G PROTEIN SIGNALING IN POLYCYSTIC KIDNEY DISEASE

多囊肾疾病中的 G 蛋白信号转导

• 批准号: 7070270
• 负责人: BRADLEY M DENKER
• 金额: $ 23.21万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7070270
• 关键词: BCL2 gene /protein; G protein; MDCK cell; apoptosis; autosomal dominant trait; biological signal transduction; cell growth regulation; cell proliferation; chimeric proteins; enzyme activity; fluorescence resonance energy transfer; gene mutation; genetic disorder; genetic manipulation; laboratory mouse; phosphoprotein phosphatase; phosphorylation; polycystic kidney; protein binding; protein protein interaction; protein sequence; protein structure; protein structure function; tissue /cell culture; transfection

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.

多囊肾脏疾病（PKD）占透析患者的5-10％，是个人和经济负担的巨大负担。常染色体显性PKD是由两个基因PKD1或PKD2及其蛋白质产物Polycystin -1和-2（PC1，PC2）突变引起的。 PKD的囊肿部分是由于细胞生长和凋亡信号通路的异常而发展。 G蛋白介导许多信号通路，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，并确定了Ser/Thre磷酸酶（PP2A）的新型激活。 PC1通过G蛋白信号，我们已经证实了GA12和PP2A与PC1的C端的结合。我们假设PC1 C末端组织了包含GA12和PP2A的多蛋白信号传导复合物，并且我们预测PC1/GA12/PP2A相互作用对于PC1功能至关重要。长期目标是确定机制（以及潜在的疗法） 这些相互作用导致细胞生长和凋亡的变化介导。该建议的目标是表征PC1/GA12/PP2A如何调节流信号传导，并影响PC1信号复合物内的蛋白质相互作用和磷酸化。在AIM 1中，将通过突变和嵌合体研究确定与相互作用所必需的GA12和PC1域，以及PC1对GA12功能的影响。 PP2A与PC1 C末端结合的机制也将得到阐明。在AIM 2中，具有诱导GA12和活化GA12（Q229L）的MDCK细胞系将与PC1和PC1 C末端结构域的腺病毒表达一起使用，以确定GA12和PP2A（具有抑制剂）在磷酸化上的作用 PC1和相互作用的蛋白质，PC2，纤维囊蛋白，E-钙粘蛋白和B-catenin。在AIM 3中，将在培养细胞中确定由PC1/GA12/PP2A介导的生长和凋亡。此外，将通过创建将越过GGT-CRE小鼠的floxed Q229L GA12转基因小鼠来建立活化GA12的近端小管动物模型。该模型将扩展从体外研究获得的发现。缺乏停止或逆转PKD患者的囊肿扩大的疗法。肾脏细胞生长和细胞死亡的障碍是囊肿形成和肾衰竭发展的基础。这些研究的结果将允许对某些通常调节细胞生长和死亡的信号在PKD中如何改变。这将导致PKD治疗的新方法。