Berhanu P, Anderson C, Hickman M, Ciaraldi T P
Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 1997 Sep 5;272(36):22884-90. doi: 10.1074/jbc.272.36.22884.
The cytoplasmic juxtamembrane domain of the human insulin receptor (hIR) contains a single copy of the tetrameric amino acid sequence Asn-Pro-Glu-Tyr (NPEY) (residues 969-972 in the exon 11-containing B-isoform), which exists in the context of NPXY. In this study, we examined the role of NPEY972 in mediating insulin signal transduction and cellular biological effects. Transfected Chinese hamster ovary cell lines expressing either the wild-type hIR-B isoform (hIR.WT) or a mutant receptor lacking the NPEY972 sequence (hIRDeltaNPEY) and control Chinese hamster ovary.Neo cells were used to comparatively analyze the following insulin effects: in vivo receptor tyrosine autophosphorylation and kinase activity, signal transduction to downstream signaling molecules, and stimulation of glycogen and DNA synthesis. The results showed that in comparison to hIR.WT, the hIRDeltaNPEY mutant demonstrated the following: (a) normal insulin-mediated receptor tyrosine phosphorylation, but approximately 50% reduction in phosphorylation of p185-(insulin receptor substrate-1) and binding of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), (b) an enhanced stimulation of PI 3-kinase enzymatic activity, (c) a complete inability to phosphorylate Shc, (d) minimal impairment of insulin sensitivity for glycogen synthesis, and (e) an augmented response to insulin-stimulated DNA synthesis via a high capacity, low sensitivity pathway. These results demonstrate the following: 1) the NPEY972 sequence is important but not absolutely essential for coupling of hIR kinase to insulin receptor substrate-1 and p85 or for mediating insulin's metabolic and mitogenic effects, 2) the NPEY972 sequence is necessary for Shc phosphorylation, and 3) the absence of Shc phosphorylation releases the constraints on maximal insulin-stimulated mitogenic response, thus indicating that alternate signaling pathway(s) exist for this insulin action. This alternate pathway appears to be associated with enhanced activation of PI 3-kinase and is of high capacity and low sensitivity.
人胰岛素受体（hIR）的胞质近膜结构域包含四聚体氨基酸序列天冬酰胺-脯氨酸-谷氨酸-酪氨酸（NPEY）的单拷贝（含第11外显子的B异构体中的第969 - 972位氨基酸残基），其存在于NPXY序列中。在本研究中，我们检测了NPEY972在介导胰岛素信号转导和细胞生物学效应中的作用。使用转染了表达野生型hIR - B异构体（hIR.WT）或缺失NPEY972序列的突变受体（hIRDeltaNPEY）的中国仓鼠卵巢细胞系，以及对照中国仓鼠卵巢.Neo细胞，来比较分析以下胰岛素效应：体内受体酪氨酸自身磷酸化和激酶活性、向下游信号分子的信号转导，以及对糖原和DNA合成的刺激。结果显示，与hIR.WT相比，hIRDeltaNPEY突变体表现出以下情况：（a）胰岛素介导的受体酪氨酸磷酸化正常，但p185-（胰岛素受体底物-1）的磷酸化及磷脂酰肌醇3激酶（PI 3激酶）p85亚基的结合减少约50%，（b）PI 3激酶酶活性的刺激增强，（c）完全无法使Shc磷酸化，（d）对糖原合成的胰岛素敏感性仅有轻微损害，以及（e）通过高容量、低敏感性途径对胰岛素刺激的DNA合成反应增强。这些结果表明：1）NPEY972序列对于hIR激酶与胰岛素受体底物-1及p85的偶联或介导胰岛素的代谢和促有丝分裂效应很重要，但并非绝对必需；2）NPEY972序列对于Shc磷酸化是必需的；3）Shc磷酸化的缺失解除了对最大胰岛素刺激的促有丝分裂反应的限制，从而表明存在该胰岛素作用的替代信号通路。这条替代通路似乎与PI 3激酶的增强激活相关，且具有高容量和低敏感性。
