The binding of thyroid hormone receptors to DNA

The behaviour of tri-iodothyronine (T3)- and thyroxine (T4)-receptor complexes when bound to native DNA-cellulose is reported. Equal and large proportions of both T3- and T4-receptor complexes bind to DNA but although T3-receptor complexes are 99% recoverable by 0.5 M NaCl buffer elution, only 60-70% of the T4-receptor complexes are regained. The balance appears as free T4, apparently released as the T4-receptor complexes bind to the DNA whilst the corresponding receptor remains bound. This effect is independent of T4-receptor complex/DNA ratio up to ca. 4 fmol/micrograms DNA, of the presence of an equal amount of unoccupied receptor and of an eight-fold concentration range of both T4-receptor complex and DNA at a fixed ratio, in the cellulose matrix. Pre-formed receptor-DNA material, likewise, only accepts some 60% of the expected quantity of T4 whereas the capacity for T3 appears to be similar to that of free receptors.     

DNA binding of thyroid hormone receptors.

Thyroid hormone receptors, isolated from rat liver nuclei, bind to purified DNA. By contrast, free triiodothyronine and plasma proteins which bind thyroid hormone do not associate with DNA. Thus, the nuclear localization of thyroid hormone in target tissues may be explained by the association of its receptors with DNA.     

A nuclear factor that enhances binding of thyroid hormone receptors to thyroid hormone response elements

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.     

Nuclear thyroid hormone receptors: evidence for association with nucleolar chromatin.

When hypothyroid rat liver nuclei labeled $\text{in vivo}$ with [125 I]L-triiodothyronine are incubated with micrococcal nuclease, the nuclear chromatin is digested and chromatin particles are released into the medium. The nuclease-treated nuclei contain intact nucleoli and a residual chromatin fraction. When this residual chromatin is purified, it contains only a small percentage of the initial nuclear DNA but is strikingly enriched in [125 I]L-triiodothyronine. This chromatin fraction has many of the characteristics of nucleolar chromatin including a high protein to DNA ratio, an abundance of nonhistone proteins, and a relatively high RNA to DNA ratio. An association of thyroid hormone receptors with a nucleolar component implicates this organelle in the early events of thyroid hormone action.     

The nature of thyroid hormone receptors. Intracellular transport and compartmentalization of chromatin receptors and thyroid hormones

It is well established that in vivo administered labelled TBPA penetrates into liver, brain and lung cells, is translocated from cytosol into the nucleus and is accepted by chromatin without being affected by modifications touching upon the antigenic determinants of this protein. Electron microscopic autoradiography demonstrated that 125I-TBPA translocated from cytosol into the nucleus is localized on the border between hetero- and euchromatin. The data obtained may serve as an additional proof of the universal structure of intracellular thyroid hormone receptors and suggest that TBPA participate in manifestation of genetic effects of thyroid hormones.     

Nuclear benzodiazepine binding: Possible interaction with thyroid hormone receptors

The biochemical and pharmacological properties of nuclear [³H]flunitrazepam in brain tissues were studied. Nuclear [³Hflunitrazepam binding is saturable for both central and peripheral binding sites. Inosine and hypoxanthine displace nuclear [³H]flunitrazepam binding with greater potency than the membrane [³H]flunitrazepam binding. Triiodothyronine (T₃) increases the maximum number of binding sites (B_max) of nuclear [³H]flunitrazepam binding in vitro while thyroxine (T₄) does not have any effect. Diazepam reduces the affinity of nuclear¹²⁵I-T₃ binding in vitro, while the B_max is not affected significantly. Mild digestion of chromatin, using micrococcal nuclease, reveals that a major portion of nuclear [³H]flunitrazepam binding sites are located on chromatin. These data suggest a functional role for nuclear benzodiazepine binding and a possible modulatory effect of benzodiazepines on T₃ binding with its nuclear receptors.     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

An assay procedure for solubilized thyroid hormone receptor: use of Lipidex

A method using Lipidex-1000 is reported for the assay of thyroid hormone receptor activity. The receptor was extracted from rat liver nuclei, incubated with [125I]T3, and applied to 1-ml disposable pipet tip columns containing a small volume of Lipidex. The resin absorbed the free hormone, and the receptor-bound hormone was recovered in the eluate. The method allowed accurate assay of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentration. The usefulness of this method was demonstrated by determining the pH optimum and the Kd and Bmax of T3 binding by the receptor. The Lipidex column can be used for the preparation of an unoccupied receptor after dissociation of the endogenously bound hormone.     

From binding proteins to hormone receptors?

Phytohormones exert in responsive plant cells specific biochemical and physiological effects. It is a widely held view that phytohormones are first recognized by specific receptors which initiate the transduction of the hormonal signal. While hormone receptors are well studied in many eukaryotes ranging from yeast to man, we are lacking a detailed understanding of phytohormone receptors. Phytohormone binding proteins have been suspected to provide candidates for such receptors. In this review recent progress towards molecular analysis of such proteins and their genes will be summarized.