ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment.
ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment.
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DOI：
10.1091/mbc.e15-05-0278
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发表时间：
2015-11-15
影响因子：
3.3
通讯作者：
Santy LC
中科院分区：
文献类型：
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作者：
Salem JC;Reviriego-Mendoza MM;Santy LC
R-Ras和cytohesin-2/ARNO协同控制上皮细胞的粘附，但其机制尚不清楚。胞浆素-2/ARNO通过ehd1阳性循环室调节交通。抑制cytohesin-2/ARNO活性可将R-Ras和整合素困在EHD1细胞室内，并损害粘附和扩散。当细胞素-2/ARNO在上皮细胞中表达时，ARF小gtp酶的鸟嘌呤核苷酸交换因子（GEF）会引起强烈的迁移反应。最近的证据表明，细胞分裂素-2/ARNO作用于GTPase R-Ras的下游，促进扩散和迁移。我们假设胞内素-2/ARNO通过ARF激活调节R-Ras的再循环来传递R-Ras信号。我们发现，在瞬时表达的HeLa细胞中，eps15同源结构域1 （EHD1）是一种与内噬循环区（ERC）相关的蛋白质，与活性R-Ras共定位。此外，我们还发现ehd1阳性循环内体是细胞胞嘧啶-2/ARNO的一种新型隔室。gef失活（E156K）胞内素-2/ARNO的敲低或表达导致R-Ras在含有EHD1的循环内体上积累，抑制细胞扩散。E156K-ARNO也会导致黏附灶大小和数量的减少。最后，我们证明了R-Ras/ARNO信号是α - 5整合素和R-Ras再循环到质膜所必需的。这些数据证实了细胞分裂素-2/ARNO作为R-Ras和整合素循环的调节因子的作用，并表明arf调节的R-Ras运输是R-Ras依赖于扩散和粘附形成的作用所必需的。
R-Ras and cytohesin-2/ARNO coordinate in the control of epithelial cell adhesion, but the mechanism has been unclear. Cytohesin-2/ARNO regulates traffic through an EHD1-positive recycling compartment. Inhibition of cytohesin-2/ARNO activity traps R-Ras and integrins within the EHD1 compartment and impairs adhesion and spreading. When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras–dependent effects on spreading and adhesion formation.