Specification of the Direction of Adhesive Signaling by the Integrin β Cytoplasmic Domain

Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes (“outside-in signaling”); second, cells can regulate the affinity of integrins (“inside-out signaling”) by such pathways. Integrin β cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to β tails, are important for integrin signaling. Here, we utilized “homology scanning” mutagenesis to identify β tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin β3 tail control the binding specificity for SFKs but have no effect on talin binding. Using β tail mutants at these residues, we found that SFK binding to integrin β tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the β3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin β tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo. Integrin adhesion receptors can signal in two directions: first, they can regulate cellular behaviors by modulating cellular signaling enzymes (“outside-in signaling”); second, cells can regulate the affinity of integrins (“inside-out signaling”) by such pathways. Integrin β cytoplasmic domains (tails) mediate both types of signaling, and Src family kinases (SFKs) and talin, which bind to β tails, are important for integrin signaling. Here, we utilized “homology scanning” mutagenesis to identify β tail mutants selectively defective in c-Src binding and found that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin β3 tail control the binding specificity for SFKs but have no effect on talin binding. Using β tail mutants at these residues, we found that SFK binding to integrin β tails is dispensable for inside-out signaling but is obligatory for cell spreading, a marker of outside-in signaling. Conversely, we found that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the β3 tail or the initiation of outside-in signaling once the integrins are in a high affinity form. Thus, we show that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin β tails. Furthermore, based on our results, it is possible to discern the relative contributions of the direction of integrin signaling on biological functions in cell culture and, ultimately, in vivo. Cell-cell and cell-matrix adhesion, mediated by integrin adhesion receptors, is essential to the development and functioning of multicellular organisms (1Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1474) Google Scholar, 2Humphries M.J. Newham P. Trends Cell Biol. 1998; 8: 78-83Crossref PubMed Google Scholar, 3Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 793-805Crossref PubMed Scopus (1859) Google Scholar, 4Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (7005) Google Scholar). In addition to supporting adhesion, these receptors are bona fide signaling receptors that inform the cell about the physical and biochemical nature of its environment, leading to regulation of gene expression and cell proliferation, differentiation, and survival (1Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1474) Google Scholar, 4Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (7005) Google Scholar, 5Hannigan G. Troussard A.A. Dedhar S. Nat. Rev. Cancer. 2005; 5: 51-63Crossref PubMed Scopus (510) Google Scholar, 6Guo W. Giancotti F.G. Nat. Rev. Mol. Cell. Biol. 2004; 5: 816-826Crossref PubMed Scopus (1231) Google Scholar, 7Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3846) Google Scholar). This process is frequently referred to as “outside-in signaling.” The ability of many integrins to bind ligands is regulated by cellular signaling mechanisms (8Mould A.P. Humphries M.J. Curr. Opin. Cell Biol. 2004; 16: 544-551Crossref PubMed Scopus (86) Google Scholar, 9Humphries M.J. McEwan P.A. Barton S.J. Buckley P.A. Bella J. Paul M.A. Trends Biochem. Sci. 2003; 28: 313-320Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (449) Google Scholar, 11Campbell I.D. Ginsberg M.H. Trends Biochem. Sci. 2004; 29: 429-435Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). This process, often called integrin “activation” or “inside-out signaling,” can arise through conformational changes in the integrin extracellular domain and/or changes in the physical distribution of these receptors on the cell surface (12Hato T. Pampori N. Shattil S.J. J. Cell Biol. 1998; 141: 1685-1695Crossref PubMed Scopus (214) Google Scholar). Thus, integrins can transmit information in both directions across the plasma membrane. A central question is to what extent each form of integrin signaling contributes to particular biological functions. Integrins are heterodimers of non-covalently associated α and β subunits. The N terminus of each subunit is extracellular, and a single transmembrane domain separates the ∼700-1200-residue extracellular domain from a usually short (5-65-residue) cytoplasmic domain, or tail (13Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9044) Google Scholar). The cytoplasmic domains of integrins play an essential role in bi-directional signaling processes, and intensive work has sought to identify cellular proteins that interact directly with these domains (14Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar). Recent studies have focused attention on two groups of proteins that bind to integrin β cytoplasmic domains and regulate integrin signaling: Src family kinases (SFKs) 1The abbreviations used are: SFK, Src family kinase; SH, Src homology; GST, glutathione S-transferase; GFP, green fluorescent protein. (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar) and talin (16Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (991) Google Scholar). SFKs are constitutively associated with integrins, and their activation is a proximal and early consequence of integrin clustering (17de Virgilio M. Kiosses W.B. Shattil S.J. J. Cell Biol. 2004; 165: 305-311Crossref PubMed Scopus (60) Google Scholar, 18Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (357) Google Scholar). The presence of SFKs is obligatory for the majority of tyrosine phosphorylation events that follow integrin-mediated adhesion (19Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (650) Google Scholar). Cell spreading, a measure of integrin signaling into cells, depends on the presence of SFKs. We recently found that SFKs can bind directly to integrin β cytoplasmic domains via their SH3 domains (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). This interaction requires the C terminus of the integrin β tail and promotes the activation of the SFKs (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar) and downstream transcriptional factors such as nuclear factor-κB (20Courter D.L. Lomas L. Scatena M. Giachelli C.M. J. Biol. Chem. 2005; 280: 12145-12151Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Thus, the binding of SFKs to integrin β tails is important for outside-in signaling; however, its role in integrin activation remains to be clarified. Talin 1 is required for many integrin functions (21Cram E.J. Clark S.G. Schwarzbauer J.E. J. Cell Sci. 2003; 116: 3871-3878Crossref PubMed Scopus (93) Google Scholar, 22Monkley S.J. Zhou X.H. Kinston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fassler R. Dev. Dyn. 2000; 219: 560-574Crossref PubMed Scopus (179) Google Scholar, 23Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev. Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), in part because it is a pivotal connection between integrins and the actin cytoskeleton. However, recent studies have also implicated talin as a key regulator of inside-out integrin signaling. Overexpression of the N terminus of talin leads to integrin activation (24Calderwood D.A. Zent R. Grant R. Rees D.J.G. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 25Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (652) Google Scholar), and short hairpin RNA knockdown of talin expression in cultured cells (16Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (991) Google Scholar) inhibits the activation of β1 and β3 integrins. Furthermore, in platelets and their megakaryocyte precursors, intracellular signals initiated by agonist binding to G-protein-coupled receptors result in integrin αIIbβ3 activation. Expression of talin short hairpin RNA, but not mismatched short hairpin RNA, blocked agonist-stimulated fibrinogen binding to megakaryocytes. Thus, talin is a downstream target of cellular signaling pathways that activate integrins. In addition, x-ray crystallography and NMR spectroscopy have indicated that a 96-residue F3 domain of talin interacts with multiple sites on the integrin β3 tail (26Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ulmer T.S. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 27Ulmer T.S. Calderwood D.A. Ginsberg M.H. Campbell I.D. Biochemistry. 2003; 42: 8307-8312Crossref PubMed Scopus (73) Google Scholar, 28Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T. Plow E. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). Thus, binding of the talin F3 domain to the β3 cytoplasmic tail is a final common step in integrin activation; however, the role of talin binding to integrin β cytoplasmic tails in outside-in signaling is less clear. Relationships between inside-out and outside-in integrin signaling remain and studies have that the conformational in the extracellular domain to both of signaling M. J.W. C. J.S. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Takagi J. Springer T.A. 2004; PubMed Scopus Google Scholar). In of the of the β3 cytoplasmic domain can inhibit cell spreading, with less of an effect on integrin activation J. Cell Biol. 2003; PubMed Scopus Google Scholar), that the two be at at the cytoplasmic of the Furthermore, talin 1 the of with effect on cell or SFK activation G. G. Critchley D.R. J. Cell Biol. 2003; PubMed Scopus Google Scholar). that the interaction of SFKs and talin with integrin β tails have separable in integrin signaling in the two we that amino acid exchanges affecting a combination of an Arg and Thr residue in the integrin β3 tail control the binding specificity for SFKs but have no effect on talin binding. Using mutants at these and cells to c-Src we that SFK binding to integrin β tails is not required for inside-out integrin signaling but is obligatory for cell Conversely, we show that point mutations that disrupt talin binding abolish integrin activation, but they do not inhibit SFK binding to the β3 tail or the initiation of outside-in signaling by SFKs. Thus, we that inside-out and outside-in integrin signaling are and the integrin β3 subunit and (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). is for the c-Src SH3 to the β1 integrin subunit from Science. PubMed Scopus Google Scholar). is for the integrin subunit K. V. Shattil S.J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar). and have Plow M.A. J. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.H. J. R. Plow Shattil S.J. PubMed Google Scholar, Plow Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). to talin from to and from to from from to from fibrinogen from from from from Cell and cell that αIIbβ3 have A.A. Shattil S.J. Plow Ginsberg M.H. Cell PubMed Scopus Google Scholar). cells in and and cells with (19Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (650) Google Scholar) from the in with and at with with as by the cells in and cultured for an in The expression and have K. V. Shattil S.J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar, L. C. J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Shattil S.J. Plow Ginsberg M.H. Science. PubMed Scopus Google Scholar). mutations and exchanges the mutagenesis and by expression and and from expression for the SH3 domains of SFKs and expression for integrin tails (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). The β tail mutations and by cells in 1 and by at for at and of from the of cell with the indicated to the or β3 integrin and to and with the indicated by integrin cytoplasmic tail proteins both a and an in at the N terminus and from as (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). A the amino acid of the integrin β3 tail in the and used as control for binding. of cell integrin tail proteins on as (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). integrin tails at to of domains of and in E. of SH3 to in and for 1 at with and for an by with and a final with and development with at of with β3 tail in the presence or of 1 by with of the cytoplasmic tail of to the β3 tail binding by binding with β3 or by with an of found to the binding of to β3 (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). of the binding binding to a single binding in which by to the of binding to for in signal at between of c-Src with β cytoplasmic tail proteins to in the presence of a Src tyrosine phosphorylation of by c-Src by of in the presence or of In the the effect of talin or β3 tail with 1 or for to the addition of as the of c-Src in the of of of to the tail of the Integrin cells with or and a marker of binding of the by as J. Shattil S.J. Plow Ginsberg M.H. Science. PubMed Scopus Google Scholar). binding on a of and Integrin surface expression of each with in presence or of or αIIbβ3 J. B. Ginsberg M.H. Mol. Biol. Cell. PubMed Scopus Google Scholar). binding in the presence of used to and with used to binding. of integrin activation, we an activation as is the of is the of binding in the presence of and is the of binding in the presence of Cell with fibrinogen at with and blocked with for 1 at the cells at in and on at for of cells by with cells with and with with and by a with a The surface of cells c-Src and Talin to in the β3 sought to discern the relative biological of c-Src and talin binding to integrin β cytoplasmic c-Src binding to integrin β3 the of β3 (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar), a with the talin binding (26Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ulmer T.S. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 27Ulmer T.S. Calderwood D.A. Ginsberg M.H. Campbell I.D. Biochemistry. 2003; 42: 8307-8312Crossref PubMed Scopus (73) Google Scholar). identify β tail mutants selectively defective in c-Src we utilized a D.A. A. Kiosses W.B. M.A. Ginsberg M.H. Nat. Cell Biol. 2001; Scopus Google Scholar) by between the β3 and cytoplasmic tails We found that c-Src to the β3 cytoplasmic but not to the or tails, and that binding to the β3 tail is of the amino acid (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). the between and β3 we that the β3 tail with the at its ability to bind the of β3 a of binding c-Src We found that the SFKs and to β1 and β3 (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google each of these kinases to of the in In which is (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar), an binding to c-Src not β3 or of c-Src binding to the β3 and mutations or to c-Src binding to the tail not control for a of the β3 cytoplasmic tail to interact with c-Src of the SFKs not these that the binding of c-Src is on the presence of the Arg and Thr of the β3 and c-Src bind to of the β3 binding for and or β3 cytoplasmic tails by from and β3 cytoplasmic tails. c-Src and used to for tail c-Src interacts with β3 via its SH3 domain (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar), we the effect of integrin β tail on the binding of to or and β3 tails The c-Src SH3 with an to the β3 tail Conversely, the of binding to of c-Src with β3 c-Src (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar). the the to activate c-Src in the Thus, the of β3 and β1 control binding to c-Src and the of the integrin tail to regulate c-Src that an of amino acid regulate c-Src binding to and activation by integrins, we the of on the binding of talin, which interacts with integrin β tails through its D.A. B. de Pereda J.M. B. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of with of β3 c-Src it no effect on the binding of the of talin or talin to the β3 Conversely, β3 mutations to abolish talin binding (16Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (991) Google Scholar, B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ulmer T.S. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) no effect on the binding of c-Src SH3 or c-Src to the integrin β3 of the talin (26Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Critchley D.R. Ulmer T.S. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, D.A. B. de Pereda J.M. B. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) to the binding of the c-Src SH3 domain to β3 or the of β3 to activate c-Src Thus, the interaction sites in β3 integrins for talin and c-Src are and talin not with c-Src for binding to the Src to Integrin β1 that of the of β3 and β1 can regulate the binding and activation of cells multiple Src family and we found in studies with (15Arias-Salgado E.G. Lizano S. Sarkar S. Brugge J.S. Ginsberg M.H. Shattil S.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13298-13302Crossref PubMed Scopus (456) Google Scholar) that and kinases are of binding to integrin and β3 tails. We that the SH3 domains of and can bind to and β3 tails to Furthermore, and associated with integrins the β1 and tails in cells that of the between and β3 can regulate the binding of it not the binding of or to be the A and of the the of the we sought a cellular in which c-Src the SFK (19Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (650) Google Scholar) have from and and that integrin signaling and cell are in these cells have also these cells with c-Src that kinases from cells and that c-Src the in the cells not The cells with the subunit in combination with integrin β3 or with the talin the cells with and of β3 the β3 extracellular and transmembrane domains to the β1 cytoplasmic domain K. V. Shattil S.J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar, T. J. Yamada K.M. Shattil S.J. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or of the cytoplasmic domains or c-Src in β3 the or tail In no c-Src associated with integrins the tail or the Thus, these cells with a to the biological role of the The β3 but the for Integrin activation is regulated by the interaction of talin with the integrin β the relative of talin and c-Src binding to the integrin β tail on integrin activation, we cells with the integrin β3 a talin or a c-Src an we a integrin α subunit in which the extracellular and transmembrane domains of to the cytoplasmic domain of J. Shattil S.J. Plow Ginsberg M.H. Science. PubMed Scopus Google Scholar). Integrin activation with the αIIbβ3 S.J. J.A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The c-Src binding to the extent as αIIbβ3 and In and as the talin to binding. integrins as by the binding of an not Thus, talin binding is c-Src binding is dispensable for integrin activation. The β3 but the for Cell adhesion the of We the that β3 integrins, but not the β1 integrins, mediate cell on J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar). cells to on fibrinogen but with the β3 integrin subunit β3 to of the cells not that the both and of cells with the or the to cell that in cells, that c-Src binding to the integrin tail required for Conversely, the of c-Src binding in the that integrin subunit to mediate cell the in the cell for cells The β3 integrins that on in a in cell relative to the that to Thus, SFK binding to the integrin β tail is required for and cell the role of talin binding to the β3 tail in cell spreading, we the of cells with to from the that of the β3 tail inhibits adhesion to fibrinogen J. Ginsberg M.H. J. Cell Biol. PubMed Scopus Google Scholar), cells however, they to as and by We that the of activation of the β3 integrin to of in a of cell addition of the Plow M.A. J. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Plow Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), cell in cells in the presence of cells on that the of these cells not to a in integrin activation. that talin contributes to cell by the but once the integrin is talin binding to the integrin β tail is no and studies that inside-out and outside-in integrin signaling are mediated by distinct and separable interactions of the integrin cytoplasmic Talin binding is required for integrin activation, and the binding of c-Src kinases is required for the signaling events that to cell Thus, it is possible to control the direction of integrin signaling. Furthermore, studies have that integrin activation and regulate the ability of these receptors to intracellular signals V. R. Ginsberg M.H. J. 2001; PubMed Scopus Google Scholar, E. M.A. Shattil S.J. S. M.A. EMBO J. 2001; PubMed Scopus Google Scholar). The studies directly point by that the by of talin binding of can be by in binding This result also that cells can talin binding to the integrin tail is blocked by or by of talin G. G. Critchley D.R. J. Cell Biol. 2003; PubMed Scopus Google Scholar). studies have integrin signaling in the and a of The studies a to the relative contributions of the direction of integrin signaling on these in cell culture and, ultimately, in vivo. with