Thyroid hormone action: in vitro characterization of solubilized nuclear receptors from rat liver and cultured GH1 cells.
We previously reported that putative nuclear receptors for thyroid hormone can be demonstrated by incubation of hormone either with intact GH(1) cells, a rat pituitary tumor cell line, or with isolated GH(1) cell nuclei and rat liver nuclei in vitro. We characterized further the kinetics of triiodothyronine (T3) and thyroxine (T4) binding and the biochemical properties of the nuclear receptor after extraction to a soluble form with 0.4 M KCl. In vitro binding of [(125)I]T3 and [(125)I]T4 with GH(1) cell and rat liver nuclear extract was examined at 0 degrees C and 37 degrees C. Equilibrium was attained within 5 min at 37 degrees C and 2 h at 0 degrees C. The binding activity from GH(1) cells was stable for at least 1 h at 37 degrees C and 10 days at - 20 degrees C. Chromatography on a weak carboxylic acid column and inactivation by trypsin and Pronase, but not by DNase or RNase, suggested that the putative receptor was a nonhistone protein. The estimated equilibrium dissociation constants (K(d)) for hormone binding to the solubilized nuclear binding activity was 1.80 x 10(-10) M (T3) and 1.20 x 10(-9) M (T4) for GH(1) cells and 1.57 x 10(-10) M (T3) and 2.0 x 10(-9) M (T4) for rat liver. These K(d) values for T3 are virtually identical to those which we previously reported with isolated rat liver nuclei and GH(1) cell nuclei in vitro. The 10-fold greater affinity for T3 compared to T4 in the nuclear extract is also identical to that observed with intact GH(1) cells. In addition, the [(125)I]T3 and [(125)I]T4 high-affinity binding in the nuclear extract were inhibited by either nonradioactive T3 or T4, which suggests that the binding activity in nuclear extract was identical for T3 and T4. In contrast, the binding activity for T4 and T3 in GH(1) cell cytosol was markedly different from that observed with nuclear extract (K(d) values were 2.87 x 10(-10) M for T4 and 1.13 x 10(-9) M for T3). Our results indicate that nuclear receptors for T3 and T4 can be isolated in a soluble and stable form with no apparent change in hormonal affinity. This should allow elucidation of the mechanisms of thyroid hormone action at the molecular level.
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D009698:Nucleoproteins / Q000378:metabolism; D010911:Pituitary Neoplasms; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013974:Thyroxine / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*
D006657:Histones; D007457:Iodine Radioisotopes; D009698:Nucleoproteins; D011956:Receptors, Cell Surface; D014284:Triiodothyronine; D012710:Serum Albumin, Bovine; D013974:Thyroxine