Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan.
Department of Molecular Biology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan.
Cell Commun Signal. 2017 Oct 2;15(1):36. doi: 10.1186/s12964-017-0193-y.
The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex.
We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges.
Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.
小分子 GTP 酶 ARF1 主要介导来自高尔基体的膜运输，对于 G 蛋白偶联受体（GPCR）介导的中性粒细胞趋化作用至关重要。在这个过程中，ARF1 被鸟嘌呤核苷酸交换因子 GBF1 激活，并被 GTP 酶激活蛋白 GIT2 失活。中性粒细胞在 GPCR 诱导的趋化作用过程中产生 Gβγ-PAK1-αPIX-GIT2 线性复合物，其中αPIX 激活 RAC1/CDC42，然后由 PAK1 作用。然而，为什么 GIT2 包含在这个复合物中仍不清楚。
我们研究了 ARF1 与 RAC1/CDC42 在 fMLP 刺激的 HL60 细胞趋化作用过程中的关联。我们发现，GBF1 的沉默显著损害了 RAC1 向前缘的募集，但不影响 PAK1、αPIX、RAC2 或 CDC42。在刺激细胞中，大量的 RAC1 与 ARF1 在前缘处共定位，而 fMLP 同时激活了 ARF1 和 ARF5。一致地，ARF1 的沉默，而不是 ARF5 的沉默，损害了 RAC1 的募集，而 RAC1 的沉默不影响 ARF1 向前缘的募集。
我们的结果表明，ARF1 的激活触发了 GPCR 介导的趋化作用中 RAC1 的质膜募集，这对于皮质肌动蛋白重塑是必不可少的。因此，在趋化作用中，前缘的膜重塑似乎先于肌动蛋白重塑。结合 GIT2 作为激活 RAC1 的机制的组成部分，它失活 ARF1 的事实，我们提出了一个模型，即 ARF1-RAC1 连接使得 ARF1 能够在 GPCR 介导的中性粒细胞趋化作用过程中通过重复的开启/关闭循环进行调节。
