Reverse endocytosis of transmembrane ephrin-B ligands via a clathrin-mediated pathway.
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.
Parker
Monica
M
Division of Rheumatology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Roberts
Richard
R
Enriquez
Miriam
M
Zhao
Xia
X
Takahashi
Takamune
T
Pat Cerretti
Douglas
D
Daniel
Tom
T
Chen
Jin
J
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D034281:Dynamins / Q000737:chemistry; D004705:Endocytosis; D036387:Ephrin-B1 / Q000737:chemistry*; D005434:Flow Cytometry; D005799:Genes, Dominant; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009154:Mutation; D011188:Potassium / Q000737:chemistry; D036081:Receptors, Eph Family / Q000737:chemistry*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D002966:Clathrin; D018076:DNA, Complementary; D036387:Ephrin-B1; D008024:Ligands; D036081:Receptors, Eph Family; D034281:Dynamins; D011188:Potassium