Thyroid Hormone Action IN VITRO CHARACTERIZATION OF SOLUBILIZED NUCLEAR RECEPTORS FROM RAT LIVER AND CULTURED GH1 CELLS

Abstract

A B S T R A C T We previously reported that putative nuclear receptors for thyroid hormone can be demonstrated by incubation of hormone either with intact GH1 cells, a rat pituitary tumor cell line, or with isolated GH1 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 KCI. In vitro binding of [lI]T3 and ['I]T4 with GH1 cell and rat liver nuclear extract was examined at 0C and 370C. Equilibrium was attained within 5 min at 370C and 2 h at 0C.The binding activity from GH1 cells was stable for at least 1 h at 370C and 10 days at -200C. 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 (Kd) for hormone binding to the solubilized nuclear binding activity was 1.80 x 10-10 M (T3) and 1.20 X 10" M (T4) for GH1 cells and 1.57 X 10`" M (T3) and 2.0 X 10' M (T4) for rat liver. These Kd values for T3 are virtually identical to those which we previously reported with isolated rat liver nuclei and GH1 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 GH1 cells. In addition, the ['I]T3 and ['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.Dr. Samuels is the recipient of a PHS Research Career Development Award AM 46546.

Influence of thyroid hormone on ADP‐ribosylation of nuclear proteins in cultured GH1 cells

“…corporation into isolated nuclei after incubating GHI cells for 24 h witll 0.25 nM and 2.5 nM T3. These T3 concentrations restllt in approximately 35070 and 80% occupancy of the thyroid hormone receptor [3,9], As we have previously observed [71, approximately IO% of the radioactivity was present in the acid-soluble histone fraction and 90% was incorporated into non-histon¢ proteins, T3 decreased [nP]ADP-ribose incorporation into both fractions and the reduction was more pr • nounced at 2,5 nM T3. The effect of T3 was also examined with 350 uM [adenylate-2,SJH]NAD and the same decrease was found (results not shown), thus excluding a=P-labeling by an alternative modification, such as protein phosphorylation.…”

“…GHI cell nuclei were isolated [9], and the ADPribosylation assay was performed as described [7] by incubation for 15 rain at 37°C in 0.5 ml of 0,1 M Tris-HCl. pH 7,9 at 25=C, 2 mM [adenylateJ=P]NAD.…”

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

“…The thyroid hormone receptors appear to be salt-extractable, acidic chromatin proteins (5,8,10,11). The possibility that thyroid hormones act at the chromatin level is also consistent with findings that these hormones influence RNA synthesis (3,17,18).…”

“…The concentrations of T3 or T4 required for binding to these receptors and for bioreactivity are similar (6), and there is a good correlation between the ability of thyroid hormone analogs to inhibit T3-receptor binding and to promote the biological effect (15). Studies of thyroid hormone analogs also suggest that T3 and T4 function through physical interactions, such as with receptors, rather than through chemical reactions involving iodine or quinones (16).The thyroid hormone receptors appear to be salt-extractable, acidic chromatin proteins (5,8,10,11). The possibility that thyroid hormones act at the chromatin level is also consistent with findings that these hormones influence RNA synthesis (3,17,18).…”

“…Recently, high-affinity, limitedcapacity binding sites for triiodothyronine (T3), and thyroxine (T4), which appear to be receptors for these hormones, have been found in target cell nuclei (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). The concentrations of T3 or T4 required for binding to these receptors and for bioreactivity are similar (6), and there is a good correlation between the ability of thyroid hormone analogs to inhibit T3-receptor binding and to promote the biological effect (15).…”