Molecular Aspects of Insulin Receptor Signaling

胰岛素的代谢和促有丝分裂作用是由激素与其细胞表面受体结合而启动的。这导致胰岛素受体β亚基的细胞内酪氨酸激酶域被激活，随后细胞底物在酪氨酸上被磷酸化。已经发现了许多与受体β亚基相互作用的接头和效应蛋白，并参与了胰岛素信号的调节。我们的实验室最近发现，当细胞用同双功能交联剂1，6-双马来亚胺己烷处理细胞时，一种名为TRAP的硫醇反应膜相关蛋白与人胰岛素受体的细胞质区域共价结合(Garant等人，2000年)。为了进一步了解TRAP在胰岛素信号转导中的生物学意义，我们对TRAP蛋白复合体进行了纯化，质谱分析表明TRAP是磷脂酶C-伽马1(PLCGamma1)。PLCGamma1是一种酶，通过促进二酰甘油和1，4，5-三磷酸肌醇的产生在跨膜信号转导中发挥关键作用，这两个第二信使分子控制着钙动员和蛋白激酶C的激活。在培养的细胞系和大鼠肝细胞的原代培养中，PLCGamma1被发现与胰岛素受体有关，这反映了潜在的生理意义。除了其催化结构域，PLCGamma1还具有离散的结构域，使其能够与许多信号分子结合。PLCGamma1和胰岛素受体之间的动态关联被发现依赖于这两种蛋白质中的特定结构域。我们已经通过表达突变形式的PLCGamma1鉴定了其中的一些基序，并分析了完整细胞中胰岛素受体-PLCGamma1的关联模式。使用siRNA降低PLCGamma1的表达可取消对丝裂原活化蛋白激酶(MAPK)的胰岛素依赖调节，但不能消除AKT的调节。相反，在缺乏PLC-Gamma1的成纤维细胞中重建PLC-Gamma1可以改善胰岛素对MAPK的激活。这些结果表明，PLCGamma1是一种硫醇反应蛋白，它与胰岛素受体的结合可能有助于胰岛素激活MAPK信号。RAS-GRP是依赖钙和二酰甘油的鸟嘌呤核苷酸交换因子家族中的一员，作用于RAS蛋白，从而将膜结合受体与MAPK激活联系起来。这些蛋白质是否对PLCGamma1信号对胰岛素的反应是重要的还有待确定。 胰岛素刺激通常导致肌动蛋白细胞骨架的快速重组，以产生形成质膜褶皱和许多其他细胞过程所需的力量，包括胰岛素可调节的葡萄糖转运体4在细胞表面的重新分布。肌动蛋白网络的稳定及其与细胞膜的附着是由肌动蛋白结合蛋白协调的。这些蛋白质中的一组称为丝蛋白，已被证明与肌动蛋白细丝和一些大分子结合，特别是整合素受体、小GTP酶和RAFT相关的小窝蛋白-1。丝素与驻留的RAFT蛋白共定位，从而为肌动蛋白细胞骨架的脂筏的组织和聚集提供了证据。第二项研究的目的是确定细丝蛋白A是否在从胰岛素受体到下游信号级联的激活途径中发挥作用，进而导致关键基因的磷酸化和转录激活。我们报道缺乏细丝素A的人黑色素瘤M2细胞表现出正常的胰岛素受体介导的IRS-1和Shc蛋白的酪氨酸磷酸化，以及在胰岛素刺激下AKT和MAPK的适当激活。相反，表达丝蛋白A的M2A7细胞不能诱导胰岛素依赖的Shc酪氨酸磷酸化、MAPK激活以及胰岛素依赖的Shc、SOS1和MAPK转位到脂筏微区。有趣的是，细丝蛋白A的表达并没有影响血清或EGF对MAPK的激活。免疫共沉淀实验和体外结合实验表明，细丝蛋白A与胰岛素受体呈结构性结合，而胰岛素和细胞松弛素D对肌动蛋白的解聚都不影响这种相互作用。在肝源性HepG2细胞表面检测到内源性细丝蛋白A与胰岛素受体共存。细丝蛋白A的羧基末端片段(FlaCT)在HepG2细胞中的过表达可阻断细丝蛋白A与胰岛素受体的相互作用。FlNaCT对胰岛素受体和IRS-1的胰岛素依赖性酪氨酸磷酸化无影响，但可选择性增加胰岛素刺激的MAPK的磷酸化和转录因子ELK-1的激活。观察到FlNaCT在HepG2细胞膜皱褶中的定位。事实上，细丝素A与胰岛素受体的结构性相互作用，沿着MAPK激活途径施加抑制音调，这一事实是新的，而且可能非常重要。根据这项工作的信息，有可能构建一个模型，通过该模型，一小段细丝A的表达可以转化为胰岛素信号的增强(例如，葡萄糖摄取、糖原合成、细胞增殖、细胞骨架重排、基因表达)。细丝蛋白A的表达或翻译后修饰的改变和/或细丝蛋白A与特定伙伴相互作用的能力是否可能导致胰岛素抵抗状态的发生将是未来研究的主题。

The metabolic and mitogenic actions of insulin are initiated by binding of the hormone to its cell-surface receptor. This results in activation of the intracellular tyrosine kinase domain of the insulin receptor beta-subunit with subsequent phosphorylation of cellular substrates on tyrosine. A number of adaptor and effector proteins have been discovered that interact with the receptor beta-subunit and participate in the mediation of insulin signaling. Our laboratory has recently shown that a thiol-reactive membrane-associated protein, termed TRAP, binds covalently to the cytoplasmic domain of the human insulin receptor when cells are treated with the homobifunctional crosslinking reagent, 1,6- bis-maleimidohexane (Garant et al., 2000). To further our understanding of the biological importance of TRAP in insulin signaling, the protein complex was purified and TRAP was found to be phospholipase C-gamma1 (PLCgamma1) by mass spectrometry analysis. PLCgamma1 is an enzyme that plays a pivotal role in transmembrane signaling by promoting the production of diacylglycerol and inositol 1,4,5-trisphosphate, two second messenger molecules that control calcium mobilization and the activation of protein kinase C. PLCgamma1 was found to be associated with the insulin receptor both in cultured cell lines and in a primary culture of rat hepatocytes, which reflects the potential for physiological significance. In addition to its catalytic domain, PLCgamma1 possesses discrete domains that allow its binding with a number of signaling molecules. The dynamic association between PLCgamma1 and the insulin receptor was found to be dependent on specific domains within both proteins. We have identified some of these motifs by expressing mutant forms of PLCgamma1 and analyzed the pattern of insulin receptor-PLCgamma1 association in intact cells. A reduction in PLCgamma1 expression using siRNA abrogates insulin-dependent regulation of mitogen-activated protein kinase (MAPK), but not that of AKT. Conversely, reconstitution of PLCgamma1 in fibroblasts lacking PLCgamma1 improved MAPK activation by insulin. These results show that PLCgamma1 is a thiol-reactive protein whose association with the insulin receptor could contribute to the activation of MAPK signaling by insulin. Ras-GRP is a member of the family of calcium- and diacylglycerol-dependent guanine nucleotide exchange factors that act on Ras proteins, thereby linking membrane-bound receptors to MAPK activation. Whether such proteins are important for PLCgamma1 signaling in response to insulin remains to be determined. Insulin stimulation often leads to rapid reorganization of the actin cytoskeleton to generate the forces necessary for plasma membrane ruffling formation and a host of other cellular processes, including the redistribution of insulin-regulatable glucose transporter 4 in the cell surface. The stabilization of actin network and its attachment to cellular membranes is orchestrated by actin binding proteins. One group of these proteins, called filamins, has been shown to bind with both actin filaments and a number of macromolecules, notably integrin receptors, small GTPases, and raft-associated caveolin-1. Filamin colocalizes with resident raft proteins, thus providing evidence for the organization and clustering of lipid rafts by the actin cytoskeleton. The aim of this second study was to determine whether filamin A plays a role in activation pathways from the insulin receptor to downstream signaling cascades, leading in turn to the phosphorylation and transcriptional activation of critical genes. We report that human melanoma M2 cells lacking filamin A exhibit normal insulin receptor-mediated tyrosine phosphorylation of IRS-1, Shc proteins, and the proper activation of AKT and MAPK upon stimulation with insulin. In contrast, filamin A-expressing M2A7 cells were unable to elicit insulin-dependent Shc tyrosine phosphorylation, MAPK activation, and insulin-dependent translocation of Shc, SOS1 and MAPK to lipid raft microdomains. Interestingly, filamin A expression did not impair MAPK activation in response to serum or EGF. Coimmunoprecipitation experiments and in vitro binding assays demonstrated that filamin A binds constitutively to the insulin receptor, and that neither insulin nor depolymerization of actin by cytochalasin D affected this interaction. The colocalization of endogenous filamin A with insulin receptor was detected at the surface of the liver-derived HepG2 cells. The interaction between filamin A and the insulin receptor was blocked by overexpression of a carboxy-terminal fragment of filamin A (FLNaCT) in HepG2 cells. FLNaCT had no effect on insulin-dependent tyrosine phosphorylation of insulin receptor and IRS-1, but caused a selective increase in insulin-stimulated phosphorylation of MAPK and activation of the transcription factor, Elk-1. The localization of FLNaCT to membrane ruffles in HepG2 was noted. The fact that filamin A interacts constitutively with the insulin receptor to exert an inhibitory tone along the MAPK activation pathway is novel and potentially very important. With the information from this work, it is possible to construct a model whereby expression of a small fragment of filamin A may translate into potentiation of insulin signaling (e.g., glucose uptake, glycogen synthesis, cell proliferation, cytoskeletal rearrangement, gene expression). The possibility that alterations in expression or posttranslational modification of filamin A and/or ability of filamin A to interact with specific partners are responsible for the development of insulin resistant states will be the subject of future investigations.