Reverse Endocytosis of Transmembrane ephrin-B Ligands via a Clathrin-Mediated Pathway

Biochem Biophys Res Commun. 2004 Oct 8;323(1):17-23. doi: 10.1016/j.bbrc.2004.07.209.

Abstract

Eph/ephrin receptors and ligands mediate cell-cell interaction through reciprocal signaling upon juxtacrine contact, and play a critical role in embryonic patterning, neuronal targeting, and vascular assembly. To study transmembrane ephrin-B ligand trafficking, we determined the cellular localization of ephrin-B1-GFP upon engagement by EphB1. Under normal culture conditions ephrin-B1-GFP is localized to the plasma membrane, mostly at the lateral cell borders. Addition of soluble EphB1-Fc receptor induces ephrin-B1-GFP clustering on the cell surface and subsequent internalization, as judged by biochemical studies, electron microscopy, and co-localization with endosomal markers. A dominant-negative mutant of dynamin or potassium depletion blocks ephrin-B1 endocytosis. These results suggest that ephrin-B1 internalization is an active receptor-mediated process that utilizes the clathrin-mediated endocytic pathway.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Biotinylation
  • Blotting, Western
  • CHO Cells
  • Cell Membrane / metabolism*
  • Cell Separation
  • Cells, Cultured
  • Clathrin / metabolism*
  • Cricetinae
  • DNA, Complementary / metabolism
  • Dynamins / chemistry
  • Endocytosis
  • Ephrin-B1 / chemistry*
  • Flow Cytometry
  • Genes, Dominant
  • Genetic Vectors
  • Humans
  • Ligands
  • Microscopy, Confocal
  • Microscopy, Electron
  • Mutation
  • Potassium / chemistry
  • Receptors, Eph Family / chemistry*
  • Signal Transduction
  • Time Factors
  • Transfection
  • Umbilical Veins / cytology

Substances

  • Clathrin
  • DNA, Complementary
  • Ephrin-B1
  • Ligands
  • Receptors, Eph Family
  • Dynamins
  • Potassium