ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment

• Published in: Molecular biology of the cell Vol. 26; no. 23; pp. 4265 - 4279
• Main Authors: Salem, Joseph C, Reviriego-Mendoza, Marta M, Santy, Lorraine C
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 15.11.2015
• Subjects: Endosomes - metabolism; Epithelial Cells - metabolism; Focal Adhesions - metabolism; GTPase-Activating Proteins - metabolism; HeLa Cells; Humans; Integrin alpha5 - metabolism; ras Proteins - metabolism; Signal Transduction; Vesicular Transport Proteins - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E15-05-0278

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.