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

Abstract

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.

FIG. 1.

(A) Activation of the Gal4-TR-VP16 (GTV) chimera by the apo-TR LBD or liganded RXR LBD. HeLa cells were transfected with 1 μg of the Gal4 reporter pMC110 and 400 ng of the GTV chimera to examine GTV-mediated transactivation. The TR LBD (2.5 μg) or RXR LBD (800 ng) or the control empty expression vector pEX0 (1.5 μg) was cotransfected as indicated. CAT activities were determined for cells without ligand (open bars) or with T3 (shaded bars) or with 9-cis-RA (hatched bars). In panel 4, Gal4-TR (GT, 400 ng) was used instead of GTV. Error bars indicate SEMs. (B) Schematic model for derepression of GTV by the apo-TR LBD. (Top) In cells transfected with GTV only, a cellular inhibitor/corepressor (Inh) associates with the TR moiety of the GTV fusion protein, which in turnmasks the transactivation function of VP16. (Middle) In cells cotransfected with the TR LBD but without ligand, the expressed apo-TR LBD competes in trans with GTV for binding to the cellular inhibitor. This results in dissociation of the inhibitor from GTV, which in turn allows VP16 to elicit its transactivation function. (Bottom) Cotransfection of the TR LBD in the presence of T3 results in the dissociation of the inhibitor from the liganded TR LBD. As a result, the inhibitor rebinds to the GTV chimera and represses VP16 activity. The TR moiety in GTV lacks helix 12 and thus is defective in ligand binding as well as ligand-induced dissociation of the inhibitor(s)/corepressor(s). Therefore, GTV alone is inactive with or without T3. (C) Schematic model for the inability of the apo-RXR LBD to activate GTV. The apo-RXR LBD has a low affinity for the inhibitor and thus cannot compete efficiently with GTV for inhibitor binding. As a result, cotransfection of the RXR LBD in the absence of ligand fails to derepress the GTV chimera.

FIG. 2.

(A) Two alternative models for derepression of GTV by the liganded RXR LBD. Left, the competition model. Although the apo-RXR LBD does not associate with the inhibitor efficiently and thus cannot activate GTV, this model proposes that a conformational change in the RXR LBD upon ligand binding increases its affinity for the inhibitor, resulting in the derepression of GTV. Right, the heterodimerization model. This model proposes that the liganded RXR LBD heterodimerizes with the TR moiety of GTV or, equivalently, the RXR component of the GTV/RXR heterodimer binds to its ligand 9-cis-RA. In doing so, it induces a conformational change in TR that decreases its affinity for the inhibitor, resulting in the derepression of GTV. (B) Activation of the mutant GTV L372R by the apo-TR LBD but not by the liganded RXR LBD. HeLa cells were transfected with 1 μg of the Gal4 reporter pMC110 and 5 μg of the mutant GTV L372R chimera. The TR LBD (5 μg) or RXR LBD (4 μg) or the control empty expression vector pEX0 (4 μg) was cotransfected as indicated. CAT activities were determined for cells without ligand (open bars) or with T3 (shaded bars) or with 9-cis-RA (hatched bars). Error bars indicate SEMs.

FIG. 3.

(A) Activation of the mutant GTV P158R by the liganded RXR LBD but not by the apo-TR LBD. HeLa cells were transfected with 2.5 μg of the Gal4 reporter pMC110 and 1 μg of the mutant GTV P158R chimera. The TR LBD (3 μg) or RXR LBD (3 μg) or the control empty expression vector pEX0 (3 μg) was cotransfected as indicated. CAT activities were determined for cells without ligand (open bars) or with T3 (shaded bars) or with 9-cis-RA (hatched bars). Error bars indicate SEMs. (B) Schematic model to account for the results in A. Left, the hinge region mutation P158R leads to a structural destabilization or conformational disruption of the mutant chimera, which renders it transcriptionally inactive regardless of the state of the cellular inhibitor. Thus, although the apo-TR LBD can efficiently bind the inhibitor, the GTV P158R remains inactive. Right, in cells cotransfected with the RXR LBD and treated with 9-cis-RA, heterodimerization between the liganded RXR LBD and the TR moiety of the GTV P158R chimera leads to structural stabilization of the mutant chimera and dissociation of the inhibitor from the TR moiety. As a result, GTV P158R is now transcriptionally active. More is discussed in the text.

FIG. 4.

Coactivator recruitment by the liganded RXR LBD is not required for its activation of GTV. (A) Expression of the nuclear receptor-interacting domain (NID) of GRIP1 blocks ligand-mediated transactivation by Gal4-RXR. HeLa cells were transfected with 1.2 μg of the Gal4 reporter pMC110 and a plasmid expressing the Gal4-RXR LBD (400 ng) to examine ligand-dependent transactivation of the RXR LBD. The plasmid pcDNA3-GRIP1 NID (2.5 μg), which expresses the nuclear receptor-interacting domain of GRIP1, or control plasmid pcDNA3 (2 μg) was cotransfected as indicated. CAT activities were determined for cells in the absence (open bars) or presence (hatched bars) of 9-cis-RA. (B) GRIP1 NID has no effect on the liganded RXR LBD-mediated activation of GTV. HeLa cells were transfected with pMC110 (1 μg), GTV (400 ng), and the RXR LBD (1.5 μg) in the absence (open bars) or presence (hatched bars) of 9-cis-RA to examine activation of GTV by the liganded RXR LBD. pcDNA3-GRIP1 NID (2.5 μg) or the control pcDNA3 (2 μg) was cotransfected as indicated. Error bars indicate SEMs.

FIG. 5.

Activation of a native TRE-bound VP16-TR chimera by RXR in the presence of 9-cis-RA. HeLa cells were transfected with the TRE-lys-CAT reporter (1 μg), together with the indicated plasmids: VP16-TR (300 ng) only, or VP16-TR (300 ng) plus hRXRβ (3 μg), or hRXRβ (3 μg) only. CAT activities were determined for cells without ligand (open bars) or with 9-cis-RA (hatched bars). Error bars indicate SEMs.

FIG. 6.

9-cis-RA enhances T3-mediated transactivation from a TRE-bound TR/RXR heterodimer. HeLa cells were transfected with 1 μg of TRE-DR4A-CAT, and 100 ng each of the TR and RXR expression plasmids. CAT activities were determined for cells without ligand, with 9-cis-RA only, with T3 only, and with T3 and 9-cis-RA. Error bars indicate SEMs.

FIG. 7.

Activation of GTV by the RXR LBD in the presence of LGD1069. HeLa cells were transfected with 1.5 μg of pMC110 together with the indicated plasmids: Gal4-RXR LBD (500 ng), GTV (500 ng), pEX0 (1.2 μg), or RXR LBD (1.5 μg). CAT activities were determined for cells without ligand, with 9-cis-RA, or with LGD1069. Error bars indicate SEMs.