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: 7413359
• 负责人: James E. Casanova
• 金额: $ 28.5万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7413359
• 关键词: ADP-Ribosylation Factors; Adaptor Signaling Protein; Amyloid; Arrestin; Arrestins; Binding; Capsid Proteins; Cell Adhesion; Cell membrane; Cells; Cholesterol Homeostasis; Class; Clathrin; Clathrin-Coated Vesicles; Complex; Condition; 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; Personal Satisfaction; Process; Protein Isoforms; Proteins; RNA Interference; Range; Rate; 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; 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（Arfs）是一个小的gtp酶家族，在所有真核细胞中调节囊泡运输。在这种情况下，Arfs的主要作用是在载体囊泡形成的位置使外壳蛋白复合物的组装成核。这些外壳复合物选择和浓缩货物进行运输，并提供能量形成囊泡。Arfs促进了高尔基体和核内体中网格蛋白包被囊泡的组装，但它们在质膜上网格蛋白介导的内吞作用中的作用尚不清楚。在哺乳动物的6种Arf亚型中，Arf6在细胞外周最为丰富，已被证明可调节g蛋白偶联受体依赖于网格蛋白的内吞作用，但不调节转铁蛋白受体的内吞作用，转铁蛋白受体的内化也依赖于网格蛋白。我们假设Arf6调节了一组质膜蛋白的内吞作用，这些蛋白需要单体衔接蛋白的参与才能将它们与网格蛋白的内吞机制连接起来。在Aim 1中，我们将研究Arf6在一组与内吞机制表现出不同相互作用模式的膜蛋白的内吞作用和内吞后运输中的作用。Arf6也在核内体上起作用，调节内吞后循环，我们假设它在每个位置的激活受到位点特异性鸟嘌呤核苷酸交换因子（gef）的差异调节。目的1还将探讨不同的Arf6 gef在内吞和内吞后运输中的作用。我们已经开始描述一类arf6特异性的gef，即BRAGs (Brefeldin Resistant Arf gef)，它可以结合网格蛋白和AP-2接头复合物。在Aim 2中，我们将定义三种自夸异构体在内吞过程中的具体功能，并进行结构/功能分析以确定其作用机制。最后，我们的初步数据表明，Arf6结合到一个含有n端磷酸酪氨酸结合域（PTBs）的单体衔接蛋白家族，该衔接蛋白已被证明将含有NPXY分选信号的蛋白与网格蛋白内吞机制连接起来。在Aim 3中，我们将确定Arf6如何调节这些蛋白与cargo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因子，因此拟议的研究将为这些过程的调节机制提供基本的见解。