Two novel and related C 2 H 2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors.COUP-TFI, 1 ARP1/COUP-TFII, and Ear2/COUP-TFIII have been grouped in the same subfamily of orphan nuclear receptors based on sequence similarity (1-3), evolutionary analysis (4), and a common capacity to repress ligand-dependent transcriptional activation of target genes mediated by other nuclear receptors, such as retinoic acid (5-10), thyroid hormone (8), estrogen (11)(12)(13)(14), and vitamin D 3 (9) receptors as well as peroxisome proliferator-activated receptor α (PPARα;Ref. 15).COUP-TFs play important roles in pattern formation in the developing nervous systems of Xenopus (16) and Drosophila (17). Deletion of the COUP-TFI gene in the mouse results in * This work was supported in part by American Heart Association Grant 9640219N; NIEHS, National Institutes of Health, Grants ES00210 and ES00040; the Oregon State University College of Pharmacy; and the Laboratory of Molecular Pharmacology. 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.© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. ∥ An established investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed: MATERIALS AND METHODS Yeast Two-hybrid Screening and cDNA CloningYeast two-hybrid screening was conducted as described previously (21) using the hinge region and putative ligand binding domain of ARP1 (amino acids 144-414) as a bait. Fragments corresponding to CTIP1 and CTIP2 (see Fig. 1A) were used to screen mouse cDNA libraries obtained from CLON-TECH and from Dr. René Hen (Columbia University), yielding several overlapping clones. These overlapping clones were then used to prepare a full-length CTIP1 construct that was inserted into the eukaryotic expression vector, pCDNA3+ (Invitrogen). Yeast β-Galactosidase Assays and GST Pull-down ExperimentsProtein-p...

The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor ␣ in a ligand-enhanced manner. The PPAR␣-interacting domain of p300 was mapped to amino acids 39 -117 which interacted strongly with PPAR␣ but did not interact with retinoic acid receptor-␥ or retinoid X receptor-␣. Amino acids within the carboxyl terminus of PPAR␣ as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPAR␣ and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPAR␣ and further characterize the molecular mechanisms of PPAR␣-mediated transcriptional regulation.

A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy. GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell. Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells. Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins. GRASP⅐GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane. Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor ␣ (PPAR␣), and PPAR␣ ligands suppressed this interaction. In contrast to the interaction of PPAR␣ with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPAR␣ ligand binding domain. NCoR was found to suppress PPAR␣-dependent transcriptional activation in the context of a PPAR␣⅐retinoid X receptor ␣ (RXR␣) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPAR␣⅐RXR␣ heterodimeric complexes in mammalian cells. NCoR appears to influence PPAR␣ signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.