G PROTEIN SIGNALING IN POLYCYSTIC KIDNEY DISEASE

多囊肾（PKD）占透析患者的5-10%，是一个巨大的个人和经济负担。常染色体显性PKD由两个基因PKD 1或PKD 2及其蛋白产物多囊蛋白-1和-2（PC 1，PC 2）的突变引起。囊肿在PKD中发展，部分是由于细胞生长和凋亡信号通路的异常。G蛋白介导包括生长/凋亡在内的多种信号通路。我们已经确定了Ga 12在上皮细胞中的重要作用，并确定了丝氨酸/苏氨酸磷酸酶（PP 2A）的新激活。PC 1通过G蛋白发出信号，我们已经证实了Ga 12和PP 2A与PC 1的C末端结合。我们假设，PC 1的C-末端组织一个多蛋白信号复合物含有Ga 12和PP 2A，我们预测，PC 1/Ga 12/PP 2A的相互作用是至关重要的PC 1功能。长期目标是确定机制（和潜在的治疗方法） 通过这些相互作用介导，导致细胞生长和凋亡的变化。该提案的目标是表征PC 1/Ga 12/PP 2A如何调节下游信号传导并影响PC 1信号复合物内的蛋白质相互作用和磷酸化。目的1：通过突变和嵌合体研究确定Ga 12和PC 1相互作用所必需的结构域，并表征PC 1对Ga 12功能的影响。PP 2A与PC 1 C-末端结合的机制也将被阐明。在目的2中，将使用具有可诱导的Ga 12和活化的Ga 12（Q229 L）的MDCK细胞系与PC 1和PC 1 C-末端结构域的腺病毒表达，以确定Ga 12和PP 2A（具有抑制剂）对磷酸化的作用 PC 1和相互作用蛋白，PC 2，纤维囊蛋白，E-钙粘蛋白和b-连环蛋白。在目的3中，将在培养的细胞中确定由PC 1/Ga 12/PP 2A介导的生长和凋亡。此外，将通过创建将与gGT-Cre小鼠杂交的floxed Q229 L Ga 12转基因小鼠来建立活化的Ga 12的近端小管动物模型。该模型将扩展从体外研究中获得的结果。目前尚缺乏阻止或逆转PKD患者囊肿扩大的治疗方法。肾脏中细胞生长和细胞死亡的紊乱是囊肿形成和肾衰竭发展的基础。这些研究的结果将使人们对正常调节细胞生长和死亡的某些信号在PKD中是如何改变的有新的认识。这将为PKD的治疗带来新的方法。

Polycystic kidney disease (PKD) accounts for 5-10% of patients on dialysis and is an enormous personal and economic burden. Autosomal dominant PKD results from mutations in two genes, PKD1 or PKD2 and their protein products polycystin-1, and -2 (PC1, PC2). Cysts develop in PKD, in part, from abnormalities in cell growth and apoptosis signaling pathways. G proteins mediate numerous signaling pathways including growth/apoptosis. We have identified important roles for Ga12 in epithelial cells and identified novel activation of ser/thre phosphatase (PP2A). PC1 signals through G proteins, and we have confirmed binding of both Ga12 and PP2A to the C-terminus of PC1. We hypothesize that the PC1 C-terminus organizes a multiprotein signaling complex containing Ga12 and PP2A, and we predict that PC1/Ga12/PP2A interactions are critical for PC1 functions. The long-term objectives are to identify mechanisms (and potential therapies) mediated by these interactions that lead to changes in cell growth and apoptosis. The goals of this proposal are to characterize how PC1/Ga12/PP2A modulates down stream signaling and affects protein interactions and phosphorylation within the PC1 signaling complex. In Aim 1, the domains of Ga12 and PC1 necessary for interaction will identified through mutatgenesis and chimera studies, and effects of PC1 on Ga12 function characterized. The mechanism of PP2A binding to PC1 C-terminus will also be elucidated. In Aim 2, MDCK cell lines with inducible Ga12 and activated Ga12 (Q229L) will be used with adenoviral expression of PC1 and the PC1 C-terminal domain to determine the role of Ga12 and PP2A (with inhibitors) on phosphorylation of PC1 and interacting proteins, PC2, fibrocystin, E-cadherin and b-catenin. In Aim 3, growth and apoptosis mediated by PC1/Ga12/PP2A will be determined in cultured cells. In addition, a proximal tubule animal model of activated Ga12 will be established by creating a floxed Q229L Ga12 transgenic mouse that will be crossed gGT-Cre mice. This model will extend findings obtained from in-vitro studies. Therapies to stop or reverse the enlarging cysts in patients with PKD have been lacking. Disturbances in cell growth and cell death in the kidney are fundamental to cyst formation and the development of kidney failure. Results from these studies will permit new understanding of how certain signals that normally regulate cell growth and death are altered in PKD. This will lead to new approaches for treatment of PKD.