Someya A, Sata M, Takeda K, Pacheco-Rodriguez G, Ferrans V J, Moss J, Vaughan M
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2413-8. doi: 10.1073/pnas.051634798.
A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP(100) accelerated [(35)S]GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas. No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.
我们鉴定出了一种人类cDNA，它编码一种针对ADP-核糖基化因子（ARFs）的841个氨基酸的鸟嘌呤核苷酸交换蛋白（GEP），命名为ARF-GEP(100)，该蛋白含有一个Sec7结构域、一个类普列克底物蛋白同源（PH）结构域和一个不完整的IQ模体。在对人类组织进行Northern印迹分析时，与ARF-GEP(100) cDNA杂交的约8 kb mRNA在外周血白细胞、脑和脾中含量丰富。ARF-GEP(100)使[(35)S]GTPγS与ARF1（I类）和ARF5（II类）的结合加速2至3倍，与ARF6（III类）的结合加速约12倍。ARF-GEP(100)的Sec7结构域含有Asp(543)和Met(555)，它们对应于酵母Sec7中与对真菌代谢产物布雷菲德菌素A（BFA）抑制作用敏感性相关的残基，但也含有与BFA不敏感性相关的Phe(535)和Ala(536)。类PH结构域在参与磷脂结合的区域与其他ARF GEP的结构域有很大差异。与其结构一致，ARF-GEP(100)的活性不受BFA或磷脂的影响。对培养的T98G人胶质母细胞瘤细胞进行亚细胞分级分离后，ARF6几乎完全存在于粗膜级分中，而用抗肽抗体检测到的100 kDa蛋白ARF-GEP(100)则存在于胞质中。在免疫荧光显微镜下，这两种蛋白在整个细胞中均呈现点状分布模式，仅在周边区域明显共定位。EEA-1在靠近细胞核区域的粗大点状分布似乎与该区域的ARF-GEP(100)分布一致。未观察到ARF-GEP(100)与AP-1、AP-2、连环蛋白、LAMP-1或58K有类似的共定位情况。这种新的人类BFA不敏感GEP可能在特定的内吞过程中与ARF6共同发挥作用。
