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 PKD 是由两个基因 PKD1 或 PKD2 及其蛋白质产物多囊蛋白-1 和 -2（PC1、PC2）突变引起的。多囊肾 (PKD) 中形成囊肿的部分原因是细胞生长和凋亡信号通路的异常。 G 蛋白介导多种信号传导途径，包括生长/细胞凋亡。我们已经确定了 Ga12 在上皮细胞中的重要作用，并确定了丝氨酸/苏氨酸磷酸酶 (PP2A) 的新激活。 PC1 通过 G 蛋白发出信号，我们已经证实 Ga12 和 PP2A 均与 PC1 的 C 末端结合。我们假设 PC1 C 末端组织了一个包含 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（和潜在的疗法） 由这些相互作用介导，导致细胞生长和凋亡的变化。该提案的目标是描述 PC1/Ga12/PP2A 如何调节下游信号传导并影响 PC1 信号传导复合物内的蛋白质相互作用和磷酸化。在目标 1 中，将通过诱变和嵌合体研究鉴定相互作用所需的 Ga12 和 PC1 结构域，并表征 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。在目标 2 中，具有诱导型 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（带有抑制剂）对磷酸化的作用 PC1 和相互作用蛋白、PC2、纤维囊肿蛋白、E-钙粘蛋白和 b-连环蛋白。在目标 3 中，将在培养细胞中测定 PC1/Ga12/PP2A 介导的生长和凋亡。此外，将通过创建 floxed Q229L Ga12 转基因小鼠与 gGT-Cre 小鼠杂交来建立活化 Ga12 的近端小管动物模型。该模型将扩展体外研究的结果。目前尚缺乏阻止或逆转 PKD 患者囊肿增大的治疗方法。肾脏细胞生长和细胞死亡的干扰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通常调节细胞生长和死亡的某些信号如何在 PKD 中发生改变有了新的认识。这将带来治疗 PKD 的新方法。