The Role of Arf GTPases in Endocytosis and Postendocytic Transport

Arf GTP 酶在内吞作用和内吞后转运中的作用

• 批准号: 7841866
• 负责人: James E. Casanova
• 金额: $ 28.2万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7841866
• 关键词: ADP-Ribosylation Factors; Adaptor Signaling Protein; Amyloid; Arrestins; Binding; Capsid Proteins; Cell Adhesion; Cell membrane; Cells; Cholesterol Homeostasis; Clathrin; Clathrin-Coated Vesicles; Complex; Data; Dominant-Negative Mutation; Endocytosis; Endosomes; Eukaryotic Cell; Event; Exhibits; Family; Fluorescence Resonance Energy Transfer; G-Protein-Coupled Receptors; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Hela Cells; Individual; Integrins; Link; Literature; Location; Low Density Lipoprotein Receptor; Measures; Mediating; Membrane; Membrane Proteins; Microscopy; Monomeric GTP-Binding Proteins; N-terminal; Neurons; PTB Domain; Pathway interactions; Process; Protein Isoforms; Proteins; RNA Interference; Recycling; Reporting; Resistance; Role; Signal Transduction; Site; Sorting - Cell Movement; Structure; TFAP2A gene; Testing; Transferrin Receptor; Transport Process; Vesicle; arrestin3; cell type; coated pit; design; insight; link protein; member; protein complex; receptor; trafficking

DESCRIPTION (provided by applicant): The ADP-ribosylation factors (Arfs) are a family of small GTPases that regulate vesicular transport in all eukaryotic cells. The primary role of Arfs in this context is to nucleate the assembly of coat protein complexes at sites of carrier vesicle formation. These coat complexes select and concentrate cargo for transport, and also provide the energy to form a vesicle. It is well established that Arfs promote the assembly of clathrin-coated vesicles in the Golgi apparatus and on endosomes, but their role in clathrin- mediated endocytosis at the plasma membrane is poorly understood. Of the six mammalian Arf isoforms, Arf6 is most abundant in the cell periphery, where it has been shown to regulate the clathrin-dependent endocytosis of G-protein coupled receptors, but not of transferrin receptor, whose internalization is also clathrin dependent. We hypothesize that Arf6 regulates the endocytosis of a subset of plasma membrane proteins that require the participation of monomeric adaptor proteins to link them to the clathrin endocytic machinery. In Aim 1, we will examine the role of Arf6 in the endocytosis and postendocytic transport of a panel of membrane proteins that exhibit different modes of interaction with the endocytic machinery. Arf6 also functions on endosomes to regulate postendocytic recycling, and we hypothesize that its activation at each location is differentially regulated by site-specific guanine nucleotide exchange factors (GEFs). Aim 1 will also explore the role of different Arf6 GEFs in endocytic and postendocytic transport. We have begun to characterize a class of Arf6-specific GEFs, the BRAGs (Brefeldin Resistant Arf GEFs) that bind both clathrin and the AP-2 adaptor complex. In Aim 2, we will define the specific functions of the three BRAG isoforms in endocytic processes, and perform a structure/function analysis to define their mechanisms of action. Finally, our preliminary data indicate that Arf6 binds to a family of monomeric adaptor proteins containing N-terminal phosphotyrosine binding domains (PTBs) that have been shown to link proteins containing NPXY sorting signals to the clathrin endocytic machinery. In Aim 3 we will determine how Arf6 may regulate interaction of these proteins with cargo and the clathrin/AP-2 complex. Because this class of cargo molecules includes key regulators of cholesterol homeostasis, cell adhesion and neuronal function, the proposed studies will provide fundamental insights into the mechanisms by which these processes are regulated.

描述（由申请人提供）：ADP-核糖基化因子（ARF）是一个小的GTP酶家族，可调节所有真核细胞中的水泡转运。在这种情况下，ARF的主要作用是对载体囊泡形成部位的外套蛋白复合物的组装成核。这些涂层配合物选择并集中货物进行运输，还提供形成囊泡的能量。众所周知，ARF促进高尔基体和内体中涂有网格蛋白涂层囊泡的组装，但是它们在质膜上的网状蛋白介导的内吞作用中的作用知之甚少。在六个哺乳动物ARF同工型中，ARF6在细胞外围最丰富，在该细胞外围，已证明它可以调节G蛋白偶联受体的网格蛋白依赖性内吞作用，但并非转移蛋白受体的内在化也依赖于网状蛋白。我们假设ARF6调节需要单体衔接蛋白参与的质膜蛋白的一部分内吞作用，以将其链接到网格蛋白内吞蛋白内吞机械。在AIM 1中，我们将研究ARF6在一系列膜蛋白的内吞和内吞和后内环传递中的作用，这些膜蛋白表现出与内吞机械相互作用的不同模式。 ARF6还对内体的作用以调节内膜后回收，并且我们假设其在每个位置的激活都由位点特异性鸟嘌呤核苷酸交换因子（GEFS）差异调节。 AIM 1还将探索不同ARF6 GEF在内吞和内膜细胞转运中的作用。我们已经开始表征一类ARF6特异性GEF，即结合网格蛋白和AP-2适配器复合物的吹牛（Brefeldin抗ARF GEFS）。在AIM 2中，我们将在内吞过程中定义三个吹牛同工型的特定功能，并执行结构/功能分析以定义其作用机理。最后，我们的初步数据表明，ARF6与含有N末端磷酸酪氨酸结合结构域（PTB）的单体衔接蛋白（PTB）结合，这些蛋白已显示出与含有NPXY分类信号的蛋白联系起来的，这些蛋白与网状蛋白质内粘蛋白内胶质机器人联系起来。在AIM 3中，我们将确定ARF6如何调节这些蛋白质与货物和网格蛋白/AP-2复合物的相互作用。由于这类货物分子包括胆固醇稳态，细胞粘附和神经元功能的关键调节剂，因此拟议的研究将提供有关调节这些过程的机制的基本见解。