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 RabGAP5, 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.

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.

The PD-L1/PD-1 signaling axis is a pivotal regulator of T-cell activity and a key mechanism by which tumors evade immune surveillance. Inhibiting this pathway has resulted in significant anti-tumor responses, establishing immune checkpoint blockade (ICB) as a crucial component of modern cancer therapy. However, many patients with high PD-L1 expression do not respond to PD-1/PD-L1 blockade, underscoring the necessity for a deeper investigation into the mechanisms underlying this resistance. Recent studies have identified DRG2 as a critical modulator of anti-PD-1 therapeutic efficacy. While DRG2 depletion enhances IFN-γ signaling and increases the overall PD-L1 levels, it disrupts the recycling of endosomal PD-L1, resulting in reduced surface expression and impaired PD-1 interaction, ultimately compromising therapeutic outcomes. Furthermore, TRAPPC4, HIP1R, and CMTM6 help stabilize PD-L1 by preventing lysosome degradation. When depleted, these proteins have been shown to boost the body’s immune response against tumors. Research into the complex regulatory mechanisms of PD-L1 suggests that targeting DRG2, TRAPPC4, HIP1R, and CMTM6 could enhance the effectiveness of PD-1/PD-L1 blockade therapies. This strategy could create exciting new possibilities for cancer immunotherapy and improve patient outcomes.

Malignant metastatic melanoma 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 metastatic melanoma compared with primary melanoma and dysplastic nevi. A correlation between DRG2 expression and poor disease‐specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed binding of HIF‐1α to the VEGF‐A promoter region, expression of VEGF‐A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited growth of primary melanoma and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF‐A expression and of growth of primary melanomas and lung metastases.