Distinct involvement of β3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling

We have investigated the structural requirements of the beta(3) integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alpha(v) beta(3)-mediated cell spreading. Using CHO cells transfected with various beta(3) mutants, we demonstrate a close correlation between alpha(v) beta(3)-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alpha(v) beta(3)-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation, The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr(747) completely abolished gp,dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITU759 motif, rather than the phosphorylation status of Tyr(759) is important for beta(3)-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta(3) post-ligand binding events.