Recently, it has been shown that peroxisome proliferators, an important class of tumor promoters, activate novel members of the steroid hormone receptor superfamily, the peroxisome proliferator-activated receptors (PPAR). Several subtypes of PPAR have been discovered (alpha, beta, gamma), although the predominant liver subtype (PPAR alpha) is the focus of the present investigation. The present proposal will examine the sequence of events initiated by peroxisome proliferators and culminated in altered gene expression. First, the appropriate molecular tools will be developed for the study of PPARalpha function. Recently, specific antibodies to PPARalpha and an epitope-labeled receptor (PPARalpha-Flag) have been developed which will help characterize the alpha subtype among the other possible forms. Photoaffinity ligand probes and stable cells lines which express PPARalpha-Flag will be pursued. Second, the subcellular localization of PPARalpha will be examined. Whether PPARalpha is primarily cytosolic in the inactivated state is an important consideration in receptor function. Confocal microscopy will be used to examine PPARalpha distribution in the inactivated and peroxisome proliferator-activated states. Third, the composition of the PPARalpha-protein complex will be determined. The specific interactions of PPARalpha with heat shock proteins and other factors often associated with steroid hormone receptors will be examined using several complementary approaches. Comparison of the receptor complex before and after peroxisome proliferator treatment will be important in the overall understanding of the signal transduction pathway. Fourth, determine if phosphorylation is an important regulator of PPARalpha function. Chemical and enzymatic digestion followed by phosphoamino and phosphopeptide analysis will be used to examine which residues are phosphorylated on PPARalpha and which are differentially affected by peroxisome proliferator treatment. The importance of each phosphorylated residue will be examined by introducing specific mutations in PPARalpha. Last, the process of transport of PPARalpha across the nuclear membrane will be examined. If confocal microscopy verifies that PPARalpha is present in the cytosol, the process that governs its transport into the nucleus will be examined. Fluorescently-labeled PPARalpha will be constructed and in vitro nuclear uptake studies implemented and identification of proteins acting as chaperones will be pursued. Taken together the proposed studies will greatly increase our knowledge of PPARalpha's mechanism of regulation and hence our understanding of an important class of carcinogen.
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