Rearrangements in thyroid hormone receptor charge clusters that stabilize bound 3,5',5-triiodo-L-thyronine and inhibit homodimer formation

In this study, we investigated how thyroid hormone (3,5',5-triiodo-l-thyronine, T3) inhibits binding of thyroid hormone receptor (TR) homodimers, but not TR-retinoid X receptor heterodimers, to thyroid hormone response elements. Specifically we asked why a small subset of TRbeta mutations that arise in resistance to thyroid hormone syndrome inhibit both T3 binding and formation of TRbeta homodimers on thyroid hormone response elements. We reasoned that these mutations may affect structural elements involved in the coupling of T3 binding to inhibition of TR DNA binding activity. Analysis of TR x-ray structures revealed that each of these resistance to thyroid hormone syndrome mutations affects a cluster of charged amino acids with potential for ionic bond formation between oppositely charged partners. Two clusters (1 and 2) are adjacent to the dimer surface at the junction of helices 10 and 11. Targeted mutagenesis of residues in Cluster 1 (Arg338, Lys342, Asp351, and Asp355) and Cluster 2 (Arg429, Arg383, and Glu311) confirmed that the clusters are required for stable T3 binding and for optimal TR homodimer formation on DNA but also revealed that different arrangements of charged residues are needed for these effects. We propose that the charge clusters are homodimer-specific extensions of the dimer surface and further that T3 binding promotes specific rearrangements of these surfaces that simultaneously block homodimer formation on DNA and stabilize the bound hormone. Our data yield insight into the way that T3 regulates TR DNA binding activity and also highlight hitherto unsuspected T3-dependent conformational changes in the receptor ligand binding domain.     

V-erba homodimers mediate the potent dominant negative activity of v-erba on everted repeats

The oncoprotein v-erbA is a mutated form of TRalpha1 that is unable to bind thyroid hormone (T3). V-erbA homodimerizes or heterodimerizes with retinoid X receptor (RXR) on core motifs arranged as direct, everted, or inverted repeats (DRs, ERs, or IRs). We created a series of v-erbA mutants in order to obtain a better understanding of the role of v-erbA homodimers versus v-erbA-RXR heterodimers in the dominant negative activity of v-erbA on ERs (the most potent v-erbA response elements). We found that one of these mutants, v-erbA mutant E325A, is able to homodimerize but unable to heterodimerize with RXR on ERs. Our data also suggest that v-erbA homodimers interact preferentially with the corepressor NCoR over SMRT and that the interaction with corepressors is stronger with v-erbA homodimers over v-erbA-RXR heterodimers. Furthermore, functional studies showed that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on ERs.     

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.     

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.