The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate-containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase-dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and Rab-GAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. INTRODUCTIONIntracellular vesicular trafficking contributes to diverse cellular processes, such as nutrient uptake and cell migration (Mellman, 1996). Small GTPase Rab proteins ensure the delivery of cargoes to their correct destinations by binding to various effectors, such as molecular motors and tethering factors (Stenmark, 2009). Rab5, a wellknown early endosome marker, recruits early endosome antigen 1 (EEA1; Christoforidis et al., 1999a) and regulates fusion and motility of early endosomes (Nielsen et al., 1999). Rab7 plays key roles in biogenesis and maintenance of lysosomes (Bucci et al., 2000) by making endosomes competent for fusion with lysosomes (Rink et al., 2005). Rab11 is the most prominent recycling endosome marker and regulates vesicular recycling to the plasma membrane (Ullrich et al., 1996;Grant and Donaldson, 2009). As cargoes move along endocytic pathways, the transition between early and late or recycling endosomes is mediated by Rab conversion, a process in which cascades of Rab guanine nucleotide exchange factors (GEFs) and Rab GTPase-activating proteins (GAPs) act as key regulators (Hutagalung and Novick, 2011). One well-studied Rab conversion is that from Rab5 to Rab7 as endosomes mature from early endosomes to late endosomes (Rink et al., 2005). Rab5 recruits the SAND-1(Mon1)/CZZ-1 heterodimer, which in turn recruits and activates Rab7 as the endosome matures (Poteryaev et al., 2010). Activated Rab7 recruits Rab5 GAP and facilitates inactivation and removal of Rab5 (Rink et al., 2005). Defects in Rab5 GAP inhibit the Rab5-to-Rab7 conversion and result in accumulation of large vesicles containing both Rab5 and Rab7 (Chotard et al., 2010). In contrast to Rab5-to-Rab7 conversion, little is known about how Rab5-to-Rab11 conversion along the recycling pathway is controlled. Krauss and Haucke, 2007). To confirm the localization of DRG2, we cotransfected MCF7 cells with DRG2 and markers for PIs, including Akt-PH for phosphatidylinositol (3,4,5)-trisphosphat...

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Malignant metastatic melanoma (MM) is the most lethal of all skin cancers, but detailed mechanisms for regulation of melanoma metastasis are not fully understood. Here, we demonstrated that developmentally regulated GTP-binding protein 2 (DRG2) is required for the growth of primary tumors and for metastasis. DRG2 expression was significantly increased in MM compared with primary melanoma (PM) and dysplastic nevi. A correlation between DRG2 expression and poor disease-specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed the binding of Hypoxia-inducible factor 1a to the VEGF-A promoter region, expression of vascular endothelial growth factor (VEGF)-A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited the growth of PM and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF-A expression and of growth of PMs and lung metastases.

Vacuolar-type H + -ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3α/DMXL2 and Rbcn3β/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3β/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3β/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3β/WDR7, and cell assays indicated that Rbcn3β/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3α, or Rbcn3β in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3β/WDR7 interactions.

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