G PROTEIN SIGNALING IN POLYCYSTIC KIDNEY DISEASE

多囊肾病(PKD)占透析患者的5%-10%，是巨大的个人和经济负担。常染色体显性遗传性PKD是由PKD1或PKD2两个基因及其蛋白产物Polycystin-1和-2(PC1，PC2)突变所致。在PKD中，囊变的部分原因是细胞生长和凋亡信号通路的异常。G蛋白介导多种信号通路，包括生长/凋亡。我们已经确定了GA12在上皮细胞中的重要作用，并发现了丝氨酸/苏氨酸磷酸酶(PP2A)的新激活。PC1通过G蛋白传递信号，我们已经证实GA12和PP2A都与PC1的C末端结合。我们假设PC1的C末端组织了一个含有GA12和PP2A的多蛋白信号复合体，我们预测PC1/GA12/PP2A的相互作用对PC1的功能至关重要。长期目标是确定机制(和潜在的治疗方法) 通过这些相互作用导致细胞生长和凋亡的变化。这项建议的目标是表征PC1/GA12/PP2A如何调节下游信号，并影响PC1信号复合体中的蛋白质相互作用和磷酸化。在目标1中，将通过突变和嵌合体研究确定相互作用所必需的GA12和PC1结构域，并表征PC1对GA12功能的影响。PP2A与PC1C末端结合的机制也将被阐明。在目标2中，带有可诱导的GA12和激活的GA12(Q229L)的MDCK细胞株将与PC1和PC1的C末端结构域的腺病毒表达一起使用，以确定GA12和PP2A(带有抑制剂)在磷酸化中的作用 PC1及其相互作用蛋白、PC2、纤维囊藻蛋白、E-钙粘蛋白和b-连环蛋白。在目标3中，将确定PC1/GA12/PP2A介导的培养细胞的生长和凋亡。此外，还将通过建立一只Q229L GA12转基因小鼠与GGT-CRE小鼠杂交，建立激活的GA12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患者囊性变大的治疗方法。肾脏中细胞生长和死亡的紊乱是囊性形成和肾功能衰竭发展的基础。这些研究的结果将使人们对正常情况下调节细胞生长和死亡的某些信号在PKD中如何改变有新的理解。这将导致治疗PKD的新方法。

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.