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: 7935868
• 负责人: James E. Casanova
• 金额: $ 12.14万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935868
• 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(申请人提供)：ADP-核糖化因子(ARF)是一个小的GTP酶家族，调节所有真核细胞中的囊泡运输。在这种情况下，ARF的主要作用是使外壳蛋白复合体在载体囊泡形成部位的组装成核。这些包衣复合体选择和集中运输的货物，并提供形成囊泡的能量。已有的研究表明，ARF能促进高尔基体和内小体组装被膜蛋白的囊泡，但它们在膜蛋白介导的质膜内吞作用中的作用却知之甚少。在六种哺乳动物Arf亚型中，Arf6在细胞外周含量最高，已被证明可以调节G蛋白偶联受体的胞吞作用，但不能调节转铁蛋白受体的内吞作用，转铁蛋白受体的内化也是依赖于笼蛋白的。我们假设Arf6调节一组质膜蛋白的内吞作用，这些蛋白需要单体接头蛋白的参与才能将它们连接到笼状蛋白的内吞机制。在目标1中，我们将研究Arf6在一组膜蛋白的内吞作用和内吞后转运中的作用，这些膜蛋白表现出与内吞机制不同的相互作用模式。ARF6也在内小体上发挥功能，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)的不同调控。目的1还将探讨不同的Arf6 GEF在细胞内和细胞后转运中的作用。我们已经开始鉴定一类Arf6特异的GEF，即结合笼蛋白和AP-2接头复合体的Brefeldin抗性Arf GEF。在目标2中，我们将定义三种BRAG亚型在内吞过程中的具体功能，并进行结构/功能分析，以确定它们的作用机制。最后，我们的初步数据表明，Arf6与一系列含有N末端磷酸酪氨酸结合域(PTB)的单体接头蛋白结合，这些PTB已被证明将含有NPXY分选信号的蛋白质连接到网状蛋白内吞机制。在目标3中，我们将确定Arf6如何调节这些蛋白质与Cargo和cathrin/AP-2复合体的相互作用。由于这类货物分子包括胆固醇稳态、细胞黏附和神经元功能的关键调节器，拟议的研究将为这些过程的调控机制提供基本的见解。