Small GTP-binding protein RalA associates with Weibel-Palade bodies in endothelial cells

In endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endo thelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage poo ls in a number of cells. In this study we have investigated whether small G TP-binding proteins are associated with Weibel-Palade bodies. We isolated W eibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were h ighly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinc t band with an apparent molecular weight of 28,000 was observed. Two-dimens ional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A mono clonal antibody directed against RalA showed reactivity with the small GTP- binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bo dies in endothelial cells.