Radhakrishna H, Klausner R D, Donaldson J G
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Cell Biol. 1996 Aug;134(4):935-47. doi: 10.1083/jcb.134.4.935.
To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.
为了研究ADP-核糖基化因子（ARF）6 GTP结合蛋白的效应器功能，我们用野生型、带有表位标签的ARF6转染了HeLa细胞。先前已证明氟化铝（AIF）可间接激活ARF1 GTP酶，对转染了ARF6的细胞进行AIF处理会导致ARF6和肌动蛋白重新分布到质膜上的离散位点，随着时间的推移，这些位点会变得越来越突出。AIF的作用是可逆的，对用野生型ARF6转染的细胞具有特异性，并且类似于在表达ARF6的GTP酶缺陷突变体的细胞中观察到的细胞突起。重要的是，用ARF6转染的细胞中观察到的突起与分别用RhoA和Rac1转染的细胞中观察到的增强的应力纤维和膜皱褶不同。在形成突起的细胞中，在突起结构内明显刺激了巨胞饮作用和膜循环。相比之下，在这些细胞中未检测到转铁蛋白摄取受阻或网格蛋白AP-2复合物分布的改变。细胞松弛素D和脂氧合酶途径抑制剂可阻断AIF诱导的、ARF6依赖性突起结构的形成。这些观察结果支持了ARF6 GTP酶在塑造质膜和潜在细胞骨架方面的新作用。
