Molecular Aspects of Insulin Receptor Signaling

这个项目的主要目标是扩展我们对控制胰岛素作用的信号事件的了解。胰岛素的代谢和促有丝分裂作用是通过激素与其细胞表面受体结合而启动的。胰岛素刺激导致肌动蛋白细胞骨架的快速重组，产生质膜皱褶和其他细胞过程所需的力量。我们已经确定了磷酸肌醇特异性磷脂酶C-γ 1（PLCg 1）作为一个结合伙伴的激活胰岛素受体（IR）在各种细胞系和大鼠肝细胞的原代培养。PLCg 1是一种在跨膜信号传导中起关键作用的酶，特别是细胞外信号调节激酶1/2（ERK）的调节。我们的数据显示，在肝源性HepG 2细胞中，通过RNA干扰敲低PLCg 1表达显著降低了胰岛素对ERK的激活，但对Akt的激活没有影响。相反，PLCg 1（-/-）小鼠胚胎成纤维细胞中PLCg 1的重建显示胰岛素刺激的ERK活化显著增加，表明PLCg 1可能参与通过Ras/ERK途径活化转导胰岛素介导的信号。PLCg 1包含几个结构域，通过这些结构域，它可以与肌动蛋白、信号蛋白和脂质产物相互作用。通过这种相互作用的网络，PLCg 1被激活并在细胞内重新分布，在哺乳动物的生长和分化中发挥重要作用。对于PLCg 1在不同细胞区室之间穿梭的信号级联反应以及胰岛素刺激后其活性的调节知之甚少。我们最近的研究表明，肌动蛋白结合蛋白细丝蛋白A组成型结合IR施加选择性抑制信号级联导致胰岛素介导的ERK及其下游靶点，转录因子Elk-1的激活。用编码细丝蛋白A的C-末端区域的质粒（FLNa-CT）转染肝源性HepG 2细胞显著降低了内源性细丝蛋白A与IR的关联，同时引起ERK介导的Elk-1响应于胰岛素的反式激活的选择性激活。共聚焦免疫荧光显微镜显示细丝蛋白A和IR在HepG 2细胞表面的强烈共定位，而异位表达FLNa-CT在膜皱褶处积累，支持细丝蛋白A在胰岛素诱导的细胞骨架重排中发挥作用的观点。然而，细丝蛋白A的功能方面的再分配和激活内源性PLCg 1响应胰岛素还不清楚。 细丝蛋白A的一个重要功能是它将质膜受体束缚在肌动蛋白细胞骨架上，从而调节它们的内化速率并指导它们的细胞内运输。它还结合筏相关的小窝蛋白-1，这为肌动蛋白细胞骨架组织和聚集脂筏微结构域提供了证据。我们目前正在测试的假设，细丝蛋白A单独或与小窝蛋白-1调节胰岛素依赖性激活PLCg 1和其细胞内运输，以促进基因表达和分化。 在衰老、肥胖和疾病状态如2型糖尿病中胰岛素抵抗发展的分子基础是复杂的，并且仍然难以捉摸。目前旨在降低高血糖症的干预措施在临床试验中的结果令人失望。因此，有效的新的治疗干预措施，以改善胰岛素的反应性，需要更好地了解蛋白质-蛋白质相互作用的胰岛素受体信号转导

The major objective of this project is to extend our knowledge of signaling events that control insulin action. The metabolic and mitogenic actions of insulin are initiated by binding of the hormone to its cell-surface receptor. Insulin stimulation leads to rapid reorganization of the actin cytoskeleton to generate the forces necessary for plasma membrane ruffling and a host of other cellular processes. We have identified phosphoinositide-specific phospholipase C-gamma 1 (PLCg1) as a binding partner to the activated insulin receptor (IR) in various cell lines and in a primary culture of rat hepatocytes. PLCg1 is an enzyme that plays a pivotal role in transmembrane signaling, notably the regulation of extracellular signal-regulated kinases 1/2 (ERK). Our data showed that knockdown of PLCg1 expression by RNA interference significantly reduces ERK activation by insulin, but not that of Akt, in the liver-derived HepG2 cells. Conversely, reconstitution of PLCg1 in PLCg1(-/-) mouse embryonic fibroblasts shows a marked increase in insulin-stimulated ERK activation, suggesting that PLCg1 may be involved in transducing insulin-mediated signals through activation of the Ras/ERK pathway. PLCg1 contains several domains through which it can interact with actin, signaling proteins and lipid products. Through this network of interactions, PLCg1 is activated and redistributed within the cell where it exerts an essential role in mammalian growth and differentiation. Little is known about the signaling cascade involved in the shuttling of PLCg1 between various cellular compartments, and regulation of its activity upon insulin stimulation. Our recent study indicates that the actin-binding protein filamin A binds constitutively to the IR to exert a selective inhibition of signaling cascades leading to the insulin-mediated activation of ERK and its downstream target, the transcription factor Elk-1. Transfection of liver-derived HepG2 cells with a plasmid encoding the C-terminal region of filamin A (FLNa-CT) markedly reduced the association of endogenous filamin A with the IR while causing a selective activation of ERK-mediated transactivation of Elk-1 in response to insulin. Confocal immunofluorescence microscopy showed strong colocalization of filamin A and the IR at the surface of HepG2 cells, whereas ectopically expressed FLNa-CT accumulated at the membrane ruffles, supporting the notion that filamin A has a role in insulin-induced cytoskeletal rearrangement. However, the functions of filamin A with regard to the redistribution and activation of endogenous PLCg1 in response to insulin are not understood. One important function of filamin A is that it tethers plasma membrane receptors to the actin cytoskeleton and, thus, modulates their rate of internalization and directs their intracellular trafficking. It also binds raft-associated caveolin-1, which provides evidence for the organization and clustering of lipid raft microdomains by the actin cytoskeleton. We are currently testing the hypothesis that filamin A alone or together with caveolin-1 modulates insulin-dependent activation of PLCg1 and its intracellular trafficking to promote gene expression and differentiation. The molecular basis for the development of insulin resistance in aging, obesity and in disease states such as type 2 diabetes is complex and remains elusive. Current interventions aimed at reducing hyperglycemia have had disappointing outcomes in clinical trials. Hence, effective new therapeutic interventions to improve insulin responsiveness require a better understanding of protein-protein interactions applied to insulin receptor signaling