Venkateswarlu Kanamarlapudi
Department of Pharmacology, The University of Bristol, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 2003 Oct 31;278(44):43460-9. doi: 10.1074/jbc.M304078200. Epub 2003 Aug 14.
The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2.
ADP核糖基化因子6（ARF6）小GTP酶在刺激肌动蛋白重组和膜皱襞形成的信号通路中作为一种受GDP/GTP调节的开关发挥作用。活性ARF6GTP的形成受到鸟嘌呤核苷酸交换因子（GEF）如细胞粘附素的刺激，细胞粘附素通过其pleckstrin同源结构域与脂质第二信使磷脂酰肌醇3,4,5 -三磷酸结合，转位至激动剂刺激细胞的质膜，随后激活ARF6。利用细胞粘附素2作为酵母双杂交筛选的诱饵，我们分离出一个编码名为细胞粘附素交换因子1相互作用蛋白（IPCEF1）的蛋白质的cDNA。通过酵母双杂交和谷胱甘肽S -转移酶下拉实验结合缺失突变分析，确定了细胞粘附素2与IPCEF1相互作用所需的特定结构域位于细胞粘附素2的卷曲螺旋结构域和IPCEF1的C末端121个氨基酸。IPCEF1还与ARF GEF细胞粘附素家族的其他成员相互作用，这表明与IPCEF1的相互作用在ARF GEF细胞粘附素家族中高度保守。通过免疫沉淀证实了细胞粘附素2与IPCEF1在哺乳动物细胞中的相互作用。免疫荧光分析显示，在未受刺激的细胞中，IPCEF1与细胞粘附素2共定位于细胞质，在表皮生长因子刺激的细胞中，通过与细胞粘附素2结合转位至质膜。然而，缺乏细胞粘附素2结合位点的IPCEF1缺失突变体在受刺激的细胞中无法与细胞粘附素2共迁移至细胞膜。通过证明IPCEF1增加细胞粘附素2在体外和体内对ARFGTP形成的刺激作用，证实了IPCEF1 -细胞粘附素2相互作用的功能意义。
