Thyroid hormone action in cell culture: domonstration of nuclear receptors in intact cells and isolated nuclei.
Triiodothyronine and thyroxine induce a 3-fold increase in the rate of growth of GH(1) cells in culture. To study further the action of these hormones, we examined the binding of [(125)I]triiodothyronine and purified [(125)I]thyroxine to cellular fractions after incubation with intact cells in serum-free medium. High-affinity, low-capacity binding sites for the hormones were demonstrated in nuclear but not in mitochondrial or cytosol fractions. Chromatographic analysis of the bound nuclear radioactivity from cells incubated with [(125)I]thyroxine demonstrated 97% thyroxine, 1% iodide, and 1% triiodothyronine. Apparent equilibrium dissociation constants, determined by Scatchard analysis, were 29 pM for triidothyronine and 260 pM for thyroxine. The maximal binding capacity was identical for both hormones, with about 5000 sites per cell nucleus. [(125)I]Thyroxine binding was competitively inhibited by triiodothyronine. These data suggest that triiodothyronine and thyroxine interact with identical nuclear receptors, and that conversion of thyroxine to triiodothyronine may not be a prerequisite for biologic activity. Similar high-affinity, low-capacity nuclear binding sites were also demonstrated by incubation of [(125)I]triidothyronine directly with isolated nuclei. Incubation of cells with increasing concentrations of nonradioactive triidothyronine results in a subsequent increase in binding when [(125)I]triiodothyronine is then incubated directly with isolated nuclei. This result suggests that nuclear receptors are not fixed, but increase after exposure of intact cells to hormone. This increase in nuclear receptor content may result from the transfer of an unstable cytosol receptor to the nucleus.
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D010911:Pituitary Neoplasms; D051381:Rats; D011956:Receptors, Cell Surface; D013347:Subcellular Fractions / Q000378:metabolism; D013974:Thyroxine / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D014284:Triiodothyronine; D013974:Thyroxine