The Role of Arf GTPases in Endocytosis and Postendocytic Transport

描述（由申请人提供）：ADP-核糖基化因子（Arfs）是调节所有真核细胞中囊泡转运的小GTP酶家族。在这种情况下，Arfs的主要作用是在载体囊泡形成的位点使外壳蛋白复合物的组装成核。这些包被复合物选择和集中货物运输，并提供能量形成囊泡。已经确定Arfs促进网格蛋白包被的囊泡在高尔基体和核内体上的组装，但是它们在质膜处的网格蛋白介导的内吞作用中的作用知之甚少。在六种哺乳动物Arf同种型中，Arf 6在细胞外周中最丰富，在细胞外周中已显示其调节G蛋白偶联受体的网格蛋白依赖性内吞作用，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是网格蛋白依赖性的。我们假设Arf 6调节胞吞作用的质膜蛋白的一个子集，需要单体衔接蛋白的参与，将它们连接到网格蛋白胞吞机制。在目的1中，我们将研究Arf 6在一组膜蛋白的内吞和胞后转运中的作用，这些膜蛋白表现出与内吞机制不同的相互作用模式。Arf 6也对内体的功能，以调节内吞后再循环，我们假设，它的激活在每个位置的差异调节位点特异性鸟嘌呤核苷酸交换因子（GEFs）。目的1也将探讨不同的Arf 6 GEFs在内吞和内吞后转运中的作用。我们已经开始表征一类Arf 6特异性GEF，BRAG（Brefeldin抗性Arf GEF），其结合网格蛋白和AP-2接头复合物。在目标2中，我们将定义内吞过程中三种BRAC亚型的具体功能，并进行结构/功能分析以定义其作用机制。最后，我们的初步数据表明，Arf 6结合到一个家庭的单体衔接蛋白含有N-末端磷酸酪氨酸结合结构域（PTB），已被证明连接蛋白含有NPXY分选信号的网格蛋白内吞机制。在目标3中，我们将确定Arf 6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，因此拟议的研究将为这些过程的调节机制提供基本见解。

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.