Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

Abstract

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...

Entry of Classical Swine Fever Virus into PK-15 Cells via a pH-, Dynamin-, and Cholesterol-Dependent, Clathrin-Mediated Endocytic Pathway That Requires Rab5 and Rab7

“…To this end, DN mutants of Rab5 (S34N), Rab7 (T22N), and Rab11 (S25N), which have been validated in previously reported entry studies with different enveloped viruses (31,32,57), were employed. The functionality of Rab WT and DN constructs was confirmed in PK-15 cells by using labeled transferrin as a control (31,58). As expected, the overexpression of all three Rab DN constructs reduced the accumulation of transferrin in comparison with cells expressing WT Rab, with minor fluorescence intensity in the cytoplasm and failure in the superposition of both proteins (Fig.…”

“…Because Rab5 depletion affected CSFV entry more than did Rab7 depletion, Rab5 may play a more critical role in CSFV infection. In addition, the functionality of Rab knockdown in PK-15 cells was confirmed by using labeled transferrin as a control (31,58). As expected, silencing of Rab5 or Rab7 reduced the accumulation of transferrin, compared to that in control cells, with minor fluorescence intensity in the cytoplasm and failure in the superposition of both proteins (Fig.…”

DRG2 supports the growth of primary tumors and metastases of melanoma by enhancing VEGF‐A expression

“…Previously, we have reported that DRG2 depletion induces cell cycle arrest and mitochondrial dysfunction , leading to inhibition of cell growth. In addition, it has been reported that DRG2 interacts with Rab5 on early endosome and DRG2 depletion inhibits endosome trafficking , enhances the Akt signal from endosome, and inhibits perinuclear Golgi stacking and cell migration . In this study, we found that DRG2 depletion inhibited growth of melanoma cells.…”

“…Previously, we found that overexpression of DRG2 affects cell proliferation and apoptosis in Jurkat human T cells , inhibits T H 17 differentiation, and ameliorates experimental autoimmune encephalomyelitis in mice . We also learned that DRG2 interacts with Rab5 on endosomes and is required for Rab5 inactivation on endosomes and for recycling of transferrin (Tfn) to the plasma membrane . Together, these data demonstrate that DRG2 is an important regulator of signal pathways for cell growth, differentiation, and/or vesicle trafficking.…”

“…DRG2 upregulates VEGF‐A by enhancing transcription. Previously, we have reported that DRG2 regulates endosome trafficking and microtubule dynamics by interacting with Rab5 on endosome. Interestingly, we here observed that DRG2 depletion inhibits nuclear translocation of HIF‐1α and HIF‐1α‐mediated transcription of VEGF‐A .…”