Polycystin-1 Regulation of Galpha 12 and Integrins in Polycystic Kidney Disease

描述(由申请人提供)： 多囊肾疾病(PKD)由于进行性肾小管囊性扩张而导致肾功能衰竭。常染色体显性遗传性PKD的85%由PKD1基因突变所致，其基因产物多囊蛋白-1(Polycystin-1，PC1)是一种复杂的大蛋白。多囊蛋白-1与多种蛋白质相互作用，存在于纤毛、连接复合体和基底面。PKD的异常包括细胞-细胞黏附和细胞-基质相互作用的改变，导致正常小管结构的丧失。PC1的C末端与G蛋白相互作用，我们已经发现GA12与PC1 C末端的结合，但没有发现Ga13与PC1 C末端的结合。GA12也在上皮细胞连接复合体中发现，在那里它调节通透性和细胞间的相互作用。初步研究表明，细胞在胶原-1上的附着和迁移受GA12和a2b1整合素的调节，在小管形成实验中，激活GA12会导致包囊的形成。在高表达全长PC1的MDCK细胞中，内源性GA12的凝血酶激活阻止了增加的小管生成，并导致囊性形成。在一项新的激活GA12的检测中，MDCK细胞中PC1水平的升高或降低显著影响GA12的激活。我们假设PC1调节GA12和整合素，以调节细胞基质相互作用，细胞-细胞黏附和细胞迁移对于正常的小管发育是必不可少的。这些研究将在培养的肾上皮细胞和小鼠肾脏中共定位GA12、整合素和PC1，并将通过免疫沉淀研究和质谱仪鉴定大分子复合体(目标1)。在目标2中，GA12将通过整合素对细胞-细胞黏附、细胞-基质相互作用和细胞迁移的调节进行探索，PC1在这些信号通路中的作用将在细胞培养模型中阐明。在目标3中，将利用PC1在小管发生试验中调节GA12/整合素信号的作用来确定这些通路在小管发育中的作用。将使用a2整合素基因敲除小鼠、PC1缺失杂合子和GGT-CRE/QLa12小鼠来探索导致体内囊性发育的机制。 公共卫生相关性：多囊肾疾病(PKD)由于进行性肾小管囊性扩张导致肾功能衰竭。正常的肾脏发育是复杂的，需要协调的事件(信号)来调节细胞与邻近细胞的相互作用和潜在的支持。在PKD中，这些信号被改变并导致囊变形成。这些研究将揭示纠正PKD中这些异常信号的新的治疗策略。

DESCRIPTION (provided by applicant): Polycystic Kidney Disease (PKD) causes renal failure due to progressive tubular cystic expansion. Mutations of PKD1 are responsible for 85% of ADPKD (autosomal dominant PKD), and the gene product (polycystin-1, PC1) is a large complex protein. Polycystin-1 interacts with numerous proteins and is found in cilia, the junctional complex and at the basal surface. Abnormalities in PKD include alterations in cell-cell adhesion and cell-matrix interactions contributing to loss of normal tubule structure. The C terminus of PC1 interacts with G proteins, and we have identified binding of Ga12, but not Ga13 to the PC1 C terminus. Ga12 is also found in the epithelial cell junctional complex where it regulates permeability and cell-cell interactions. Preliminary studies reveal that cell attachment and migration on collagen-1 are regulated by Ga12 and a2b1 integrins, and activating Ga12 leads to cyst formation in tubulogenesis assays. In MDCK cells over expressing full length PC1, increased tubulogenesis is blocked by thrombin activation of endogenous Ga12 and leads to cyst formation. In a novel assay of activated Ga12, increasing or decreasing PC1 levels in MDCK cells significantly affects Ga12 activation. We hypothesize that PC1 regulates Ga12 and integrins to modulate cell matrix interactions, cell-cell adhesion and cell migration essential for normal tubule development. These studies will colocalize Ga12, integrins and PC1 in cultured renal epithelial cells and mouse kidney, and will identify the macromolecular complex through immunoprecipitation studies and mass spectrometry (Aim 1). Ga12 regulation of cell-cell adhesion, cell-matrix interaction and cell migration via integrins will be explored in Aim 2, and the role of PC1 in these signaling pathways will be elucidated in cell culture models. In Aim 3, the effect of PC1 on regulating Ga12/integrin signaling in tubulogenesis assays will be used to identify the role of these pathways in tubule development. The mechanisms leading to cyst development in vivo will be explored using a2 integrin knockout mice, PC1 null heterozyotes and gGT-Cre/QLa12 mice. PUBLIC HEALTH RELEVANCE: Polycystic Kidney Disease (PKD) causes renal failure due to progressive tubular cystic expansion. Normal kidney development is complex and requires coordinated events (signals) that regulate cell interactions with neighboring cells and the underlying support. In PKD, these signals are altered and lead to cyst formation. These studies will reveal new treatment strategies to correct these abnormal signals in PKD.