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

Abstract

We have investigated the structural requirements of the β3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during α(v)β3-mediated cell spreading. Using CHO cells transfected with various β3 mutants, we demonstrate a close correlation between α(v)β3-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 α(v)β3- 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 Tyr747 completely abolished α(v)β3 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 NITY759 motif, rather than the phosphorylation status of Tyr759 is important for β3-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 β3 post-ligand binding events.