Retinoid X Receptor α (RXRα) Helix 12 Plays an Inhibitory Role in the Recruitment of the p160 Co-activators by Unliganded RXRα/Retinoic Acid Receptor α Heterodimers

Abstract

Retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers control gene expression through recruitment of co-repressors or co-activators, depending on their hormone binding status. We show that the helix 12 of RXR␣ and RAR␣ is critical for recruitment of the co-regulators and transcriptional regulation by RXR␣, RAR␣, and RXR␣/RAR␣. LG268, an RXR-specific agonist, was able to promote co-activator association with the heterodimers, but was unable to dissociate corepressors. Reconstitution experiments in yeast demonstrated that LG268 was capable of activating transcription by RXR␣/RAR␣ through recruitment of the coactivator. We hypothesize that the inability to release co-repressors from RXR␣/RAR␣ is responsible for the inability of LG268 to activate RXR␣/RAR␣ heterodimers in mammalian cells. Deletion of RAR␣ helix 12 (RXR␣/ RAR␣ ⌬403) abolished both hormone-dependent dissociation from co-repressors and hormone-dependent association with co-activators. Deletion of RXR␣ helix 12 (RXR␣ ⌬443/RAR␣) resulted in a higher binding affinity for co-repressors. Unexpectedly, RXR␣ ⌬443/RAR␣ also gained hormone-independent co-activator binding activity. Moreover, LG268 became an antagonist to RXR␣ ⌬443/RAR␣ heterodimers. These data suggest that the helix 12 of RXR␣ plays an inhibitory role in the recruitment of co-activators by unliganded RXR␣/RAR␣.The nuclear hormone receptors comprise a large family of transcription factors that regulate animal development, cell proliferation, and cell differentiation through the control of a network of genes involved in these processes (1, 2). Nuclear receptors harbor two evolutionarily conserved domains, DNA binding domains (DBDs) 1 and ligand binding domains (LBDs).Whereas the DBD recognizes specific DNA sequences, the LBD dictates transcriptional activity by nuclear receptors through recruitment of co-repressors such as silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor co-repressor or co-activators, including p160 family proteins (3, 4).Nuclear hormone receptors have been further subdivided into four classes based on their mode of action and DNA binding status (1). Retinoic acid receptors (RARs) and Retinoid X receptors (RXRs) are class II receptors that mediate retinoid signaling by regulating a complex gene network. RARs primarily form heterodimers with RXRs on DR5 (direct repeat spaced by 5 base pairs) or DR1-containing sequences. Conversely, RXRs are capable of forming homodimers on DR1 or heterodimers with other class II nuclear hormone receptors (1). The ability of RXRs to form homodimers or heterodimers with other receptors generates a complex mechanism by which the ligands of RXR may or may not exert effects on target genes, depending on the dimerization partner of the receptor (5). Receptors such as RARs and TRs (thyroid hormone receptors) dimerize with RXRs, generating a non-permissive complex in which RXR ligands are not capable of activating heterodimers. In such heterodimeric complexes, RXRs are referred to as silent partne...

Aberrant Association of Promyelocytic Leukemia Protein-Retinoic Acid Receptor-α with Coactivators Contributes to Its Ability to Regulate Gene Expression

“…To our surprise, we found that unliganded PML-RAR␣ interacted with coactivators both in mammalian cells and in vitro. This result is reminiscent of our previous study in which RXR␣/RAR␣(⌬443), a helix 12 deletion mutant, acquired not only hormone-resistant dissociation with the corepressors, but also hormone-independent association with the coactivators (46). In addition, hormone further promoted the association between PML-RAR␣ and coactivators, as observed in GST pulldown assays (Fig.…”

“…These data suggest that the LBD of the RAR␣ portion of PML-RAR␣ is the major contributor to the hormone-independent interactions with coactivators. To confirm this, we tested the binding of ACTR to a PML-RAR␣ mutant containing a mutation at a site shown previously to abolish the interaction of RAR␣ with coregulators in vivo by a mammalian two-hybrid assay (46). We found that F584A (corresponding to F249A in RAR␣) completely abolished the interaction of PML-RAR␣ with ACTR in comparison with wild-type binding (lane 6).…”

The retinoid X receptors and their ligands

“…Reconstitution experiments in yeast, which did not express the CoA SRC-3 (pCIP/AIB-1/RAC-3/TRAM-1/ACTR) or the CoR SMRT, reporter assays, and EMSAs were used to demonstrate that binding of the RXR agonist LG100268 to the wild-type RXRα–RARα heterodimer induced the recruitment of the SRC-3 receptor interacting domain (RID) peptide and activation of a DR-5 reporter construct [65]. However, this complex was unable to induce the release of the SMRT RID peptide, which only occurred in the presence of the RAR agonist ATRA, which also had recruited the CoA peptide [65].…”

“…However, this complex was unable to induce the release of the SMRT RID peptide, which only occurred in the presence of the RAR agonist ATRA, which also had recruited the CoA peptide [65]. The RXRαΔ(403) mutant lacked the RXR H11–H12 loop and H12.…”

Familial Focal Segmental Glomerulosclerosis (FSGS)-linked α-Actinin 4 (ACTN4) Protein Mutants Lose Ability to Activate Transcription by Nuclear Hormone Receptors

“…HA-PPAR␥ was generated by PCR PPAR␥ and cloning to CMX-1H vector. HA-RAR␣ was described previously (46).…”