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 ␤ 3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during ␣ v ␤ 3 -mediated cell spreading. Using CHO cells transfected with various ␤ 3 mutants, we demonstrate a close correlation between ␣ v ␤ 3 -mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY 747 and NITY 759 motifs in these signaling processes. Deletion of the NITY 759 motif alone was sufficient to completely prevent ␣ v ␤ 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 ␣ v ␤ 3 -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 receptorligand interaction, our results suggest that the structural integrity of the NITY 759 motif, rather than the phosphorylation status of Tyr 759 is important for ␤ 3 -mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY 747 motif is required for optimal ␤ 3 post-ligand binding events.Anchorage of cells to the extracellular matrix is mediated in part by integrins, a large family of heterodimeric cell surface receptors, that regulate numerous aspects of cell behavior, such as cell motility, proliferation, differentiation, and apoptosis (1). Cell engagement with extracellular matrix ligands induces integrin translocation to subcellular structures known as focal adhesion plaques that form at regions of close contact between the cell and its underlying substratum (2). Integrin clustering at focal contact sites in turn triggers major intracellular events, including cytoskeleton reorganization, intracellular ion transport, phosphoinositide turnover, kinase activation, and tyrosine phosphorylation of intracellular proteins (3). A large number of tyrosine-phosphorylated proteins have been identified within focal adhesion plaques. These include cytoskeletal proteins, kinases and adaptor proteins, growth factor receptors, and growth factor receptor-related signaling molecules, thus emphasizing the potential role of integrins as recruiting centers for molecules involved in various signaling pathways.Although the link of integrins with focal adhesions is well established, the precise mechanism by which integrins associate with cytoskeletal proteins, regulate focal adhesion plaque assembly, and participate in the activation of intracellular signaling cascades is still unclear. There is convincing evidence that integrin ␤ subunits are likely to play a major role in these processes: (i) truncation of the ␤ subuni...

Distinct Ligand-binding Modes for Integrin αvβ3-Mediated Adhesion to Fibronectinversus Vitronectin

“…Extensive analysis of cytoplasmic domain mutants of ␤ 3 has lead to the conclusion that this domain is critical for the regulation of ligand binding (33)(34)(35). This has been extended using the spinning-disc analysis showing that Tyr-747 and Tyr-759 make specific quantitative contributions to the ␣ v ␤ 3 bond strength (18).…”

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

“…HB-19 and Nucant 6L pseudopeptides were from Polypeptide Laboratories (Strasbourg, France). Plasmids encoding human wild-type ␤ 3 , ␤ 3 Y773F, ␤ 3 Y785F, ␤ 3 Y773F/ Y785F, and ␣ have been previously described (29).…”

“…Stable CHO cell clones expressing wildtype ␤ 3 , ␤ 3 Y773F, ␤ 3 Y785F, or ␤ 3 Y773F/Y785F were generated as previously described (29). Cell culture reagents were from BiochromKG (Seromed, Germany).…”

Probing Conformational Changes in the I-like Domain and the Cysteine-rich Repeat of Human β3 Integrins following Disulfide Bond Disruption by Cysteine Mutations

“…The presence of each mutation was verified by DNA sequencing of the mutant ␤ 3 cDNA. Wild type and mutant ␤ 3 integrin cDNA constructs were transfected into CHO cells expressing recombinant human ␣ IIb using the LipofectAMINE method (Life Technologies, Inc.) as previously described (28). Positive colonies were isolated by cylinder cloning and further subcloned by limiting dilution.…”

“…Flow Cytometry Analysis-Cell surface expression of recombinant ␣ IIb ␤ 3 was analyzed by flow cytometry with a panel of anti-␣ IIb , anti-␤ 3 and complex-specific anti-␣ IIb ␤ 3 monoclonal antibodies as previously described (28). Cells were detached from culture plates with EDTA buffer, pH 7.4, and washed twice with serum-free Iscove's modified Dulbecco's medium.…”