Mechanisms and consequences of affinity modulation of integrin α(v)β3 detected with a novel patch-engineered monovalent ligand

Abstract

Integrin α(v)β3 mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which α(v)β3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post- receptor events is unknown. Affinity regulation of the related integrin, α(IIb)β3, has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of α(v)β3, a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single α(v) integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated avα(v)β3, but not to α(IIb)β3, in receptor and cell binding assays, α(v)β3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K(d) = 2.4 μM), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K(d) = 80 nM), with no change in receptor number. In contrast, α(v)β3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of β3 cytoplasmic tails. Up-regulation of α(v)β3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that α(v)β3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.