Department of Host Defense and Biochemical Research, Juntendo University, School of Medicine, Tokyo 113-8421, Japan.
J Biol Chem. 2010 Oct 1;285(40):30698-707. doi: 10.1074/jbc.M110.107458. Epub 2010 Jul 2.
Phagocytosis is a complex multistep process requiring diverse signaling and regulatory molecules. ADP-ribosylation factor 6 (ARF6), a small GTPase, is known to regulate membrane trafficking and the actin cytoskeketon at the plasma membrane and functions as a regulatory molecule of phagocytosis. ARF activity is regulated by cycling between GDP-bound and GTP-bound forms. ARF activation is catalyzed by guanine nucleotide exchange factors (GEFs) that facilitate GTP binding. We had earlier reported a 100-kDa ARF-GEF, termed ARF-guanine nucleotide exchange protein 100, GEP100, that preferentially activates ARF6 and was also described by Dunphy et al. (Dunphy, J. L., Moravec, R., Ly, K., Lasell, T. K., Melancon, P., and Casanova, J. E. (2006) Curr. Biol. 16, 315-320) as brefeldin A-resistant ARF-GEF2 (BRAG2). We have now examined a role for GEP100 in phagocytosis. Stable depletion of GEP100 decreased phagocytosis of serum-treated zymosan and IgG-coated latex beads by human monocyte-macrophage-like U937 cells differentiated with phorbol 12-myristate 13-acetate. Decrease of phagocytic activity by RNAi was not rescued by GEP100ΔSec7, a deletion mutant lacking the ARF-activating domain. GEP100-depleted cells also exhibited reduced F-actin fibers around internalized particles. Attachment of these particles to cells and amounts of C3bi and Fcγ receptors, however, were not affected by GEP100 depletion. On immunofluorescence microscopy, GEP100 and ARF6 were concentrated and partially colocalized around internalized particles. Phagocytosis by GEP100-depleted cells was not further affected by depletion of ARF6. Phagocytic activity of GEP100-depleted cells was, however, rescued by expression of the constitutively active ARF6Q67N mutant but not by the dominant-negative ARF6T27N mutant. These data are consistent with the conclusion that GEP100 functions in phagocytosis via its role in ARF6-dependent actin remodeling.
吞噬作用是一个复杂的多步骤过程，需要多种信号和调节分子。ADP-核糖基化因子 6（ARF6）是一种小 GTP 酶，已知其调节质膜的膜运输和肌动蛋白细胞骨架，并作为吞噬作用的调节分子发挥作用。ARF 活性通过 GDP 结合形式和 GTP 结合形式之间的循环调节。ARF 的激活由鸟嘌呤核苷酸交换因子（GEF）催化，促进 GTP 结合。我们之前报道了一种 100kDa 的 ARF-GEF，称为 ARF-鸟嘌呤核苷酸交换蛋白 100（GEP100），它优先激活 ARF6，并且也被 Dunphy 等人描述为布雷菲德菌素 A 抗性 ARF-GEF2（BRAG2）（Dunphy，J.L.，Moravec，R.，Ly，K.，Lasell，T.K.，Melancon，P.，和 Casanova，J.E.（2006）Curr Biol. 16, 315-320）。我们现在研究了 GEP100 在吞噬作用中的作用。稳定耗尽 GEP100 会降低人单核细胞-巨噬细胞样 U937 细胞吞噬经血清处理的酵母聚糖和 IgG 包被的乳胶珠的能力，这些细胞是用佛波醇 12-肉豆蔻酸 13-醋酸盐分化的。RNAi 降低吞噬活性不能被缺乏 ARF 激活结构域的缺失突变体 GEP100ΔSec7 挽救。GEP100 耗尽的细胞也表现出内化颗粒周围 F-肌动蛋白纤维减少。然而，这些颗粒与细胞的附着以及 C3bi 和 Fcγ 受体的量不受 GEP100 耗尽的影响。在免疫荧光显微镜下，GEP100 和 ARF6 集中并部分共定位在内化颗粒周围。GEP100 耗尽的细胞吞噬作用进一步不受 ARF6 耗尽的影响。然而，用组成性激活的 ARF6Q67N 突变体而不是显性负性 ARF6T27N 突变体表达可挽救 GEP100 耗尽的细胞的吞噬活性。这些数据与以下结论一致，即 GEP100 通过其在 ARF6 依赖性肌动蛋白重塑中的作用在吞噬作用中发挥作用。
