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• W2059207612 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 α (mPPARα) in transfection experiments were tested for the ability to induce conformational changes within mPPARα in vitro. WY-14,643, 5,8,11,14-eicosatetraynoic acid, LY-171883, and clofibric acid all directly induced mPPARα conformational changes as evidenced by a differential protease sensitivity assay. Carboxyl-terminal truncation mutagenesis of mPPARα 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, mPPARα 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 mPPARα. 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 α (mPPARα) in transfection experiments were tested for the ability to induce conformational changes within mPPARα in vitro. WY-14,643, 5,8,11,14-eicosatetraynoic acid, LY-171883, and clofibric acid all directly induced mPPARα conformational changes as evidenced by a differential protease sensitivity assay. Carboxyl-terminal truncation mutagenesis of mPPARα 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, mPPARα 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 mPPARα. INTRODUCTIONPeroxisome proliferator-activated receptors (PPARs) 1The abbreviations used are: PPARperoxisome proliferator-activated receptormPPARαmurine peroxisome proliferator-activated receptor αETYA5,8,11,14-eicosatetraynoic acidDR1direct repeat separated by one nucleotideRXRretinoid X receptorPPREperoxisome proliferator-activated response elementhPPARαhuman peroxisome proliferator-activated receptor αDPSAdifferential protease sensitivity assaymRXRαmurine retinoid X receptor αEMSAelectrophoretic mobility shift assayACO-PPREacyl-CoA oxidase peroxisome proliferator response elementLBDligand binding domainRARγretinoic acid receptor γPFproteolytic fragmentGSTglutathione S-transferasePBSphosphate-buffered saline. are members of a large family of ligand-inducible transcription factors that includes receptors for retinoids, vitamin D, and thyroid and steroid hormones (1Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 2Green S. Chambon P. Trends Genet. 1988; 4: 309-314Abstract Full Text PDF PubMed Scopus (830) Google Scholar, 3Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 4Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar, 5Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar). The mammalian PPAR family is composed of at least three genetically and pharmacologically distinct subtypes, PPARα, -γ, and -δ (reviewed in 6Green S. Mutat. Res. 1995; 333: 101-109Crossref PubMed Scopus (158) Google Scholar). Murine PPAR α (mPPARα) was originally isolated from a mouse liver cDNA library by Issemann and Green (7Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar) 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 PPARα in transient transfection experiments. Particularly noteworthy among these compounds are: 1) lipids such as arachidonic acid (8Gottlicher M. Widmark E. Li Q. Gustafsson J.-A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (796) Google Scholar, 9Dreyer C. Keller H. Mahfoudi A. Laudet V. Krey G. Wahli W. Biol. Cell. 1993; 77: 67-76Crossref PubMed Scopus (236) Google Scholar, 10Issemann I. Prince R.A. Tugwood J.D. Green S. J. Mol. Endocrinol. 1993; 11: 37-47Crossref PubMed Scopus (283) Google Scholar, 11Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (849) Google Scholar) and its synthetic analog 5,8,11,14-eicosatetraynoic acid (ETYA,Refs .9Dreyer C. Keller H. Mahfoudi A. Laudet V. Krey G. Wahli W. Biol. Cell. 1993; 77: 67-76Crossref PubMed Scopus (236) Google Scholar, 11Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (849) Google Scholar, 12Mukherjee R. Low L. Noonan D. McDonnell D.P. J. Steroid Biochem. Mol. Biol. 1994; 51: 157-166Crossref PubMed Scopus (262) Google Scholar, 13Hsu M.-H. Palmer C.N. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1995; 48: 559-567PubMed Google Scholar), 8-[S]-hydroxyeicosatetraenoic acid (14Yu K. Bayona W. Kallen C.B. Harding H.P. Ravera C.P. McMahon G. Brown M. Lazar M.A. J. Biol. Chem. 1995; 270: 23975-23983Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar), a lipoxygenase metabolite of arachidonic acid, and linoleic acid (8Gottlicher M. Widmark E. Li Q. Gustafsson J.-A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (796) Google Scholar, 9Dreyer C. Keller H. Mahfoudi A. Laudet V. Krey G. Wahli W. Biol. Cell. 1993; 77: 67-76Crossref PubMed Scopus (236) Google Scholar, 10Issemann I. Prince R.A. Tugwood J.D. Green S. J. Mol. Endocrinol. 1993; 11: 37-47Crossref PubMed Scopus (283) Google Scholar, 11Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (849) Google Scholar, 14Yu K. Bayona W. Kallen C.B. Harding H.P. Ravera C.P. McMahon G. Brown M. Lazar M.A. J. Biol. Chem. 1995; 270: 23975-23983Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 15Rodriguez J.C. Gil-Gomez G. Hegardt F.G. Haro D. J. Biol. Chem. 1994; 269: 18767-18772Abstract Full Text PDF PubMed Google Scholar); 2) fibric acid anti-hyperlipidemic drugs (WY-14,643, clofibric acid, gemfibrozil, ciprofibric acid; 10Issemann I. Prince R.A. Tugwood J.D. Green S. J. Mol. Endocrinol. 1993; 11: 37-47Crossref PubMed Scopus (283) Google Scholar, 16Castelein H. Gulick T. Declercq P.E. Mannaerts G.P. Moore D.D. Baes M.I. J. Biol. Chem. 1994; 269: 26754-26758Abstract Full Text PDF PubMed Google Scholar, 17Varanasi U. Chu R. Huang Q. Catellon R. Yeldandi A.V. Reddy J.K. J. Biol. Chem. 1996; 271: 2147-2155Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) that represent a class of therapeutic agents useful in the treatment of hypertriglyceridemia (18Witztum J.L. Hardman J.G. Gilman A.G. Limbird L.E. The Pharmacological Basis of Therapeutics. 9th Ed. McGraw-Hill, Inc., New York1995: 892-894Google Scholar); and 3) a leukotriene D4 antagonist, LY-171883 (19Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1269) Google Scholar). 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 20Lake B.G. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 483-507Crossref PubMed Google Scholar). While chemically distinct, most of these compounds have been demonstrated to induce proliferation of peroxisomes leading to hepatic hyperplasia and hepatocarcinogenesis in many species (20Lake B.G. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 483-507Crossref PubMed Google Scholar). Peroxisome proliferator-induced alteration of hepatocyte phenotype is believed to result from activation of PPARα and subsequent modulation of gene expression downstream of this nuclear receptor (reviewed in 6Green S. Mutat. Res. 1995; 333: 101-109Crossref PubMed Scopus (158) Google Scholar, 20Lake B.G. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 483-507Crossref PubMed Google Scholar; see below). The central role of PPARα in xenobiotic-induced peroxisomal proliferation was recently demonstrated by the absence of hepatomegaly and peroxisome proliferation in mice null for expression of this gene (21Lee S.S.-T. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1492) Google Scholar).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, 11Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (849) Google Scholar, 17Varanasi U. Chu R. Huang Q. Catellon R. Yeldandi A.V. Reddy J.K. J. Biol. Chem. 1996; 271: 2147-2155Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 22Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1514) Google Scholar, 3Leid M. Kastner P. Chambon P. Trends Biochem. Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar, 24Issemann I. Prince R.A. Tugwood J.D. Green S. Biochimie (Paris). 1993; 75: 251-256Crossref PubMed Scopus (101) Google Scholar). PPAR·RXR heterodimeric complexes appear to be responsive to both PPAR activators and 9-cis-retinoic acid, the endogenous ligand for RXR (11Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (849) Google Scholar, 17Varanasi U. Chu R. Huang Q. Catellon R. Yeldandi A.V. Reddy J.K. J. Biol. Chem. 1996; 271: 2147-2155Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 22Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1514) Google Scholar, 23Gearing K.L. Gottlicher M. Teboul M. Widmark E. Gustafsson J.-A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1440-1444Crossref PubMed Scopus (343) Google Scholar, 24Issemann I. Prince R.A. Tugwood J.D. Green S. Biochimie (Paris). 1993; 75: 251-256Crossref PubMed Scopus (101) Google Scholar).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 (17Varanasi U. Chu R. Huang Q. Catellon R. Yeldandi A.V. Reddy J.K. J. Biol. Chem. 1996; 271: 2147-2155Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 25Tugwood J.D. Issemann I. Anderson R.G. Bundell K.R. McPheat W.L. Green S. EMBO J. 1992; 11: 433-439Crossref PubMed Scopus (801) Google Scholar), bifunctional enzyme (26Bardot O. Aldridge T.C. Latruffe N. Green S. Biochem. Biophys. Res. Commun. 1993; 192: 37-45Crossref PubMed Scopus (232) Google Scholar, 27Zhang B. Marcus S.L. Sajjadi F.G. Alvares K. Redyy J.K. Subramani S. Rachubinski R.A. Capone J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7541-7545Crossref PubMed Scopus (234) Google Scholar), malic enzyme (16Castelein H. Gulick T. Declercq P.E. Mannaerts G.P. Moore D.D. Baes M.I. J. Biol. Chem. 1994; 269: 26754-26758Abstract Full Text PDF PubMed Google Scholar), liver fatty acid binding protein (28Issemann I. Prince R. Green S. Biochem. Soc. Trans. 1992; 20: 824-827Crossref PubMed Scopus (143) Google Scholar), 3-hydroxy-3-methylglutaryl-CoA synthase (15Rodriguez J.C. Gil-Gomez G. Hegardt F.G. Haro D. J. Biol. Chem. 1994; 269: 18767-18772Abstract Full Text PDF PubMed Google Scholar), and cytochrome P450 fatty acid ω-hydroxylase (29Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar). Such findings indicate a prominent regulatory role for the PPAR receptor family in lipid metabolism and homeostasis. In addition, overexpression of PPARα and -γ in cultured fibroblasts and subsequent exposure to PPAR ligands has been shown to confer adipogenicity (30Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3092) Google Scholar, 31Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (407) Google Scholar), 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 PPARα have identified: 1) a Glu282→ Gly point mutation in mPPARα that ameliorates transcriptional responses to WY-14,643 and ETYA (13Hsu M.-H. Palmer C.N. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1995; 48: 559-567PubMed Google Scholar), and 2) a Leu433→ Arg point mutation in human PPARα (hPPARα) that abolishes heterodimerization with RXR (32Juge-Aubry C.E. Gorla-Bajszczak A. Pernin A. Lemberger T. Wahli W. Burger A.G. Meier C.A. J. Biol. Chem. 1995; 270: 18117-18122Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The present studies were undertaken to identify mPPARα carboxyl-terminal receptor regions that are important for both ligand responsiveness and heterodimerization with mRXRα and to determine if structurally diverse PPAR ligands induce similar conformational changes within mPPARα. To our knowledge, these studies provide the first direct biochemical evidence demonstrating that peroxisome proliferators induce conformational changes within mPPARα. Ligand-induced stabilization of particular mPPARα conformational states likely underlies the molecular basis for the ability of these compounds to activate the receptor and to modulate expression of mPPARα target genes including those implicated in peroxisome proliferation.RESULTSCarboxyl-terminal truncation mutants of PPARΔAB were constructed to define regions of the receptor required for interaction with RXR and to determine if diverse ligands require distinct mPPARα structural features. Based on the crystal structures of RXRα (37Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1051) Google Scholar, 38Renaud J.-P. Rochel N. Ruff M. Vivant V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1021) Google Scholar) and retinoic acid receptor γ (RARγ, 38Renaud J.-P. Rochel N. Ruff M. Vivant V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1021) Google Scholar) LBDs and the predicted structural similarity of these receptors to mPPARα (39 and data not shown) two PPARΔAB carboxyl-terminal truncation mutants were prepared as follows: 1) PPARΔAB/Δ448 that lacks a portion of putative helix H11 and all of helix H12, and 2) PPARΔAB/Δ425 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, 39Wurtz J.-M. Bourguet W. Renaud J.-P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (674) Google Scholar), neither would be expected to activate transcription in a ligand-dependent manner.mPPARα Carboxyl-terminal Truncation Mutants Define PPAR·RXR Heterodimerization InterfaceEMSAs were conducted to compare the ability of PPARΔAB, PPARΔAB/Δ448, and PPARΔAB/Δ425 to bind two degenerate DR1 probes: a DR1 retinoid responsive element described previously (35Mader S. Chen J.-Y. Chen Z. White J. Chambon P. Gronemeyer H. EMBO J. 1993; 12: 5029-5041Crossref PubMed Scopus (195) Google Scholar) 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 (25Tugwood J.D. Issemann I. Anderson R.G. Bundell K.R. McPheat W.L. Green S. EMBO J. 1992; 11: 433-439Crossref PubMed Scopus (801) Google Scholar). 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 mRXRα resulted in mRXRα·PPARΔAB (Fig. 2, lanes 4 and 12) and mRXRα·PPARΔAB/Δ448 (Fig. 2, lanes 6 and 14) heterodimeric complex formation on both probes. PPARΔAB/Δ425 did not interact with mRXRα on either probe (Fig. 2, lanes 8 and 16). RXR homodimeric complexes have previously been demonstrated to bind DR1 response elements (36Leid M. J. Biol. Chem. 1994; 269: 14175-14181Abstract Full Text PDF PubMed Google Scholar, 40Mangelsdorf D.J. Umesono M. Kliewer S.A. Borgmeyer U. Ong E.S. Evans R.M. Cell. 1991; 66: 555-561Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 40Mangelsdorf D.J. Umesono M. Kliewer S.A. Borgmeyer U. Ong E.S. Evans R.M. Cell. 1991; 66: 555-561Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 41Zhang X.-K. Lehmann J. Hoffmann B. Dawson M.I. Cameron J. Graupner G. Hermann T. Tran P. Pfahl M. Nature. 1992; 358: 587-591Crossref PubMed Scopus (519) Google Scholar, 42Durand B. Saunders M. Leroy P. Leid M. Chambon P. Cell. 1992; 71: 73-85Abstract Full Text PDF PubMed Scopus (358) Google Scholar, 43Lehmann J.M. Zhang X.-K. Graupher G. Lee M.-O. Hermann T. Hoffmann B. Pfahl M. Mol. Cell. Biol. 1993; 13: 7698-7707Crossref PubMed Scopus (91) Google Scholar), 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 mRXRα·PPARΔAB/Δ448 complex formation on both probes was reduced approximately 2-fold relative to that of mRXRα·PPARΔAB, suggesting that mPPARα 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 mPPARα carboxyl-terminal residues (amino acids 425-468; PPARΔAB/Δ425) abolished the ability of the receptor to interact with mRXRα on either probe (Fig. 2, lanes 8 and 16).Protein-protein interaction experiments were carried out to investigate the ability of PPARα carboxyl-terminal truncation mutants to interact with RXR independently of DNA binding. GST-mRXRα fusion protein, immobilized on glutathione-Sepharose, was used in standard GST pull-down experiments for this purpose. In vitro translated 35S-PPARΔAB and 35S-PPARΔAB/Δ448 both interacted with GST-mRXRα (Fig. 3, lanes 4 and 5) while an interaction between 35S-PPARΔAB/Δ425 and GST-mRXRα was not detected (Fig. 3, lane 6). The efficiency of 35S-PPARΔAB/Δ448 interaction with GST-mRXRα was reduced approximately 2-fold relative to that of 35S-PPARΔAB with GST-mRXRα 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. 3PPAR interactions with an immobilized GST-mRXRα fusion protein. GST-mRXRα (lanes 4-6) or GST (lanes 7-9) immobilized on glutathione-Sepharose beads was incubated with 35S-PPARΔAB, 35S-PPARΔAB/Δ448, and 35S-PPARΔAB/Δ425 (∼100 fmol) and extensively washed as described under “Materials and Methods.” The beads were resuspended in 30 μl of 2 x SDS sample buffer and boiled, and 15 μl were loaded on a 12.5% SDS-polyacrylamide gel. Input lanes represent ∼10 fmol of 35S-PPARΔAB, 35S-PPARΔAB/Δ448, and 35S-PPARΔAB/Δ425 (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Sensitivity of mPPARα to Chymotryptic Digestion Is Altered by Interaction with Ligands That Activate the ReceptorWe have adapted a differential protease sensitivity assay (DPSA, 36Leid M. J. Biol. Chem. 1994; 269: 14175-14181Abstract Full Text PDF PubMed Google Scholar) for use with 35S-PPARΔAB 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-PPARΔAB 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-PPARΔAB at concentrations up to 1 mM (data not shown). Moreover, none of the mPPARα activators examined affected the protease sensitivity of other nuclear receptors such as mRXRα (data not shown), indicative of the specificity of these observations. These results suggest that mPPARα undergoes a ligand-induced conformational change upon interaction with compounds previously demonstrated to activate the receptor in transient transfection experiments (7Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar, 13Hsu M.-H. Palmer C.N. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1995; 48: 559-567PubMed Google Scholar, 19Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1269) Google Scholar, 44Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-820Abstract Full Text PDF PubMed Scopus (1859) Google Scholar). 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 mPPARα may be induced by all five PPAR activators. The following rank order of efficacy of the five PPAR activators for induction of PFs within PPARΔAB, at concentrations of 100 μM, was determined using quantitative densitometric scanning of autoradiographs from DPSAs (as described previously in 36Leid M. J. Biol. Chem. 1994; 269: 14175-14181Abstract Full Text PDF PubMed Google Scholar; data not shown): WY-14,643 ≫ ETYA > LY-171883 ≫ clofibric acid > clofibrate.Fig. 4Ligand-induced mPPARα conformational change. A, 35S-PPARΔAB subjected to DPSA. 35S-PPARΔAB (∼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-PPARΔAB/Δ448 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-PPARΔAB/Δ425 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DPSAs were carried out using truncation mutants 35S-PPARΔAB/Δ448 and 35S-PPARΔAB/Δ425 (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-PPARΔAB/Δ448 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-PPARΔAB and 35S-PPARΔAB/Δ448 appeared to be qualitatively indistinguishable for all ligands examined except that the proteolytic fragments derived from 35S-PPARΔAB/Δ448 were of smaller mass reflecting the truncation of 21 carboxyl-terminal amino acids (see arrows in Fig. 4B; termed PF33Δ448, PF31Δ448, and PF27Δ448). However, in contrast to 35S-PPARΔAB, the protease sensitivity of 35S-PPARΔAB/Δ448 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 PPARΔAB/Δ448, 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 mPPARα residues (448-468 corresponding to all of putative helix 12 and a portion of helix 11; see Fig. 1A) are important for mPPARα responsiveness to ETYA. With the possible exception of WY-14,643, 35S-PPARΔAB/Δ425 did not exhibit a differential proteolytic pattern in the presence of any PPAR ligands examined (Fig. 4C) suggesting that the extreme carboxyl-terminal mPPARα amino acids may be required for responsiveness to many PPAR ligands (see below).Induction of Proteolytic Fragments Is Dependent on Ligand ConcentrationDPSAs were conducted using 35S-PPARΔAB 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-PPARΔAB was clearly ligand-dependent in all cases (Fig. 5A-D), and the relative potencies with which these compounds induced 35S-PPARΔAB conformational change in vitro was generally consistent with previously reported transcriptional activation studies (7Issemann I. Green S. Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar, 13Hsu M.-H. Palmer C.N. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1995; 48: 559-567PubMed Google Scholar, 19Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1269) Google Scholar; see “Discussion”).Fig. 5Dose-response relationships for PPAR ligands with PPARΔAB. A, 35S-PPARΔAB subjected to DPSA in the presence of increasing concentrations of WY-14,643. 35S-PPARΔAB (∼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-PPARΔAB subjected to DPSA in the presence of increasing concentrations of ETYA. Experiments were conducted as in A. C, 35S-PPARΔAB subjected to DPSA in the presence of increasing concentrations of LY-171883. Experiments were conducted as in A. D, 35S-PPARΔAB 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.View Large Image Figur" @default.
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