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PLOS ONE  2012 

Integrin β3 Crosstalk with VEGFR Accommodating Tyrosine Phosphorylation as a Regulatory Switch

DOI: 10.1371/journal.pone.0031071

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Integrins mediate cell adhesion, migration, and survival by connecting intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the importance of the interaction between β3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. Here we present in vitro evidence of the direct association between the cytoplasmic tails (CTs) of β3 and VEGFR2. Specifically, the membrane-proximal motif around 801YLSI in VEGFR2 mediates its binding to non-phosphorylated β3CT, accommodating an α-helical turn in integrin bound conformation. We also show that Y747 phosphorylation of β3 enhances the above interaction. To demonstrate the importance of β3 phosphorylation in endothelial cell functions, we synthesized β3CT-mimicking Y747 phosphorylated and unphosphorylated membrane permeable peptides. We show that a peptide containing phospho-Y747 but not F747 significantly inhibits VEGF-induced signaling and angiogenesis. Moreover, phospho-Y747 peptide exhibits inhibitory effect only in WT but not in β3 integrin knock-out or β3 integrin knock-in cells expressing β3 with two tyrosines substituted for phenylalanines, demonstrating its specificity. Importantly, these peptides have no effect on fibroblast growth factor receptor signaling. Collectively these data provide novel mechanistic insights into phosphorylation dependent cross-talk between integrin and VEGFR2.


[1]  Hynes RO (1987) Integrins: a family of cell surface receptors. Cell 48: 549–554.
[2]  Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687.
[3]  Ma YQ, Qin J, Plow EF (2007) Platelet integrin alpha(IIb)beta(3): activation mechanisms. J Thromb Haemost 5: 1345–1352.
[4]  Calderwood DA, Zent R, Grant R, Rees DJ, Hynes RO, et al. (1999) The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation. J Biol Chem 274: 28071–28074.
[5]  Vinogradova O, Velyvis A, Velyviene A, Hu B, Haas T, et al. (2002) A structural mechanism of integrin alpha(IIb)beta(3) “inside-out” activation as regulated by its cytoplasmic face. Cell 110: 587–597.
[6]  Anthis NJ, Haling JR, Oxley CL, Memo M, Wegener KL, et al. (2009) {beta} integrin tyrosine phosphorylation is a conserved mechanism for regulating talin-induced integrin activation. J Biol Chem 284: 36700–36710.
[7]  Cowan KJ, Law DA, Phillips DR (2000) Identification of shc as the primary protein binding to the tyrosine-phosphorylated beta 3 subunit of alpha IIbbeta 3 during outside-in integrin platelet signaling. J Biol Chem 275: 36423–36429.
[8]  Phillips DR, Nannizzi-Alaimo L, Prasad KS (2001) Beta3 tyrosine phosphorylation in alphaIIbbeta3 (platelet membrane GP IIb-IIIa) outside-in integrin signaling. Thromb Haemost 86: 246–258.
[9]  Deshmukh L, Gorbatyuk V, Vinogradova O (2010) Integrin beta3 phosphorylation dictates its complex with Shc PTB domain. J Biol Chem 285: 34875–34884.
[10]  Blystone SD, Williams MP, Slater SE, Brown EJ (1997) Requirement of integrin beta3 tyrosine 747 for beta3 tyrosine phosphorylation and regulation of alphavbeta3 avidity. J Biol Chem 272: 28757–28761.
[11]  Boettiger D, Huber F, Lynch L, Blystone S (2001) Activation of alpha(v)beta3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta3. Mol Biol Cell 12: 1227–1237.
[12]  Chandhoke SK, Williams M, Schaefer E, Zorn L, Blystone SD (2004) Beta 3 integrin phosphorylation is essential for Arp3 organization into leukocyte alpha V beta 3-vitronectin adhesion contacts. J Cell Sci 117: 1431–1441.
[13]  Butler B, Williams MP, Blystone SD (2003) Ligand-dependent activation of integrin alpha vbeta 3. J Biol Chem 278: 5264–5270.
[14]  Datta A, Huber F, Boettiger D (2002) Phosphorylation of beta3 integrin controls ligand binding strength. J Biol Chem 277: 3943–3949.
[15]  Byzova TV, Goldman CK, Pampori N, Thomas KA, Bett A, et al. (2000) A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell 6: 851–860.
[16]  Mahabeleshwar GH, Feng W, Reddy K, Plow EF, Byzova TV (2007) Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis. Circ Res 101: 570–580.
[17]  Soldi R, Mitola S, Strasly M, Defilippi P, Tarone G, et al. (1999) Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J 18: 882–892.
[18]  Mahabeleshwar GH, Feng W, Phillips DR, Byzova TV (2006) Integrin signaling is critical for pathological angiogenesis. J Exp Med 203: 2495–2507.
[19]  Napione L, Cascone I, Mitola S, Serini G, Bussolino F (2007) Integrins: a flexible platform for endothelial vascular tyrosine kinase receptors. Autoimmun Rev 7: 18–22.
[20]  Serini G, Valdembri D, Bussolino F (2006) Integrins and angiogenesis: a sticky business. Exp Cell Res 312: 651–658.
[21]  Byzova TV, Kim W, Midura RJ, Plow EF (2000) Activation of integrin alpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein. Exp Cell Res 254: 299–308.
[22]  Mahabeleshwar GH, Chen J, Feng W, Somanath PR, Razorenova OV, et al. (2008) Integrin affinity modulation in angiogenesis. Cell Cycle 7: 335–347.
[23]  Vaynberg J, Qin J (2006) Weak protein-protein interactions as probed by NMR spectroscopy. Trends Biotechnol 24: 22–27.
[24]  Leppanen VM, Prota AE, Jeltsch M, Anisimov A, Kalkkinen N, et al. (2010) Structural determinants of growth factor binding and specificity by VEGF receptor 2. Proc Natl Acad Sci U S A 107: 2425–2430.
[25]  Harris PA, Cheung M, Hunter RN 3rd, Brown ML, Veal JM, et al. (2005) Discovery and evaluation of 2-anilino-5-aryloxazoles as a novel class of VEGFR2 kinase inhibitors. J Med Chem 48: 1610–1619.
[26]  Deshmukh L, Gorbatyuk V, Vinogradova O (2010) Integrin {beta}3 phosphorylation dictates its complex with the Shc phosphotyrosine-binding (PTB) domain. The Journal of biological chemistry 285: 34875–34884.
[27]  Deshmukh L, Meller N, Alder N, Byzova T, Vinogradova O (2011) Tyrosine Phosphorylation as a Conformational Switch: A CASE STUDY OF INTEGRIN beta3 CYTOPLASMIC TAIL. The Journal of biological chemistry 286: 40943–40953.
[28]  Davis GE, Stratman AN, Sacharidou A, Koh W (2011) Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting. International review of cell and molecular biology 288: 101–165.
[29]  Law DA, DeGuzman FR, Heiser P, Ministri-Madrid K, Killeen N, et al. (1999) Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature 401: 808–811.
[30]  Reynolds LE, Wyder L, Lively JC, Taverna D, Robinson SD, et al. (2002) Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins. Nature medicine 8: 27–34.
[31]  Holmqvist K, Cross MJ, Rolny C, Hagerkvist R, Rahimi N, et al. (2004) The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J Biol Chem 279: 22267–22275.
[32]  Takahashi T, Yamaguchi S, Chida K, Shibuya M (2001) A single autophosphorylation site on KDR/Flk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J 20: 2768–2778.
[33]  Schaffner-Reckinger E, Gouon V, Melchior C, Plancon S, Kieffer N (1998) Distinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling. J Biol Chem 273: 12623–12632.
[34]  Czuchra A, Meyer H, Legate KR, Brakebusch C, Fassler R (2006) Genetic analysis of beta1 integrin “activation motifs” in mice. J Cell Biol 174: 889–899.
[35]  Vallar L, Melchior C, Plancon S, Drobecq H, Lippens G, et al. (1999) Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein. J Biol Chem 274: 17257–17266.
[36]  Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, et al. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6: 277–293.
[37]  Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, et al. (2005) The CCPN data model for NMR Spectroscopy. Proteins 59: 687–696.
[38]  Wuthrich K (1986) NMR of proteins and nucleic acids. New York: John Wiley & Sons.
[39]  Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160: 65–73.
[40]  Ward C, Kuehn D, Burden RE, Gormley JA, Jaquin TJ, et al. (2010) Antibody targeting of cathepsin S inhibits angiogenesis and synergistically enhances anti-VEGF. PLoS One 5:
[41]  Passaniti A (1992) Extracellular matrix-cell interactions: Matrigel and complex cellular pattern formation. Lab Invest 67: 804; author reply 804–808.


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