Mechanisms and consequences of affinity modulation of integrin alpha(v)beta(3) detected with a novel patch-engineered monovalent ligand

Integrin alpha(v)beta(3) mediates diverse responses in vascular cells, rang ing from cell adhesion, migration, and proliferation to uptake of adenoviru ses. However, the extent to which alpha(v)beta(3) is regulated by changes i n receptor conformation (affinity), receptor diffusion/clustering (avidity) , or post-receptor events is unknown. Affinity regulation of the related in tegrin, alpha(IIb)beta(3), has been established using a monovalent ligand-m imetic antibody, PAC1 Fab. To determine the role of affinity modulation of alpha(v)beta(3), a novel monovalent ligand-mimetic antibody (WOW-1) was cre ated by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single alpha(v) integrin binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated alpha( v)beta(3), but not to alpha(IIb)beta(3), in receptor and cell binding assay s. alpha(v)beta(3) affinity varied with the cell type. Unstimulated B-lymph oblastoid cells bound WORT-1 Fab poorly (apparent K-d = 2.4 mu M), but acut e stimulation with phorbol 12-myristate 13-acetate increased receptor affin ity >30-fold (K-d = 80 nM), With no change in receptor number. In contrast, alpha(v)beta(3) in melanoma cells was constitutively active, but ligand bi nding could be suppressed by overexpression of beta(3) cytoplasmic tails. U p-regulation of alpha(v)beta(3) affinity had functional consequences in tha t it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that alpha(v)beta(3) is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.