Negative regulation by thyroid hormone receptor requires an intact coactivator-binding surface
Tania M. Ortiga-Carvalho, Nobuyuki Shibusawa, Amisra Nikrodhanond, Karen J. Oliveira, Danielle S. Machado, Xiao-Hui Liao, Ronald N. Cohen, Samuel Refetoff, Fredric E. Wondisford
Tania M. Ortiga-Carvalho, Nobuyuki Shibusawa, Amisra Nikrodhanond, Karen J. Oliveira, Danielle S. Machado, Xiao-Hui Liao, Ronald N. Cohen, Samuel Refetoff, Fredric E. Wondisford
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Research Article Endocrinology

Negative regulation by thyroid hormone receptor requires an intact coactivator-binding surface

Abstract

Thyroid hormone (TH) action is mediated by TH receptors (TRs), which are members of the nuclear hormone receptor superfamily. In vitro studies have demonstrated that TR activity is regulated by interactions with corepressor and coactivator proteins (CoRs and CoAs, respectively). TH stimulation is thought to involve dissociation of CoRs and recruitment of CoAs to the liganded TR. In contrast, negative regulation by TH is thought to occur via recruitment of CoRs to the liganded TR. The physiological role of CoAs bound to TRs, however, has yet to be defined. In this study, we used gene-targeting techniques to mutate the TR-β locus within its activation function–2 (AF-2) domain (E457A). This mutation was chosen because it completely abolished CoA recruitment in vitro, while preserving normal triiodothyronine (T3) binding and CoR interactions. As expected, TH-stimulated gene expression was reduced in homozygous E457A mice. However, these animals also displayed abnormal regulation of the hypothalamic-pituitary-thyroid axis. Serum thyroxine, T3, and thyroid-stimulating hormone (TSH) levels and pituitary Tshb mRNA levels were inappropriately elevated compared with those of WT animals, and L-T3 treatment failed to suppress serum TSH and pituitary Tshb mRNA levels. Therefore, the AF-2 domain of TR-β is required for positive and, paradoxically, for negative regulation by TH in vivo.

Authors

Tania M. Ortiga-Carvalho, Nobuyuki Shibusawa, Amisra Nikrodhanond, Karen J. Oliveira, Danielle S. Machado, Xiao-Hui Liao, Ronald N. Cohen, Samuel Refetoff, Fredric E. Wondisford

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Figure 1

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In vitro characterization of TR-β (E457A). (A) Schematic representation ...
In vitro characterization of TR-β (E457A). (A) Schematic representation of the TR-β isoforms and the location of the E457A mutation. Amino acid numbers are given for the human mutation in the mouse TR-β locus. DBD, DNA-binding domain; LBD, ligand-binding domain. (B and C) Gel mobility shift assay showing that the E457A receptor mutant binds to CoR (NCoR) but not to CoA (SRC-1) protein. RXR was added to all the lanes shown in C. A positive TRE (DR+4 probe) was radiolabeled, and T3 was added at a concentration of 10 nM. HD, heterodimer; UP, unprogrammed or non-specific. (D) Gel mobility shift assay showing that the E457A receptor mutant does not bind to RIP140 in the presence of T3 on the same radiolabeled DR+4 element. RXR and T3 were added to all lanes. (E) Effect of mutant TR-β (E457A) on positive and negative TREs. Ligand-dependent transcriptional activity of positively regulated reporter gene DR+4 LUC and negatively regulated reporter genes TRH LUC (human TRH –900 to +55 bp) and TSH-β LUC (human TSH –1192 to +37 bp). TK 109, a minimal thymidine kinase promoter in pA3Luc, was used as a control. Equal amounts of WT or mutant TR-β2–expressing plasmids or empty vector (SG5) were cotransfected into HEK 293 T cells with a luciferase reporter gene. The data are presented as fold change in activity compared with the level in the absence of T3. Data represent 5 separate experiments performed in triplicate. *P < 0.001 vs. no T3; **P < 0.001; #P < 0.01.