Abstract
Receptor-interacting protein 140 (RIP140) contains multiple receptor interaction domains and interacts with retinoic acid receptors in a ligand-dependent manner. Nine LXXLL receptor-interacting motifs are organized into two clusters within this molecule, each differentially interacting with retinoic acid receptor (RAR) and retinoid X receptor (RXR). RAR interacts with the 5' cluster, whereas RXR interacts with both clusters. Additionally, a third ligand-dependent receptor-interacting domain is assigned to the very C terminus of this molecule, which contains no LXXLL motif. In mammalian cells, receptor heterodimerization is required for efficient interaction of RAR/RXR with RIP140. Furthermore, the heterodimeric, holoreceptors cooperatively interact with RIP140, which requires the activation function 2 domains of both receptors. By using different retinoic acid reporter systems, it is demonstrated that RIP140 strongly suppresses retinoic acid induction of reporter activities, but coactivator SRC-1 enhances it. Furthermore, an intrinsic repressive activity of RIP140 is demonstrated in a GAL4 fusion system. Unlike receptor corepressor, which interacts with antagonist-bound RAR/RXRs, RIP140 does not interact with antagonist-occupied RAR/RXR dimers. These data suggest that RIP140 represents a third coregulator category that is able to suppress the activation of certain agonist-bound hormone receptors.
Highlights
Ceptors, such as retinoic acid (RAR) thyroid, and vitamin D receptors, and oxysterol and fatty acid receptors (PPARs) heterodimerize with a common partner, retinoid X receptor (RXR) [2, 3]
The ligand binding domain (LBD) of both retinoic acid receptor (RAR) and RXR was each fused to the C terminus of glutathione S-transferase (GST) protein and expressed in E. coli
We have utilized molecular approaches to understand the domain requirement for Receptor-interacting protein 140 (RIP140) interaction with RAR/RXR, the biological effects of this interaction in terms of RA induction of target gene expression, and the mechanisms underlying this biological activity of RIP140
Summary
Construction of Expression Vectors and Reporters—The DEF domains (the ligand binding domains) of both mouse RAR␣ (primers 5Ј-GAATTCATGTCCAAGGAGTCGGTG-3Ј and 5Ј-ACTCGAGTCATGGGGATTGCGT-3Ј) and RXR (primers 5Ј-GGGAATTCAAAAGGGAGGCGGTT-3Ј and 5Ј-CCCTCGAGTCAGGCTAGCTGGT-3Ј) were generated in polymerase chain reactions and cloned into the EcoRI and SalI sites of the pGEX-2T vector (Amersham Pharmacia Biotech) for the production of glutathione S-transferase (GST) fusion proteins in Escherichia coli. The GAL4BD fusions of all the RIP140 expression vectors constructed in pM vector (CLONTECH) were as described previously [21], and the BD-N-CoR containing the C-terminal portion of mouse N-CoR (residues 1843–2453) was cloned into the EcoRI and SalI site of the pM vector as described [3].The SRC-1 expression vector (under the control of a cytomegalovirus promoter) was kindly provided by Dr H. For mammalian two-hybrid tests, COS-1 cells were co-transformed with different combinations of GAL4BD and VP16 fusions (50 ng each), a GAL4-tk-luciferase reporter (400 ng), and an SV40-lacZ internal control (25 ng). Cells were harvested 24 h later, and luciferase and lacZ activities, means, and S.E. values were determined as described previously [4, 21]
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