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

ADP核糖化因子(ARF)是一类GTP结合蛋白家族 它调节细胞内的膜运输和细胞器结构。我们 一直在研究ARF6的细胞功能。使用瞬变 表达ARF6野生型和突变型的HeLa细胞 已经表明，ARF6通过其GTP循环调节 质膜(PM)进出一种新的内涵体 车厢。质膜上的ARF6-GTP刺激 富含肌动蛋白的突起的形成，以及在GTP水解时，ARF6-GDP 膜被内化，形成可循环的内涵体； 膜回到PM需要激活ARF6。这 在没有ARF6的情况下，存在ARF6调节的新的循环途径 过度表达和内源性PM蛋白，如MHC I类，是 被发现在其中穿行。这种膜转运途径及其作用 肌动蛋白聚合可用于改变细胞形状。在支持中 在这其中，我们在两个实例中确定了对ARF6的需求。1) 激活的rac1，另一种GTP结合蛋白，诱导形成 与ARF6诱导的突起不同的是， 细胞表面。RAC刺激的褶皱依赖于激活 ARF6及其调控的膜循环途径的功能。 相反，ARF6刺激的突起独立于RAC形成 活动。2)内源性ARF6定位于多种细胞类型 内部、内体结构，以及在细胞的边缘，在 皮质肌动蛋白细丝和膜褶皱。在传播期间 圆形细胞放在培养皿上，细胞形成突起，含有 ARF6和肌动蛋白。这些突起的形成和细胞的扩散 当ARF6的显性阴性突变体T27N表达时被抑制 在这些细胞中，从而牵涉到ARF6及其对膜的调节 细胞扩散的循环利用。

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. Using transient transfection of HeLa cells expressing wild type and mutants of ARF6, we have shown that ARF6 regulates, through its GTP cycle, the movement of plasma membrane (PM) into and back out of a novel, endosomal compartment. ARF6-GTP at the plasma membrane (PM) stimulates the formation of actin-rich protrusions, and upon GTP hydrolysis, ARF6-GDP and membrane is internalized, to form the recycling endosome; recycling of membrane back to the PM requires activation of ARF6. This ARF6-regulated, novel recycling pathway exists in the absence of ARF6 overexpression, and endogenous PM proteins such as MHC class I, are found to cycle through it. This membrane traffic pathway and its effect on actin polymerization may be utilized to alter cell shape. In support of this, we have identified a requirement for ARF6 in two instances. 1) Activated Rac1, another GTP binding protein, induces the formation of membrane ruffles, distinct from ARF6-induced protrusions, over the surface of cells. Rac-stimulated ruffling is dependent upon activation of ARF6 and the functioning of its regulated membrane recycling pathway. In contrast, ARF6-stimulated protrusions form independently of Rac activity. 2) Endogenous ARF6 is localized in a number of cell types to internal, endosomal structures, and at the edges of cells, at sites of cortical actin filaments and membrane ruffling. During spreading of rounded cells onto culture dishes, cells form protrusions containing ARF6 and actin. Formation of these protrusions and cell spreading is inhibited when the dominant negative, T27N mutant of ARF6 is expressed in these cells, thus implicating ARF6 and its regulation of membrane recycling for cell spreading.