Negative regulation by thyroid hormone receptor requires an intact coactivator-binding surface
Tania M. Ortiga-Carvalho, … , Samuel Refetoff, Fredric E. Wondisford
Tania M. Ortiga-Carvalho, … , Samuel Refetoff, Fredric E. Wondisford
Published September 1, 2005
Citation Information: J Clin Invest. 2005;115(9):2517-2523. https://doi.org/10.1172/JCI24109.
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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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Characterization of TR-β (E457A) mice. (A) Introduction of E457A mutatio...
Characterization of TR-β (E457A) mice. (A) Introduction of E457A mutation into the mouse TR-β gene by homologous recombination. KI, knock-in. (B) Homologous integration of the targeting vector was confirmed by Southern blot analysis of genomic DNA digested with BamHI and probed with a probe located outside the targeting vector. (C) RT-PCR of TR-β from pituitary RNA using primers described in Methods. A 950-bp fragment from the TR-βWT/WT, TR-βE457A/WT, and TR-βE457A/E457A mice was observed. Two different animal tissues from each group were analyzed. (D) DNA sequence of the 950-bp fragment. The mutated amino acid is underlined. (E) Thrb and Thra mRNA levels in the anterior pituitary of TR-βWT/WT, TR-βE457A/WT, and TR-βE457A/E457A mice according to quantitative RT-PCR analysis. Data were normalized for each mRNA level relative to the values for the WT animals (n = 5–8).