Israel D I, Kaufman R J
Genetics Institute, Cambridge, MA 02140.
Proc Natl Acad Sci U S A. 1993 May 1;90(9):4290-4. doi: 10.1073/pnas.90.9.4290.
A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [3H]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.
构建了一个嵌合基因，其编码完整的小鼠二氢叶酸还原酶（DHFR）蛋白，并带有一个羧基末端延伸部分，该延伸部分包含大鼠糖皮质激素受体（GR）的第494 - 795位氨基酸。通过将缺乏DHFR的中国仓鼠卵巢（CHO）细胞转化为DHFR阳性表型的能力来衡量，该嵌合DHFR/GR基因编码一种功能性DHFR蛋白。DHFR/GR蛋白与[3H]地塞米松结合的亲和力与野生型GR相似。在浓度不断增加的甲氨蝶呤中选择稳定的CHO转化体，导致DHFR/GR的表达增加。添加合成糖皮质激素激动剂地塞米松后，通过在选择培养基中形成集落、荧光甲氨蝶呤的结合以及对DHFR的直接酶活性测定来衡量，嵌合蛋白的活性降低。添加糖皮质激素拮抗剂RU486可拮抗地塞米松的作用。在没有地塞米松的情况下，嵌合蛋白主要定位于细胞质。在存在地塞米松或RU486的情况下，DHFR/GR易位进入细胞核。然而，RU486并未降低DHFR活性，这表明亚细胞定位与功能活性不同。这些结果表明糖皮质激素对DHFR/GR的功能有负面影响。
