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

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-beta was mutated within its P-box (GS mutant) using gene targeting techniques. This mutation in vitro completely abolishes TR-beta DNA-binding, while preserving ligand (T3) and cofactor interactions with the receptor. Homozygous mutant (TR-betaGS/GS) mice displayed abnormal T3 regulation of the hypothalamic-pituitary-thyroid axis and retina identical to abnormalities previously observed in TR-beta KO (TR-beta-/-) mice. However, TR-betaGS/GS mutant mice maintained normal hearing at certain frequencies and did not display significant outer hair cell loss, in contrast to TR-beta-/- 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.

Spectral tuning in the mammalian short-wavelength sensitive cone pigments.

The wild-type mouse ultraviolet (UV) and bovine blue cone visual pigments have absorption maxima of 358 and 438 nm, respectively, while sharing 87% amino acid identity. To determine the molecular basis underlying the 80 nm spectral shift between these pigments, we selected several amino acids in helices II and III for site-directed mutagenesis. These amino acids included: (1) those that differ between mouse UV and bovine blue; (2) the conserved counterion, Glu113; and (3) Ser90, which is involved in wavelength modulation in avian short-wavelength sensitive cone pigments. These studies resulted in the identification of a single amino acid substitution at position 86 responsible for the majority of the spectral shift between the mouse UV and bovine blue cone pigments. This is the first time that this amino acid by itself has been shown to play a major role in the spectral tuning of the SWS1 cone pigments. A single amino acid substitution appears to be the dominant factor by which the majority of mammalian short-wavelength sensitive cone pigments have shifted their absorption maxima from the UV to the visible regions of the spectrum. Studies investigating the role of the conserved counterion Glu113 suggest that the bovine and mouse SWS1 pigments result from a protonated and unprotonated Schiff base chromophore, respectively.