Functions and mechanisms of the endocytic adaptor Dab2

描述(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，通过细胞表面受体调节信号，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。失活蛋白2(DAB2)是一种“磷酸酪氨酸结合”(PTB)结构域蛋白，可与笼状蛋白和其他内源性蛋白结合，并定位于笼状蛋白包被的凹坑。我们现在已经证明，DAB2基因敲除在体内和体外都能抑制内吞作用。DAB2作为低密度脂蛋白受体(LDLRs)的网状蛋白相关分类蛋白(CLAP)发挥作用。与所有以前表征的CLASPS不同，DAB2的功能独立于AP-2。我们还发现，正常细胞中DAB2的缺失会影响黏附、迁移和整合素内吞作用。已知DAB2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。DAB2的缺失使癌细胞能够抵抗失巢(脱落诱导的细胞凋亡)。我们想要了解DAB2是如何通过其在内吞作用中的作用而引起这种表型变化的。这项拟议的研究建立在一项新技术的基础上，该技术允许我们识别由给定接头蛋白运输的受体。这种方法揭示了整合素的内吞作用需要DAB2。整合素水平的变化与DAB2诱导的细胞扩散和迁移过程中局部黏附周转的变化有关。DAB2是第一个被发现的整合素内吞作用的接头蛋白。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黏附、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，这可能是DAB2缺陷细胞迁移减少和抗失巢凋亡增加的原因。我们现在提出两个目标，一个是全球的，一个是具体的。在第一个目标中，我们将鉴定需要不同PTB接头蛋白DAB2、ARH和Numb的货物，确定货物是如何选择的，并鉴定内化信号。我们将探索特定的货物如何与DAB2、ARH和Numb的细胞表型相关，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。我们将询问整合素是如何被识别为内吞作用的；DAB2是介导基础内化还是黏附依赖性内化；DAB2介导的整合素摄取是受生长因子还是细胞骨架的调节；整合素内吞作用与细胞迁移、黏附和细胞骨架重塑有何关系。我们将测试DAB2下调的癌细胞表面整合素水平是否增加，以及整合素上调在癌症表型中的作用。这些研究将加深我们对受体和整合素是如何被选择进行内吞作用的理解，以及细胞表面蛋白在细胞生物学和癌症进展中的作用。细胞与环境之间的适当相互作用是由细胞外表面调节的，细胞外表面不断地更新来自细胞内的新蛋白质和返回细胞内的旧蛋白质。表面蛋白内化缺陷会导致发育缺陷，并在癌症中起重要作用。这项应用提出了理解细胞表面蛋白被识别为内化的机制。这一结果将对影响细胞存活和运动的癌细胞变化提供信息。 公共卫生：细胞与环境之间的适当相互作用是由细胞外表面调节的，细胞外表面不断地更新来自细胞内的新蛋白质和返回细胞内的旧蛋白质。表面蛋白内化缺陷会导致发育缺陷，并在癌症中起重要作用。这项应用提出了理解细胞表面蛋白被识别为内化的机制。这一结果将对影响细胞存活和运动的癌细胞变化提供信息。

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.