Activation of the integrin alpha v beta 3 involves a discrete cation-binding site that regulates conformation.
"Activation" of integrins is involved in the dramatic transition of leukocytes and platelets from suspension to adhesion. The integrin alpha v beta 3 is not known to take part in this sort of transition, even though it shares its beta subunit with alpha IIb beta 3, the activable integrin on platelets. In the context of a constitutively adhered cell, changes in activation state may be more subtle in their effects, but nonetheless important in regulating cell behavior. We hypothesized that alpha v beta 3 can undergo conformational changes analogous to those associated with alpha IIb beta 3 activation. Accordingly, we examined alpha v beta 3 on the surface of M21 cells (a human melanoma cell line) and found that, like alpha IIb beta 3, it can undergo conformational changes upon binding of a ligand analog and can be activated for ligand binding and migration by a monoclonal antibody directed against beta 3. Modulation of the binding of this activating antibody, AP5, ligand binding, and antibody-mediated activation all are associated with a discrete cation-binding site shared in both alpha IIb beta 3 and alpha v beta 3. Based on a measured Ki, this site has an apparent Kd for calcium of approximately 20 microM. At physiological levels of calcium, about 40% of the total alpha v beta 3 on a cell's surface is in a conformation detected by AP5. The data suggest a model for both alpha v beta 3 and alpha IIb beta 3 function in which the molecule can exist in either of (at least) two conformational states, one stabilized either by AP5 or ligand binding, refractory to calcium binding, and enhanced for ligand recognition, the other stabilized by calcium binding and refractory to AP5 and ligand binding. Functional analysis suggests that AP5 activates alpha v beta 3 by preventing occupation of this calcium site, and that the activated form of alpha v beta 3 differs functionally from the basal form. The active form is more conducive to migration and the basal to tight adhesion.
Pelletier
A J
AJ
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Kunicki
T
T
Quaranta
V
V
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002465:Cell Movement; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008545:Melanoma; D011487:Protein Conformation; D019030:Receptors, Vitronectin / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D002412:Cations; D005456:Fluorescent Dyes; D019030:Receptors, Vitronectin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium