Attachment, Targeting and Localization of Galpha Subunits

描述（由申请人提供）：G蛋白的G12/13家族与酪氨酸激酶和Rho信号通路偶联，并调节多种上皮细胞功能。上皮细胞紧密连接（TJ）提供细胞旁屏障并维持极性。TJ的破坏是缺血/再灌注（I/R）损伤的早期（可逆）事件，TJ的丢失启动了导致进行性间质纤维化的其他途径。我们已经就G蛋白在紧密连接调节中的作用进行了大量的观察。我们现在将这些观察扩展到G蛋白的作用及其在急性肾损伤中对紧密连接的调节。我们鉴定了Ga 12与TJ支架蛋白ZO-1的直接结合，并阐明了Ga 12-Src介导的导致TJ破坏的信号传导。此外，Ga 12和Ga 13与PP 2A相互作用，PP 2A是TJ内调节TJ组装的关键磷酸酶。我们推测，Ga 12/13通过酪氨酸激酶和Rho途径调节TJ，以介导肾损伤反应的早期步骤。因此，Ga 12和Ga 13信号传导是调节TJ和从急性肾损伤中恢复的关键治疗靶点。在MDCK细胞中，Ga 12和Ga 13的激活通过不同的机制导致屏障功能丧失和延迟TJ组装。缺氧处理的MDCK细胞显示延迟的TJ组装，并且在缺氧小鼠的肾裂解物中检测到活化的Ga 13。我们的目标是确定导致TJ损失的Ga 12/13信号传导机制，以及这些途径如何与肾损伤一起调节。在目标1中，将使用体外测定完成调节Ga 12/ZO-1和PP 2A相互作用的结合结构域和机制。目的2：在MDCK细胞中研究Ga 12和Ga 13通过Src、PP 2A和Rho调节TJ的信号通路。Ga 12将被沉默以确定是否可以预防TJ的损失，并在损伤模型（ATP耗竭和ROS）中研究Ga 12/13信号传导。在目标3中，缺氧、I/R和输尿管梗阻的动物模型将用于确定肾小管中Src和Rho通路的Ga 12/13活化。将使用在近端小管中具有活化的Ga 12的条件性表达的小鼠来确定Ga 12如何调节TJ和肾小管功能。肾细胞紧密相连，以防止尿液中的废物重新进入体内。这些连接在肾脏疾病中受损，这些研究将确定这些连接在损伤后如何重建。这将有助于发现新的治疗方法来预防肾衰竭。

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.