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: 8372587
• 负责人: Lorraine C Santy
• 金额: $ 29.21万
• 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/8372587
• 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. PUBLIC HEALTH RELEVANCE: HGF-dependent migration of epithelial cells is important for wound healing, normal development and has been implicated in metastatic progression. The proposed research will test a model for a signaling circuit involved in this migration. Understanding the molecular details of this circuit will allow the development of tools to alter it activity, which could be used to promote kidney repair after injury and fight metastatic progression.

描述（由申请人提供）：急性肾脏损伤是一种常见的医院并发症，如果恢复不完整，会导致死亡或长期肾脏损伤。肝细胞生长因子（HGF）在多个步骤中促进了肾脏损伤的恢复。尽管已知许多由HGF激活的近端信号和重塑细胞形状的下游靶标是已知的，但尚不清楚将它们连接在一起的电路的细节。这限制了选择性调节HGF信号传导而不会影响其他生长因素的作用的可能性。申请人实验室中的工作最近表明，蛋白质蛋白相互作用对于建立临时迁移回路至关重要，该传播电路将cytohesin-2与Dock180的RAC的下游激活联系起来。初步数据表明，HGF刺激的RAC激活和迁移需要此信号传导电路。该应用的总体目的是确定该细胞凝聚依赖性ARF-RAC信号传导模块的分子细节，并测试其介导HGF诱导的运动性以及从急性肾脏损伤中恢复的程度。这是朝着理解小型GTPase级联体在上皮细胞中运动开始的长期目标的下一步。 该应用程序将测试依赖于细胞粘着蛋白的ARF-RAC信号传导模块的假设模型。该模块将通过追求4个特定目的来测试：1）确定如何控制细胞粘着素-2的活性； 2）确定细胞粘着蛋白2和DOCK180相互作用的机制，并测试这种相互作用介导HGF依赖性RAC激活的程度； 3）确定活性ARF6在细胞粘着蛋白依赖性RAC激活中的作用； 4）确定细胞粘着依赖性RAC活化的程度，调节HGF刺激的伤口愈合和从缺血再灌注损伤中恢复肾脏。 AIM 1将检验以下假设：细胞粘着蛋白-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破坏。 AIM 2将使用诱变来确定细胞粘着结合支架与Dock180相互作用所需的区域。这种相互作用对HGF诱导的RAC激活的重要性将通过敲低的抓握和抓握突变体的表达来测试。 AIM 3将检验以下假设：ARF6依赖性脂质修饰促进Dock180促进RAC激活。 AIM 4将使用siRNA，突变体和药理学的调节，对细胞粘着蛋白依赖性ARF-TO-RAC信号传导来测试其参与HGF刺激的伤口愈合和从缺血再灌注损伤中恢复肾脏的恢复。该亲迁移回路的验证模型将允许设计干预措施可以调节该电路，而不会影响其他ARF6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素的作用。 公共卫生相关性：上皮细胞的HGF依赖性迁移对于伤口愈合，正常发育很重要，并且与转移性进展有关。拟议的研究将测试与此迁移有关的信号传导电路的模型。了解该电路的分子细节将使工具的开发改变IT活性，可用于促进受伤后肾脏修复和打击转移性进展。