G PROTEIN SIGNALING IN POLYCYSTIC KIDNEY DISEASE

多囊肾病（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由两个基因的突变引起，PKD1或PKD2及其蛋白产物多囊蛋白-1和-2 （PC1, PC2）。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，并确定了丝氨酸/三磷酸酶（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端结合。我们假设PC1 c端组织了一个包含Ga12和PP2A的多蛋白信号复合物，我们预测PC1/Ga12/PP2A相互作用对PC1功能至关重要。长期目标是确定机制（和潜在的治疗方法）

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.