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

Thromb Haemost. 1999 Sep;82(3):1177-81.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Electrophoresis, Gel, Two-Dimensional
  • Endothelium, Vascular / enzymology*
  • Endothelium, Vascular / ultrastructure
  • GTP Phosphohydrolases / chemistry
  • GTP Phosphohydrolases / isolation & purification
  • GTP Phosphohydrolases / metabolism*
  • Humans
  • Isoelectric Point
  • Molecular Weight
  • Weibel-Palade Bodies / enzymology*
  • ral GTP-Binding Proteins*

Substances

  • GTP Phosphohydrolases
  • RALA protein, human
  • ral GTP-Binding Proteins