Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor α (TRα) from ligand-treated HeLa (α-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRα, and these interactions are mediated through the C terminus of TRα and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors [vitamin D receptor, retinoic acid receptor α, retinoid X receptor α, peroxisome proliferation-activated receptor (PPAR) α, PPARγ and, to a lesser extent, estrogen receptor] in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor α, PPARα, and PPARγ. Consistent with these results, TRAP220 moderately stimulates human TRα-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRα) and in cell free transcription systems (TRα and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRα during the function of the TRα–TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily.

The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.

The TRAP (thyroid hormone receptor-associated proteins) transcription coactivator complex (also known as Mediator) was first isolated as a group of proteins that facilitate the function of the thyroid hormone receptor. This complex interacts physically with several nuclear receptors through the TRAP220 subunit, and with diverse activators through other subunits. TRAP220 has been reported to show ligand-enhanced interaction with peroxisome proliferator-activated receptor gamma(2) (PPAR gamma(2)), a nuclear receptor essential for adipogenesis. Here we show that Trap220(-/-) fibroblasts are refractory to PPAR gamma(2)-stimulated adipogenesis, but not to MyoD-stimulated myogenesis, and do not express adipogenesis markers or PPAR gamma(2) target genes. These defects can be restored by expression of exogenous TRAP220. Further indicative of a direct role for TRAP220 in PPAR gamma(2) function via the TRAP complex, TRAP functions directly as a transcriptional coactivator for PPAR gamma(2) in a purified in vitro system and interacts with PPAR gamma(2) in a ligand- and TRAP220-dependent manner. These data indicate that TRAP220 acts, via the TRAP complex, as a PPAR gamma(2)-selective coactivator and, accordingly, that it is specific for one fibroblast differentiation pathway (adipogenesis) relative to another (myogenesis).

The TRAP220 component of the TRAP/SMCC complex, a mammalian homologof the yeast Mediator that shows diverse coactivation functions, interacts directly with nuclear receptors. Ablation of the murine Trap220 gene revealed that null mutants die during an early gestational stage with heart failure and exhibit impaired neuronal development with extensive apoptosis. Primary embryonic fibroblasts derived from null mutants show an impaired cell cycle regulation and a prominent decrease of thyroid hormone receptor function that is restored by ectopic TRAP220 but no defect in activation by Gal4-RARalpha/RXRalpha, p53, or VP16. Moreover, haploinsufficient animals show growth retardation, pituitary hypothyroidism, and widely impaired transcription in certain organs. These results indicate that TRAP220 is essential for a wide range of physiological processes but also that it has gene- and activator-selective functions.

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A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified. This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex. The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD. These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes.

Many peptide hormone and neurotransmitter receptors belonging to the seven membrane-spanning G protein-coupled receptor family have been shown to transmit ligand-dependent mitogenic signals in vitro. However, the physiological roles of the mitogenic activity through G protein-coupled receptors in vivo remain to be elucidated. Here we have generated G protein-coupled cholecystokinin (CCK)-B/ gastrin receptor deficient-mice by gene targeting. The homozygous mice showed a remarkable atrophy of the gastric mucosa macroscopically, even in the presence of severe hypergastrinemia. The atrophy was due to a decrease in parietal cells and chromogranin A-positive enterochromaffin-like cells expressing the H+,K+-ATPase and histidine decarboxylase genes, respectively. Oral administration of a proton pump inhibitor, omeprazole, which induced hypertrophy of the gastric mucosa with hypergastrinemia in wild-type littermates, did not eliminate the gastric atrophy of the homozygotes. These results clearly demonstrated that the G protein-coupled CCK-B/gastrin receptor is essential for the physiological as well as pathological proliferation of gastric mucosal cells in vivo.Cell proliferation and differentiation are regulated by a wide array of factors such as growth factors, cytokines, and hormones (1). Several peptide hormones such as bombesin/ gastrin-releasing peptide, angiotensin, and endothelin, and neurotransmitters such as serotonin and adrenaline have been shown to stimulate cell proliferation through their own seventransmembrane, heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors in vitro (2-5). Certain serotonin, acetylcholine, or adrenergic receptor subtypes were reported not only to stimulate cell proliferation but also to transform 3T3 fibroblasts in a ligand-dependent manner as do growth factor receptors (3-5). Very recently, G proteincoupled receptors have also been shown to involve tyrosine kinases and the Ras-mitogen-activating protein kinase pathway in their intracellular signaling as do growth factor and cytokine receptors (6-9). Although some peptides could promote the proliferation of a variety of human tumor cell lines in vivo as well as in vitro (10,11), the physiological significance of the mitogenic activity through the G protein-coupled receptor superfamily remains to be clarified.The peptide hormone, gastrin, is well characterized as a stimulant of gastric acid secretion. In addition, there is circumstantial evidence that gastrin presumably functions as a trophic factor for the gastrointestinal tissues (11, 12). Another peptide hormone, cholecystokinin (CCK), is also isolated as a stimulant of enzyme secretion by the pancreas (13). Because of the abundant expression of CCK in the central nervous system as well as in digestive organs, this hormone is also thought to act as a neurotransmitter or modulator in the brain (14). Moreover, the specific receptors for CCK and/or gastrin have been pharmacologically shown to be expressed in various human tumor cells and to stimulat...

Mediator is a general coactivator complex connecting transcription activators and RNA polymerase II. Recent work has shown that the nuclear receptor-interacting MED1/TRAP220 subunit of Mediator is required for peroxisome proliferator-activated receptor ␥ (PPAR␥)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPAR␥-Mediator interaction that requires the two LXXLL nuclear receptor recognition motifs on MED1/TRAP220 and, furthermore, we show that the intact LXXLL motifs are essential for optimal PPAR␥ function in a reconstituted cell-free transcription system. Surprisingly, a conserved N-terminal region of MED1/TRAP220 that lacks the LXXLL motifs but gets incorporated into Mediator fully supports PPAR␥-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPAR␥-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPAR␥ shows significantly reduced transcriptional activity in cells deficient for a subunit (MED24/ TRAP100) important for the integrity of the Mediator complex, indicating a general Mediator requirement for PPAR␥ function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPAR␥ and Mediator through MED1/TRAP220 is not essential either for PPAR␥-stimulated adipogenesis or for PPAR␥ target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPAR␥ transcriptional activity, our data indicate the presence of alternative mechanisms for Mediator recruitment, possibly through intermediate cofactors or other cofactors that are functionally redundant with MED1/TRAP220.Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a key regulator of transcriptional pathways important for adipogenesis (34). PPAR␥ Ϫ/Ϫ mice show a total absence of both brown and white adipose tissue. Furthermore, retrovirus vector-mediated ectopic expression of PPAR␥ alone can stimulate mouse embryonic fibroblasts (MEFs) to undergo adipogenesis. In such cells, the expression of CCAAT/enhancer-binding protein ␣ (C/EBP␣), another key transcriptional regulator of adipogenesis, and adipogenesis markers such as aP2, fatty acid synthase (FAS), and adipsin are induced in a PPAR␥-dependent manner.PPAR␥ and other nuclear hormone receptors comprise a superfamily of DNA binding transcription factors. However, they also require various transcriptional coactivators to activate, in a ligand-dependent manner, transcription of the specific target genes important for cell growth, homeostasis, and differentiation (36). These transcription coactivators often exist as multiprotein complexes. They may act either through chromatin remodeling and histone modification, after recruitment by promoter-bound nuclear receptors, or at steps involving subsequent preinitiation complex formation or function (transcription initiation and elongation). Transcripti...