Attachment, Targeting and Localization of Galpha Subunits

Galpha 亚基的附着、靶向和定位

• 批准号: 7316295
• 负责人: BRADLEY M DENKER
• 金额: $ 35.77万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7316295
• 关键词: A Mouse; Acute; Acute Renal Failure with Renal Papillary Necrosis; Affect; Animal Model; Apical; Apoptosis; Binding; Brain Hypoxia-Ischemia; Cell physiology; Cultured Cells; Development; Disruption; Epithelial; Epithelial Cells; Epithelium; Etiology; Eukaryotic Cell; Event; Family; Fibrosis; GTP-Binding Proteins; Goals; Heterotrimeric GTP-Binding Proteins; Hypoxia; In Vitro; Injury; Ischemia; Kidney; Kidney Diseases; Kidney Failure; Kidney Transplantation; Lead; Localized; MDCK cell; Mediating; Mesenchymal; Modeling; Molecular; Movement; Mus; Numbers; Pathway interactions; Permeability; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological reperfusion; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Tyrosine Kinase; Proteins; Recovery; Regulation; Renal tubule structure; Reperfusion Injury; Reperfusion Therapy; Research Personnel; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Staging; Structure; Tight Junctions; Time; Transgenic Mice; Transgenic Organisms; Tubular formation; Ureteral obstruction; Urine; base; cell growth; cell growth regulation; cell injury; cell motility; in vitro Assay; in vivo; injured; insight; interstitial; kidney cell; novel; prevent; programs; response; response to injury; rho; therapeutic target; wasting

DESCRIPTION (provided by applicant): The G12/13 family of G proteins couple to tyrosine kinase and Rho signaling pathways and regulate numerous epithelial cell functions. Epithelial cell tight junctions (TJs) provide the paracellular barrier and maintain polarity. Disruption of TJs is an early (and reversible) event in ischemia/reperfusion (I/R) injury, and loss of TJs initiates additional pathways contributing to progressive interstitial fibrosis. We have made a number of observations regarding the role of G proteins in the regulation of tight junctions. We are now extending these observations into the role of G proteins and their regulation of tight junctions in acute renal injury. We identified direct binding of Ga12 with ZO-1, a TJ scaffolding protein, and elucidated Ga12-Src mediated signaling leading to TJ disruption. In addition, Ga12 and Ga13 interact with PP2A, a phosphatase within the TJ critical for regulating TJ assembly. We hypothesize that Ga12/13 regulate TJs through tyrosine kinase and Rho pathways to mediate early steps in the renal injury response. Therefore, Ga12 and Ga13 signaling are key therapeutic targets for regulating TJs and the recovery from acute kidney injury. In MDCK cells, activation of Ga12 and Ga13 leads to loss of barrier function and delayed TJ assembly through distinct mechanisms. Hypoxia-treated MDCK cells show delayed TJ assembly, and activated Ga13 is detected in kidney lysates from hypoxic mice. Our goals are to define Ga12/13 signaling mechanisms leading to loss of TJs, and how these pathways are modulated with renal injury. In Aim 1, the binding domains and the mechanisms regulating Ga12/ZO-1 and PP2A interactions will be completed using in vitro assays. In Aim 2, Ga12 and Ga13 signaling pathways through Src, PP2A and Rho regulating TJs will be studied in MDCK cells. Ga12 will be silenced to determine if loss of TJs can be prevented, and Ga12/13 signaling investigated in models of injury (ATP depletion and ROS). In Aim 3, animal models of hypoxia, I/R and ureteral obstruction will be used to determine Ga12/13 activation of Src and Rho pathways in renal tubules. A mouse with conditional expression of activated Ga12 in proximal tubules will be used to determine how Ga12 regulates TJs and renal tubular function. Kidney cells are tightly connected to prevent waste in the urine from reentering the body. These connections are injured in kidney disease, and these studies will determine how these connections are rebuilt after injury. This will aid discovery of new treatments to prevent kidney failure.

描述（由申请人提供）：G12/13 G蛋白夫妇与酪氨酸激酶和Rho信号通路途径，并调节众多上皮细胞功能。上皮细胞紧密连接（TJS）提供细胞屏障并保持极性。 TJS的破坏是缺血/再灌注（I/R）损伤的早期（且可逆的）事件，而TJS的损失启动了额外的途径，导致渐进性间质纤维化。我们已经对G蛋白在紧密连接处的调节中的作用进行了许多观察。现在，我们将这些观察结果扩展到G蛋白的作用及其对急性肾脏损伤中紧密连接的调节。我们确定了GA12与ZO-1，TJ支架蛋白的直接结合，并阐明了GA12-SRC介导的信号传导，导致TJ破坏。此外，GA12和GA13与PP2A相互作用，PP2A是调节TJ组装至关重要的TJ中的磷酸酶。我们假设GA12/13通过酪氨酸激酶和RHO途径调节TJ，以介导肾脏损伤反应的早期步骤。因此，GA12和GA13信号传导是调节TJS和急性肾脏损伤恢复的关键治疗靶标。在MDCK细胞中，GA12和GA13的激活导致屏障功能的丧失，并通过不同的机制延迟TJ组装。缺氧处理的MDCK细胞显示出延迟的TJ组装，并且在低氧小鼠的肾脏裂解物中检测到活化的GA13。我们的目标是定义GA12/13信号传导机制，导致TJ损失，以及这些途径如何通过肾脏损伤调节。在AIM 1中，使用体外测定将完成调节GA12/ZO-1和PP2A相互作用的结合域和调节GA12/ZO-1和PP2A相互作用的机制。在AIM 2中，将在MDCK细胞中研究通过SRC，PP2A和RHO调节TJ的GA12和GA13信号通路。 GA12将被沉默以确定是否可以防止TJ的丢失，并在损伤模型（ATP耗竭和ROS）中研究了GA12/13信号传导。在AIM 3中，缺氧动物模型，I/R和输尿管阻塞将用于确定肾小管中SRC和RHO途径的GA12/13激活。近端小管中活化GA12的有条件表达的小鼠将用于确定GA12如何调节TJ和肾小管功能。肾细胞紧密连接，以防止尿液中的浪费重新进入人体。这些连接在肾脏疾病中受伤，这些研究将确定受伤后如何重建这些连接。这将有助于发现新的治疗方法，以防止肾脏衰竭。