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Thyroid Hormone Receptors from Liver Nuclei: Characteristics of Receptor from Normal, Thyroidectomized, and Triiodothyronine-Treated Rats; Measurement of Occupied and Unoccupied Receptors, and Chromatin Binding of Receptors^*

JUAN BERNAL, ALDO H. COLEONI and LESLIE J. DEGROOT

Thyroid Study Unit, Department of Medicine, University of Chicago Chicago, Illinois 60637

Address requests for reprints to: Dr. Leslie J DeGroot, Department of Medicine, Thyroid Study Unit, University of Chicago, Division of the Biological Sciences and Pritzker School of Medicine, 950 East 59th Street, Chicago, Illinois 60637.

Abstract

The capacity and characteristics of liver nuclear receptors for T₃ have been compared in normal rats, thyroidectomized rats, and thyroidectomized rats treated with different doses of T₃. Receptor capacity was unchanged, with the exception of a decrease of total nuclear receptors in rats treated with 15 µg T₃/day, which is believed due to lack of complete dissociation of the endogenously bound hormone during incubation in vitro. During incubation of the isolated nuclei in 0.32 M sucrose plus 1 mM MgCl₂ plus 20 mM Tris-Cl buffer, pH 7.85, about 50% of the nuclear receptors were released to the medium and no effect of T₃ treatment on the amount released was found. The affinity of the receptors free in solution was 50% higher than that of receptors retained in the nuclei. Receptors from normal and thyroidectomized rats were studied by ultracentrifugation through sucrose gradients. No difference in sedimentation coefficient was found between the groups of rats, whether the receptors were filled with the labeled hormone before or after centrifugation and separation of the gradient fractions.

Occupied and unoccupied receptor capacity was estimated by incubation of receptor preparations at 20 and 0 C for 2 h. Observed capacity at 20 C represents unoccupied plus approximately 80% of occupied sites, while observed capacity at 0 C represents unoccupied plus 10% of occupied site.

Binding of receptors to the chromatin was studied by incubating nuclear extract from normal rats with chromatin and measuring the amount of receptor that was not bound and remained free in the supernatant. Receptor not bound to chromatin was incubated at 0 and 20 C, with a saturating concentration of [¹²⁵I]T₃, to measure occupied and unoccupied sites. Both occupied and unoccupied receptor decrease in the supernatant after incubation with the chromatin when the concentration of chromatin in the incubation medium is increased. No saturation of the chromatin-binding sites was achieved by increasing the concentration of receptor several-fold, and no difference in the binding of occupied and unoccupied receptors to chromatin was observed.

These data rule out the possibility that the concentration of receptors in the nucleus is under control of the hormone. Major molecular changes of the receptor that could be induced by T₃, such as dissociation into subunits, are also ruled out. Binding studies to the chromatin are not necessarily conclusive, as nonspecific binding may be masking the physiological binding of the receptors to a limited number of acceptor sites.

Footnotes

^* This work was supported by USPHS Grants AM-13377, CA-19266, AM-07011, and TW-02155.

Recipient of a Fogarty International Fellowship (F05-TW02155). Present address: Departamento de Endocrinologia Experimental, C.S.I.C, Facultad de Medicina, Universidad Autonoma, Madrid 34, Spain.

Received May 31, 1977.