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: 8547062
• 负责人: Lorraine C Santy
• 金额: $ 28.15万
• 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/8547062
• 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，如果恢复不完全，会导致死亡或长期肾损伤。肝细胞生长因子（HGF）促进肾脏损伤的多步恢复。虽然已知许多由HGF激活的近端信号和响应HGF重塑细胞形状的下游靶点，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。申请人的实验室中的工作最近已经证明，蛋白质-蛋白质相互作用对于构建将Arf 6通过细胞粘连蛋白-2的激活与Rac通过Dock 180的下游激活联系起来的促迁移回路是至关重要的。初步数据表明，这个信号通路是HGF刺激的Rac激活和迁移所必需的。本申请的总体目标是确定这种细胞粘连素依赖性Arf至Rac信号传导模块的分子细节，并测试其介导HGF诱导的运动性和从急性肾损伤中恢复的程度。这是下一步的长期目标，了解启动运动的上皮细胞的小GT3级联。 本申请将测试一个假设模型的细胞粘连蛋白依赖性的Arf到Rac信号传导模块。该模块将通过追求4个具体目标进行测试：1）确定细胞粘连蛋白-2活性如何被控制; 2）确定细胞粘连蛋白-2和Dock 180的相互作用机制，并测试这种相互作用介导HGF依赖性Rac活化的程度; 3）确定活性Arf 6在细胞粘连蛋白依赖性Rac活化中的作用; 4）确定细胞粘连素依赖性Rac活化调节HGF刺激的伤口愈合和肾从缺血再灌注损伤的恢复的程度。目的1将测试的假设，即细胞粘连素-2分子内的相互作用，防止其与膜的相互作用，直到这些相互作用被磷酸化破坏。目的2将使用诱变来鉴定细胞粘连素结合支架GRASP与Dock 180相互作用所必需的区域。将使用GRASP的敲低和GRASP突变体的表达来测试这种相互作用对于HGF诱导的Rac活化的重要性。目的3将检验Arf 6依赖性脂质修饰促进Dock 180激活Rac的假设。目的4将使用siRNA、突变体和细胞胶素依赖性Arf至Rac信号传导的药理学调节来测试其参与HGF刺激的伤口愈合和肾缺血再灌注损伤的恢复。这种亲迁移回路的经验证的模型将允许设计干预措施来调节该回路，而不影响其他Arf 6或Rac依赖性功能。这种治疗可以促进或抑制HGF依赖性迁移，而不会破坏广泛的生长因子的作用。