title: ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Salem
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802.
authors.forename: Marta M
authors.initials: MM
authors.lastname: Reviriego-Mendoza
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802 lcsanty@psu.edu.
authors.forename: Lorraine C
authors.initials: LC
authors.lastname: Santy
mesh_terms: D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D039482:Integrin alpha5 / Q000378:metabolism*; D015398:Signal Transduction; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
chemical_list: C120700:EHD1 protein, human; D020690:GTPase-Activating Proteins; D039482:Integrin alpha5; D033921:Vesicular Transport Proteins; C102474:cytohesin-2; C085929:RRAS protein, human; D018631:ras Proteins