Endocrine Abstracts

Background: Resistance to thyroid hormone (TH) alpha (RTHα), caused by heterozygous mutations in THRA, is characterized by abnormal thyroid function tests and features due to tissue-specific hypothyroidism, including disproportional short stature, variable motor and cognitive defects, macrocephaly and macroglossia, constipation and anemia. Mutant receptors display defective T3 binding resulting in impaired transcriptional activity. Dominant negative inhibition of the WT receptor results from a failure of corepressor complex dissociation from mutant receptors. The residues interacting with corepressors and the mechanisms underlying dominant negative inhibition have not been elucidated.

Methods: We describe a family, carrying a heterozygous H175R TRα variant, that lack most classical features of RTHα, except short stature. In vitro, T3-dependent transcriptional activity of WT and H175R mutant TRα1 were measured using three TRE-luciferase reporters (MAL-DR4, PAL-IR0, F2-ER6) in Jeg-3 cells. T3-dependent cofactor (NCoR1, SMRT-γ and SRC1) interactions with WT and H175R mutant TRα1 were tested in mammalian two-hybrid assays and in co-immunoprecipitations. Artificial mutations (H175A, H175F, H175K) were generated to investigate the required amino acid properties at position 175.

Results: The H175R showed impaired T3-dependent transcriptional activity on all TRE tested (EC50 H175R vs WT: 5.4 [3.9-7.4] vs 0.28 [0.26-0.29] nM, P < 0.001 for MAL; 5.4 [4.7-6.2] vs 0.29 [0.19-0.43] nM, P < 0.01 for PAL; 1.21 [0.94-1.56] vs 0.23 [0.13-0.41] nM, ns for F2). However, when co-transfected with WT TRα1 we found no significant dominant negative effect for TRα1-H175R. In mammalian two-hybrid assays, recruitment of GAL4-SRC1 was concomitantly impaired (EC50 H175R vs WT: 207 [147-290] vs 0.53 [0.41-0.68] nM, P < 0.001). Strikingly, T3-induced GAL4-NCoR1 and GAL4-SMRT-γ dissociation curves showed complete absence of NCoR1 and SMRT-γ interactions with the H175R mutant, which was confirmed by co-immunoprecipitation. Substitution of His175 with Lysine (H175K), but not with Alanine or Phenylalanine (H175A and H175F), similarly reduced T3-dependent transcriptional activity and abolished corepressor recruitment.

Conclusion: Our data indicate His175 as an important residue for corepressor binding in human TRα1, compatible with previous in vitro studies using artificial mutants. The substitution for a large positively charged residue rather than the absence of His175 causes lack of corepressor binding to H175R TRα1. At present, it is unclear if the H175R variant is linked to the phenotype. Studies in animal models carrying this variant will be performed to address this question.