In vivo interaction of steroid receptor coactivator (SRC)-1 and the activation function-2 domain of the thyroid hormone receptor (TR) beta in TRbeta E457A knock-in and SRC-1 knockout mice

Abstract

The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-beta is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRbeta, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRbeta(E457A/E457A)) mice worsened the degree of resistance to TH, resulting in increased serum T(4) and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRbeta or the TRalpha to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain.

Figure 1

TT₄RI. The product of serum TSH and TT₄ is plotted against each genotype in the order of increasing number of deficient/mutant alleles (SRC-1 KO and TRβ ^E457A/E457A). The higher the TT₄RI, the more severe is the resistance. Homozygotes TRβ ^E457A/E457A are shown as A/A and heterozygotes TRβ^E457A/WT as E/A. n = 8–15 for each group.

Figure 2

Serum TSH after TH withdrawal and l-T₃ treatment. Serum TSH concentrations are reported for each genotype after 14 d PTU/LoI diet (black bars) and then after 4 d l-T₃ (0.8 μg/100 g) treatment (gray bars). The data are expressed as mean ± se. Number of mice per group is indicated inside the bar. Statistical significance at P ≤ 0.05: a, vs. WT; b, TRβ^E457A/E457A vs. TRβ^E457A/WT-SRC-1 KO; c, SRC-1 KO vs. TRβ^E457A/WT-SRC-1 KO; d, TRβ^E457A/E457A vs. TRβ^E457A/WT-SRC-1 KO.

Figure 3

Expression of Dio1 (A) and Gst (B) in liver of transgenic mice. Real-time PCR was used to measure mRNA in the liver of various groups of transgenic mice. Livers were extracted from mice after 14 d PTU/LoI diet and then after 4 d l-T₃ (0.8 μg/100 g) treatment. Values are reported relative to the mean value ± se of WT mice after PTU treatment expressed as 100%. n = 5–6 mice per group. Statistical significance at P ≤ 0.05: a, vs. WT PTU treated; b, vs. WT T₃ treated; c, vs. SRC-1 KO PTU treated; d, vs. SRC-1 KO T₃ treated.

Figure 4

Proposed mechanism of SRC-1 and AF-2 interaction in ligand-independent TRβ-mediated up-regulation of TSH (upper) and ligand-dependent down-regulation of TSH (lower). The model shown in this diagram illustrates that in mice with disruption of the AF-2 domain and an intact SRC-1, the amount of TSH up-regulation during TH withdrawal (upper) is greater than when SRC-1 is absent. During TH treatment (lower), the disruption of the AF-2 domain with or without the presence of SRC-1 results in a similar resistance to TSH suppression. See Discussion for more details. DBD, DNA-binding domain.