Distinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.
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.
Schaffner-Reckinger
E
E
Laboratoire Franco-Luxembourgeois de Recherche Biomédicale (CNRS and CRP-Santé), Centre Universitaire, 162A, avenue de la Faïencerie, L-1511 Luxembourg, Grand Duchy of Luxembourg.
Gouon
V
V
Melchior
C
C
Plançon
S
S
Kieffer
N
N
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
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