Cloning and Characterization of Mouse RIP140, a Corepressor for Nuclear Orphan Receptor TR2

Abstract

The mouse homologue of the human receptor-interacting protein 140 (RIP140) was isolated from a mouse embryonic cDNA library in yeast two-hybrid screening experiments by using the ligand binding domain (LBD) of nuclear orphan receptor TR2 as the bait. The receptor-interacting domains of mouse RIP140 were mapped to the regions containing the LXXLL motif (where L is leucine and X is any amino acid), and the RIP140-interacting domain of TR2 was mapped to its C-terminal 10-to 20-amino-acid sequence, a putative activation function 2 (AF-2) region. In a GAL4 reporter system and a reporter driven by the proximal region of the TR2 promoter, RIP140 functioned as a corepressor for both a GAL4 DNA binding domain (BD)-TR2 fusion and the wild-type receptor. When tethered to the BD of GAL4, RIP140 exerted a trans-repressive effect on the GAL4 reporter. In addition, RIP140 suppressed the retinoic acid (RA) receptor-mediated RA induction in a dosedependent manner. Finally, it was demonstrated that in the presence of RIP140, a cytosolic, green fluorescent protein-tagged TR2 LBD translocated into the nucleus, and TR2 and RIP140 were coimmunoprecipitated from the cell extract, indicating that the interaction between RIP140 and the LBD of TR2 occurred in vivo. The potential biological role of RIP140 in TR2-modulated transcriptional activity is discussed.Nuclear receptors regulate target gene expression by binding to their cognate DNA response elements and recruiting associate proteins to the transcription machinery (19, 45). Recently, different coactivators and corepressors for several nuclear receptors have been identified (16). For instance, the nuclear receptor corepressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) have been shown to function as corepressors for retinoic acid (RA) receptor ␣ (RAR␣), thyroid hormone receptors (T 3 Rs), and orphan receptor COUP-TFI (4, 15, 36). The newly identified nuclear protein Nab1 is a corepressor for the orphan receptor NGFI-A family (38), and SUN-CoR is able to potentiate transcriptional repression by T 3 Rs and RevErb (44). In the coactivator category, the transcriptional mediator/intermediary factor 2 and steroid receptor coactivator 1 (SRC-1) are known to mediate transcriptional activation of RAR, estrogen receptor (ER), retinoid X receptor (RXR), and T 3 Rs (8,14,33,39,42). With respect to the working mechanism of corepressors and coactivators, it has been demonstrated that their actions involve the alteration of chromatin structure, such as the acetylation status of histone proteins (13,34,37).The orphan receptor TR2 belongs to the superfamily of nuclear receptors (21,31). This receptor gene is expressed most abundantly in the testes of adult animals, particularly the developing germ cell populations (22,24,25). During embryonic stages, TR2 expression is highest between embryonic day 8 (E8) and E12 (22). It is also known that this gene encodes two isoform receptors, one retaining the entire ligand binding domain (LBD) and the other truncated at ...

The Transcriptional Corepressor, PELP1, Recruits HDAC2 and Masks Histones Using Two Separate Domains

“…Several bifunctional coregulators of NR have been reported to exist, including ZAC1, NSD1, and RIP140, which tend to behave either as coactivators or as corepressors under distinct circumstances (17)(18)(19)(20). Although the switching mechanisms responsible for the bifunctionality of these coregulators have yet to be elucidated, mouse variant ZAC1 (mZac1b), which binds to many other coregulators including GRIP1, CBP, and p300, has FIG.…”

“…Some transcriptional coregulators for NR-mediated transcription, such as ZAC1, NSD1, and RIP14, have been demonstrated to possess dual functions, acting as both coactivators and corepressors (17)(18)(19)(20). Recently, the nuclear protein PELP1 (proline-, glutamic acid-, and leucine-rich protein 1) has been proposed to be another dual-function coregulator for NR-mediated transcription; i.e.…”

Constitutive Activation of Retinoic Acid Receptor β2 Promoter by Orphan Nuclear Receptor TR2

“…COS-1 cells were used in co-transfection experiments to determine the effects of added nuclear receptors on the reporter gene activity as well as mammalian two-hybrid interaction experiments as described (16,20). All the COS-1 cultures were maintained in Dulbecco's modified Eagle's medium containing dextran charcoal-treated serum (DCC medium).…”

“…In addition, a novel receptor heterodimeric pathway was identified that involved heterodimers of TR2 and TR4 (19). Recently, the mouse nuclear receptor-interacting protein 140 (RIP140) was cloned and demonstrated as a co-repressor for TR2 by interacting with its LBD (20).…”

Limiting Effects of RIP140 in Estrogen Signaling

“…The coregulator RIP140 contains 10 LXXLL motifs and like a classic coactivator interacts preferentially with ligand-bound nuclear receptors (11). However, RIP140 inhibits the transactivation function of ligand-bound nuclear receptors in reporter assays (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). RIP140 is now recognized as one of the first proteins of a unique class of coregulators that are able to repress ligand-bound nuclear receptors (23).…”