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 β3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during αvβ3-mediated cell spreading. Using CHO cells transfected with various β3mutants, we demonstrate a close correlation between αvβ3-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 NITY759motif alone was sufficient to completely prevent αvβ3-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 αvβ3-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 β3post-ligand binding events. 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 αvβ3-mediated cell spreading. Using CHO cells transfected with various β3mutants, we demonstrate a close correlation between αvβ3-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 NITY759motif alone was sufficient to completely prevent αvβ3-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 αvβ3-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 β3post-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 (1Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar). 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 (2Burridge K. Fath K. Kelly T. Nuckolls G. Turner C. Annu. Rev. Cell Biol. 1988; 4: 487-525Crossref PubMed Scopus (1684) Google Scholar). 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 (3Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2802) Google Scholar). 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 β subunits are likely to play a major role in these processes: (i) truncation of the β subunit cytoplasmic domain impairs integrin recruitment to focal contacts (4Solowska J. Guan J.L. Marcantonio E.E. Trevithick J.E. Buck C.A. Hynes R.O. J. Cell Biol. 1989; 109: 853-861Crossref PubMed Scopus (129) Google Scholar, 5Hayashi Y. Haimovich B. Reszka A. Boettiger D. Horwitz A. J. Cell Biol. 1990; 110: 175-184Crossref PubMed Scopus (188) Google Scholar, 6Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (240) Google Scholar), and (ii) information contained in β subunit cytoplasmic tails coupled to the transmembrane and extracellular domains of the interleukin-2 receptor is sufficient to target these chimeric receptors to focal contacts (7LaFlamme S.E. Akiyama S.K. Yamada K.M. J. Cell Biol. 1992; 117: 437-447Crossref PubMed Scopus (241) Google Scholar) and to activate the focal adhesion kinase (FAK) 1The abbreviations used are: FAK, focal adhesion kinase; BSA, bovine serum albumin; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; ICAP-1, integrin cytoplasmic domain-associated protein-1; IMDM, Iscove's modified Dulbecco's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TBS, Tris-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.1The abbreviations used are: FAK, focal adhesion kinase; BSA, bovine serum albumin; CHO, Chinese hamster ovary; FITC, fluorescein isothiocyanate; ICAP-1, integrin cytoplasmic domain-associated protein-1; IMDM, Iscove's modified Dulbecco's medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; TBS, Tris-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.signaling pathway (8Akiyama S.K. Yamada S.S. Yamada K.M. LaFlamme S.E. J. Biol. Chem. 1994; 269: 15961-15964Abstract Full Text PDF PubMed Google Scholar). Based on mutational analysis of the cytoplasmic domain of the β1 integrin, three motifs have been identified that are important for the recruitment of integrins to adhesion plaques; these motifs correspond to the highly conserved acidic membrane-proximal domain and to two C-terminal NPXY motifs (6Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (240) Google Scholar, 9Marcantonio E.E. Guan J.L. Trevithick J.E. Hynes R.O. Cell Regul. 1990; 1: 597-604Crossref PubMed Scopus (116) Google Scholar), which constitute typical recognition sites for tyrosine kinases and adaptor proteins (10Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1154) Google Scholar). Subsequent complementary studies (based on a combination of deletion analysis, single amino acid substitution, and the use of cytoplasmic domain synthetic peptides) have provided evidence that these highly conserved cytoplasmic motifs in the various integrin β subunits have similar functional properties (11Cone R.I. Weinacker A. Chen A. Sheppard D. Cell Adhes. Commun. 1994; 2: 101-113Crossref PubMed Scopus Google Scholar, C. J. Cell Biol. 1995; PubMed Scopus Google Scholar, J. J. Ginsberg M.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. PubMed Scopus Google Scholar, C. J. Cell 110: Google Scholar) and binding sites for the structural cytoskeletal proteins and the adaptor paxillin, as well as proteins including FAK, Cell Biol. PubMed Scopus Google Scholar), T. M. M. Ginsberg M.H. J. Cell Biol. 1995; PubMed Scopus Google Scholar), J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and integrin cytoplasmic domain-associated C. J. J. Cell Biol. PubMed Scopus Google Scholar). The of tyrosine phosphorylation of focal adhesion proteins during focal contact formation is well as tyrosine kinase prevent the of focal adhesion plaques and K. Turner J. Cell Biol. 1992; PubMed Scopus Google Scholar), and of cells with which tyrosine phosphorylation of FAK and paxillin C. K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). the precise by which integrin β subunits tyrosine phosphorylation of focal adhesion proteins during cell and spreading are well to amino of the β3 cytoplasmic domain involved in the signaling induced by LaFlamme S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have various β3 cytoplasmic domains as tails to extracellular Using the role of events, such as cell and cell spreading, in the FAK signaling LaFlamme S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the we have used to the structural requirements of the β3 subunit cytoplasmic domain necessary to intracellular tyrosine phosphorylation during cell spreading. various cytoplasmic domain which αvβ3-dependent CHO cell spreading, we demonstrate a close correlation between a conserved β3 integrin cytoplasmic cell spreading and FAK/paxillin phosphorylation, as C-terminal truncation to cell spreading, to tyrosine phosphorylation. highlight major in the involvement of the cytoplasmic domain tyrosine in β3-mediated binding events. The presence of residue Tyr759 in the is necessary for β3-mediated focal contact formation, cell spreading, and tyrosine phosphorylation of FAK paxillin, whereas residue the membrane-proximal NPLY747 motif is required for optimal receptor function. the NPLY747 and NITY759 motifs in the β3 cytoplasmic domain necessary for binding signaling events. The Chinese hamster cell in the was the The cells in Iscove's modified Dulbecco's with and serum and The cells with The J. T. J. 1989; PubMed Scopus Google Scholar), and and the The was a of D. and provided by M. and The by J. and the by M. for and of and bovine serum The wild type β3 was the of the as C. Cell Adhes. Commun. 4: PubMed Google Scholar). The and mutations in the β3 by the in wild type β3 was the and the and used for the of the the of a in to the was to the The β3 was the with and of the cell The the and subunits by of the of the at the at of the β3 for and at the at for the The was by of a by polymerase chain The used to the The for the and was a to the β3 The for the and was a to the β3 The for the was a to the β3 a contained a by The and and was used as a for while the was the the the was with and and the which the wild type been the the with and the β3 which the wild been β3 was by the to the β3 as a β3 in and of with of in a of of and to CHO cells to in serum was to the and the cells in used as for cell surface of the integrin β3 subunit the and fluorescein transfected cells by and for cell surface of the transfected β3 integrin of the transfected was by the and with and in phosphate-buffered The cells for on with the with PBS, and for on with a and in and on was cells to the of and Scopus Google Scholar). of was with the as a The to the cytoplasmic domain of the β3 integrin subunit was The by gel and the of in and in of was by to the at a of The for at in PBS, and with in for at The and in the CHO cells with and in a of at in the presence of of the the cells to of at with at in IMDM, and to for at The to the at a a at with the with in of the cells a with as with J. 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PubMed Scopus Google Scholar). cells with and in The cells to of with at in at and cell was to at the cells in the at of the of of cells cells was on the to cell cells and the as of three in of cells was at with of in The cells with with IMDM, and in of the The cells for at with in PBS, with PBS, and with in PBS, for at was by the for with a to β3 to the in three the for with in the presence of to tetramethylrhodamine in the of the The three in and in The with a a at with of in The with in for at and with cells with with IMDM, in IMDM, and in to the in the presence of a at cells at for and with the cells in with the by at for at and the with the cells with in and for in of by at for at and the was to the of PubMed Scopus Google Scholar). cell of of for at with for tyrosine phosphorylation with by a at with of a in The three with and the by the in of in the presence of cell of by gel and a The The for in and for tyrosine phosphorylation and with the in to in the for with to in at The in and to the to the for tyrosine phosphorylation by a at in of and with a to FAK the of binding was by and the results as the of The for cell to the for CHO cells on as to the β3 integrin cytoplasmic domain tyrosine phosphorylation during cell spreading, a of subunit that to β3 cell spreading of wild type β3 CHO cell by for surface of the chimeric to β3 and the As in the cell for the similar of cell surface of the chimeric for which only analysis of the β3 subunit in cell the the surface of deletion a in to that for wild type β3 in CHO The of deletion and as with wild type β3 of with the β3 subunit was for deletion by the As in two to and β3 with similar for deletion including to that cell the β3 integrin subunit with the cytoplasmic was the transfected cell and The the cytoplasmic domain was β3 and the these demonstrate that the cell on cell surface the β3 subunit with the and that deletion of the cytoplasmic domain of the integrin β3 subunit with surface of the heterodimeric integrin analysis of chimeric in CHO cells transfected with CHO transfected with the β3 subunits in to in IMDM, with and in The cells with of a to β3 to the and with The the number of cells and the the in analysis of β3 integrin substitution in CHO CHO cells to in IMDM, with and in Cell and as of CHO cells by to and with a to β3 of was in as a a with CHO cell and analysis of β3 integrin deletion in CHO CHO cell as in the of the of CHO cells and β3 of by The to the subunit was by with the of CHO cells and β3 of used for with a to β3 The by to and with a to J. T. J. 1989; PubMed Scopus Google Scholar), with hamster and and with the with The strong with the of in to of was in as a to the β3 the to with a cell binding was CHO cells completely with and a type the cells with binding of the to the cells The binding of was with the the known to in its domain B. M. Y. J. J. 1994; PubMed Scopus Google Scholar) the cell the to a similar as CHO that the β3 cytoplasmic domain mutations the functional role of the tyrosine in the membrane-proximal NPLY747 and NITY759 in αvβ3-mediated cell spreading on of CHO cells the β3 in was a adhesion The analysis of cell spreading is in and of CHO cells wild type β3 was a at in to CHO cells that the αvβ3-dependent on that CHO cell spreading on completely on the transfected of the three deletion and on that a deletion of C-terminal amino the NITY759 motif was sufficient to completely prevent β3 cell spreading. the single tyrosine 747 a of cell spreading on was with and a strong was with the the cell spreading as the the more conservative, but still of tyrosine by and of cell spreading was for whereas only of the cells the mutation Y747F as with CHO to cell spreading was to cell spreading a was and cell spreading As in spreading of CHO and CHO cells a at only of the CHO cells cell spreading of at of the cells the β3 receptor as by cell the that the in cell spreading on cell that during the to three cell and the spreading in cell spreading was with of these evidence that the structural integrity of the β3 subunit cytoplasmic is a for β3-mediated cell spreading and that the presence of residue but in the motifs is required for the spreading We the of the β3 to to focal contacts and to was a of transfected CHO cells on The cells and with a to the β3 integrin subunit with and to As in none of the to to focal adhesion plaques. The use of the that deletion including heterodimeric with the of β3 integrins and in CHO cell The wild type β3 subunit was in focal contacts at the of well of the transfected cells the the of but and the of β3 focal adhesions as by the of the cells with the and was with the deletion and The cell formation and β3 focal contact recruitment in cells that to on whereas cells a wild type with was in the cells that β3 integrin was in focal adhesion plaques, but the number of focal adhesion plaques was and the at the cell these results the for cell spreading of β3 integrins and in transfected CHO cells on CHO cells on as in the of and the cells with a to β3 and with and of the cells cytoskeleton and β3 integrin to cell spreading with tyrosine kinase activation and intracellular we investigated the of the cytoplasmic domain mutations on β3 events, tyrosine phosphorylation of the intracellular proteins FAK and As tyrosine phosphorylation of FAK and paxillin is only but by growth the transfected cells in to of a at on cells in and the used for FAK paxillin of the with a and with a a in of tyrosine phosphorylation of FAK was transfected CHO cells wild type β3 to on whereas only tyrosine phosphorylation was the cells in for CHO cells on that the in FAK tyrosine phosphorylation to the cell a strong correlation between β3-mediated cell spreading and FAK phosphorylation was the cell that to on to FAK phosphorylation CHO CHO as well as CHO and CHO The to tyrosine phosphorylation of FAK, with the spreading of CHO the more conservative, but still substitution of Tyr759 restored FAK tyrosine phosphorylation, while the Y747F substitution phenotype, that the presence of and phosphorylation of is required to FAK tyrosine phosphorylation. These that β3 integrins with a structural of the cytoplasmic to substitution of to FAK tyrosine of the to FAK tyrosine phosphorylation. The of FAK tyrosine phosphorylation was as The of three are to the of paxillin phosphorylation during β3 cell spreading, CHO cells on in the presence of known to prevent cell spreading by and As in in the of CHO cell spreading was of on and a to the paxillin was identified with the that paxillin was to a but and abolished β3-mediated cell spreading and tyrosine phosphorylation of The of paxillin cells in the presence of was the the β3 the results with for FAK the C-terminal deletion and abolished paxillin phosphorylation the substitution only the was to paxillin phosphorylation to wild type the and the results as the to paxillin was of on the The of paxillin transfected cell was the as and of the with These results that the presence of the conserved amino acid Tyr759 of the NITY759 within the β3 cytoplasmic domain is required for of β3 mutations on paxillin tyrosine phosphorylation. CHO cell as and paxillin was and paxillin tyrosine phosphorylation was by tyrosine-phosphorylated the and with a to Integrin cytoplasmic domains are in function. cell the and β subunit cytoplasmic domains integrin for extracellular and play a role in signaling J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Ginsberg M.H. Science. PubMed Scopus Google Scholar, Y. K. J. Ginsberg M.H. J. Cell Biol. 1994; PubMed Scopus Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). cell spreading, in to on integrin β as the β subunit cytoplasmic by sufficient information to target integrins to focal adhesions (7LaFlamme S.E. Akiyama S.K. Yamada K.M. J. Cell Biol. 1992; 117: 437-447Crossref PubMed Scopus (241) Google Scholar) and to tyrosine phosphorylation of intracellular proteins (8Akiyama S.K. Yamada S.S. Yamada K.M. LaFlamme S.E. J. Biol. Chem. 1994; 269: 15961-15964Abstract Full Text PDF PubMed Google Scholar). the of three regions within the cytoplasmic of the β1 integrin subunit necessary for focal contact recruitment of integrins (6Reszka A.A. Hayashi Y. Horwitz A.F. J. Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (240) Google Scholar), numerous studies on β1 and β3 subunits have on the functional role of two of these highly conserved NPXY and which constitute typical recognition sites for tyrosine kinases and are by a single known to S.K. Horwitz A.F. Cell Biol. PubMed Scopus Google Scholar). of these domains to for integrin receptor studies of various β3 integrin subunits as with as a a major have β3 with a deletion of the NITY759 to amino acid completely cell spreading on J. J. Ginsberg M.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), while deletion of the C-terminal domain to amino acid αvβ3-dependent cell spreading on C. J. 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Cell Biol. 1990; PubMed Scopus Google Scholar). results demonstrate a close correlation between αvβ3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and in these binding events. the NITY759 motif, deletion of motif was sufficient to completely prevent αvβ3-dependent focal contact formation, cell spreading, as well as FAK/paxillin tyrosine phosphorylation. A Y759A substitution in a strong inhibitory the more conservative, but still Y759F mutation was to wild type receptor function. These suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of is important for β3-mediated cytoskeleton reorganization tyrosine phosphorylation of FAK and the membrane-proximal our studies demonstrate that substitution of the highly conserved residue at 747 completely abolished αvβ3-dependent formation of focal adhesion plaques and cell spreading, and FAK and paxillin tyrosine phosphorylation, while a Y747F substitution, with the Y759F substitution, only partially restored these receptor that phosphorylation of residue Tyr747 required for optimal β3-mediated signaling events. These the used by to cell adhesion J. A. PubMed Scopus Google Scholar). with results on the of of Tyr747 on cell adhesion and spreading C. J. Cell Biol. 1995; PubMed Scopus Google Scholar, J. J. Ginsberg M.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), the that the of the β3 cytoplasmic domain by the NPLY747 and NITY759 motifs is for β3-mediated cytoskeletal and FAK/paxillin phosphorylation. These are in with the results of LaFlamme S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and with the with the A. A. M. G. J. Cell Biol. 1994; PubMed Scopus Google Scholar) and (8Akiyama S.K. Yamada S.S. Yamada K.M. LaFlamme S.E. J. Biol. Chem. 1994; 269: 15961-15964Abstract Full Text PDF PubMed Google Scholar), to in FAK tyrosine phosphorylation. our to a that the β3 is necessary for tyrosine phosphorylation of FAK M.A. Ginsberg M.H. J. Cell 1995; Google Scholar). cell spreading, our results are in with on cell spreading J. J. Ginsberg M.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and demonstrate that the structural requirements of the β3 cytoplasmic domain necessary for αvβ3-mediated cell spreading are the is by the that the which is to the NITY759 motif and for a phenotype, in and while a mutation wild type cell spreading for J. J. Ginsberg M.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) as well as C. Cell Adhes. Commun. 4: PubMed Google Scholar). β subunit cytoplasmic binding proteins have been which with the C-terminal of β1 and β3 integrin cytoplasmic tails T. M. M. Ginsberg M.H. J. Cell Biol. 1995; PubMed Scopus Google Scholar, C. J. J. Cell Biol. PubMed Scopus Google Scholar, B. B. Full Text Full Text PDF PubMed Scopus Google Scholar). is that and ICAP-1, which binding to the β3 and the β1 cytoplasmic on a motif for integrin as a Tyr substitution been to completely prevent these while a Tyr substitution only inhibitory C. J. J. Cell Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). the provided evidence that the presence of residue Tyr747 of the membrane-proximal NPLY747 motif is required for β3 tyrosine phosphorylation and for αvβ3-mediated adhesion in cells S.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are in with these and the distinct involvement of the NPLY747 and NITY759 in FAK/paxillin phosphorylation, as demonstrate that Tyr759 is required for the results of evidence that of the of the β3 cytoplasmic to deletion of the C-terminal amino to a structural within the membrane-proximal NPLY747 and to a within the NITY759 impairs β3-mediated cell spreading and formation as well as paxillin FAK tyrosine phosphorylation. of residue the NITY759 is necessary for the investigated β3 integrin receptor while phosphorylation of Tyr747 required to these The CHO cell various β3 a to of the β3 subunit cytoplasmic with signaling proteins, and to the involvement of distinct β3 cytoplasmic in various signaling pathways. We J. M. M. and D. for of We of for with