Intracellular signaling by the insulin receptor kinase

胰岛素受体激酶的细胞内信号传导

• 批准号: 8001225
• 负责人: JEFFREY E. PESSIN
• 金额: $ 23.55万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8001225
• 关键词: Actins; Adipocytes; Alleles; Binding; Binding Proteins; Biological; Biological Assay; CD29 Antigen; Caveolae; Caveolins; Cell membrane; Cells; Chimeric Proteins; Complex; Data; Diffusion; Docking; F-Actin; Fluorescence Recovery After Photobleaching; GLUT4 gene; Goals; In Vitro; Insulin; Insulin Receptor; Integrins; Knockout Mice; Lateral; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Microtubules; Mus; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Process; Protein Kinase C; Proteins; Reaction; Regulation; Research Proposals; Role; SNAP receptor; Signal Transduction; Signal Transduction Pathway; Site; Small Interfering RNA; Structure; Talin; Testing; Vesicle; adipocyte differentiation; c-Ha-ras p21; cellubrevin; computerized data processing; depolymerization; genetic regulatory protein; homologous recombination; in vivo; insulin signaling; mouse Stxbp4 protein; mutant; phosphatidylinositol 4-phosphate; polymerization; prevent; receptor; reconstitution; research study; scaffold; syntaxin 4; target SNARE proteins; trafficking; transmission process; vesicular SNARE proteins

DESCRIPTION (provided by applicant): The overall goals of this research proposal are to identify and characterize the insulin signal transduction pathways leading to functional regulation of the protein components directly involved in the intracellular trafficking, plasma membrane tethering, docking and fusion of GLUT4-containing vesicles. Substantial evidence has indicated that plasma membrane lipid raft microdomains provide an important spatially restricted compartment necessary for insulin biological action. These microdomains are self-organized into large rosette-like structures highly enriched in caveolin that serve as scaffolding or platform for the assembly of plasma membrane cortical actin (Cav-actin). Although it is well-established that dynamic actin remodeling plays an essential role in the GLUT4 translocation process the mechanism(s) responsible are completely unknown. Our preliminary data indicates that a certain subset of actin assembly proteins (beta1 integrin receptor subunit, talin and the type I phosphatidylinositol-4-phosphate 5-kinase) but not others, are co-localized to these Cav-Actin domains. In addition, GLUT4 vesicle fusion proteins (SNARE) have also been localized to these domains and appear to functionally interact with F-actin. To further examine the functional role of adipocyte lipid raft microdomains in mediating insulin signaling, we propose four specific aims, 1. We will reduce expression of the a1 integrin receptor subunit, talin and PI4P5 kinase by the use of siRNA in culture and by homologous recombination in vivo. In these experiments, we will assess the role of these proteins during the adipocyte differentiation process both in vivo and in vitro in terms of the assembly of actin and acquisition of insulin specific signaling processes. 2 We will assess the role of several SNARE proteins in GLUT4 vesicle docking versus fusion, effects on actin assembly and requirement for lipid raft microdomain localization. 3. We will reconstitute membrane vesicle fusion and determine the effect of these regulatory proteins on the in vitro fusion reaction. The effect of direct kinase phosphorylation and phosphorylation deficient mutants will be determined. As efforts to establish a plasma membrane fusion assay, we will assess the fusion reaction between these reconstituted membrane vesicles with adipocytes plasma membrane sheets. 4. We will use FRAP to assess the diffusion rates in isolated plasma membrane sheets and intact cells under a variety of conditions including disruption and/or stabilization of lipid rafts, cortical actin and microtubules. In a complimentary approach, we will also take advantage of the newly developed photoactivable GFP construct (PA-GFP) to examine diffusion from non-lipid raft and caveolin-enriched plasma membrane microdomains.

描述（由申请人提供）：该研究建议的总体目标是识别和表征胰岛素信号转导途径，从而导致直接参与细胞内运输，质膜支架，对接和含GLUT4辅助囊泡的蛋白质成分的功能调节。大量证据表明，质膜脂质筏微区域为胰岛素生物学作用所需的重要空间限制室提供了重要的。这些微域被自组织成高度富集在小窝蛋白中的大型玫瑰花结状结构，这些结构是质膜皮质肌动蛋白（Cav-actin）组装的脚手架或平台。尽管有良好的公认，动态肌动蛋白重塑在GLUT4易位过程中起着至关重要的作用，但负责的机制是完全未知的。我们的初步数据表明，肌动蛋白组装蛋白的一定子集（Beta1整合素受体亚基，Talin和I型I磷脂酰肌醇-4-磷酸5-激酶），但没有共同定位于这些Cav-actin域。另外，Glut4囊泡融合蛋白（SNARE）也位于这些结构域，似乎在功能上与F-肌动蛋白相互作用。为了进一步研究脂肪细胞脂质筏微域在介导胰岛素信号传导中的功能作用，我们提出了四个具体目标，即1。我们将通过在培养和同源性重组中使用sirNA在培养物中使用sirNA在培养物中使用sirNA来降低A1整合素受体亚基，塔林和PI4P5激酶。在这些实验中，我们将根据肌动蛋白的组装和胰岛素特异性信号传导过程在体内和体外评估这些蛋白质在脂肪细胞分化过程中的作用。 2我们将评估几种核心蛋白在Glut4囊泡对接与融合，对肌动蛋白组装的影响以及对脂质筏微域定位的需求的作用。 3。我们将重建膜囊泡融合，并确定这些调节蛋白对体外融合反应的影响。将确定直接激酶磷酸化和磷酸化缺陷突变体的影响。随着建立质膜融合测定法的努力，我们将评估这些重构膜囊泡与脂肪细胞质膜片之间的融合反应。 4。我们将使用FRAP评估在各种条件下分离的质膜和完整细胞中的扩散速率，包括破坏和/或稳定脂质筏，皮质肌动蛋白和微管。在一种免费方法中，我们还将利用新开发的光活性GFP构建体（PA-GFP）来检查非脂形筏和小窝蛋白含有富含质膜的质膜微域的扩散。