Molecular Aspects of Insulin Receptor Signaling

这个项目的主要目标是扩大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由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，以产生质膜褶皱和一系列其他细胞过程所需的力量。在不同的细胞系和大鼠肝细胞的原代培养中，我们发现磷脂酰肌醇特异性磷脂酶C-γ1(PLCG1)是激活的胰岛素受体(IR)的结合伙伴。PLCG1是一种在跨膜信号转导中起关键作用的酶，尤其是对细胞外信号调节激酶1/2(ERK)的调节。我们的数据显示，通过RNA干扰抑制PLCG1的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，PLCG1在PLCG1(-/-)小鼠胚胎成纤维细胞中的重组显示胰岛素刺激的ERK激活显著增加，提示PLCG1可能通过激活RAS/ERK通路参与胰岛素介导的信号转导。PLCG1包含几个结构域，通过这些结构域可以与肌动蛋白、信号蛋白和脂质产物相互作用。通过这种相互作用的网络，PLCG1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。PLCG1在不同的细胞室之间穿梭，以及它在胰岛素刺激下的活性调节所涉及的信号级联，目前还知之甚少。我们最近的研究表明，肌动蛋白结合蛋白细丝蛋白A结构性地结合到IR上，选择性地抑制信号通路，导致胰岛素介导的ERK及其下游靶点--转录因子ELK-1的激活。将编码细丝蛋白A C端区的质粒(FLNA-CT)导入肝脏来源的HepG2细胞，可显著降低内源性细丝蛋白A与IR的联系，同时选择性激活ERK介导的ELK-1反式激活以响应胰岛素。共聚焦免疫荧光显微镜显示，细丝蛋白A与IR在细胞表面强烈共存，而异位表达的Flna-CT聚集在膜褶皱处，支持细丝蛋白A在胰岛素诱导的细胞骨架重排中起作用的观点。然而，细丝蛋白A在内源性PLCG1对胰岛素的反应中重新分布和激活的功能尚不清楚。 细丝蛋白A的一个重要功能是它将质膜受体连接到肌动蛋白细胞骨架上，从而调节它们的内化速度，并指导它们在细胞内的运输。它还与RAFT相关的小窝蛋白-1结合，这为肌动蛋白细胞骨架对脂筏微域的组织和聚集提供了证据。我们目前正在测试这一假设，即细丝素A单独或与小窝蛋白-1一起调节PLCG1的胰岛素依赖激活及其细胞内转运，以促进基因表达和分化。 在衰老、肥胖和2型糖尿病等疾病状态下发生胰岛素抵抗的分子基础是复杂的，仍然难以捉摸。目前旨在降低高血糖的干预措施在临床试验中的结果令人失望。因此，提高胰岛素反应性的有效新的治疗干预措施需要更好地了解应用于胰岛素受体信号转导的蛋白质-蛋白质相互作用。

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