Nemoto T, Mason G G, Wilhelmsson A, Cuthill S, Hapgood J, Gustafsson J A, Poellinger L
Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, Sweden.
J Biol Chem. 1990 Feb 5;265(4):2269-77.
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.
对二噁英受体和糖皮质激素受体从非DNA结合状态激活为DNA结合状态的体外激活过程进行了表征。无配体的二噁英受体和糖皮质激素受体从大鼠肝脏胞质溶胶中部分共纯化，在用各自的配体标记后，两种受体均在9S处沉降。二噁英受体和糖皮质激素受体的9S形式先前已被证明代表含有分子量约为90,000的热休克蛋白的异源复合物。即使在存在稳定剂钼酸盐的情况下，将9S无配体或配体结合的糖皮质激素受体暴露于0.4M NaCl后，也会转化为单体4-5S形式。在相同条件下，9S无配体和配体结合的二噁英受体形式基本保持完整。然而，在没有钼酸盐的情况下，二噁英受体在暴露于高浓度盐时可转化为4-5S形式。这些结果表明，即使在没有激素的情况下，糖皮质激素受体也很容易从9S解离为4-5S形式，而无配体和有配体占据的9S二噁英受体形式都代表更稳定的物种。凝胶迁移率变动实验表明，4-5S糖皮质激素受体在无配体和有配体的情况下均与糖皮质激素反应元件相互作用。另一方面，配体占据二噁英受体极大地增强了受体被激活为与其靶增强子元件结合形式的能力。一旦解离，二噁英受体的单体形式即使在没有配体的情况下也能够与其DNA靶序列相互作用。因此，配体结合有效地促进了二噁英受体的亚基解离，但本身不是DNA结合的先决条件。鉴于其非DNA结合9S形式的明显稳定性，二噁英受体系统可能是研究可溶性受体蛋白激活机制的有用模型。
