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: 6162665
• 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/6162665
• 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. 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.

ADP-核糖基化因子（ARF）是GTP结合蛋白的一个家族 调节细胞膜运输和细胞器结构。 我们 研究ARF 6的细胞功能 利用瞬态 转染表达野生型和ARF 6突变体的HeLa细胞，我们 已经表明，ARF 6通过其GTP循环调节 质膜（PM）进入和退出一个新的，内体 车厢 质膜（PM）上的ARF 6-GTP刺激了 形成富含肌动蛋白的突起，GTP水解后，ARF 6-GDP 膜被内化，形成循环内体， 将膜返回PM需要激活ARF 6。 这 在缺乏ARF 6的情况下，存在ARF 6调节的新型再循环途径 过度表达，和内源性PM蛋白如MHC I类， 这种膜运输途径及其影响 可以利用肌动蛋白聚合来改变细胞形状。 支持 其中，我们在两个实例中确定了对ARF 6的要求。第一章 另一种GTP结合蛋白Rac 1被激活后， 膜皱褶，不同于ARF 6诱导的突起，在 细胞的表面。 RAC刺激的皱褶依赖于激活 ARF 6及其调节的膜再循环途径的功能。 相反，ARF 6刺激的突起形成独立于Rac 活动 2)内源性ARF 6定位于许多细胞类型中， 内部、核内体结构以及细胞边缘、 皮层肌动蛋白丝和膜皱褶。 在传播过程中， 圆形细胞置于培养皿上，细胞形成含有 ARF 6和肌动蛋白。 这些突起的形成和细胞扩散是 当ARF 6的显性阴性T27 N突变体表达时， 在这些细胞中，因此涉及ARF 6及其对膜的调节 循环用于细胞扩散。