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 endothelial 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 pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal 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 bodies in endothelial cells.
de Leeuw
H P
HP
Department of Blood Coagulation, CLB, Amsterdam, The Netherlands.
Wijers-Koster
P M
PM
van Mourik
J A
JA
Voorberg
J
J
D002458:Cell Fractionation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D020302:Weibel-Palade Bodies / Q000201:enzymology*; D020725:ral GTP-Binding Proteins
D020558:GTP Phosphohydrolases; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins