RAB23 facilitates clathrin-coated nascent vesicle formation at the plasma membrane and modulates cell signaling.

RAB23 is known to regulate signaling by several growth factors during organogenesis. RABs and other small GTPases function as molecular switches during cellular membrane trafficking. However, what has not been established is how RAB23 functions during cellular membrane trafficking and how this influences cell signaling. To address this, we characterized RAB23's localization in the endocytic pathway and determined the route of endocytosis. We find that RAB23 interacts with β-adaptin (AP2β1) subunit of the clathrin adaptor protein 2 (AP-2) complex, suggesting RAB23's involvement in clathrin-dependent endocytosis at the plasma membrane. Our results show that RAB23 might function at multiple steps during clathrin-coated nascent vesicle formation. We find that RAB23 interacts with clathrin assembly protein PICALM, vesicle curvature protein endophilin A2, and a protein linked with vesicle scission, cortactin. To understand the functionality of RAB23, we performed time-lapse live cell imaging of transferrin uptake, which showed that clathrin-dependent endocytosis is affected in RAB23 deficient osteoprogenitors with inefficient cargo internalization. We normalized transferrin uptake in RAB23 knockdown human osteosarcoma cells (MG-63) by overexpressing RAB23. Our results show that deficiency of RAB23 reduced the interaction between β-adaptin and clathrin. We demonstrate that vesicle formation upon BMP stimulation and subsequent signal transduction is aberrant in RAB23-deficient cells. We further show evidence by providing microarray data-driven hypergeometric test of differentially expressed genes in WT and RAB23-deficient samples which suggests RAB23's participation in vesicle formation, endocytosis and cell signaling. Collectively, our data indicate a role for RAB23 in vesicle formation, membrane trafficking, and cell signaling.