Functions and mechanisms of the endocytic adaptor Dab2

描述（由申请人提供）：内吞作用将必需营养素和其他分子带入细胞，通过细胞表面受体调节信号传导，并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（Dab 2）是一种“磷酸酪氨酸结合”（PTB）结构域蛋白，其结合网格蛋白和其他内吞蛋白并定位于网格蛋白包被的凹坑。我们现在已经证明Dab 2敲除在体内和体外抑制内吞作用。Dab 2作为低密度脂蛋白受体（LDLR）的网格蛋白相关分选蛋白（CLASP）起作用。与所有先前表征的CLASP不同，Dab 2功能独立于AP-2。我们还发现，Dab 2从正常细胞的耗竭影响粘附，迁移和整合素内吞作用。已知Dab 2在许多不同的人类癌症中强烈下调，特别是卵巢和乳腺肿瘤。Dab 2的缺失允许癌细胞抵抗失巢凋亡（凋亡诱导的细胞凋亡）。我们希望了解Dab 2如何通过其在内吞作用中的作用引起这种表型变化。这项拟议中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的衔接蛋白贩运的受体。这种方法揭示了整合素内吞作用需要Dab 2。整合素水平的变化与细胞铺展和迁移过程中Dab 2诱导的粘着斑周转变化相关。Dab 2是第一个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，研究整合素内吞作用的重要性，细胞粘附，细胞骨架组织和运动。我们推测，Dab 2依赖的细胞表面蛋白可能是上调的表面时，Dab 2是不存在的，并可能是负责减少迁移和增加失巢凋亡的Dab 2缺陷细胞的抗性。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Aim中，我们将识别需要不同PTB衔接蛋白（Dab 2、ARH和Numb）的货物，确定如何选择货物，并识别内化信号。我们将探索特定货物如何与Dab 2，ARH和Numb的细胞表型相关，包括Dab 2在癌细胞中重新表达的影响。在第二个目标中，我们将研究Dab 2介导的整合素内吞作用。我们将询问整合素是如何被识别为内吞作用的; Dab 2是否介导基础或粘附依赖性内化; Dab 2介导的整合素摄取是否受生长因子或细胞骨架调节;整合素内吞作用如何与细胞迁移、粘附和细胞骨架重塑相关。我们将测试Dab 2下调的癌细胞是否具有增加的整合素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被选择用于内吞作用的理解，以及细胞表面蛋白在细胞生物学和癌症进展中的作用。细胞与其环境之间的适当相互作用由细胞的外表面介导，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更新。表面蛋白质内化的缺陷导致发育缺陷，在癌症中很重要。本申请旨在了解细胞表面蛋白被识别用于内化的机制。结果将提供有关影响细胞存活和运动性的癌细胞变化的信息。 公共卫生部门：细胞与其环境之间的适当相互作用由细胞的外表面介导，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更新。表面蛋白质内化的缺陷导致发育缺陷，在癌症中很重要。本申请旨在了解细胞表面蛋白被识别用于内化的机制。结果将提供有关影响细胞存活和运动性的癌细胞变化的信息。

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.