Thyroid hormone receptors. Binding characteristics and lack of hormonal dependency for nuclear localization

Bj Spindler,Km Macleod,J Ring,Jd Baxter

doi:10.1016/s0021-9258(19)41393-8

Abstract

Thyroid hormones have diverse effects on growth and metabolism. Specific "receptor" proteins which bind triiodothyronine and other biologically active analogs and which may be involved in thyroid hormone action have been recently found in nuclei of responsive tissues. This report presents studies of these receptors in rat liver nuclei. Confirming previous reports, a Scatchard analysis of the binding data suggests the reaction, triiodothyronine + specific receptor in equilibrium with triiodothyronine-receptor complex, with an apparent equilibrium dissociation constant (Kd) at 22 degrees of about 190 pM and a capacity of about 1 pmol of triiodothyronine-binding sites per mg of DNA. The kinetics of the binding were also examined. Triiodothyronine-receptor complex formation is second order and dissociation is first order. The apparent association (k+1) and dissociation (k minus 1) rate constants at 22 degrees are, respectively, 4.7 times 10-7 m-minus 1 min-minus 1 and 7.6 times 10-minus 3 min-minus 1. The apparent Kd, estimated from the ratio of the rate constants (k minus 1:k+1), was about 150 pM, similar to that determined from the equilibrium data. These data support the expression written above for the interaction of thyroid hormone with its receptor. Additional kinetic experiments indicate that some of the triiodothyronine binding by cell-free nuclei is to sites previously occupied by hormone in the intact animal, providing further evidence that the intact cell and cell-free reactions are the same. It was previously found that nuclear-bound triiodothyronine is localized in chromatin. We found that isolated chromatin retains specific binding activity similar to that of isolated nuclei. Thus, binding may not require cytoplasmic, nucleoplasmic, or nuclear membrane factors. These findings may imply that chromatin localization of the receptor does not depend on the hormone. This idea is supported by an earlier finding that binding activity is present in nuclei from thyroidectomized animals. However, many stimuli such as steroid hormones, bacterial inducers, and cyclic adenosine 3':5'-monophosphate in bacteria influence regulatory proteins at the gene level by promoting the protein's addition to or removal from chromatin. Thus, we studied the effect of thyroid hormone on the nuclear content of receptors under assay conditions of receptor stability and reversible binding. Receptor levels in hypothyroid animals are identical with those in euthyroid animals. These data suggest that the hormone does not influence the nuclear localization of receptors. Thus, the basis for thyroid hormone action may be to regulate the activity of receptors resident in chromatin rather than to promote receptor addition to or removal from chromatin.

Highlights

Thyroid hormones have diverse effects on growth and metabolism
Since the thyroid hormone receptor may be a DNA-associated chromatin protein, we investigated whether the hormone itself exerts any influence on the nuclear content of these chromatin receptors
Most experiments were performed at 22”, since the binding kinetics were found to be more rapid at this temperature than at 0”

Summary

Methods

Many of the procedures were performed using modifications of techniques previously described by Samuels and Tsai [5,9].MateriaZs-[1251]Triiodothyronine (Abbott, 496 mCi/mg) was about 92yo pure as determined by Sephadex column chromatography [24] and was either purified by this technique or used without further purification.Identical nuclear binding results were obtained with either preparation.Further, over 987, of the specific radioactivity bound to nuclei was extracted as described elsewhere [5] and found to migrate with authentic triiodothyronine in the Sephadex column [24]. Many of the procedures were performed using modifications of techniques previously described by Samuels and Tsai [5,9]. MateriaZs-[1251]Triiodothyronine (Abbott, 496 mCi/mg) was about 92yo pure as determined by Sephadex column chromatography [24] and was either purified by this technique or used without further purification. Identical nuclear binding results were obtained with either preparation. Over 987, of the specific radioactivity bound to nuclei was extracted as described elsewhere [5] and found to migrate with authentic triiodothyronine in the Sephadex column [24]. Nonradioactive triiodothyronine purchased from Sigma and made in solution at 5 rnM with 0.1 M NaOH was used without further purification.

Results

Discussion

Conclusion