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Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2
C H Lee1, C Chinpaisal, L N Wei
1Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
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 dose-dependent 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.
Insights
Mouse receptor-interacting protein 140 (RIP140) acts as a corepressor for the nuclear orphan receptor TR2. This interaction, occurring in vivo, influences TR2-mediated transcriptional activity and retinoic acid signaling.
Area of Science:
- Molecular Biology
- Nuclear Receptor Signaling
- Gene Regulation
Background:
- Receptor-interacting protein 140 (RIP140) is a key transcriptional coregulator.
- Nuclear orphan receptor TR2 plays roles in various cellular processes.
- Understanding RIP140's interaction with TR2 is crucial for elucidating gene regulation.
Purpose of the Study:
- To identify and characterize the interaction between mouse RIP140 and nuclear orphan receptor TR2.
- To investigate the functional consequences of this interaction on transcriptional activity.
- To determine the in vivo relevance of the RIP140-TR2 interaction.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- Mapping of receptor-interacting domains using deletion mutants.
- Reporter gene assays (GAL4 system) to assess transcriptional activity.
- Co-immunoprecipitation and fluorescence microscopy to confirm in vivo interaction.
Main Results:
- Mouse RIP140 was identified as an interacting protein of TR2's ligand-binding domain (LBD).
- RIP140 contains LXXLL motifs, and TR2's C-terminal region (AF-2) mediates the interaction.
- RIP140 functions as a corepressor for TR2, suppressing TR2-driven and retinoic acid receptor-mediated transcription.
- RIP140 promotes the nuclear translocation of TR2 LBD and co-immunoprecipitates with TR2 in vivo.
Conclusions:
- RIP140 directly interacts with TR2's LBD in vivo.
- RIP140 acts as a transcriptional corepressor for TR2, modulating its activity.
- The findings provide insights into the role of RIP140 in TR2-regulated biological processes.