Alpha v integrins mediate the rise in intracellular calcium in endothelial cells on fibronectin even though they play a minor role in adhesion.
We have investigated which integrins mediate the elevation of intracellular calcium ([Ca2+]i) triggered by spreading of endothelial cells on fibronectin (FN). Specific anti-integrin monoclonal antibodies immobilized on glass surfaces were used as agonists to trigger cell spreading. These experiments demonstrated that an antibody to alpha v could induce the rise in [Ca2+]i, whereas two antibodies to alpha 5 beta 1 were inactive, despite their ability to induce cell spreading and elevation of intracellular pH. Antibodies in solution were then used as agonists to block association of specific integrins with FN. These experiments also showed that alpha v integrin(s) but not alpha 5 beta 1 mediated the rise in [Ca2+]i in cells spreading on FN. Adhesion assays in the presence of function-blocking anti-integrin antibodies and affinity chromatography on FN columns of surface-labeled cell extracts were carried out to characterize the integrins that bind to FN. Both methods showed that alpha v integrin(s) and alpha 5 beta 1 participate in FN binding; however, the contribution from alpha v integrin(s) was much less than that of alpha 5 beta 1. These results demonstrate that two receptors for FN on the same cells can trigger distinct intracellular signaling pathways and, furthermore, that an integrin whose contribution to adhesion is barely detectable can have a major effect on cellular responses. The results also suggest that the specificity for activation of the calcium signaling pathway resides primarily in the integrin alpha subunit.
Schwartz
M A
MA
Scripps Research Institute, Committee on Vascular Biology, La Jolla, California 92037.
Denninghoff
K
K
D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D004730:Endothelium, Vascular / Q000378:metabolism / Q000502:physiology*; D005353:Fibronectins; D016257:Fura-2; D006801:Humans; D039564:Integrin alphaV; D016023:Integrins / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D017469:Receptors, Fibronectin; D014471:Umbilical Veins
D000906:Antibodies; D005353:Fibronectins; D039564:Integrin alphaV; D016023:Integrins; D017469:Receptors, Fibronectin; D002118:Calcium; D016257:Fura-2