Functions and mechanisms of the endocytic adaptor Dab2

内吞接头 Dab2 的功能和机制

• 批准号: 8072191
• 负责人: Jonathan A Cooper
• 金额: $ 41.05万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072191
• 关键词: Adaptor Signaling Protein; Adhesions; Affect; Anoikis; Apoptosis; Binding; Binding Sites; Biological; Biological Assay; Biology; Carcinoma; Cell Adhesion; Cell Surface Proteins; Cell Surface Receptors; Cell Survival; Cell surface; Cells; Cellular biology; Clathrin; Cytoskeletal Modeling; Cytoskeleton; Defect; Development; Disabled Persons; Endocytosis; Environment; Focal Adhesions; Goals; Growth Factor; Human; In Vitro; Integrins; Knock-out; Low Density Lipoprotein Receptor; Malignant Neoplasms; Mammary Neoplasms; Maps; Mediating; Membrane Proteins; Normal Cell; Nutrient; PTB Domain; Phenotype; Phosphotyrosine; Positioning Attribute; Protein Binding; Proteins; Public Health; Recycling; Regulation; Research; Resistance; Role; Signal Transduction; Sorting - Cell Movement; Surface; TFAP2A gene; Testing; Tumor Suppressor Proteins; Up-Regulation; cancer cell; cell motility; coated pit; disease phenotype; extracellular; hypercholesterolemia; in vivo; interest; migration; new technology; novel; ovarian neoplasm; receptor; trafficking; tumor progression; uptake

DESCRIPTION (provided by applicant): Endocytosis brings essential nutrients and other molecules into cells, regulates signaling by cell-surface receptors, and modulates interactions with the extracellular environment. Our general goal is to understand how endocytosis affects phenotypes and disease. Disabled 2 (Dab2) is a "phosphotyrosine-binding" (PTB) domain protein that binds to clathrin and other endocytic proteins and localizes to clathrin-coated pits. We have now shown that Dab2 knockout inhibits endocytosis in vivo and in vitro. Dab2 functions as a clathrin- associated sorting protein (CLASP) for low density lipoprotein receptors (LDLRs). Unlike all previously characterized CLASPs, Dab2 function is independent of AP-2. We also found that Dab2 depletion from normal cells affects adhesion, migration and integrin endocytosis. Dab2 is known to be strongly down-regulated in many different human carcinomas, particularly ovarian and mammary tumors. Loss of Dab2 allows cancer cells to resist anoikis (detachment-induced apoptosis). We would like to understand how Dab2 could cause such phenotypic changes through its role in endocytosis. The proposed research builds on a new technology that allows us to identify receptors trafficked by a given adaptor protein. This approach has revealed that integrin endocytosis requires Dab2. Changes in integrin levels correlate with Dab2-induced changes in focal adhesion turnover during cell spreading and migration. Dab2 is the first adaptor protein found for integrin endocytosis. We are now in a unique position to study the importance of integrin endocytosis for cell adhesion, cytoskeletal organization and motility. We hypothesize that Dab2-dependent cell surface proteins may be up-regulated on the surface when Dab2 is absent, and may be responsible for reduced migration and increased anoikis-resistance of Dab2-deficient cells. We now propose two aims, one global and one specific. In the first Aim, we will identify cargoes requiring different PTB adaptor proteins, Dab2, ARH and Numb, determine how cargoes are selected, and identify internalization signals. We will explore how specific cargoes relate to the cellular phenotypes of Dab2, ARH and Numb, including the effects of Dab2 re-expression in cancer cells. In the second Aim, we will investigate Dab2-mediated endocytosis of integrins. We will ask how integrins are recognized for endocytosis; whether Dab2 mediates basal or adhesion-dependent internalization; whether Dab2-mediated integrin uptake is regulated by growth factors or the cytoskeleton; how integrin endocytosis relates to cell migration, adhesion and cytoskeletal remodeling. We will test whether cancer cells in which Dab2 is down-regulated have increased surface levels of integrins and the role of integrin up-regulation in the cancer phenotype. These studies will enhance our understanding of how receptors and integrins are selected for endocytosis, and the role of cell surface proteins in cell biology and the progression of cancer. Proper interactions between cells and their environment are mediated by the outer surface of the cell, which is continually renewed by new proteins coming from, and old proteins returning to, the inside of the cell. Defects in internalization of surface proteins cause defects in development and are important in cancer. This application proposes to understand the mechanisms by which cell surface proteins are recognized for internalization. The results will be informative regarding changes in cancer cells that affect cell survival and motility. PUBLIC HEALTH REVELANCE: Proper interactions between cells and their environment are mediated by the outer surface of the cell, which is continually renewed by new proteins coming from, and old proteins returning to, the inside of the cell. Defects in internalization of surface proteins cause defects in development and are important in cancer. This application proposes to understand the mechanisms by which cell surface proteins are recognized for internalization. The results will be informative regarding changes in cancer cells that affect cell survival and motility.

描述（由申请人提供）：内吞作用将必需的营养物质和其他分子带入细胞，调节细胞表面受体的信号传导，调节与细胞外环境的相互作用。我们的总体目标是了解内吞作用如何影响表型和疾病。Disabled 2 （Dab2）是一种“磷酸酪氨酸结合”（PTB）结构域蛋白，与网格蛋白和其他内吞蛋白结合，并定位于网格蛋白包被的凹坑。我们现在已经证明，在体内和体外敲除Dab2抑制内吞作用。Dab2是一种低密度脂蛋白受体（ldlr）的网格蛋白相关分选蛋白（CLASP）。与所有先前表征的CLASPs不同，Dab2功能独立于AP-2。我们还发现正常细胞中的Dab2缺失会影响粘附、迁移和整合素内吞。已知Dab2在许多不同的人类肿瘤中强烈下调，特别是卵巢和乳腺肿瘤。Dab2的缺失使癌细胞能够抵抗脱落诱导的细胞凋亡。我们想了解Dab2如何通过其在内吞作用中的作用引起这种表型变化。这项拟议的研究建立在一项新技术的基础上，该技术使我们能够识别由给定接头蛋白运输的受体。该方法揭示了整合素内吞作用需要Dab2。在细胞扩散和迁移过程中，整合素水平的变化与dab2诱导的局灶黏附翻转变化有关。Dab2是第一个被发现的整合素内吞作用的衔接蛋白。我们现在处于一个独特的位置来研究整合素内吞作用对细胞粘附、细胞骨架组织和运动的重要性。我们推测，当Dab2缺失时，依赖Dab2的细胞表面蛋白可能在表面上调，并可能导致Dab2缺失细胞的迁移减少和抗氧化性增加。我们现在提出两个目标，一个是全球目标，一个是具体目标。在第一个目标中，我们将确定需要不同的PTB适配器蛋白Dab2， ARH和Numb的货物，确定如何选择货物，并识别内化信号。我们将探讨Dab2、ARH和Numb的细胞表型与特异性货物的关系，包括Dab2在癌细胞中重新表达的影响。在第二个目标中，我们将研究dab2介导的整合素内吞作用。我们会问整合素是如何被识别为内吞作用的；Dab2是否介导基础内化或粘连依赖性内化；dab2介导的整合素摄取是否受到生长因子或细胞骨架的调节；整合素内吞作用与细胞迁移、粘附和细胞骨架重塑的关系。我们将测试Dab2下调的癌细胞是否增加了整合素的表面水平，以及整合素上调在癌症表型中的作用。这些研究将增强我们对受体和整合素如何选择内吞作用的理解，以及细胞表面蛋白在细胞生物学和癌症进展中的作用。细胞和环境之间的正常相互作用是由细胞的外表面调节的，细胞的外表面不断被来自细胞内部的新蛋白质和返回细胞内部的旧蛋白质所更新。表面蛋白内化缺陷导致发育缺陷，在癌症中也很重要。该应用程序旨在了解细胞表面蛋白被内化识别的机制。研究结果将对影响细胞存活和运动的癌细胞变化提供信息。

项目成果

FCH domain only-2 organizes clathrin-coated structures and interacts with Disabled-2 for low-density lipoprotein receptor endocytosis.

• DOI: 10.1091/mbc.e11-09-0812
• 发表时间: 2012-04
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Mulkearns EE;Cooper JA
• 通讯作者: Cooper JA

The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis.

网格蛋白适配器 Dab2 招募 EH 结构域支架蛋白来调节整合素 β1 内吞作用。

• DOI: 10.1091/mbc.e11-12-1007
• 发表时间: 2012
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Teckchandani,Anjali;Mulkearns,ErinE;Randolph,TimothyW;Toida,Natalie;Cooper,JonathanA
• 通讯作者: Cooper,JonathanA

Disabled-2 is required for efficient hemostasis and platelet activation by thrombin in mice.

在小鼠体内，Disabled-2 是凝血酶有效止血和血小板激活所必需的。

• DOI: 10.1161/atvbaha.114.302602
• 发表时间: 2014
• 期刊: Arteriosclerosis, thrombosis, and vascular biology
• 作者: Tsai,Hui-Ju;Huang,Chien-Ling;Chang,Yao-Wen;Huang,Ding-Yuan;Lin,Chung-Ching;Cooper,JonathanA;Cheng,Ju-Chien;Tseng,Ching-Ping
• 通讯作者: Tseng,Ching-Ping