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

我们一直在研究ADP-核糖化的细胞功能 哺乳动物细胞中的ARF-6蛋白。ARF6是ARF的成员 RAS相关GTP结合蛋白家族。瞬时转染法 带有表位标记的野生型ARF6和ARF6突变体的细胞表明 ARF6根据其核苷酸分布在细胞内 状态；它定位于质膜(PM)，处于GTP结合状态 以及与其GDP结合的独特的管状内膜隔间 州政府。然而，在转基因细胞中，野生型蛋白同样 沿着质膜分布并与管状内小体相关，我们 已经确定了两种药物治疗方法，可以改变这种情况 分发。用G蛋白激活剂处理这些细胞 氟化铝导致蛋白质向质膜转移，导致 PM处细胞突起的形成，富含肌动蛋白细丝 以及许多肌动蛋白相关蛋白。这些外周膜 突起类似于在细胞中观察到的表达 成分活性(GTP结合)ARF6突变体。相比之下，治疗 含有肌动蛋白聚合抑制物的细胞，如细胞松弛素 D，导致ARF6野生型蛋白将其分布从 PM到内膜室。这个管状内体 隔室不是转铁蛋白受体阳性的，因此不是 受体介导的内吞作用途径，然而，我们表明 PM蛋白进出这个隔间。因此，它似乎 代表了一种新的回收隔间，PM蛋白质通过它循环。 ARF6与这个PM回收隔间的关联表明 ARF6通过其GTP循环可能调节膜运动和肌动蛋白 在细胞外围聚合，可能影响细胞形状和 迁移。通过ARF-6特异性抗肽抗体，我们发现 ARF6在所检测的所有细胞和组织中均有表达。有了这个 抗体后，内源性ARF6蛋白已定位于 外围PM和内部结构，类似于在 转基因细胞。

We have been studying the cellular function of the ADP-ribosylation factor (ARF) 6 protein in mammalian cells. ARF6 is a member of the ARF family of Ras-related GTP-binding proteins. Transient transfection of cells with epitope-tagged, wild type ARF6 and mutants of ARF6, suggests that ARF6 is distributed within the cell according to its nucleotide status; it localizes to the plasma membrane (PM) in its GTP-bound state and to a unique, tubular, endosomal membrane compartment in its GDP-bound state. Whereas, the wild type protein in transfected cells is equally distributed along the PM and associated with the tubular endosomes, we have identified two pharmacologic treatments which shift this distribution. Treatment of these cells with the G protein activator aluminum fluoride causes a shift of the protein to the PM and results in the formation of cell protrusions at the PM, enriched in actin filaments as well as many actin associated proteins. These peripheral membrane protrusions resemble those observed in cells expressing the constitutively active (GTP-bound) ARF6 mutant. In contrast, treatment of cells with inhibitors of actin polymerization, such as cytochalasin D, causes the ARF6 wild type protein to shift its distribution off the PM to the internal membrane compartment. This tubular endosomal compartment is not transferrin receptor-positive and thus is not the well-studied receptor-mediated endocytosis pathway, and yet, we show that PM proteins move into and out of this compartment. Thus, it appears to represent a novel recycling compartment through which PM proteins cycle. The association of ARF6 with this PM recycling compartment suggests that ARF6, through its GTP cycle, may regulate membrane movement and actin polymerization at the cell periphery, possibly influencing cell shape and migration. With ARF-6 specific anti-peptide antibodies, we have found ARF6 expressed in all cell and tissue types examined. With this antibody, the endogenous ARF6 protein has been localized to the peripheral PM and internal structures, similar to that observed in the transfected cells.