Binding of thyroid hormones to nuclear extracts of thyroid cells

Binding of T3 and T4 to soluble nuclear extracts of FRTL-5 cells, rabbit thyroid glands, and rat liver was studied. [125I]Iodo-T3 or [125I]iodo-T4 in concentration ranges of 100-fold (10-fold on each side of measured Kd) was incubated with extract at 4 C, pH 8.2, and the quantity of bound hormone was determined by collection on nitrocellulose filters. The results were corrected for nonspecific binding. Steady state (equilibrium) binding was achieved by 36 h. Apparent dissociation constants (Kd) were determined from Scatchard analysis of data pertaining to extent of binding at 36 h as a function of hormone concentration and were also calculated from kinetics of binding as the ratio of rate constants. A single class of saturable, high affinity hormone-binding sites was found. Kd values for T3 and nuclear extracts of FRTL-5 cells, rabbit thyroid gland, and rat liver were, respectively, 3.9 X 10(-11) M, 2.8 X 10(-11) M, and 4.3 X 10(-11) M from Scatchard analysis; when calculated from kinetics of hormone association, the value was 3.6 X 10(-11) M for both FRTL-5 cell and rat hepatic nuclear extract. No analysis of the time course of binding of T3 to rabbit thyroid nuclear extract was made. Kd values for T4 and FRTL-5 cell extract were 6.2 X 10(-10) M from Scatchard analysis and 5.0 X 10(-10)M from kinetic data. Half-times (t1/2) of dissociation of T3 from FRTL-5 cell and rat liver nuclear extract, calculated from association curves, were 7 and 5 h, respectively, while corresponding values determined directly and experimentally were 10.5 and 13 h. For T4 and FRTL-5 cell extract, the t1/2 of dissociation calculated from kinetics of association was 5 h; no direct experimental determination of the value was made. Numbers of T3-binding sites of FRTL-5 cell, rabbit thyroid gland, and rat liver nuclear extracts were, respectively, 71 X 10(-15), 62 X 10(-15), and 208 X 10(-15) mol/mg protein. For T4 and FRTL-5 cell extract, the value was 70 X 10(-15) mol/mg protein. The data indicate that the reaction of T3 and T4 with the various nuclear extracts can be described as reversible and bimolecular. The presence in thyroid cells of thyroid hormone nuclear binding sites suggests that they may be receptors that mediate cellular actions of these hormones within the gland itself.