RalB uncoupling from exocyst is required for endothelial Weibel-Palade body exocytosis.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multisubunit tethering complex exocyst. Here, we show that Ral isoform RalB, but not RalA, is necessary for regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in GDP-bound state. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that protein kinase C (PKC)-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its interaction with exocyst. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling a RalB exocyst-binding site mutant. In summary, uncoupling of exocyst from RalB promotes endothelial WPB exocytosis. Our data show that RalB may be more dynamically regulated by phosphorylation and may confer distinct functionality given the high degree of homology and the shared set of effector protein between the two Ral isoforms.