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

多囊肾病中多囊蛋白 1 对 Galpha 12 和整合素的调节

• 批准号: 7677482
• 负责人: Tianqing Kong
• 金额: $ 15.77万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-20 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7677482
• 关键词: Affect; Architecture; Autosomal Dominant Polycystic Kidney; Binding; Biological Assay; Cell Communication; Cell Culture Techniques; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Cilia; Collagen; Complex; Confocal Microscopy; Cyst; Development; E-Cadherin; Epithelial Cells; Event; Figs - dietary; Focal Adhesions; GTP-Binding Proteins; Immunoprecipitation; Integrins; Kidney; Kidney Failure; Knockout Mice; Laboratories; Lead; Length; Link; MDCK cell; Macromolecular Complexes; Mass Spectrum Analysis; Modeling; Morphology; Mus; Mutation; Pathway interactions; Permeability; Polycystic Kidney Diseases; Protein Binding; Proteins; Reagent; Regulation; Renal tubule structure; Role; Signal Pathway; Signal Transduction; Site; Structure; Surface; Thrombin; Tubular formation; cell motility; in vivo; insight; migration; mouse model; nephrogenesis; novel; polycystic kidney disease 1 protein; protein complex; public health relevance; scaffold; treatment strategy

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.

描述（由申请人提供）： 多囊肾病（PKD）是一种慢性肾脏病，是一种慢性肾脏病。85%的ADPKD（常染色体显性PKD）是由PKD 1突变引起的，基因产物（多囊蛋白-1，PC 1）是一种大型复杂蛋白质。多囊蛋白-1与许多蛋白质相互作用，并在纤毛、连接复合体和基底表面发现。PKD的异常包括细胞-细胞粘附和细胞-基质相互作用的改变，导致正常小管结构的丧失。PC 1的C末端与G蛋白相互作用，并且我们已经鉴定了Ga 12而不是Ga 13与PC 1 C末端的结合。Ga 12也存在于上皮细胞连接复合体中，在那里它调节渗透性和细胞-细胞相互作用。初步研究表明，胶原蛋白-1上的细胞附着和迁移受Ga 12和a2 b1整联蛋白的调节，并且激活Ga 12导致小管形成测定中的囊肿形成。在过表达全长PC 1的MDCK细胞中，增加的小管形成被内源性Ga 12的凝血酶激活阻断，并导致囊肿形成。在激活的Ga 12的新测定中，增加或降低MDCK细胞中的PC 1水平显著影响Ga 12激活。我们假设，PC 1调节Ga 12和整合素，以调节细胞基质相互作用，细胞间粘附和细胞迁移的正常小管发育所必需的。这些研究将在培养的肾上皮细胞和小鼠肾脏中共定位Ga 12、整合素和PC 1，并将通过免疫沉淀研究和质谱法鉴定大分子复合物（Aim 1）。Ga 12通过整联蛋白调节细胞-细胞粘附、细胞-基质相互作用和细胞迁移将在Aim 2中探索，PC 1在这些信号传导途径中的作用将在细胞培养模型中阐明。在目的3中，将使用PC 1在小管发生测定中调节Ga 12/整联蛋白信号传导的作用来鉴定这些途径在小管发育中的作用。将使用α 2整联蛋白敲除小鼠、PC 1无效杂合子和gGT-Cre/QLa 12小鼠探索导致体内囊肿发展的机制。 公共卫生相关性：多囊肾病（PKD）由于进行性肾小管囊性扩张导致肾衰竭。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肿形成。这些研究将揭示新的治疗策略，以纠正PKD中的这些异常信号。