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: 6109173
• 负责人: Julie 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/6109173
• 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, an ARF that affects plasma membrane (PM) traffic and the actin cytoskeleton. ARF6 regulates the movement of PM into and out of a novel, endosomal recycling pathway. The return of membrane to the PM requires ARF6 activation, occurs at discrete sites along the peripheral edges of cells and is associated with the formation of actin containing protrusions and membrane ruffling. Formation of protrusions is exaggerated in HeLa cells overexpressing ARF6 upon the addition of aluminum fluoride (AlF), an activator of heterotrimeric G proteins. The AlF treatment results in an accumulation of ARF6 at the PM, formation of protrusive structures, and a block in internalization of PM into the endosomal compartment. These are also characteristics of the effect of expressing the constitutively active mutant of ARF6 in cells. To investigate whether a G protein was responsible for this effect of AlF and thus a potential upstream regulator of ARF6, we cotransfected HeLa cells with ARF6 and constitutively active, candidate G alpha proteins. G alpha q, but not Gs, Gi2, or G12, appears to specifically activate ARF6 and recreates effects observed with AlF. We are currently investigating the mechanism whereby Gq maintains ARF6 in the activated state. In order to identify domains in ARF6 responsible for its cellular localization and function, we have constructed chimeras between ARF6 and ARF1 and expressed them in HeLa cells. ARF1 associates with Golgi membranes and regulates traffic through and maintenance of the Golgi complex. We found that the carboxyl- terminal halves of ARF6 and ARF1 contain information for targeting them to the correct membrane compartment, whereas the amino-terminal halves contain information for coupling to effector functions. Although the 1-6 chimera (amino-terminal half of ARF1, carboxyl-terminal half of ARF6) resembles ARF6 in it localization, it does not induce protrusions upon the addition of AlF. However, substitution of 2 amino acid residues (QS) in the amino-terminal half of this 1-6 chimera results in a gain-of- function, where protrusions now form. Mapping this effector domain to these two amino acids in ARF6 will facilitate the identification of interacting effector molecules and development of inhibitory peptides or antibodies that can be used to block ARF6 function.

ADP-核糖基化因子 (ARF) 是一个家族 调节膜运输和细胞器的 GTP 结合蛋白 细胞内的结构。我们一直在研究细胞的功能 ARF6，一种影响质膜 (PM) 流量的 ARF 肌动蛋白细胞骨架。 ARF6 调节 PM 的移动 一种新颖的内体回收途径。返回的 PM 的膜需要 ARF6 激活，发生在离散的 沿细胞外围边缘的位点，并与 形成含有肌动蛋白的突起和膜褶皱。 HeLa 细胞中突出物的形成被夸大 添加氟化铝后过表达 ARF6 (AlF)，异源三聚体 G 蛋白的激活剂。 AlF处理 导致 ARF6 在 PM 处积累，形成 突出的结构，以及 PM 内化的障碍 内体室。这些也是效果的特点 在细胞中表达ARF6的组成型活性突变体。到 研究 G 蛋白是否对这种效应负责 AlF 因此是 ARF6 的潜在上游调节因子，我们 与 ARF6 共转染 HeLa 细胞并具有组成型活性， 候选Gα蛋白。 G alpha q，但不是 Gs、Gi2 或 G12， 似乎专门激活 ARF6 并重现观察到的效果 与AlF。我们目前正在研究其机制 Gq维持ARF6处于激活状态。为了识别 ARF6 中负责其细胞定位的域和 函数，我们构建了ARF6和ARF1之间的嵌合体 并在 HeLa 细胞中表达它们。 ARF1 与高尔基体相关 膜并调节通过和维护的交通 高尔基复合体。我们发现羧基末端的一半 ARF6 和 ARF1 包含将它们定位到的信息 正确的膜区室，而氨基末端一半 包含与效应器功能耦合的信息。虽然 1-6嵌合体（ARF1的氨基末端一半，羧基末端一半 ARF6）在定位方面与 ARF6 相似，它不会诱导 添加 AlF 后会出现突起。然而，替换2 该1-6的氨基末端一半的氨基酸残基（QS） 嵌合体导致功能获得，现在形成突起。 将此效应结构域映射到 ARF6 中的这两个氨基酸 将有助于识别相互作用的效应分子 开发可用于 阻止 ARF6 功能。