Cytohesin dependent ARF to Rac signaling in HGF mediated motility

HGF 介导的运动中细胞粘附素依赖性 ARF 至 Rac 信号传导

• 批准号: 8721404
• 负责人: Lorraine C Santy
• 金额: $ 29.13万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-20 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8721404
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Affect; Area; Binding; Cell Shape; Cell Survival; Cells; Cessation of life; Complication; Cytosol; Data; Development; Epithelial; Epithelial Cells; Event; Fibrosis; Goals; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Hepatocyte Growth Factor; Hospitals; Immigration; Injury; Kidney; Knowledge; Link; Lipids; Mediating; Membrane; Modeling; Modification; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Movement; Mutagenesis; Neoplasm Metastasis; Normal Cell; Pathway interactions; Patients; Phosphatidylinositols; Phosphorylation; Process; Production; Publishing; Recovery; Recruitment Activity; Renal function; Reperfusion Injury; Research; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Small Interfering RNA; Staging; Surface; Testing; Work; Wound Healing; base; cell motility; fighting; kidney repair; migration; mutant; prevent; protein protein interaction; renal ischemia; repaired; research study; response; scaffold; therapeutic target; therapy design; therapy development; tool development; tumor

DESCRIPTION (provided by applicant): Acute kidney injury is a common hospital complication that leads death or long-term kidney damage if recovery is not complete. Hepatocyte growth factor (HGF) promotes the recovery from kidney damage at multiple steps. While many of the proximal signals activated by HGF and the downstream targets that remodel cell shape in response to HGF are known, the details of the circuits that link them together are unclear. This limits the possibility of selectively modulating HGF signaling without impacting the actions of other growth factors. Work in the applicant's lab has recently demonstrated that protein- protein interactions are critical for building a pro-migratory circuit that links activatin of Arf6 by cytohesin-2 to the downstream activation of Rac by Dock180. Preliminary data suggest that this signaling circuit is required for HGF-stimulated Rac activation and migration. The overall objective of this application is to determine the molecular details of this cytohesin-dependent Arf-to-Rac signaling module and to test the degree to which it mediates HGF-induced motility and the recovery from acute kidney injury. This is the next step toward the long-term goal of understanding the initiation of motility in epithelial cells by small GTPase cascades. This application will test a hypothesized model for the cytohesin-dependent Arf-to-Rac signaling module. This module will be tested by pursuing 4 specific aims: 1) Determine how cytohesin-2 activity is controlled; 2) Determine the mechanism of interaction of cytohesin-2 and Dock180 and test the degree to which this interaction mediates HGF dependent Rac activation; 3) Determine the role of active Arf6 in cytohesin-dependent Rac activation; 4) Determine the extent to which cytohesin-dependent Rac activation regulates HGF-stimulated wound healing and recovery of the kidney from ischemia reperfusion injury. Aim 1 will test the hypothesis that cytohesin-2 intramolecular interactions prevent its interaction with membranes until these interactions are disrupted by phosphorylation. Aim 2 will use mutagenesis to identify the regions necessary for the interaction of the cytohesin-binding scaffold GRASP with Dock180. The importance of this interaction for HGF- induced Rac activation will be tested using knockdown of GRASP and expression of GRASP mutants. Aim 3 will test the hypothesis that Arf6-dependent lipid modifications promote activation of Rac by Dock180. Aim 4 will use siRNA, mutant and pharmacologic modulation of cytohesin-dependent Arf-to- Rac signaling to test its involvement in HGF-stimulated wound healing and recovery of the kidney from ischemia reperfusion injury. A proven model of this pro-migratory circuit will allow the design of interventions to modulate this circuit without impacting other Arf6 or Rac-dependent functions. Such treatments could promote or inhibit HGF-dependent migration without disrupting the actions of a broad range of growth factors.

描述(由申请人提供)：急性肾损伤是一种常见的医院并发症，如果恢复不彻底，会导致死亡或长期的肾脏损害。肝细胞生长因子(HGF)从多个步骤促进肾脏损伤的恢复。虽然许多由HGF激活的近端信号和响应HGF重塑细胞形状的下游靶点是已知的，但将它们连接在一起的电路的细节尚不清楚。这限制了选择性地调节HGF信号而不影响其他生长因子的作用的可能性。申请人实验室的工作最近证明，蛋白质-蛋白质相互作用对于建立一个促进迁移的回路至关重要，该回路将细胞粘附素-2激活Arf6与Dock180激活RAC下游联系起来。初步数据表明，该信号通路是HGF刺激的RAC激活和迁移所必需的。这项应用的总体目标是确定这种细胞内酯依赖的Arf-to-Rac信号模块的分子细节，并测试它在介导HGF诱导的运动和急性肾损伤恢复的程度。这是通向理解小GTP酶级联启动上皮细胞运动的长期目标的下一步。 这个应用程序将测试一个假设的模型，用于细胞内黄素依赖的Arf-to-RAC信号模块。该模块将通过以下4个特定目标进行测试：1)确定细胞粘附素-2活性是如何被控制的；2)确定细胞粘附素-2与Dock180相互作用的机制，并测试这种相互作用在多大程度上介导HGF依赖的RAC激活；3)确定活性Arf6在细胞粘附素依赖的RAC激活中的作用；4)确定细胞粘附素依赖的RAC激活在多大程度上调节HGF刺激的伤口愈合和肾脏缺血再灌注损伤的恢复。目的1将验证一种假设，即细胞内切蛋白-2分子内相互作用阻止其与膜的相互作用，直到这些相互作用被磷酸化中断。目的2将使用突变来确定细胞粘附素结合的支架GRAP与Dock180相互作用所必需的区域。这种相互作用对于HGF诱导的RAC激活的重要性将通过敲除GRAP和表达GRAP突变体来测试。目的3将验证依赖Arf6的脂质修饰促进Dock180激活RAC的假设。目的4将利用siRNA、突变体和药物调节细胞内切蛋白依赖的Arf-to-RAC信号来测试其在HGF刺激的伤口愈合和肾脏缺血再灌注损伤恢复中的作用。经过验证的这种促迁移回路模型将允许设计干预措施来调节这一回路，而不会影响其他Arf6或RAC依赖的功能。这样的治疗可以促进或抑制HGF依赖的迁移，而不会扰乱一系列生长因子的作用。