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，调节多种上皮细胞功能。上皮细胞紧密连接（TJs）提供细胞旁屏障并维持极性。TJs的破坏是缺血/再灌注（I/R）损伤的早期（可逆）事件，TJs的丧失启动了促进进行性间质纤维化的其他途径。我们对G蛋白在紧密连接调节中的作用做了一些观察。我们现在将这些观察扩展到G蛋白在急性肾损伤中的作用及其对紧密连接的调节。我们发现了Ga12与ZO-1（一种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TJ内的一种磷酸酶，对调节TJ组装至关重要。我们假设Ga12/13通过酪氨酸激酶和Rho途径调节TJs，介导肾损伤反应的早期步骤。因此，Ga12和Ga13信号是调控TJs和急性肾损伤后恢复的关键治疗靶点。在MDCK细胞中，Ga12和Ga13的激活通过不同的机制导致屏障功能丧失和TJ组装延迟。缺氧处理的MDCK细胞显示TJ组装延迟，并且在缺氧小鼠的肾裂解物中检测到活化的Ga13。我们的目标是确定导致TJs丢失的Ga12/13信号机制，以及这些途径如何在肾损伤中被调节。在Aim 1中，结合域和调节Ga12/ZO-1和PP2A相互作用的机制将通过体外实验完成。在Aim 2中，我们将在MDCK细胞中研究通过Src、PP2A和Rho调控TJs的Ga12和Ga13信号通路。Ga12将被沉默以确定是否可以阻止TJs的丢失，并在损伤模型（ATP耗尽和ROS）中研究Ga12/13信号传导。在Aim 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Ga12/13活化Src和Rho通路。在近端小管中条件表达活化Ga12的小鼠将被用来确定Ga12如何调节TJs和肾小管功能。肾细胞紧密相连，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。这将有助于发现预防肾衰竭的新疗法。

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.