Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR.

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 PPARgamma 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 PPARgamma. Deletion of the last 12 amino acids from the carboxyl terminus of PPARgamma resulted in the abolition of the interaction between PRIP and PPARgamma. PRIP also binds to PPARalpha, RARalpha, RXRalpha, ER, and TRbeta1, and this binding is increased in the presence of specific ligands. PRIP acts as a strong coactivator for PPARgamma in the yeast and also potentiates the transcriptional activities of PPARgamma and RXRalpha 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.

expression of target genes, in particular those associated with lipid metabolism (1)(2)(3). PPARs, which derive the designation by virtue of their ability to mediate predictable pleiotropic effects in response to peroxisome proliferators (2,4), consist of three isotypes, namely PPAR␣, PPAR␦ (also called PPAR␤), and PPAR␥, which are products of separate genes (1,(5)(6)(7). These PPAR isotypes appear to exhibit distinct patterns of tissue distribution and differ considerably in their ligand-binding domains, implying that they possibly perform different functions in different cell types (5)(6)(7)(8)(9). 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 (8), and evidence derived from mice with PPAR␣ gene disruption indicates that this receptor is essential for the pleiotropic responses induced by peroxisome proliferators (10). Also worth noting is that the fatty acyl-CoA oxidase, the first and the rate-limiting enzyme of the peroxisomal fatty acid ␤-oxidation system, plays a critical role in the metabolism of biological/natural ligands of PPAR␣ in that mice with fatty acyl-CoA oxidase gene disruption exhibit sustained spontaneous up-regulation of PPAR␣-responsive genes (11). PPAR␦ isotype is ubiquitously expressed in the adult tissues and binds the same ligands as other PPARs (7,12,13). Recent evidence suggests that nonsteroidal anti-inflammatory drugs inhibit colon carcinogenesis through inhibition of PPAR␦ and that PPAR␦ functions as a target for adenomatous polyposis coli (14). PPAR␥, which is expressed predominantly in adipose tissue, as well as in certain epithelial cells such as those of the colonic mucosa, mammary epithelium, and urinary bladder (8,9), plays a pivotal role in adipocyte development and lipid homeostasis (15)(16)(17). Although a great deal is known about genes regulated by PPAR␣, especially in conjunction with peroxisome proliferator-induced pleiotropic responses (2), there is very little information on PPAR␥-and PPAR␦-regulated genes in specific cell types, despite the fact that many of the ligands identified to date interact with one or more PPAR isotypes (12,13).
Evidence accumulated so far indicates that the transcriptional activation of nuclear receptors after ligand binding involves the participation of cofactors termed nuclear receptor corepressors and coactivators (18,19). The current model suggests that unliganded receptors are maintained in a repressed state by nuclear receptor corepressors such as N-CoR (20) and SMRT (21). Upon ligand binding, the corepressor(s) dissociates from the nuclear receptor, thus enabling the liganded nuclear receptor to recruit a complex of proteins, called nuclear receptor coactivators, that bridge the nuclear receptors with basal transcription machinery (18). The coactivators identified in recent years include the SRC-1 family, which has three member ((SRC-1 (22-24), TIF-2 (SRC-2, GRIP1) (25, 26); p/CIP (ACTR, AIB1, RAC3, and SRC-3) (26 -28, 30)); CBP/p300 (31, * This work was supported by National Institutes of Health Grants R37 GM23750 (to J. K. R.) and DK28492 (to Y. S. K.), by Department of Veterans Affairs merit review grants (to A. V. Y. and M. S. R.), and by the Joseph L. Mayberry, Sr. Endowment Fund. This work was presented at the Keystone Symposium on PPARs in April 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF216186.
The identification and functional analysis of many of the known nuclear receptor coactivators resulted from studies utilizing the yeast two-hybrid screening (22,24,34,35). Alternative strategies that focused on the identification and purification of the putative transcriptional complexes yielded valuable information regarding the transcriptional activation of thyroid hormone receptor (TR), vitamin D receptor, and other transcriptional units (41)(42)(43)(44). The thyroid hormone receptor-associated proteins (TRAPs) can increase the transactivation activity of the liganded TR (42). Likewise, multiple protein complexes such as DRIP (vitamin D3 receptor interacting protein) and ARC (activator-recruited cofactor) are identified and found to be required for the transcriptional activation of multiple transcription factors including SP1, p53, NF-B, vp16, and nuclear receptors (43,44). It is of special interest that this complex acts on the nuclear receptors by binding to PBP (PPARBP), a nuclear receptor coactivator we originally identified as a coactivator for PPAR (34). More recently, we demonstrated that the PBP gene (PPARBP) is overexpressed in about 50% of breast cancers examined, and the PBP gene amplification was noted in about 25% of breast tumors (45). These observations underscore the importance of nuclear receptor coactivators in transcriptional activation and also point to their possible role in neoplastic conversion. Here we report the characterization of a new nuclear receptor coactivator, designated PRIP identified during yeast two-hybrid screening. PRIP contains two LXXLL signature motifs at amino acid positions 892-896 and 1496 -1500, but the latter motif did not appear necessary for binding to PPAR␥. PRIP acts as a strong coactivator for PPAR␥ in yeast and also potentiates transcriptional activities of PPAR␥ and RXR␣ in mammalian cells. We further show that a truncated form of PRIP (amino acids 786 -1132) functions as a dominant-negative repressor, suggesting that PRIP is a genuine coactivator.
Isolation of Mouse PRIP cDNA-A partial cDNA encoding a peptide that interacts with PPAR␥ was isolated by yeast two-hybrid screening of a mouse liver cDNA library as described previously (24,34). We then obtained the full-length cDNA from a ZAP newborn mouse kidney cDNA library with RACE PCR using a kit from Life Technologies, Inc. according to the manufacturer's protocol. The PCR products were cloned into pGEM-T (Promega), and three independent clones were sequenced. The full-length cDNA that we cloned has been designated PRIP, or peroxisome proliferator activated receptor interacting protein to reflect its ability to bind PPARs and to distinguish it from PBP (PPARBP), which we cloned earlier (34).
Northern Blot Analysis-Mouse multiple tissue Northern blot (CLONTECH) containing 2 g of poly(A) RNA in each lane was probed with 32 P-labeled PRIP full-length cDNA according to the conditions outlined by the manufacturer.
Characterization of Mouse PRIP Gene-By screening a BAC mouse library (Genome Systems, St. Louis, MO) with PCR primers 5Ј-AGAG-AATGCCCGTGCAGCAG-3Ј and 5Ј-TTGCTGGGGAGTTGCACCTT-3Ј, one BAC clone (#21803) containing the PRIP gene was obtained. The BAC clone was digested with the appropriate restriction enzymes and subcloned as necessary. The intron sizes and exon/intron boundaries were determined by Southern blotting, restriction enzyme mapping, PCR, sequencing, and a comparison of sequences between the cDNA and genomic DNA.
Quantitative ␤-Galactosidase Assays-Appropriate plasmids were cotransformed into yeast HF7C, plated on selective media plates in the presence or absence of 10 Ϫ5 M BRL49653, a PPAR␥ ligand, and then incubated for 4 days at 30°C. For each assay, five colonies were suspended in 150 l of buffer Z (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 35 mM 2-mercaptoethanol). Equal number of cells in suspension was pelleted by centrifugation, and ␤-galactosidase activity was determined by a chemiluminescent reporter protocol (Galacto-light kit, Tropix, Bedford, MA).
GST Pull-down Assays-The GST alone and GST-fusion proteins GST-PRIP, GST-RXR␣, GST-RAR␣, and GST-PPAR␥ were produced in Escherichia coli DH5␣ and bound to glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Pharmacia Biotech). In vitro translation was performed using rabbit reticulocyte lysate (Promega) and [ 35 S]methionine. In a GST pull-down assay, a 10-l aliquot of GST fusion protein loaded on glutathione-Sepharose beads was incubated with 5 l of [ 35  Immunoprecipitation-EBNA cells (293) were transfected with pcDNA3.1-HA-PRIP, PFLAG-PRIP-T, or PFLAG-PRIP-M2 along with PCMV-PPAR␥ by the calcium precipitation method. 24 h after transfection, the cell was harvested. The lysate was immunoprecipitated with anti-PPAR␥ or control serum. The precipitates were resolved on SDS-PAGE and subjected to Western blot analysis.
Cell Culture and Transfection-CV-1 cells (1 ϫ 10 5 ) were plated in six-well plates and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 24 h before transfection. Cells were transfected for 5 h with 1.25 g of luciferase reporter plasmid DNA, 0.75 g of the appropriate expression plasmid DNA, and 0.5 g of ␤-galactosidase expression vector pCMV␤ (CLONTECH) DNA using the N- [1-(2,3-dioleoyloxy)propyl]-N,N,N,-trimethylammonium methylsufate-mediated transfection method (Roche Molecular Biochemicals). Cell extracts were prepared 36 h after transfection and assayed for luciferase and ␤-galactosidase activities. (Tropix). For immunofluorescence localization, COS-1 cells were fixed in ice-cold methanol for 5 min and washed twice with Tris-buffered saline (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM KCl). After incubating with antibody for 1 h at 27°C, cells were washed three times with Tris-buffered saline, and mounted with a Prolong anti-fade kit (Molecular Probes).

Molecular
Cloning and Expression of PRIP-Using the PPAR␥ ligand-binding domain as a bait in the yeast two-hybrid system, we isolated from a mouse liver cDNA library a partial cDNA encoding a PPAR-interacting protein, designated PRIP. The full-length PRIP cDNA was cloned through RACE PCR. It has an open reading frame of 6204 nucleotides that encodes a protein of 2068 amino acids ( Fig. 1) with an estimated molecular mass of ϳ 220 kDa. It contains two LXXLL motifs that have been shown to be essential for transcriptional coactivators to interact with nuclear receptors (26,36). One motif is at amino acid positions 892-896, and the second is located at amino acid positions 1496 -1500. The PRIP cDNA fragment directly recovered by yeast two-hybrid screening encodes amino acids 786 -1132 and contains the first LXXLL motif. When the amino acid sequence of PRIP was compared with known sequences in GeneBank TM , it showed 96% homology to KIAA0181 except that the first 58 amino acids were missing in the KIAA0181 cDNA. The KIAA0181 cDNA was isolated from human myeloid KG-1 cells (46). The function of this protein has not been investigated. Mouse PRIP contains a stretch of 31 glutamine residues from amino acids Glu 261 -Glu 291 , whereas its human homologue has 25 contiguous glutamines. The PRIP was expressed as an 8.5-kb transcript in all tissues examined including blood, spleen, prostate, thymus, colon, testis, ovary (Fig. 2), liver, and kidney, among others (data not shown). A high level of expression of PRIP mRNA has been noted in the testis. A second ϳ4-kb transcript is detected in the testis, which may represent an isoform for PRIP. Transfection of a plasmid containing two HA epitopes linked to the carboxyl-terminal portion of PRIP protein into COS-1 cells revealed the nuclear localization of the expressed PRIP protein (Fig. 3).
Genomic Organization of the Mouse PRIP Gene-To isolate the PRIP gene, a BAC mouse library was first screened by PCR using a pair of oligonucleotide primers. One clone covered the entire PRIP gene. The size of the introns and the exon/intron boundaries were determined by XL-PCR and by sequencing. The PRIP gene consists of 13 exons and spans about 47.5 kb (Table I). Similar to the gene structures of mSRC-1 and PBP-1, about half of the PRIP cDNA is encoded by a single large exon (exon 9). The sequences of all of the splice acceptor and donor sequences conformed to the GT-AG consensus rule for splicing (47).
Interaction of PRIP with PPAR␥ in Yeast-We investigated the influence of PPAR␥ ligand BRL49653 on the binding of PRIP to PPAR␥ in yeast. PGADH10-PRIP-T (amino acids 786 -1132), which was isolated by yeast two-hybrid screening and expressed as the fusion protein with the GAL4 activation domain, was cotransformed with Gal-PPAR␥ into yeast HF7c and the ␤-galactosidase activity measured as an indication of the relative strength of interaction in the presence or absence of ligand. In the absence of ligand, we observed an interaction between PRIP and PPAR␥, which resulted in an ϳ20-fold increase in the ␤-galactosidase activity (Fig. 4). The presence of the ligand BRL49653 further potentiated this interaction between PRIP and PPAR␥, leading to an ϳ8-fold increase in the ␤-galactosidase activity (Fig. 4).
The extreme carboxyl-terminal region of the ligand-binding domain conserved among the nuclear receptors has been shown to be essential for the ligand-dependent transcriptional activation (48). To determine whether this region is important for the binding of PPAR␥ to PRIP, GAL-PPAR␥⌬12, which lacks the last 12 amino acids from the carboxyl terminus of PPAR␥, was cotransformed with PGADH10-PRIP-T into HF7c. The presence of PRIP did not lead to an increase in ␤-galactosidase activity either in the presence or absence of the ligand, indicating that the mutation eliminates the ability of PPAR␥ to bind PRIP (Fig. 4). As LXXLL is the signature motif for the binding of cofactor to nuclear receptors, we tested whether the second LXXLL in the PRIP can bind to PPAR␥. Plasmid PGADH10-PRIP-M2, expressing a fusion protein between GAL activation domain and the partial PRIP cDNA (amino acids FIG. 2. Northern blot analysis of PRIP mRNA. A mouse multiple tissue Northern blot (CLONTECH) containing 2 g of poly(A) RNA for each tissue was probed with 32 P-labeled full-length PRIP cDNA and then exposed to film at Ϫ80°C with intensifier screens for 24 h. The transcript size of PRIP is ϳ8.5 kb in all tissues examined. An additional ϳ4-kb transcript is present in the testis. 1440 -1556) containing the second LXXLL, was cotransformed with GAL-PPAR␥ into HF7C. The presence of the second LXXLL motif did not increase the activity of ␤-galactosidase (Fig. 4). Therefore, we conclude that the second LXXLL motif is not necessary for the interaction between PRIP and PPAR␥, but the first LXXLL motif appears necessary and sufficient.
Interaction of PRIP with Nuclear Receptor in Vitro-The direct interaction between PRIP and PPAR␥ was tested further by an in vitro GST binding assay with bacterially generated GST-PPAR␥ fusion protein and in vitro translated PRIP. Although the immobilized GST-PPAR␥, but not GST alone, retained the [ 35 S]methionine-labeled PRIP in the presence and absence of PPAR␥ ligand BRL49653, the addition of the ligand strongly enhanced this interaction (Fig. 5). Moreover, PRIP also showed the ligand-dependent interaction with RXR␣, RAR␣, ER, and TR␤1, as well as PPAR␣ (Fig. 5).
Interaction between PRIP and PPAR␥ in Intact Cells-To determine whether PPAR␥ and PRIP form a complex in the context of intact cells, a vector encoding PRIP with a carboxylterminal HA epitope was constructed and expressed in 293 cell line derived from primary embryonal human kidney (ATCC CRL 1573). The potential complex was immunoprecipitated with anti-PPAR␥ and analyzed by immunoblotting using anti-HA. This study established the ligand-dependent interaction between PPAR␥ and PRIP in vivo (Fig. 6). A similar study revealed that PRIP-T (amino acids 786 -1132) containing the first LXXLL but not PRIP-M2 (amino acids 1440 -1556), which contains the second LXXLL motif, interacts with PPAR␥ in vivo (Fig. 6).
PRIP Acts as a Strong Coactivator for PPAR␥ in Yeast-To elucidate if PRIP acts as a coactivator in the yeast, the GAL-PPAR␥ was coexpressed with full-length PRIP in yeast strain HF7c. PRIP moderately increases the transcriptional activity of PPAR␥ in the absence of ligand (ϳ3-fold), whereas the increase is ϳ22-fold in the presence of ligand BRL49653 (Fig. 7).
PRIP Potentiates the Transcriptional Activities of PPAR␥ and RXR␣ in Mammalian Cells-To determine whether PRIP serves as a coactivator for PPAR␥ in mammalian cells, we overexpressed PRIP in CV-1 cells along with PPAR␥ and monitored the transcriptional activity of PPAR␥ with expression of the peroxisome proliferator response element-linked reporter luciferase gene. PRIP moderately increased the transcription of the luciferase gene by about 1.9-fold in the presence of BRL49653 (Fig. 8). When PRIP was co-expressed with RXR␣, it increased the transactivation capacity of RXR␣ by 3.9-fold.
Truncated PRIP with the First LXXLL Motif Functions as a Dominant-negative Form-To further confirm PRIP as a coactivator for PPAR␥, we overexpressed in CV-1 cells, a truncated form of PRIP (PRIP-T, amino acids 786 -1132), which contains the first LXXLL and has been shown to interact with PPAR␥. Cotransfection of PRIP-T resulted in a decrease in a PPAR␥mediated transcription of reporter in the presence of ligand, whereas no significant change was detected in the absence of the ligand (Fig. 9A). PRIP-M2 (amino acids1440 -1556), which contains the second LXXLL motif that does not bind PPAR␥, cannot inhibit the transcriptional activity of PPAR␥. Moreover, the suppressive effect of PRIP-T can be reversed by cotransfection with wild type PRIP. When tested with RXR␣, the inhibitory effect of truncated PRIP on the transcriptional activity was stronger in comparison with PPAR␥ (Fig. 9B).

DISCUSSION
Using a yeast two-hybrid system with Gal4-PPAR␥ as bait to screen a mouse liver cDNA library, we isolated a cDNA designated as PRIP, which is a nuclear protein with 2068 amino acids. In this report, we present data on the initial characterization of this coactivator protein, which reveals a strong ligand and AF-2-dependent interaction with PPAR␥ and several other nuclear receptors (Fig. 5). The entire PRIP gene consists of 13 exons and extends ϳ47.5 kb. Northern analysis showed that   TTGTC PRIP mRNA is ubiquitously expressed in adult mouse tissues. PRIP failed to interact with mutated PPAR␥, which does not contain the last 12 amino acids at the extreme carboxyl terminus of the ligand-binding domain. This AF-2 region is important for the transcriptional activation function of the nuclear receptors but dispensable for the hormone binding and heterodimerization function (48). Our observations support the role for PRIP as a coactivator for PPAR␥, because many coactivators specifically interact with the AF-2 domain of nuclear receptors. PRIP contains two LXXLL motifs that have been reported to be necessary and sufficient for the binding of several cofactors to nuclear receptors (26,36). However, we found that only one of these LXXLL motifs, located at residues 892-896, is necessary for the interaction of PRIP with the nuclear receptors. We found that the second LXXLL motif is unable to bind to PPAR. Although truncated PRIP, which contains the first LXXLL motif, binds to PPAR␥, detailed studies on mapping of the binding site and the mutational analysis of this motif are needed to ascertain the regions in PRIP protein that might play a role in protein-protein interactions. In PRIP. we found a continuous stretch of 31 glutamine residues (Fig. 1). CAG repeats, detected in a number of proteins, are critical to inherent neurodegenerative conditions such as Huntington disease (49). In these conditions, the number of CAG repeats is more than 40, and the abnormal proteins accumulate as aggregates forming intranuclear inclusions. Also of interest is the fact that a continuous stretch of 29 CAG repeats has been detected in ACTR/AIB1, another nuclear receptor coactivator (27,28). Likewise, TATA-binding protein and androgen receptor reveal continuous stretches of 38 and 22 glutamines, respectively (50,51). It is possible that whereas the LXXLL motifs facilitate interaction between coactivators and nuclear receptors, the polyglutamine tracts might promote high affinity interactions between polypeptides carrying polyglutamines, especially with TATA-binding protein (50).
We found that PRIP functions as a much stronger coactivator in the yeast when compared with its coactivator activity in the mammalian cells used in this study. This finding may be explained in part by the attenuation of the coactivator function by other factors involved in mammalian cell transcription and the relative lack of abundance of these factors in yeast; It could also be due to the fact that mammalian cells already contains endogenous PRIP, whereas data bank searches failed to reveal a PRIP homologous protein in yeast. We also found no PRIP homologue in the Caenorhabditis elegans, although this orga- nism contains many different nuclear receptors, implying that PRIP possibly emerged later during the evolutionary period as a component of a more complex transcriptional regulatory process in mammals.
In CV-1 cells, PRIP was able to increase the transcriptional activity of RXR␣ to a higher degree than that of PPAR␥. Consistent with this observation is the finding that the truncated form of PRIP exerts a stronger inhibitory effect on the transcriptional activation of RXR␣ as compared with PPAR␥. Therefore, it is possible that PRIP may contribute more to the RXR transcriptional activation than to PPAR␥ under certain circumstances. As PRIP also binds to PPAR␣, RAR␣, RXR␣, ER, and TR␤1, and as this binding is increased in the presence of specific ligands, we expect PRIP to function as a coactivator for a variety of nuclear receptors. The effectiveness of the coactivator function may, to some extent, depend on the nature of a given nuclear receptor and the cell type involved.
There is now increasing evidence to support the contention that the transcriptional activation of nuclear receptors involves at least two classes of nuclear receptor cofactors, termed corepressors and coactivators (1). Corepressors are associated with unliganded receptors to mediate repression; the ligand binding causes the dissociation of corepressor from the nuclear receptor. Subsequently, the liganded nuclear receptors actively recruit coactivators to facilitate transcription. The coactivators identified so far can be broadly categorized into two groups. The first category of coactivators, such as those belonging to the p300/CBP and SRC-1 family, possess histone acetyltransferase activities and as a result are involved in the modification of chromatin (37)(38)(39)(40). The second group of coactivators, which are devoid of histone acetyltransferase activity, appear to serve as the facilitators linking the receptor complex to the basal transcription machinery (1). In this context, PBP (PPARBP), which we cloned and characterized earlier as a nuclear receptor coactivator (34), has been found to be a critical component of the TRAP/DRIP/CRSP/ARC complexes (41)(42)(43)(44) and appears more than likely to be involved in the second step of coactivation, i.e. linking the receptors with the basal transcription machinery. Whether PRIP, which we have now cloned, is part of these, or other, multiprotein transcriptional complexes needs to be ascertained.
The reason for the existence of a multitude of coactivators remains elusive. One possibility is that different coactivators may preferentially participate in the transcription of specific target genes, as exemplified by the finding that CBP and p300 tend to exhibit target gene preference (52). It is also possible that specific nuclear receptors use only a distinct subset of coactivators for optimal transcriptional activity and that such a subset of coactivators may not function effectively for other members of the nuclear receptor superfamily because of novel sequence determinants in peroxisome proliferator signaling (53) and other complex cross-talk mechanisms that control transcription (54). For example, SRC-1 null mice are viable and fertile and exhibit only a subtle, or no, phenotypic alterations when evaluated for certain nuclear receptor functions (29,55), suggesting functional redundancy among coactivators. On the other hand, mice with PBP (PPARBP) gene ablation exhibited embryonic lethality (56). Recently, we also found overexpression and amplification of the PBP gene in breast cancer (45). Another nuclear receptor coactivator, AIB1/ACTR (27,28), was also found amplified and overexpressed in breast cancer (28). These observations raise interesting possibilities regarding the role of coactivators in cell proliferation, differentiation, and CV-1 cells were cotransfected with 1 g of reporter construct PPRE-TK-LUC, 0.25 g of PCMV-mPPAR␥, and 0.5 g of PCMV␤ with 10 Ϫ5 M BRL49653. In addition, 0.25 g of PFLAG-PRIP-T or PFLAG-PRIP-M2, along with different amounts of PCMV-PRIP, was included as indicated. The empty pcDNA 3.1 was added to make the total amount of the plasmid for each transfection equal. Luciferase activity is presented as percent where induced mPPAR␥ in the presence of ligand is arbitrarily set at 100. Results are the mean of four independent transfections normalized to the internal controls of ␤-galactosidase expression. B, RXR␣. The transfection assay was as done for PPAR␥ except using RARE-TK-LUC and PCMX-RXR␣. neoplastic change. Additional studies are needed to determine the functional role of PRIP in embryonic development and whether this gene is also amplified and overexpressed in certain neoplasms.