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Vol. 19, Issue 3, 765-775, March 2008

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A Unique Platform for H-Ras Signaling Involving Clathrin-independent Endocytosis

Natalie Porat-Shliom^*,, Yoel Kloog, and Julie G. Donaldson^*

*Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and Department of Neurobiochemistry, Tel Aviv University, Tel Aviv, Israel

Submitted August 27, 2007; Revised November 5, 2007; Accepted December 7, 2007
Monitoring Editor: Sandra Lemmon

Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatiylinositol 3,4,5-trisphosphate (PIP₃). PIP₂ loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP₃ loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP₂/PIP₃, PIP₃/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.

Address correspondence to: Julie G. Donaldson (jdonalds{at}helix.nih.gov)

Abbreviations used: CD, cytochalasin D; CDE, clathrin-dependent endocytosis; CIE, clathrin-independent endocytosis; EGF, epidermal growth factor; MHCI, class I major histocompatibility complex; PH, pleckstrin homology; PIP₂, phosphatidylinositol 4,5-bisphophate; PIP₃, phosphatidylinositol 3,4,5-trisphosphate; PI 3-kinase, phosphotidylinositol 3-kinase; PIP 5-kinase, phosphatiylinositol 4-phosphate 5-kinase; PL, phospholipase; PM, plasma membrane; RBD, Ras binding domain; Tfn, transferrin; TfR, transferrin receptor.