Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor alpha.
The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331<WY14643=ciprofibrate<L165041<gemfibrozil. Similarly, for all ligands tested, the rank order of EC(50) for peptide recruitment was CBP<PGC-1<SRC-1. These data suggest that for these LXXLL coactivator peptides, the ligands do not substantially differ in their preferences. Partial agonism was observed with ciprofibrate and PGC-1 and gemfibrozil and CBP giving a lower FRET at saturation than with the other ligands. This suggests that ciprofibrate and gemfibrozil induce a different conformation to the receptor-PGC-1 and receptor-CBP complex, respectively. In cotransfection assays, unexpected differences in potencies and efficacies were observed and the rank order of EC(50)s for activation differed from that predicted by FRET assays. In most cases, the presence of a coactivator peptide led to decrease in the EC(50)s seen in FRET assays compared to the K(i)s observed in binding to receptor only, consistent with the lower EC(50)s obtained in the transfection assays. Our data demonstrate that ligand induced coactivator preferences of PPARalpha contribute to transcription potency and efficacy.
Mukherjee
Ranjan
R
Cardiovascular Diseases Research, Bristol-Myers Squibb Company, Experimental Station, E400 Wilmington, DE 19880, USA. ranjan.mukherjee@bms.com
Sun
Shaoxian
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Santomenna
Linda
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Miao
Bowman
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Walton
Harry
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Liao
Boshan
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Locke
Kenneth
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Zhang
Ji-Hu
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Nguyen
Sonny H
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Li Tao
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Murphy
Kathleen
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Ross
Harold O
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Xia
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Teleha
Christopher
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Chen
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Selling
Bernard
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Wynn
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D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli / Q000378:metabolism; D051548:Histone Acetyltransferases; D006801:Humans; D007700:Kinetics; D008024:Ligands; D056920:Nuclear Receptor Coactivator 1; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D013053:Spectrophotometry; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D008024:Ligands; D010455:Peptides; D016271:Proto-Oncogene Proteins c-myc; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1