Cell polarity was quantified by a percentage of cells exhibiting correct orientation at the leading edge (polarity index). highlights a possible role of Btk inhibitor 1 (R enantiomer) recycling endosomal pH in regulating receptor-mediated signaling through vesicular trafficking. == INTRODUCTION == The MET protein is a receptor tyrosine kinase (RTK) and the receptor for hepatocyte growth factor (HGF)/scatter factor (Boccaccio and Comoglio, 2006). Binding of HGF to MET initiates various biological responses, including cell proliferation and survival, detachment from adjacent cells, epithelial to mesenchymal transition, and degradation of and migration through extracellular matrices (Trusolinoet al., 2010). While HGF/MET signaling is important for organogenesis and other physiological processes, aberrant activation of HGF/MET signaling facilitates metastasis in malignant tumors (Gherardiet al., 2012), and elevated expression of MET is associated with poor Btk inhibitor 1 (R enantiomer) prognosis of glioma (Nabeshimaet al., 1997; Konget al., 2009; Liuet al., 2011; Miyamotoet al., 2011). As activation of RTKs typically occurs on the cell surface, postendocytic degradation and recycling of RTKs play a crucial role in determining receptor availability and coordinating downstream signaling (Lemmon and Schlessinger, 2010). Endocytosis/recycling of oncogenic MET mutant proteins is required for oncogenicity (Joffreet al., 2011), whereas decreased MET recycling attenuates mitogen-activated protein kinase (MAPK) signaling and cell migration (Parachoniaket al., 2011). Moreover, endocytic recycling of MET facilitates sustained Rac1 signaling for optimal membrane ruffling and cell migration and invasion, and recruitment of the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and a Rac GDP/GTP exchange factor Vav2 to recycling endosomes was suggested as Btk inhibitor 1 (R enantiomer) an underlying mechanism (Menardet al., 2014). Thus MET targeting to and from recycling endosomes may account for a significant step linking HGF stimuli with actin cyoskeletal reorganization during cell migration. It was also reported that tumor-derived mutant p53 proteins enhance MET recycling and signaling, which drive invasion and scattering of human nonsmall cell lung carcinoma, H1299 cells, further signifying the role of MET recycling in metastatic progression (Mulleret al., 2013). These observations collectively suggest that up-regulated MET recycling promotes tumor invasion and metastasis. Increasing evidence points to the promigratory and proinvasive role of the acidic luminal pH of endosomes. Vacuolar-type H+-ATPases (V-ATPases) are expressed in most eukaryotic cells and play a pivotal role in establishing the acidic luminal pH of endosomal and secretory organelles by pumping protons into the lumen (Forgac, 2007). Treatment of human breast cancer cells with a V-ATPase inhibitor disrupted the leading-edge association of epidermal growth factor receptor (EGFR; Wiedmannet al., 2012). Moreover, inhibition of V-ATPases attenuates Rab5-mediated Rac1 activation in early endosomes, perturbs actin cytoskeletal remodeling, and impedes cell migration. Intravenous injection of an inhibitor against V-ATPases drastically reduced metastasis of mouse breast cancer cells (Wiedmannet al., 2012). Chloride intracellular channels (CLICs) residing in endosomal membranes are another group of acidifiers that shunt the KLRK1 proton current created by V-ATPases (Stauberet al., 2012). CLIC3, a proposed late endosomal acidifier, plays a proinvasive role in tumor metastasis by facilitating targeting of membrane-type matrix metalloproteinase (Macphersonet al., 2014) and integrins (Dozynkiewiczet al., 2012) to the plasma membrane. Recycling endosomes possess slightly acidic luminal pH (Presleyet al., 1997; DSouzaet al., 1998), but the role of the acidic pH of recycling endosomes in tumor cell migration remains poorly understood. We now show that the neuron-enriched Na+/H+exchanger NHE5 potently acidifies Btk inhibitor 1 (R enantiomer) recycling endosomes of C6 rat glioma cells and demonstrate that NHE5 is required for MET recycling. Silencing NHE5 impairs MET targeting to.
