Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90.
Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90.
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发表时间：
1990-11
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通讯作者：
E. R. Sánchez;M. Hirst;L. Scherrer;H. Tang;M. Welsh;J. Harmon;S. Simons;G. Ringold;W. Pratt
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作者：
E. R. Sánchez;M. Hirst;L. Scherrer;H. Tang;M. Welsh;J. Harmon;S. Simons;G. Ringold;W. Pratt
在这项工作中，我们研究了在中国仓鼠卵巢（CHO）细胞中过表达的小鼠糖皮质激素受体的细胞定位和蛋白质相互作用（Hirst，M.一、诺斯罗普，J. P.，Danielsen，M.，Ringold，G. M.（1990）Mol.年. 4，162-170）。我们证明，野生型unliganded小鼠糖皮质激素受体，这是在CHO细胞中表达的水平约10倍的L细胞，是本地化完全与BuGR antireceptor单克隆抗体的间接免疫荧光的细胞核。由于点突变而没有激素结合活性或没有DNA结合活性的过表达受体也定位于细胞核，提供了遗传学证据，即细胞核定位不能反映类固醇介导的受体从细胞质向细胞核的转移，并且细胞核定位不需要DNA结合活性。与未配体的孕酮受体一样，其也以松散结合的“对接”复合物与细胞核相关联，在CHO细胞中过表达的小鼠糖皮质激素受体与hsp 90和hsp 70两者相关联。这与L细胞胞质溶胶中未转化的小鼠糖皮质激素受体相反，其与hsp 90而不是hsp 70相关。细胞类型之间hsp 70相关性的差异可以反映CHO细胞中受体的过表达。然而，与CHO细胞中选择用于非常高水平过表达的受体一样，CHO细胞中选择用于与L细胞相当的中间水平受体表达的受体也与hsp 70结合。这一观察结果反对纯粹基于受体过表达的热休克蛋白70协会的解释，我们推测，协会的unliganded糖皮质激素受体与热休克蛋白70可能是其在CHO细胞中的核定位的结果。尽管CHO细胞和L细胞中的小鼠受体之间存在差异，但未转化的小鼠受体的核定位信号与从两种细胞类型制备的细胞质中的抗NL 1的AP 64抗体反应等同。
In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.