Identification of a Ca2(+)-dependent cell-cell adhesion molecule in endothelial cells.
Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)-dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non-reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.
Heimark
R L
RL
Department of Pathology, University of Washington, Seattle 98195.
Degner
M
M
Schwartz
S M
SM
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001011:Aorta / Q000166:cytology; D015820:Cadherins / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000502:physiology; D007150:Immunohistochemistry; D014357:Trypsin
D000911:Antibodies, Monoclonal; D015820:Cadherins; D015815:Cell Adhesion Molecules; D014357:Trypsin; D002118:Calcium