator of mPPARalpha in transient transfection experiments, suggesting that the enhanced potency of ETYA reported by Keller and co-workers (11) may be conferred by species-specific receptor activity, cell-specific factors, and/or mechanism(s) other than direct binding of this arachidonic acid analog to the receptor.

It has been hypothesized that Hsp72, which has been demonstrated to interact directly with rat PPARalpha , may bind PPAR activators and, in turn, allosterically activate the associated receptor protein (65). Presently, we cannot exclude this possibility; however, results from DPSAs in which mPPARalpha carboxyl-terminal truncation mutants were used suggest an alternative molecular mechanism. For example, deletion of 21 mPPARalpha carboxyl-terminal amino acids (PPARDelta AB/Delta 448) compromises the responsiveness of the receptor to ETYA but not other PPAR ligands. If Hsp72 binds these ligands directly and allosterically transduces a signal that alters the conformation of mPPARalpha , it seems unlikely that this process would be attenuated by carboxyl-terminal truncation of mPPARalpha unless the deleted amino acids are required for mPPARalpha -Hsp72 interaction. In such a case, one would expect this truncation to abolish responsiveness to all ligands. Of course, it is possible that some ligands selectively interact with mPPARalpha , Hsp72, or both proteins in the context of an mPPARalpha ·Hsp72 complex. The latter possibility would be reminiscent of ecdysone receptor in which ecdysone binding activity is associated with a complex of ecdysone receptor and ultraspiracle (61). In any event, ligand-induced conformational change likely underlies the molecular basis of ligand activation of the putative mPPARalpha transcriptional activation function, AF-2, and identification of ligand-induced mPPARalpha proteolytic fragments will be of critical importance to our understanding of the dynamic process of PPAR activation by ligands and interaction of liganded receptor with putative transcriptional intermediary factors.


FOOTNOTES

*   This work was financially supported by grants from the Oregon Affiliate of the American Heart Association (OR-94-GS-16) and the National Institute of Environmental Health Sciences (ES00210, through the Oregon State University Environmental Health Sciences Center) to M. L. Additional support from the National Institute of Environmental Health Sciences Program Project Grant ES00040 is gratefully acknowledged. 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 predoctoral fellowships from the American Foundation for Pharmaceutical Education and Sigma Xi.
par    To whom correspondence should be addressed. Tel.: 541-737-5809; Fax: 541-737-3999; E-mail: leidm{at}bcc.orst.edu.
1    The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; mPPARalpha , murine peroxisome proliferator-activated receptor alpha ; ETYA, 5,8,11,14-eicosatetraynoic acid; DR1, direct repeat separated by one nucleotide; RXR, retinoid X receptor; PPRE, peroxisome proliferator-activated response element; hPPARalpha , human peroxisome proliferator-activated receptor alpha ; DPSA, differential protease sensitivity assay; mRXRalpha , murine retinoid X receptor alpha ; EMSA, electrophoretic mobility shift assay; ACO-PPRE, acyl-CoA oxidase peroxisome proliferator response element; LBD, ligand binding domain; RARgamma , retinoic acid receptor gamma ; PF, proteolytic fragment; GST, glutathione S-transferase; PBS, phosphate-buffered saline.

Acknowledgments

We thank Ph. Kastner, P. Chambon, T. Lufkin, J. D. Tugwood, S. Green, and W. Dougherty for plasmid constructs; R. McParland and B. Robbins for oligonucleotide synthesis; B. Hettinger-Smith and J. E. Ishmael for useful discussions and critical review of the manuscript; and the Communications Media Center for figure preparation.


Note Added in Proof

Consistent with some of the data presented herein, Devchand et al. recently published results demonstrating direct binding of [3H]leukotriene B4 to a bacterially expressed and purified, GST-Xenopus PPARalpha fusion protein and inhibition of this binding by unlabeled WY-14,643 (Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43).


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