Abstract. Although putatively identified more than 10 years ago, thyroid hormone receptors in human tissues remain poorly characterized. As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line, IM-9 cells. In whole cell experiments at 37°C, nuclear binding of [125I]T3 was saturable (Kd 34 ± 6 pmol/l) and of finite capacity (≈ 350 sites/cell). The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication. Separation of bound from free [125I]T3 in the extracts was achieved using the calcium phosphate matrix, hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry. Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5% Triton X-100. Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 ± 8.6 fmol/mg protein and showed an affinity of (Kd, room temperature) 140 ± 10 pmol/l. The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues. Calling T3 100, the order of potencies observed was: Triac, 500; 3,5-diiodo-3′-isopropylthyronine, 89; T4, 32; 35-dimethyl-3′-isopropylthyronine, 2; 3,2-T2, 0.7, rT3, 0.4; 3′5′-T2, < 0.01. These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors. These cells may be a useful tool to increase our understanding of human T3 receptors. The use of hydroxyapatite to separate bound from free hormone may be adapted for use with extracts of other tissues to characterize these receptors further.
Purification and characterization of rat liver nuclear thyroid hormone receptors.
