To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR 'y (mPPARy) gene. This gene extends > 105 kb and gives rise to two mRNAs (mPPARy1 and mPPARy2) that differ at their 5' ends. The mPPARy2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPARyl and reveals a different 5' untranslated sequence.We show that mPPARy1 mRNA is encoded by eight exons, whereas the mPPARy2 mRNA is encoded by seven exons.Most of the 5' untranslated sequence of mPPARy1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPARy2 are encoded by one exon, which is located between the second and third exons coding for mPPARyl. The last six exons of mPPARy gene code for identical sequences in mPPARyl and mPPARy2 isoforms. The mPPARyl and mPPARy2 isoforms are transcribed from different promoters. The mPPARy gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Gal4-PPAR␥ as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator-1 (SRC-1) as a PPAR transcriptional coactivator. We now report the isolation of a cDNA encoding a 165-kDa PPAR␥-binding protein, designated PBP which also serves as a coactivator. PBP also binds to PPAR␣, RAR␣, RXR, and TR␤1, and this binding is increased in the presence of specific ligands. Deletion of the last 12 amino acids from the carboxyl terminus of PPAR␥ results in the abolition of interaction between PBP and PPAR␥. PBP modestly increased the transcriptional activity of PPAR␥, and a truncated form of PBP (amino acids 487-735) acted as a dominantnegative repressor, suggesting that PBP is a genuine coactivator for PPAR. In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. In situ hybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation.The peroxisome proliferator-activated receptors (PPARs) 1 are a group of transcription factors that regulate the expression of target genes, in particular those associated with lipid metabolism (1, 2). PPARs, which derive the designation by virtue of their ability to mediate predictable pleiotropic effects in response to peroxisome proliferators (1,3,4), are members of the nuclear receptor superfamily (5, 6). Three isotypes of PPARs, namely PPAR␣, PPAR␦ (also called ␤ or NUC-1), and PPAR␥ have been identified as products of separate genes from Xenopus, rodents, and humans (1, 7-12). These PPAR isotypes appear to exhibit distinct patterns of tissue distribution and differ considerably in their ligand binding domains, suggesting that they possibly perform different functions in different cell types (7,13,14). Indeed, of the three isotypes, PPAR␣ expression is relatively high in hepatocytes, enterocytes, and the proximal tubular epithelium of kidney when compared with other cell types (13,14), and evidence derived from mice with PPAR␣ gene disruption indicates that this receptor is essential for the pleiotropic responses induced by peroxisome proliferators (15). Several structurally diverse peroxisome proliferators, specific fatty acids, and eicosanoids act as ligands for PPAR␣ (4, 16 -19). Although PPAR␦ isotype is ubiquitously expressed and binds the same ligands as PPAR␣ (18,19), its functional significance remains largely elusive. PPAR␥ exists as two isoforms, PPAR␥1 and PPAR␥2, as a consequence of alternate promoter usage in the gene encoding this receptor (8,20,21). While PPAR␥1 isoform expression is restricted to liver and few other organs (8, 14), the PPAR␥2 i...

We previously isolated and identified steroid receptor coactivator-1 (SRC-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/PPARBP) as coactivators for PPAR, using the ligand-binding domain of PPAR␥ as bait in a yeast two-hybrid screening. As part of our continuing effort to identify cofactors that influence the transcriptional activity of PPARs, we now report the isolation of a novel coactivator from mouse, designated PRIP (peroxisome proliferator-activated receptor interacting protein), a nuclear protein with 2068 amino acids and encoded by 13 exons. Northern analysis showed that PRIP mRNA is ubiquitously expressed in many tissues of adult mice. PRIP contains two LXXLL signature motifs. The amino-terminal LXXLL motif (amino acid position 892 to 896) of PRIP was found to be necessary for nuclear receptor interaction, but the second LXXLL motif (amino acid position 1496 to 1500) appeared unable to bind PPAR␥. Deletion of the last 12 amino acids from the carboxyl terminus of PPAR␥ resulted in the abolition of the interaction between PRIP and PPAR␥. PRIP also binds to PPAR␣, RAR␣, RXR␣, ER, and TR␤1, and this binding is increased in the presence of specific ligands. PRIP acts as a strong coactivator for PPAR␥ in the yeast and also potentiates the transcriptional activities of PPAR␥ and RXR␣ in mammalian cells. A truncated form of PRIP (amino acids 786 -1132) acts as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.

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The nuclear receptor coactivators participate in the transcriptional activation of specific genes by nuclear receptors. In this study, we report the isolation of a nuclear receptor coactivator-interacting protein from a human liver cDNA library by using the coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) (ASC2͞AIB3͞RAP250͞NRC͞TRBP) as bait in a yeast twohybrid screen. Human PRIP-interacting protein cDNA has an ORF of 2,556 nucleotides, encodes a protein with 852 amino acids, and contains a 9-aa VVDAFCGVG methyltransferase motif I and an invariant GXXGXXI segment found in K-homology motifs of many RNA-binding proteins. The gene encoding this protein, designated PRIP-interacting protein with methyltransferase domain (PIMT), is localized on chromosome 8q11 and spans more than 40 kb. PIMT mRNA is ubiquitously expressed, with a high level of expression in heart, skeletal muscle, kidney, liver, and placenta. Using the immunofluorescence localization method, we found that PIMT and PRIP proteins appear colocalized in the nucleus. PIMT strongly interacts with PRIP under in vitro and in vivo conditions, and the PIMT-binding site on PRIP is in the region encompassing amino acids 773-927. PIMT binds S-adenosyl-L-methionine, the methyl donor for methyltransfer reaction, and it also binds RNA, suggesting that it is a putative RNA methyltransferase. PIMT enhances the transcriptional activity of peroxisome proliferator-activated receptor ␥ and retinoid-X-receptor ␣, which is further stimulated by coexpression of PRIP, implying that PIMT is a component of nuclear receptor signal transduction apparatus acting through PRIP. Definitive identification of the specific substrate of PIMT and the role of this RNA-binding protein in transcriptional regulation remain to be determined.