HIGH-LEVEL EXPRESSION OF WILD-TYPE AND VARIANT MOUSE GLUCOCORTICOID RECEPTORS IN CHINESE HAMSTER OVARY CELLS
HIGH-LEVEL EXPRESSION OF WILD-TYPE AND VARIANT MOUSE GLUCOCORTICOID RECEPTORS IN CHINESE HAMSTER OVARY CELLS
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DOI：
10.1210/mend-4-1-162
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发表时间：
1990-01-01
影响因子：
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通讯作者：
RINGOLD, GM
中科院分区：
文献类型：
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作者：
HIRST, MA;NORTHROP, JP;RINGOLD, GM
我们使用选择性二氢叶酸还原酶 (dhfr) cDNA 共扩增技术，分离了表达升高水平的野生型 (W) 和突变型糖皮质激素受体 (GR) 的中国仓鼠卵巢 (CHO) 细胞系。通过蛋白质印迹法或用[3H]-甲磺酸地塞米松标记，在用W、激素结合突变体(NA)和DNA结合突变体(NB)转染的细胞的提取物中观察到在90/92千道尔顿处的显着双倍。通过[3H]地塞米松结合对受体数量进行定量显示，在产生W和NB的细胞系中，每个细胞存在大约106个受体。这表明与未转染GR的对照CHO细胞相比，受体密度增加了25至50倍。对表达 GR 的细胞提取物进行蛋白质印迹比较定量显示，产生 NA 的细胞的水平约为对照 CHO 细胞的 500 倍。通过用糖皮质激素响应报告质粒pMMTV-氯霉素乙酰转移酶(CAT)瞬时转染来检查扩增受体的功能。我们的结果表明，诱导型 CAT 活性随着 W 受体的丰度而增加，即使在最高水平的受体下也没有观察到饱和性的证据。这支持了之前的建议，即激素调节转录因子的浓度对于最大转录效率肯定是有限的。有趣的是，即使表达高度扩增水平的 NA-GR 或 NB-GR 的细胞也没有表现出高于对照 CHO 细胞的诱导反应。因此，这些突变是非常无泄漏的，并且对于 CHO GR 的低内源活性来说并不占主导地位。 CHO 细胞中各种形式 GR 的过度表达可作为对该转录因子和其他激素调节转录因子进行详细生化分析的范例。
We have isolated Chinese hamster ovary (CHO) cell lines expressing elevated levels of wild-type (W) and mutant forms of the glucocorticoid receptor (GR) using the technique of coamplification with a selectable dihydrofolate reductase (dhfr) cDNA. A prominent double at 90/92 kilodaltons was observed by Western blotting or labeling with [3H]-dexamethasone mesylate in extracts from cells transfected with W, the hormone binding mutant (NA), and the DNA binding mutant (NB). Quantification of receptor number by [3H]dexamethasone binding revealed the presence of approximately 106 receptors per cell in the W and NB-producing lines. This represents a 25- to 50-fold increase in receptor density over control CHO cells which were not transfected with GR. Comparative quantitation by Western blotting of extracts from cells expressing GR showed that cells producing NA contain a level approximately 500-fold over control CHO cells. Function of the amplified receptors was examined by transient transfection with the glucocorticoid-responsive reporter plasmid pMMTV-chloramphenicol acetyl transferase (CAT). Our results indicate that inducible CAT activity increases with the abundance of W receptor and no evidence of saturability was observed even at the highest levels of receptor. This supports previous suggestions that the concentration of the hormone-regulated transcription factor is definitely limiting with regard to maximal transcription efficiency. Interestingly, cells expressing even highly amplified levels of NA-GR or NB-GR showed no inducible response above that seen with control CHO cells. Thus these mutations are exceedingly nonleaky and are not dominant over the low endogenous activity of the CHO GR. The overexpression of various forms of the GR in CHO cells serves as a paradigm for detailed biochemical analysis of this and other hormone-regulated transcription factors.