Yamauchi K, Pessin J E
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Mol Cell Biol. 1994 Jul;14(7):4427-34. doi: 10.1128/mcb.14.7.4427-4434.1994.
Insulin treatment of Chinese hamster ovary (CHO) cells expressing high levels of the insulin receptor (CHO/IR cells) activates both c-fos serum response element and activator protein 1 (AP-1) reporter genes approximately 10-fold. In contrast, parental CHO cells display only two- to threefold insulin stimulation of reporter gene activity. Transient transfection of parental CHO cells with an insulin receptor substrate 1 (IRS1) expression plasmid enhanced insulin downstream signaling in a biphasic manner, whereas IRS1 transfection of CHO/IR cells inhibited insulin signaling in a dose-dependent fashion. Further, expression of Grb2 in parental CHO cells had no effect on insulin signaling, whereas Grb2 increased insulin activation of reporter gene expression in CHO/IR cells. These data suggest that the expression levels of various effector molecules can either enhance or inhibit insulin downstream signaling events. To assess the relative effects of various insulin receptor, IRS1, and Grb2 levels on insulin signaling, parental CHO cells were transiently transfected with various combinations of expression plasmids encoding these proteins. Although expression of IRS1 resulted in a biphasic increase of insulin signaling in parental CHO cells, coexpression of IRS1 with the insulin receptor resulted in inhibition of signaling. This inhibition of insulin signaling directly correlated with an increased association of Grb2 with IRS1 and a concomitant sequestration of Grb2 away from Shc. Consistent with the Shc-Grb2 pathway as the major route for insulin-stimulated c-Fos and AP-1 transcriptional activation, the IRS1-mediated inhibition was reversed by transfection with an expression plasmid for Grb2. These data demonstrate that the extent of insulin-stimulated downstream signaling was dependent not only on the levels of individual signaling molecules but also on the formation of multiprotein complexes with specific stoichiometries.
用胰岛素处理过表达胰岛素受体的中国仓鼠卵巢（CHO）细胞（CHO/IR细胞），可使c-fos血清反应元件和激活蛋白1（AP-1）报告基因的活性激活约10倍。相比之下，亲本CHO细胞对报告基因活性的胰岛素刺激仅为2至3倍。用胰岛素受体底物1（IRS1）表达质粒瞬时转染亲本CHO细胞，可使胰岛素下游信号以双相方式增强，而对CHO/IR细胞进行IRS1转染则以剂量依赖性方式抑制胰岛素信号。此外，在亲本CHO细胞中表达Grb2对胰岛素信号没有影响，而Grb2可增强CHO/IR细胞中报告基因表达的胰岛素激活。这些数据表明，各种效应分子的表达水平可增强或抑制胰岛素下游信号事件。为了评估各种胰岛素受体、IRS1和Grb2水平对胰岛素信号的相对影响，用编码这些蛋白质的表达质粒的各种组合瞬时转染亲本CHO细胞。虽然IRS1的表达导致亲本CHO细胞中胰岛素信号的双相增加，但IRS1与胰岛素受体的共表达导致信号抑制。这种胰岛素信号的抑制与Grb2与IRS1的结合增加以及Grb2与Shc的分离直接相关。与Shc-Grb2途径作为胰岛素刺激的c-Fos和AP-1转录激活的主要途径一致，用Grb2表达质粒转染可逆转IRS1介导的抑制。这些数据表明，胰岛素刺激的下游信号的程度不仅取决于单个信号分子的水平，还取决于具有特定化学计量的多蛋白复合物的形成。
