title: Distinct involvement of beta3 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 beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-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 alphav beta3-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 alphav beta3-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 beta3-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 beta3 post-ligand binding events.
authors.affiliation: Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS and CRP-Santé), Centre Universitaire, 162A, avenue de la Faïencerie, L-1511 Luxembourg, Grand Duchy of Luxembourg.
authors.forename: E
authors.initials: E
authors.lastname: Schaffner-Reckinger
authors.forename: V
authors.initials: V
authors.lastname: Gouon
authors.forename: C
authors.initials: C
authors.lastname: Melchior
authors.forename: S
authors.initials: S
authors.lastname: Plançon
authors.forename: N
authors.initials: N
authors.lastname: Kieffer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002465:Cell Movement; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases; D039661:Integrin beta3; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D039661:Integrin beta3; D010980:Platelet Membrane Glycoproteins; D019000:Phosphotyrosine; D005340:Fibrinogen; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases