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: 2441399
• 负责人: J 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/2441399
• 关键词: ADP ribosylation; actins; cell motility; guanine nucleotide binding protein; immunologic assay /test; intracellular transport; protein structure function; protein transport; transfection

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.

我们一直在研究ADP-核糖基化的细胞功能 因子（ARF）6蛋白在哺乳动物细胞中的表达。 ARF 6是ARF的成员 Ras相关GTP结合蛋白家族。 瞬时转染 带有表位标记的野生型ARF 6和ARF 6突变体的细胞表明， ARF 6根据其核苷酸在细胞内分布， 状态;它定位于质膜（PM）在其GTP结合状态 和一个独特的，管状的，内体膜隔室在其GDP结合 状态 然而，转染细胞中的野生型蛋白质同样是 分布沿着PM和相关的管状内体，我们 已经确定了两种药物治疗方法， 分布 用G蛋白激活剂处理这些细胞 氟化铝导致蛋白质转移到PM， 在PM处形成细胞突起，富含肌动蛋白丝 以及许多肌动蛋白相关蛋白。 这些外周膜 突起类似于在表达 组成型活性（GTP结合）ARF 6突变体。 相反，治疗 用肌动蛋白聚合抑制剂，如细胞松弛素 D，导致ARF 6野生型蛋白质将其分布从 PM至内部膜隔室。 这种管状内体 隔室不是转铁蛋白受体阳性，因此不是转铁蛋白受体阳性。 受体介导的内吞途径，然而，我们表明， PM蛋白质进出该隔室。 因此， 代表了PM蛋白循环的一个新的循环隔间。 ARF 6与这种PM再循环室的关联表明， ARF 6通过其GTP循环，可以调节膜运动和肌动蛋白， 聚合在细胞周边，可能影响细胞的形状， 迁移 利用ARF-6特异性抗肽抗体，我们发现 ARF 6在所有检查的细胞和组织类型中表达。 与此 抗体，内源性ARF 6蛋白已被定位于 外围PM和内部结构，类似于在 转染细胞