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

ADP核糖化因子(ARF)是一类 调节细胞膜运输和细胞器的GTP结合蛋白 结构。我们一直在研究细胞功能 ARF6，一种影响质膜(PM)流量和 肌动蛋白细胞骨架。ARF6调节PM进入和移动 走出了一条新颖的内体循环途径。新时代的回归 膜到PM需要ARF6的激活，发生在离散的 沿单元格外围边缘的位置，并与 含有突起的肌动蛋白的形成和膜的褶皱。 HeLa细胞中突起的形成被夸大了 添加氟化铝后ARF6的过表达 (Alf)，异三聚体G蛋白的激活剂。ALF疗法 导致ARF6在PM积累，形成 突出的结构，以及PM内化到 内胚体隔室。这些也是效果的特点 在细胞中表达ARF6的结构性活性突变体。至 研究G蛋白是否对这种效应负责 ALF，从而成为ARF6的潜在上游调节因子，我们 将ARF6与HeLa细胞共转染后， 候选Gα蛋白。G Alpha Q，但不是Gs、GI2或G12， 似乎专门激活了ARF6，并重现了观察到的效果 和阿尔夫在一起。我们目前正在调查这一机制 GQ将ARF6维持在激活状态。为了辨认 ARF6中负责其细胞定位的结构域和 函数，我们已经在ARF6和ARF1之间构建了嵌合体 并在HeLa细胞中表达。ARF1与高尔基联手 对通过和维护的交通进行膜和管理 高尔基情结。我们发现，羧基末端的一半 ARF6和ARF1包含将它们定位到 正确的膜间隔，而氨基末端减半 包含耦合到效应器函数的信息。尽管 1-6嵌合体(ARF1的氨基末端一半，羧基末端一半 ARF6)在其定位方面与ARF6相似，它不诱导 添加Alf后的突起。然而，2的替换 1-6氨基末端的氨基酸残基(QS) 嵌合体导致了功能的增加，现在突起形成了。 将这个效应结构域映射到ARF6中的这两个氨基酸 将有助于识别相互作用的效应分子和 抑制肽或抗体的发展，可用于 阻止ARF6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.