METABOLIC EFFECTS OF THYROID HORMONE IN CELL CULTURE

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Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.