Lebwohl D E, Nunez I, Chan M, Rosen O M
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1991 Jan 5;266(1):386-90.
We have transfected mouse L cells with a recombinant membrane-anchored insulin receptor kinase (called MARK), under the transcriptional control of a glucocorticoid-responsive element. The transfected construct includes only 15 extracellular residues, with the transmembrane and intracellular kinase domains of the human insulin receptor cDNA (amino acid residues -27 to 12 and 915 to 1343 (see Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761), predicted Mr = 56,000 with signal sequence, 53,000 without). Transfected cells which are exposed to dexamethasone express two proteins of approximate Mr = 54,000 which (a) react with anti-peptide antisera raised to human insulin receptor sequences, (b) localize to the membrane fraction, and (c) possess ligand (insulin)-independent tyrosine kinase activity. In extracts of steroid-treated MARK cells, a phosphotyrosine-containing protein of Mr = 185,000 is detected, which corresponds in size to a known endogenous substrate for the insulin receptor. Control studies were performed with the nontransfected parent line, and with L cells transfected with an inactive human insulin receptor protein tyrosine kinase. Dexamethasone induced no change in the proteins detected by an anti-phosphotyrosine antibody in the two control lines. In studies of deoxyglucose uptake, dexamethasone stimulated increase in deoxyglucose uptake 2-fold in the MARK cells compared to the same cells studied in the absence of dexamethasone. Dexamethasone had no effect in the control cell lines. These studies demonstrate that a membrane-anchored insulin receptor kinase, devoid of virtually the entire extracellular domain of the insulin receptor, is sufficient to induce enhanced deoxyglucose uptake.
我们用一种重组的膜锚定胰岛素受体激酶（称为MARK）转染了小鼠L细胞，该激酶受糖皮质激素反应元件的转录控制。转染构建体仅包含15个细胞外残基，以及人胰岛素受体cDNA的跨膜和细胞内激酶结构域（氨基酸残基-27至12以及915至1343（见Ullrich，A.，Bell，J.R.，Chen，E.Y.，Herrera，R.，Petruzzelli，L.M.，Dull，T.J.，Gray，A.，Coussens，L.，Liao，Y.C.，Tsubokawa，M.，Mason，A.，Seeburg，P.H.，Grunfeld，C.，Rosen，O.M.，和Ramachandran，J.（1985）《自然》313，756 - 761），预测有信号序列时Mr = 56,000，无信号序列时Mr = 53,000）。暴露于地塞米松的转染细胞表达两种近似Mr = 54,000的蛋白质，这些蛋白质（a）与针对人胰岛素受体序列产生的抗肽抗血清反应，（b）定位于膜部分，并且（c）具有不依赖配体（胰岛素）的酪氨酸激酶活性。在经类固醇处理的MARK细胞提取物中，检测到一种Mr = 185,000的含磷酸酪氨酸的蛋白质，其大小与胰岛素受体的一种已知内源性底物相对应。用未转染的亲本细胞系以及用无活性的人胰岛素受体蛋白酪氨酸激酶转染的L细胞进行了对照研究。地塞米松在两个对照细胞系中未引起抗磷酸酪氨酸抗体检测到的蛋白质发生变化。在脱氧葡萄糖摄取研究中，与在无地塞米松情况下研究的相同细胞相比，地塞米松刺激MARK细胞中的脱氧葡萄糖摄取增加了2倍。地塞米松对对照细胞系没有影响。这些研究表明，一种几乎没有胰岛素受体整个细胞外结构域的膜锚定胰岛素受体激酶足以诱导增强的脱氧葡萄糖摄取。
