Application of a novel method for the comparison of DNA binding parameters of the two human thyroid hormone receptor subtypes hTR alpha 1 and hTR beta 1.
DNA-binding characteristics of the two human thyroid hormone receptors alpha 1 and beta 1 (hTR alpha 1 and hTR beta 1) 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 hTR alpha 1 and hTR beta 1 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 hTR alpha 1 and hTR beta 1. No significant effects of T3 on the amplitude of DNA binding of either hTR alpha 1 or hTR beta 1 to the single site response elements could be demonstrated. Receptor binding to direct repeats was stimulated by the hormone in the case of the hTR beta 1. The hTR alpha 1 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 dynamic range for the affinity constants (IC50) as compared to the affinity constants (Kd) 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.
Carlsson
B
B
Karo Bio AB, Novum, Huddinge, Sweden.
Ahola
H
H
Häggblad
J
J
D001483:Base Sequence; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014284:Triiodothyronine / Q000378:metabolism
D009841:Oligonucleotides; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D001710:Biotin; D004247:DNA