Cheatham B, Shoelson S E, Yamada K, Goncalves E, Kahn C R
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215.
Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7336-40. doi: 10.1073/pnas.90.15.7336.
The mechanism through which insulin binding to the extracellular domain of the insulin receptor activates the intrinsic tyrosine kinase in the intracellular domain of the protein is unknown. For the c-neu/erbB-2 (c-erbB-2) protooncogene, a single point mutation within the transmembrane (TM) domain converting Val-664 to Glu (erbB-2V-->E) results in elevated levels of tyrosine kinase activity and cellular transformation. We report the construction of a chimeric insulin receptor in which the TM domain of the receptor has been substituted with that encoded by erbB-2V-->E. When expressed in Chinese hamster ovary cells this chimeric receptor displays maximal levels of autophosphorylation and kinase activity in the absence of insulin. This activity results in an increase in the level of insulin-receptor substrate 1 phosphorylation but a down-regulation in insulin-receptor substrate 1 protein and desensitization to insulin stimulation of glycogen synthesis. By contrast, basal levels of DNA synthesis are elevated to levels approximately 60% of those observed in serum-stimulated cells. Over-expression of chimeric insulin receptors containing the c-erbB-2 TM domain or a single point mutation in the insulin receptor TM domain of Val-938-->Asp, on the other hand, shows none of these alterations. Thus, the TM domain encoded by erbB-2V-->E contains structural features that can confer ligand-independent activation in a heterologous protein. Constitutive activation of the insulin receptor results in a relative increase in basal levels of DNA synthesis, but an apparent resistance to the metabolic effects of insulin.
胰岛素与胰岛素受体细胞外结构域结合从而激活该蛋白细胞内结构域中内在酪氨酸激酶的机制尚不清楚。对于c-neu/erbB-2（c-erbB-2）原癌基因，跨膜（TM）结构域内的一个单点突变将缬氨酸-664转换为谷氨酸（erbB-2V→E）会导致酪氨酸激酶活性水平升高和细胞转化。我们报道了一种嵌合胰岛素受体的构建，其中该受体的TM结构域已被erbB-2V→E编码的结构域所取代。当在中国仓鼠卵巢细胞中表达时，这种嵌合受体在没有胰岛素的情况下显示出最大水平的自磷酸化和激酶活性。这种活性导致胰岛素受体底物1磷酸化水平增加，但胰岛素受体底物1蛋白下调以及对胰岛素刺激糖原合成的脱敏。相比之下，DNA合成的基础水平升高至约为血清刺激细胞中观察到水平的60%。另一方面，含有c-erbB-2 TM结构域的嵌合胰岛素受体或胰岛素受体TM结构域中缬氨酸-938→天冬氨酸的单点突变的过表达均未显示出这些改变。因此，erbB-2V→E编码的TM结构域包含可在异源蛋白中赋予非配体依赖性激活的结构特征。胰岛素受体的组成性激活导致DNA合成基础水平相对增加，但对胰岛素的代谢作用表现出明显抗性。
