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Regulation of mast cell function by the membrane trafficking proteins RabGEF1 and Rabaptin-5 [electronic resource]

Cell surface receptor regulation maintains proper responses to external signals and disregulation of receptors can have drastic effects. We found that RabGEF1, a guanine exchange factor (GEF) for the endocytic regulating small GTPase Rab5, regulates IgE+antigen (Ag)-induced Ras signaling in mast cells and skin homeostasis in vivo. To further understand RabGEF1's role in mast cell function we investigated if RabGEF1 generally regulates mast cell signaling by examining a number of modes of mast cell activation. Like IgE+Ag stimulation, RabGEF1 deficient (-/-) mast cells demonstrated enhanced activation after exposure to stem cell factor (SCF), the ligand for the receptor tyrosine kinase c-kit. Enhanced responses correlated with delayed c-kit and IgE receptor (Fc[epsilon]RI) internalization suggesting a proximal role of RabGEF1 in regulating mast cell signaling events. To dissect the roles of RabGEF1 and its domains in regulating mast cell biology we lentivirally expressed a panel of RabGEF1 mutants in RabGEF1-/- mast cells. We found that RabGEF1 (1) harbors ubiquitin (Ub) ligase activity within an N-terminal A20 zinc finger domain, (2) binds to Ub and localizes to membranes through amino acids 1-150, (3) interacts with Rabaptin-5, a Rab5 effector, through a C-terminal coiled coil (CC) domain and, (4) activates Rab5 with a central VPS9 domain. Despite containing a number of domains, only the VPS9 was crucial to restore wildtype activation of RabGEF1-/- cells after IgE+Ag stimulation. Moreover, lack of the VPS9 or CC domain diminished Rabaptin-5 levels and reduced surface Fc[epsilon]RI and [beta]1 expression on mast cells suggesting that these domains are important for receptor homeostasis. Using shRNA knockdown of Rabaptin-5 we identified a novel role for Rabaptin-5 in maintaining Fc[epsilon]RI and [beta]1 surface expression by increasing surface stability and receptor half-life. The reduced Fc[epsilon]RI and [beta]1 surface levels in Rabaptin-5 deficient cells attenuated sensitivity to antigen induced adhesion, migration, and cytokine production in these cells. Despite Rabaptin-5's in vitro documented role as a Rab5 effector, Rab5 dependant processes (i.e., receptor internalization or endosome fusion) were unaffected by Rabaptin-5 deficiency and challenge the current thoughts of Rab5 processes in intact cells. These data show the importance of receptor trafficking in regulating mast cell activation.