Discrete Roles for Peroxisome Proliferator-Activated Receptor γ and Retinoid X Receptor in Recruiting Nuclear Receptor Coactivators

Peroxisome proliferator-activated receptor γ (PPARγ) plays pivotal roles in mediating adipocyte differentiation and in modulating insulin sensitivity. Overexpression of PPARγ in fibroblasts and myoblasts drive these cells to differentiate into adipocytes (34, 45). In addition, PPARγ inhibits the growth of several human tumor cell lines in culture in response to various synthetic PPARγ ligands (2, 14, 35). For example, PPARγ is able to induce the growth arrest and differentiation of human liposarcoma cells (10, 46). PPARγ is a member of the nuclear hormone receptor superfamily. Like other nuclear receptors, PPARγ is comprised of an amino-terminal ligand-independent transactivation region (AF-1) (50), a central DNA-binding domain (DBD), and a carboxy-terminal ligand-binding domain (LBD) that contains a second, ligand-dependent transactivation surface (AF-2). Typical of many nuclear receptors, PPARγ associates with DNA targets as a heterodimer with the 9-cis-retinoic acid receptor, RXR; transactivation requires the high-affinity binding of PPARγ- or RXR-specific ligands to their respective receptors in the context of the heterodimer. The first natural PPAR-responsive element (PPRE) was identified in the promoter of the acyl coenzyme A (acyl-CoA) oxidase gene (13, 48), and the analyses of all identified PPREs have revealed a consensus sequence, 5′-AACTAGGNCA A AGGTCA-3′ (39). The properties of the PPRE, including an extented 5′ half-site and an adenine as the spacing nucleotide between two half-sites, contribute to the discrimination between PPRE and other direct repeat (DR-1)-type nuclear receptor response elements (19, 26, 29, 49). Further characterization of PPAR-RXR binding to the consensus PPRE has revealed a polarity in binding, such that PPAR and RXR occupy the 5′ and 3′ half-sites, respectively (12); this polarity is the opposite of that observed for other nuclear receptor-RXR heterodimers. PPARγ, and its related subtypes, PPARα and PPARδ, were originally discovered as orphan receptors, but this was followed by the relatively rapid identification of both natural and synthetic ligands (for a review, see reference 51). The natural ligands for PPARγ include a number of fatty acid and eicosanoid derivatives. In addition, the J-series of prostagladins derived from PGD2 have also been established as potent PPARγ ligands (53). The terminal metabolite 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) binds and activates PPARγ at micromolar concentrations and is currently the best-characterized, naturally occurring PPARγ ligand (16, 28). The components of oxidized low-density lipoprotein, such as 13-hydroxyoctadecadienoic acid and 15-hydroxyeicosatetraenoic acid, are also considered as naturally occurring PPARγ ligands (23). A class of antidiabetic agents known as thiazolidinediones (TZDs) were the first identified synthetic compounds that bound with high affinity to PPARγ (31). TZDs are able to induce PPRE-directed transactivation in adipocytes and can promote adipocyte differentiation (24, 27). More importantly, the potency of TZDs to bind PPARγ is closely related to their glucose-lowering activity in rodents, suggesting that the antidiabetic effects of TZDs occur primarily through PPARγ (1, 31, 52). Recently, a series of tyrosine-based PPARγ agonists, exemplified by GW1929, were reported (8, 9, 22). These compounds are non-TZD antidiabetic drugs and are among the most potent PPARγ agonists. For example, GW1929 exhibited equal antihyperglycemic activity at a concentration 1,000-fold lower than troglitazone in ZDF rats (3, 22), and this activity closely matches their differences in PPARγ affinity and transactivation. Other structurally diverse PPARγ agonists have also been described. Hypolipidemic agents, such as the fibrate analogue GW2331, have been shown to have activity on PPARγ. Besides PPARγ-specific ligands, PPARγ-mediated responses can also be elicited through RXR-specific ligands. Unlike many other nuclear receptors, RXR is viewed as a permissive partner for PPARγ, whereby the former can activate transcription in response to RXR-specific ligands in the context of the PPARγ-RXR heterodimer (for a review, see reference 30). Overexpression of PPARγ and RXR leads to transcriptional activation from a PPRE in response to 9-cis-retinoic acid, and simultaneous administration of both PPAR- and RXR-specific ligands to transfected cells results in an additive induction of reporter gene expression (19, 26, 29). Moreover, RXR agonists can enhance the sensitivity of diabetic and obese mice to insulin, demonstrating the ability of RXR to activate PPAR-RXR signaling pathway in vivo (36). The mechanisms by which nuclear receptors regulate transcription from target genes are not yet fully elucidated. However, compelling evidence indicates that ligand binding results in a conformational change within the receptor that permits the dissociation of corepressors, such as NCoR and SMRT, that bridge the binding of histone deacetylases, and the concomitant association of coactivators, such as the p160 class that link histone acetyltransferases (HAT) such as CBP/p300 and PCAF to the receptor (reviewed in references 17 and 20). Using an immobilized vitamin D3 receptor (VDR) LBD affinity column, we previously isolated a complex of at least 15 VDR interacting proteins (DRIPs), ranging in size from 30 to 250 kDa, from Namalwa B-cell nuclear extracts (41, 42). These proteins selectively bind as a complex to VDR in a 1,25(OH)2D3-dependent manner. A single subunit, DRIP205 (also known as TRAP220), anchors the other 14 proteins comprising the DRIP complex to nuclear receptors. DRIP is essentially identical to the thyroid receptor-interacting TRAP-SMCC complex (15, 21) and the ARC coactivator complex (37). Importantly, the mouse homologue of DRIP205 was also identified as a PPARγ-binding protein (PBP-PPARBP) in a yeast two-hybrid screen using the PPARγ LBD as a bait (55). Overexpression of PBP moderately enhanced PPARγ-mediated transcriptional induction in a ligand-dependent manner, suggesting a regulatory role for the DRIP complex in PPARγ-mediated transactivation. The DRIP complex contains several subunits found within the RNA polymerase II (Pol II)-interacting mediator-SRB complexes. We have recently demonstrated that DRIP is physically distinct from the p160-CBP complex (40). This suggests that p160-CBP coactivators might act initially to remodel nucleosomes through histone modifying activities and that DRIP, perhaps through mediator-SRB subunits, might function by directly targeting RNA Pol II holoenzyme to promoters. Moreover, the fact that both the p160 and DRIP coactivators utilize the same surface within the receptor LBD (i.e., the AF-2) suggests that they do not interact simultaneously with nuclear receptors, and a key question is what factors are influencing the recruitment by the receptor of one complex over the other. In the work presented here, we report that PPARγ and RXR in the context of a DNA-bound heterodimer play distinct roles in recruiting DRIP versus p160 coactivators. We find that PPARγ and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner, where RXR-specific ligands only induced p160 binding to RXR, and PPARγ-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARγ. This selectivity was not observed in interaction assays off DNA, implying that the specificity of the coactivator binding in response to ligand is strongly influenced by allosteric effects of DNA-bound heterodimers. These results also suggest that the cooperativity of PPARγ- and RXR-specific ligands on transactivation may occur at the level of coactivator recruitment and provides an explanation for how one complex is recruited over the other despite the similarities they appear to share in the resident determinants for nuclear receptor interaction.