Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer

Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.     

The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificites of the DNA binding domains.

We show here that, in addition to generating an increase in DNA binding efficiency, heterodimerization of retinoid X receptor (RXR) with either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) alters the binding site repertoires of RAR, RXR and TR homodimers. The binding site specificities of both homo- and heterodimers appear to be largely determined by their DNA binding domains (DBDs), and are dictated by (i) homocooperative DNA binding of the RXR DBD, (ii) heterocooperative DNA binding of RXR/RAR and RXR/TR DBDs, and (iii) steric hindrance. No homodimerization domain exists in the DBDs of TR and RAR. The dimerization function which is located in the ligand binding domain further stabilizes, but in general does not change, the repertoire dictated by the corresponding DBD(s). The binding repertoire can be further modified by the actual sequence of the binding site. We also provide evidence supporting the view that the cooperative binding of the RXR/RAR and RXR/TR DBDs to directly repeated elements is anisotropic, with interactions between the dimerization interfaces occurring only with RXR bound to the 5' located motif. This polarity, which appears to be maintained in the full-length receptor heterodimers, may constitute a novel parameter in promoter-specific transactivation.     

Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.     

Mutations in retinoid X receptor that impair heterodimerization with specific nuclear hormone receptor

Retinoid X receptor (RXR) serves as a promiscuous heterodimerization partner for many nuclear receptors through the identity box, a 40-amino acid subregion within the ligand binding domain. In this study, we randomly mutated two specific residues within the human RXRalpha identity box region previously identified as important determinants in heterodimerization (i.e. Ala(416) and Arg(421)). Interestingly, most of these mutants still retained wild type interactions with thyroid hormone receptor (TR), retinoic acid receptor, peroxisome proliferator-activated receptor alpha, small heterodimer partner, and constitutive androstane receptor. However, RXR-A416D and R421L were specifically impaired for interactions with TR, whereas RXR-A416K lost both TR and retinoic acid receptor interactions. Accordingly, RXR-A416D did not support T3 transactivation in mammalian cells, whereas RXR-A416K was not supportive of transactivation by retinoids or T3. These results provide a basis upon which to further design mutant RXRs highly selective in heterodimerization, potentially useful tools to probe nuclear receptor function in vivo.     

Functional evidence for retinoid X receptor (RXR) as a nonsilent partner in the thyroid hormone receptor/RXR heterodimer

Many members of the thyroid hormone/retinoid receptor subfamily (type II nuclear receptors) function as heterodimers with the retinoid X receptor (RXR). In heterodimers which are referred to as permissive, such as peroxisome proliferator activated receptor/RXR, both partners can bind cognate ligands and elicit ligand-dependent transactivation. In contrast, the thyroid hormone receptor (TR)/RXR heterodimer is believed to be nonpermissive, where RXR is thought to be incapable of ligand binding and is often referred to as a silent partner. In this report, we used a sensitive derepression assay system that we developed previously to reexamine the TR/RXR interrelationship. We provide functional evidence suggesting that in a TR/RXR heterodimer, the RXR component can bind its ligand in vivo. Ligand binding by RXR does not appear to directly activate the TR/RXR heterodimer; instead, it leads to a (at least transient or dynamic) dissociation of a cellular inhibitor(s)/corepressor(s) from its TR partner and thus may serve to modulate unliganded TR-mediated repression and/or liganded TR-mediated activation. Our results argue against the current silent-partner model for RXR in the TR/RXR heterodimer and reveal an unexpected aspect of cross regulation between TR and RXR.     

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.     

RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR), thyroid hormone receptor (T3R) and vitamin D3 receptor (VD3R) differ from steroid hormone receptors in that they bind and transactivate through responsive elements organized as direct rather than inverted repeats. We now show that recombinant RAR and T3R are monomers in solution and cannot form stable homodimeric complexes on their responsive elements. Stable binding of the receptors to their responsive elements requires heterodimerization with a nuclear factor. This auxiliary factor is tightly associated with RAR and T3R in the absence of DNA and co-purifies with both receptors. As demonstrated by extensive purification, the same auxiliary factor is required for stable DNA binding of RAR as for that of T3R; the factor also facilitates the formation of a stable VD3R-DNA complex. The auxiliary factor is identical to the retinoid X receptor alpha (RXR alpha) by biochemical and functional criteria. The identification of RXR alpha as a dimerization partner for the RARs, T3Rs and VD3R has important implications as to the function of these receptors and their ligands in development, homeostasis and neoplasia.     

Dimerization interfaces formed between the DNA binding domains determine the cooperative binding of RXR/RAR and RXR/TR heterodimers to DR5 and DR4 elements.

We have previously reported that the binding site repertoires of heterodimers formed between retinoid X receptor (RXR) and either retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bound to response elements consisting of directly repeated PuG(G/T)TCA motifs spaced by 1-5 bp [direct repeat (DR) elements 1-5] are highly similar to those of their corresponding DNA binding domains (DBDs). We have now mapped the dimerization surfaces located in the DBDs of RXR, RAR and TR, which are responsible for cooperative interaction on DR4 (RXR and TR) and DR5 (RXR and RAR). The D-box of the C-terminal CII finger of RXR provides one of the surfaces which is specifically required for the formation of the heterodimerization interfaces on both DR4 and DR5. Heterodimerization with the RXR DBD on DR5 specifically requires the tip of the RAR CI finger as the complementary surface, while a 7 amino acid sequence encompassing the 'prefinger region', but not the TR CI finger, is specifically required for efficient dimerization of TR and RXR DBDs on DR4. Importantly, DBD swapping experiments demonstrate not only that the binding site repertoires of the full-length receptors are dictated by those of their DBDs, but also that the formation of distinct dimerization interfaces between the DBDs are the critical determinants for cooperative DNA binding of these receptors to specific DRs.