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

基本信息

批准号：
7531909
负责人：
Tianqing Kong
金额：
$ 12.77万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
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/7531909
关键词：
Affect Architecture Autosomal Dominant Polycystic Kidney Binding Biological Assay Cell Communication Cell-Cell Adhesion Cell-Matrix Junction Cells Cilia Collagen Complex Confocal Microscopy Cultured Cells Cyst Development Disruption E-Cadherin Epithelial Cells Event Figs - dietary Focal Adhesions GTP-Binding Proteins Immunoprecipitation Integrins Kidney Kidney Failure Knockout Mice Laboratories Lead Length Link Localized MDCK cell Macromolecular Complexes Mass Spectrum Analysis Modeling Morphology Mus Mutation Pathway interactions Permeability Polycystic Kidney Diseases Protein Binding Proteins Public Health Reagent Regulation Renal tubule structure Role Signal Pathway Signal Transduction Site Structure Surface Thrombin Tubular formation cell motility in vivo insight intracellular protein transport migration mouse model nephrogenesis novel polycystic kidney disease 1 protein protein localization location scaffold

项目摘要

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）：多囊肾脏疾病（PKD）导致肾小管囊性扩张引起的肾衰竭。 PKD1的突变负责ADPKD的85％（常染色体显性PKD），而基因产物（Polycystin-1，PC1）是一种大型复杂蛋白。 Polycystin-1与多种蛋白质相互作用，并在纤毛，连接络合物和基底表面发现。 PKD的异常包括改变细胞 - 细胞粘附和细胞矩阵相互作用，导致正常小管结构的丧失。 PC1的C末端与G蛋白相互作用，我们已经鉴定出GA12的结合，而不是GA13与PC1 C末端的结合。 GA12也在上皮细胞连接络合物中发现，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GA12和A2B1整合素的调节，并且激活GA12会导致微管发生测定中的囊肿形成。在表达全长PC1的MDCK细胞中，增加的微管发生通过内源性GA12的凝血酶激活阻断，并导致囊肿形成。在激活的GA12的新型测定中，MDCK细胞中PC1水平的增加或降低会显着影响GA12激活。我们假设PC1调节GA12和整联蛋白，以调节细胞基质相互作用，细胞 - 细胞粘附和细胞迁移，对正常小管发育必不可少。这些研究将在培养的肾上皮细胞和小鼠肾脏中共定位GA12，整联蛋白和PC1，并通过免疫沉淀研究和质谱法鉴定大分子复合物（AIM 1）。在AIM 2中将探索GA12细胞细胞粘附，细胞 - 矩阵相互作用和细胞迁移的调节，并且PC1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AIM 3中，PC1对调节GA12/整合素信号在微管发生测定中的影响将用于确定这些途径在小管发育中的作用。将使用A2整联蛋白基因敲除小鼠，PC1无效杂合子和GGT-CRE/QLA12小鼠探索导致体内囊肿发育的机制。公共卫生相关性：多囊性肾脏疾病（PKD）导致肾脏囊性扩张引起的肾衰竭。正常的肾脏发育很复杂，需要调节与相邻细胞和基础支持的细胞相互作用的协调事件（信号）。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中这些异常信号。