Novel Role of Cytohesin‐2 in Regulation of Macropinocytosis Pathway and Cell Proliferation

Recently, we uncovered four V‐ATPase a‐subunit isoforms derived peptides a1N1–17, a2N1–17, a3N1–17 and a4N1–17 as a potent inhibitors of the GDP/GTP‐exchange activity of cytohesin‐2 in vitro. Here, we used these peptides to study the role of cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation in vivo. Treatment of HeLa and MTC cells by cell‐permeable and fluorescently labeled peptides (FITC‐a2N1–17‐TAT or CMTR‐a2N1–17‐TAT) give rise to cell shape remodeling and activation of the macropinocytosis pathway. Uptake of RITC‐dextran but not albumin‐Alexa555 by this pathway was insensitive to inhibitors of NHE‐exchanger (amiloride, DMA, EIPA) and V‐ATPase (bafilomycin A1). Pulse‐chase and confocal time‐lapse imaging revealed that anti‐cytohesin peptides are targeted to endosomal/lysosomal compartments and slow down vesicular trafficking. We also showed that all four anti‐cytohesin peptides are potent inhibitors of the EGFR/cytohesin‐2 dependent proliferation of human lung cancer A549 cells. In summary, here we demonstrated an important role of signaling between V‐ATPase and cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation. Importantly, these anti‐cytohesin peptides could be used to treat EGFR/cytohesin‐2 dependent cancers and to prevent development of diabetic nephropathy. Supported by grants NIH DK038452 and BADERC DK05752–08 to VM.