Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin α(IIb)β(3)

Integrin α(IIb)β(3) mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to α(IIb)β(3) function, α(IIb) was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified α(IIb) subunits were expressed with β(3) in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when α(IIb)β(3) affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of α(IIb)β(3) also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72(Syk) and fibrinogen-dependent phosphorylation of pp125(FAK), even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in α(IIb)β(3) function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of α(IIb)β(3) in platelets.