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Abstract：
RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.
摘要：
RalGPS2是RalA的不依赖Ras的鸟嘌呤核苷酸交换因子（GEF），包含PH域和SH3结合区，它参与多种细胞过程。如胞质分裂，控制细胞周期进程，分化,细胞骨架组织和重排。到目前为止,关于RalGPS2在癌细胞中的作用，其与膀胱癌的关系尚未确定。在本文中，我们证明了RalGPS2在尿路上皮癌衍生的5637癌细胞中表达，并且对于细胞生长至关重要。这些细胞产生薄膜突起，显示出富含肌动蛋白的隧道纳米管（TNTs）的特征，在这里我们表明RalGPS2参与了这些细胞突起的形成。实际上，RalGPS2或其PH结构域的过表达显着增加了纳米管的数量和长度，而RalGPS2的击倒导致这些结构的强烈减少。此外，使用一系列与不同下游成分（Sec5，Exo84，RalBP1）相互作用受损的RalA突变体，我们证明了RalA与Sec5的相互作用是TNTs形成所必需的。此外，我们发现RalGPS2与跨膜MHCIII类蛋白白细胞特异性转录物1（LST1）和RalA相互作用，导致形成促进TNTs生成的复合物。这些发现使我们能够为先前提出的关于TNTs形成的分子模型添加新元素。
