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Vol. 18, Issue 5, 1850-1860, May 2007

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R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

Akiyuki Takaya^*,, Takahiro Kamio^*, Michitaka Masuda, Naoki Mochizuki, Hirofumi Sawa, Mami Sato, Kazuo Nagashima, Akiko Mizutani^||, Akira Matsuno^¶, Etsuko Kiyokawa, and Michiyuki Matsuda^*,

*Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Osaka 565-0871, Japan; Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan; Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan; Laboratory of Molecular and Cellular Pathology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan; ^||Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Kanagawa 259-1193, Japan; and ^¶Department of Neurosurgery, Teikyo University Ichihara Hospital, Chiba 299-0111, Japan

Submitted August 29, 2006; Revised February 16, 2007; Accepted February 28, 2007
Monitoring Editor: Mark Ginsberg

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Michiyuki Matsuda (matsudam{at}path1.kyoto-u.ac.jp)

Abbreviations used: EGF, epidermal growth factor; FRET, fluorescence (Förster's) resonance energy transfer; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; NPY, neuropeptide Y; PI3K, p110 subunit of phosphoinositide-3-kinase.

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