Thyroid Hormone Receptors BINDIXG CHARACTERISTICS AND LACK OF HORRlOn-AL DEPEXDENCY FOR NUCLEAR LOCALIZATION *

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 = triiodothyronine-receptor complex, with an apparent equilibrium dissociation constant (Kd) at 22” 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+l) and dissociation (k-l) rate constants at 22’ are, respectively, 4.7 x lo7 M-I min-’ and 7.6 x 1O-3 min-I. The apparent Kd, estimated from the ratio of the rate constants (k-,: k+l), 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 pro.