Our recent work has shown that the integrin alpha-V-beta-3 like alpha-IIb- beta-3. can exist in different conformations or """"""""activation states,"""""""" that differ in ligand-binding properties. It is our hypothesis that activation of this integrin can be modulated by the cell, and that different states play a role in modulating cell behavior such as adhesion and migration. To examine this, we propose first to determine the effects of alpha-V-beta-3 activation on the adhesion and migration properties of cells and compare these to the effects of alpha-IIb-beta-3 activation in the same cell context. Activation will be achieved with the use of an activating monoclonal antibody we have described, AP5. Second, we will examine the mechanism of activation by this antibody. Our data suggest that AP5- mediated activation is related to apparently competitive inhibition between binding of AP5 and binding of calcium to a discrete binding site involving beta3. A calcium-binding site with similar properties has been implicated in the negative regulation of alpha-V-beta-3-ligand interactions (Smith et al., 1994; D'Souza et al., 1994). The competitive nature of the interaction allows us to use AP5-binding to screen for mutations to this calcium binding site, since mutations to beta3 that lose the calcium-binding site would be expected not to show calcium inhibition of AP5 binding. We expect that these mutations will have some effect on the activation state, or the activatability, of the receptor. These mutants will he transfected into 293 cells (ATCC 1573) paired either with alpha-IIb or alpha-V to determine their effects on receptor function. The phenotypes of these mutations should provide insight into the mechanisms of activation of alpha-V-beta-3 and alpha-IIb-beta-3 and into the role of alpha-V-beta-3 activation in regulation of cell behavior. Finally, our recent data have suggested a link between the activation state of beta3 integrins and the """"""""outside-in"""""""" signals they transmit. We will examine this phenomenon further with the aid of the activation antibody and the mutants we construct. We will compare signaling, in particular protein-tyrosine phosphorylation and calcium-mobilization, among the mutants paired with alpha-V or alpha-IIb in an attempt to understand the mechanisms by which activation states can regulate cell behavior.
| Pelletier, A J; van der Laan, L J; Hildbrand, P et al. (2000) Presentation of chemokine SDF-1 alpha by fibronectin mediates directed migration of T cells. Blood 96:2682-90 |
| Faccio, R; Grano, M; Colucci, S et al. (1998) Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin. Biochem Biophys Res Commun 249:522-5 |
| Lin, E C; Ratnikov, B I; Tsai, P M et al. (1997) Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain. J Biol Chem 272:14236-43 |
