Abstract
The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.
Highlights
In view of the many similarities in crude physicochemical parameters between the glucocorticoid and dioxin receptors (Wilhelmsson et al, 1986; Cuthill et al, 1987 and references therein), we examined the possibility of whether the ligand-free dioxin receptor co-chromatographed with the corresponding form of glucocorticoid receptor
We have previously shown that manipulations such as immunoprecipitation in the absence of sodium molybdate result in the dissociation of the heteromeric glucocorticoid receptor complex and unmasking of the DNA binding activity even in the absence of ligand (Denis et al, 198813).As shown in Fig. 3A, the 9 S ligand-free glucocorticoid was sensitive to exposure to salt, resulting in conversion to the 4-5 S monomeric form
Interaction of the Dioxin Receptor with Its Target Enhancer Element Is a Ligand-dependent Event--In this study, we have demonstrated that ligand is required for efficient binding of the dioxin receptor to its cognate response element in uitro
Summary
Most of the biological effects produced by TCDD and related compounds are mediated by the intracellular, soluble dioxin receptor protein (reviewed in Poland and Knutson, 1982) Studies in this and other laboratories have shown distinct similarities in the physicochemical characteristics as well as the functions of the dioxin and glucocorticoid receptors (Wilhelmsson et al, 1986; Cuthill et al, 1987; Cuthill and Poellinger, 1988; Perdew, 1988; Denis et al, 1988c and references therein). Dissociation of hsp has been suggested to trigger the process termed activation by which the glucocorticoid receptor complex is converted from the 9 S (~300 kDa) non-DNA binding form to the 4 S DNA binding species of a94 kDa before specific effects on gene expression are exerted (reviewed in Denis and Gustafsson, 1989). It might facilitate a detailed in vitro dissection of the molecular events leading to functional forms of receptor
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