Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ
Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ
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DOI：
10.1152/ajpcell.00250.2003
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发表时间：
2004-04-01
影响因子：
5.5
通讯作者：
Marshansky, V
中科院分区：
文献类型：
--
作者：
El Annan, J;Brown, D;Marshansky, V
ADP-核糖基化因子 (Arfs) 是一种小型 GTP 酶，可调节外吞和内吞途径中的囊泡运输。作为了解 Arfs 在肾脏生理学中作用的第一步，我们进行了免疫细胞化学和蛋白质印迹来表征 Arf1 和 Arf6 在上皮细胞中的原位表达和靶向。 Arf1 和 Arf6 与近端小管的顶膜和顶下囊泡相关，它们与巨蛋白共定位。 Arf1 还在远端小管、连接段和集合管 (CD) 中表达。 Arf1 在闰细胞 (IC) 中含量丰富，并与 A-IC（顶端）和 B-IC（顶端和/或基底外侧）中的 V-ATP 酶共定位。相比之下，Arf6 只与 CD 中的基底外侧膜和囊泡相关。在髓质中，基底外侧 Arf6 主要在 A-IC 中可检测到。整个外髓质主细胞中的表达变弱，在内髓质主细胞中未检测到 Arf6。在一些肾上皮细胞中，Arf1（而非 Arf6）也被靶向核周斑块，在那里它与 TGN38（跨高尔基体网络的标记）共定位。定量Western blotting显示内源性Arf1的表达量比Arf6高26-180倍。这些数据表明 Arf GTP 酶在肾上皮细胞中以细胞和膜特异性模式原位表达和靶向。这些结果提供了一个框架，以此为基础和解释未来关于 Arf GTPases 在肾小管上皮细胞中发生的多种细胞运输事件中的作用的研究。
ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct ( CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC ( apical) and B-IC ( apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26 - 180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.