Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo
Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford
Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford
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Article Endocrinology

Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo

Abstract

Thyroid hormone action is mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. DNA-binding is presumed to be essential for all nuclear actions of thyroid hormone. To test this hypothesis in vivo, the DNA-binding domain of TR-β was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-β DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-βGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-β KO (TR-β–/–) mice. However, TR-βGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-β–/– mice. DNA-binding, therefore, is essential for many functions of the TR, including retinal development and negative feedback regulation by thyroid hormone of the hypothalamic-pituitary-thyroid axis. Inner ear development, although not completely normal, can occur in the absence of TR DNA-binding, suggesting that an alternative and perhaps novel thyroid hormone-signaling pathway may mediate these effects.

Authors

Nobuyuki Shibusawa, Koshi Hashimoto, Amisra A. Nikrodhanond, M. Charles Liberman, Meredithe L. Applebury, Xiao Hui Liao, Janet T. Robbins, Samuel Refetoff, Ronald N. Cohen, Fredric E. Wondisford

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(a) The amino acid sequence of the DNA-binding domain of TR-β. The boxed...
(a) The amino acid sequence of the DNA-binding domain of TR-β. The boxed region outlines the DNA recognition α-helix. The shaded circles indicate the P-box amino acids. The exchange of glutamic acid 125 and glycine 126 to glycine and serine in the GS125 mutation is indicated. (b) Liver RNA was amplified by RT-PCR using primers located in sequences corresponding to exons 2 and 4 (A-1, A-2) and exons 3 and 4 (B-1, B-2), which are indicated by arrows. (c) RT-PCR results from liver RNA using the indicate primers. A 445-bp fragment from WT (+/+) and TR-βGS/GS animals was observed with the A primer set. A short fragment (344 bp) from the A primer set and no band from the B set were obtained from RNA transcripts from TR-β–/– mice. (d) DNA sequence of the 445-bp RT-PCR fragment. The two mutated amino acids are indicated. The deleted mRNA from TR-β–/– encoded a putative peptide that terminated after eight bases in exon 4 at a TAG stop codon. This was confirmed by sequencing the 344-bp fragment from TR-β–/– mice (data not shown). (e) Western blot analysis of liver total cellular protein extracts from WT, TR-β–/–, and TR-βGS/GS animals (a C-terminal TR-β monoclonal antibody was used), indicating that the GS125 mutation did not affect expression from the TR-β locus.