CELLULAR FUNCTION OF THE ADP-RIBOSYLATION FACTOR 6 GTP BINDING PROTEIN

ADP-核糖基化因子 6 GTP 结合蛋白的细胞功能

• 批准号: 6432644
• 负责人: Julie G Donaldson
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432644
• 关键词: ADP ribosylation; actins; cell motility; guanine nucleotide binding protein; immunologic assay /test; intracellular transport; protein structure function; protein transport; transfection

The ADP-ribosylation factors (Arfs) are a family of GTP binding proteins that regulate membrane traffic and organelle structure in the cell. We have been studying the cellular function of Arf6, an Arf that affects membrane movement between the plasma membrane and an endosomal compartment and the actin cytoskeleton. In many cells this pathway is the route followed by membrane proteins that lack clathrin localization sequences and hence are endocytosed into cells by non-clathrin mechanisms. Recycling of these membrane proteins back to the plasma membrane requires Arf6 activation whereas entry of these proteins into the endosomal recycling system requires hydrolysis of GTP-bound Arf6. While this pathway has been shown to influence plasma membrane remodeling events such as phagocytosis, and cell spreading and migration, the molecules involved in mediating Arf6 responses remain to be identified. Membrane phosphoinositides, such as phosphatidylinositol-4,5-bisphosphate (PIP2), have been implicated in multiple processes such as membrane trafficking and actin remodeling. We have investigated the subcellular distribution of PIP2 and its role in Arf6 function. We find that Arf6 regulates the localization of PIP2 within the cell. There is also evidence that Arf6, when activated, may stimulate the activity of an enzyme, phosphatidylinositol 4 phosphate 5-kinase, responsible for generating PIP2 on membranes. Indeed, we find that overexpression of this enzyme in cells recreates a phenotype similar to that observed when activated Arf6 mutants are expressed in cells. Notably, these cells accumulate massive amounts of PIP2-enriched plasma membrane in vacuolar-like structures that are coated with actin. Ordinarily, the inactivation of Arf6, would prevent such aberrant vacuole accumulations from forming. In a collaboration with Paul Randazzo at the NCI, we have identified two new Arf GAP molecules ACAP1 and ACAP2, that function to inactivate Arf6. Thus, Arf6 activation and inactivation may mediate the regulation of PIP2 synthesis and/or PIP2 trafficking at defined membrane locations. Another project in the lab concerns the fate of proteins that move through the Arf6 endosome. We find that proteins that traverse this pathway get degraded in lysosomes and gain access to the lysosomes via the Arf6 endosome. This alternative route to the lysosome contrasts to the route followed by proteins that are endocytosed into cells by clathrin-mediated mechanisms. Both pathways do converge, however, in a late endosomal compartment. We are in the process of further characterizing this alternative route to the lysosome since many important molecules, including MHC class I, integrins, src, and catenins, have been observed to traffic through this pathway. This route to degradation may be utilized or subverted by viruses or pathogenic bacteria known to influence lysosomal targeting mechanisms.

ADP-核糖基化因子（Arfs）是GTP结合蛋白家族，其调节细胞中的膜运输和细胞器结构。我们一直在研究Arf 6的细胞功能，Arf影响质膜和内体区室之间的膜运动和肌动蛋白细胞骨架。在许多细胞中，该途径是缺乏网格蛋白定位序列的膜蛋白遵循的途径，因此通过非网格蛋白机制被内吞到细胞中。这些膜蛋白再循环回到质膜需要Arf 6激活，而这些蛋白进入内体再循环系统需要GP结合的Arf 6水解。虽然该途径已显示影响质膜重塑事件，如吞噬作用和细胞扩散和迁移，但参与介导Arf 6应答的分子仍有待鉴定。 膜磷酸肌醇，如磷脂酰肌醇-4，5-二磷酸（PIP 2），已涉及多个过程，如膜运输和肌动蛋白重塑。我们研究了PIP 2的亚细胞分布及其在Arf 6功能中的作用。我们发现Arf 6调节PIP 2在细胞内的定位。也有证据表明，Arf 6，当激活时，可以刺激酶的活性，磷脂酰肌醇4磷酸5-激酶，负责产生PIP 2的膜。事实上，我们发现这种酶在细胞中的过表达重建了一种表型，类似于在细胞中表达激活的Arf 6突变体时观察到的表型。 值得注意的是，这些细胞在包被肌动蛋白的液泡样结构中积累了大量富含PIP 2的质膜。通常，Arf 6的失活将阻止这种异常液泡积累的形成。在与NCI的Paul兰达佐的合作中，我们已经鉴定了两种新的Arf GAP分子ACAP 1和ACAP 2，它们的功能是抑制Arf 6。因此，Arf 6的激活和失活可以介导PIP 2合成和/或PIP 2在限定的膜位置的运输的调节。实验室的另一个项目涉及通过Arf 6内体移动的蛋白质的命运。我们发现，穿过这一途径的蛋白质在溶酶体中降解，并通过Arf 6内体进入溶酶体。这种进入溶酶体的替代途径与通过网格蛋白介导的机制被内吞到细胞中的蛋白质所遵循的途径相反。然而，这两种途径在晚期内体区室中确实会聚。我们正在进一步表征这种通往溶酶体的替代途径，因为已经观察到许多重要的分子，包括MHC I类、整合素、src和连环蛋白通过该途径进行运输。 这种降解途径可能被已知影响溶酶体靶向机制的病毒或病原菌利用或破坏。