Application of a Novel Method for the Comparison of DNA Binding Parameters of the Two Human Thyroid Hormone Receptor Subtypes hTRα₁ and hTRβ₁

Abstract

DNA-binding characteristics of the two human thyroid hormone receptors α₁ and β₁ (hTRα₁ and hTRβ₁) were studied by applying the recently developed solid-phase scintillation technique. Biotinylated double stranded oligonucleotides containing thyroid hormone response elements (TRE) were immobilized to streptavidin coated scintillating microtiter plates. The TRE:s consisted of variants of the consensus core sequence AGGTCA as monomers or as dimers in direct repeats. Equilibrium binding of radioactive labelled hTRα₁ and hTRβ₁ were studied. Metabolically ³⁵S-labelled hTR (in vitro translated cDNA) as well as hTR expressed in the baculovirus-system and labelled with ¹²⁵I-triiodothyronine (¹²⁵I-T₃) were used. In binding saturation experiments, the affinity for the TRE:s investigated did not differ greatly between hTRα₁ and hTRβ₁. No significant effects of T₃ on the amplitude of DNA binding of either hTRα₁ or hTRβ₁ to the single site response elements could be demonstrated. Receptor binding to direct repeats was stimulated by the hormone in the case of the hTRβ₁. The hTRα₁ binding to direct repeats was not significantly altered by T₃. The single site octameric variant of a TRE. TAAGGTCA, was observed to bind tighter to the hTR:s as compared to the hexameric variant AGGTCA. In the binding competition format, with one response element immobilized and other (un-biotinylated) added to the reaction mixture, there was a larger dymanic range for the affinity constants (IC50) as compared to the affinity constants (K_d) obtained in the binding saturation experiments. The present quantitative results confirm previous reports obtained with qualitative methods like gel shift assays. The method described here is applicable in basic research concerning characterisation of DNA binding of nuclear receptors. It also lends itself to automatization in high capacity formats.