Association of the 3,5,3'-triiodo-L-thyronine nuclear receptor with the nuclear matrix of cultured growth hormone-producing rat pituitary tumor cells (GC cells).
The iodothyronine nuclear receptor, a nonhistone chromatin protein, mediates growth hormone gene transcription in cultured GC cells (Yaffe, B.M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). To determine whether the 3,5,3'-triiodo-L-thyronine (T3) receptor was localized to the nuclear matrix, we studied the subnuclear distribution of [125I]T3-receptor complexes after treatment of nuclei with DNase I and 2 M NaCl to facilitate removal of histones. After incubation with 5 nM [125I]T3 to exchange with 80-90% of nuclear T3 receptors, the nuclear matrix fraction contained less than 1% of nuclear DNA, 16.5% of nuclear protein, and 30-50% (mean, 40.0 +/- 2.3%) of specifically bound [125I]T3. Control experiments showed that nuclear matrix [125I]T3 did not appear exchangeable with added 5 nM T3 and did not result from release and nonspecific precipitation of [125I]T3 or [125I]T3-receptor complexes during nuclear matrix preparation. Studies with the T3 photoaffinity probe, N-2-diazo-3,3,3-trifluoropropionyl-[125I]T3 resulted in the finding of limited capacity receptor forms, 58,000 and 46,000 kDa, in the nuclear matrix. These receptor forms were identical to those observed when N-2-diazo-3,3,3-trifluoropropionyl-[125I]T3-labeled receptors were solubilized directly from nuclei. Lastly, limited capacity [125I]T3 binding was demonstrated in 0.4 M KCl buffer extracts of nuclear matrix. Binding displacement studies suggested that 46% of the binding activity solubilized from nuclear matrix exchanged with [125I]T3 and that the apparent equilibrium association constant of this binding activity was similar to that of 0.4 M KCl extracts of isolated nuclei. These results suggest that an appreciable fraction of nuclear T3 receptors is localized to the nuclear matrix and may influence the expression of thyroid hormone action.
D000818:Animals; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011988:Receptors, Thyroid Hormone; D013997:Time Factors
D002868:Chromosomal Proteins, Non-Histone; D011956:Receptors, Cell Surface; D011988:Receptors, Thyroid Hormone; D013006:Growth Hormone; D004492:Edetic Acid; D003850:Deoxyribonuclease I; D008274:Magnesium; D004229:Dithiothreitol