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

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 细胞中的结构。我们一直在研究 ARF 6，一种影响质膜（PM）运输和细胞膜的ARF。 肌动蛋白细胞骨架ARF 6调节PM进入和 一种新的内体循环途径。的回归 膜到PM需要ARF 6激活，发生在离散的 沿着细胞的外围边缘，并与 含有肌动蛋白的突起和膜褶皱的形成。 HeLa细胞中突起的形成被夸大 加入氟化铝后过表达ARF 6 (AlF)，异源三聚体G蛋白的激活剂。AlF治疗 导致ARF 6在PM处积累， 组织结构，并阻止PM内化为 内体区室这些也是效应的特征 在细胞中表达ARF 6的组成型活性突变体。到 研究G蛋白是否负责这种效应， AlF和因此一个潜在的上游调节ARF 6，我们 用ARF 6共转染HeLa细胞并具有组成型活性， 候选G α蛋白。G α q，但不是Gs、Gi 2或G12， 似乎特异性激活ARF 6并重现观察到的效应 在Alf。我们目前正在研究 Gq使ARF 6保持激活状态。以便识别 ARF 6中负责其细胞定位的结构域， 功能，我们构建了ARF 6和ARF 1之间的嵌合体 并在HeLa细胞中表达。ARF 1与高尔基体相关 膜和调节交通通过和维护的 高尔基复合体。我们发现， ARF 6和ARF 1包含将其定位到 正确的膜室，而氨基末端的一半， 包含与效应子功能偶联的信息。虽然 1-6嵌合体（ARF 1的氨基末端一半，羧基末端一半 ARF 6）在其定位上类似于ARF 6，它不诱导 在添加AlF时的突起。但是，替代2 氨基酸残基（QS）在氨基末端的一半，这1-6 嵌合体导致功能获得，此时形成突起。 将该效应域定位到ARF 6中的这两个氨基酸 将促进相互作用效应分子的鉴定， 开发抑制肽或抗体， 阻断ARF 6功能。

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.