Paxillin Binding to a Conserved Sequence Motif in the α4 Integrin Cytoplasmic Domain

α4β1integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other β1 integrins. Paxillin, a signaling adapter protein, binds tightly to the α4 cytoplasmic domain and is implicated in α4 integrin signaling. We now report the mapping of a paxillin-binding site in the α4 cytoplasmic domain and an assessment of its role in the α4tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu983-Tyr991) within the α4 cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr991 and Glu983 as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length α4 tail sequence. Y991A or E983A substitution disrupted the interaction of α4 integrins with paxillin. These same two point mutations reversed the effects of the α4 tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all α4 integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of α4 integrins. α4β1integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other β1 integrins. Paxillin, a signaling adapter protein, binds tightly to the α4 cytoplasmic domain and is implicated in α4 integrin signaling. We now report the mapping of a paxillin-binding site in the α4 cytoplasmic domain and an assessment of its role in the α4tail-specific integrin functions. By using truncation mutants and a peptide competition assay, we found that a region of 9 amino acid residues (Glu983-Tyr991) within the α4 cytoplasmic domain contains a minimal sequence sufficient for paxillin binding. Alanine scanning of this region implicated Tyr991 and Glu983 as critical residues. The role of these residues was confirmed by introducing these Ala substitutions into the full-length α4 tail sequence. Y991A or E983A substitution disrupted the interaction of α4 integrins with paxillin. These same two point mutations reversed the effects of the α4 tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all α4 integrins sequenced to date, including that from Xenopus laevis. The maintenance of this sequence motif suggests that paxillin binding is an evolutionarily conserved function of α4 integrins. Chinese hamster ovary cells glutathione S-transferase bovine serum albumin fibrinogen fibronectin 1,4-piperazinediethanesulfonic acid phenylmethylsulfonyl fluoride polyacrylamide gel electrophoresis phosphate-buffered saline focal adhesion kinase Integrin adhesion receptors are heterodimers of α and β subunits that contain a large extracellular domain responsible for ligand binding, a single transmembrane domain, and a cytoplasmic domain that in most cases consists of 20–70 amino acid residues (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar, 2Sastry S.K. Horwitz A.F. Curr. Opin. Cell Biol. 1993; 5: 819-831Crossref PubMed Scopus (407) Google Scholar). Integrins mediate cell adhesion and play roles in cell migration and cytoskeletal organization (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar, 3Schwartz M.A. Schaller M.D. Ginsberg M.H. Ann. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar). The α4β1integrin is expressed on leukocytes and their precursors, neural crest cells, and in developing skeletal muscle (4Hemler M. Lobb R. Curr. Opin. Hematol. 1995; 2: 61-67Crossref PubMed Scopus (45) Google Scholar, 5Shimizu Y. Rose D.M. Ginsberg M.H. Adv. Immunol. 1999; 72: 325-380Crossref PubMed Google Scholar, 6Rosen G.D. Sanes J.R. LaChance R. Cunningham J.M. Roman J. Dean D.C. Cell. 1992; 69: 1107-1119Abstract Full Text PDF PubMed Scopus (316) Google Scholar). It plays important roles in embryogenesis, hematopoiesis, myogenesis, and immune responses (5Shimizu Y. Rose D.M. Ginsberg M.H. Adv. Immunol. 1999; 72: 325-380Crossref PubMed Google Scholar, 7Stewart M. Hogg N. J. Cell. Biochem. 1996; 61: 554-561Crossref PubMed Scopus (223) Google Scholar). The α4 integrin subunit is indispensable for these biological processes, possibly because α4 regulates cell migration, cytoskeletal organization, and gene expression differently from other integrin α subunits (8Hemler M.E. Kassner P.D. Chan B.M.C. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 213-220Crossref PubMed Scopus (28) Google Scholar). α4integrins increase cell migration and oppose cell spreading and focal adhesion formation. These unusual biological properties depend on the α4 cytoplasmic domain (8Hemler M.E. Kassner P.D. Chan B.M.C. Cold Spring Harbor Symp. Quant. Biol. 1992; 57: 213-220Crossref PubMed Scopus (28) Google Scholar, 9Newton R.A. Thiel M. Hogg N. J. Leukocyte Biol. 1997; 61: 422-426Crossref PubMed Scopus (54) Google Scholar). Indeed, this region of α4 markedly stimulates cell migration and opposes cell spreading and focal adhesion formation when joined to other integrin α subunits (10Chan B.M.C. Kassner P.D. Schiro J.A. Byers H.R. Kupper T.S. Hemler M.E. Cell. 1992; 68: 1051-1060Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 11Kassner P.D. Alon R. Springer T.A. Hemler M. Mol. Biol. Cell. 1995; 6: 661-674Crossref PubMed Scopus (96) Google Scholar, 12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar). To investigate the unusual biological properties of α4integrins, we previously analyzed the binding of cellular proteins to the α4 cytoplasmic domain using α4 tail affinity chromatography (12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar). We reported that the α4cytoplasmic tail binds tightly to the signaling adaptor protein, paxillin. In the current study, we have characterized the α4 cytoplasmic domain sequences required for paxillin binding and examined effects of mutations that disrupt paxillin binding on an α4 tail-specific function. Here we report that a region of 9 amino acid residues (Glu983-Tyr991) within the α4 cytoplasmic domain is sufficient for paxillin binding. An alanine substitution at either Glu983or Tyr991 within this region can disrupt the α4 tail-paxillin association. Furthermore, introduction of E983A or Y991A point mutation into the α4 tail can abolish the effects of the α4 tail on cell spreading. Thus, the integrity of the paxillin-binding site is required for some of the unusual biological responses to ligation of the α4integrins. Human Jurkat T cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1680 with 10% fetal bovine serum, 50 units of penicillin/ml, and 50 μg of streptomycin sulfate/ml in a 37 °C tissue culture incubator. All Chinese hamster ovary (CHO)1 cell lines were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% non-essential amino acids (Sigma), penicillin, and streptomycin (12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar, 13Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). CHO cells expressing chimeric αIIbβ3 integrins were created and cultured as described (12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar, 14Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Briefly, the extracellular and transmembrane domains of the αIIb integrin subunit were joined to the cytoplasmic domain of α4, α4(Y991A), α4(E983A), or α4(R985A), respectively. The extracellular and transmembrane domains of the β3integrin subunit were joined to the cytoplasmic domain of the β1A integrin subunit. The αIIb and β3 chimeric subunits were co-transfected into CHO cells. CHO cells expressing the chimeric αIIbβ3integrin were selected with neomycin, and clonal lines were isolated by single cell fluorescence-activated cell sorting using αIIbβ3-specific antibody d-57 (15Huttenlocher A. Ginsberg M.H. Horwitz A.F. J. Cell Biol. 1996; 134: 1551-1562Crossref PubMed Scopus (313) Google Scholar). αIIbα6Aβ3β1A-expressing CHO cells have been described previously (14Hughes P.E. Renshaw M.W. Pfaff M. Forsyth J. Keivens V.M. Schwartz M.A. Ginsberg M.H. Cell. 1997; 88: 521-530Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). The following antibodies were obtained commercially: monoclonal antibodies against paxillin (clone 349, Transduction Laboratory, reactive with both paxillin and Hic-5) and against HA-tag (12CA5, ATCC). Biotin-labeled anti-paxillin antibody was prepared by labeling anti-paxillin antibody (clone 349) with N-hydroxysuccinimide-biotin (Pierce) following the manufacturer's instructions. Monoclonal antibody against human αIIbβ3 (d-57) has been described previously (15Huttenlocher A. Ginsberg M.H. Horwitz A.F. J. Cell Biol. 1996; 134: 1551-1562Crossref PubMed Scopus (313) Google Scholar). Synthetic peptides used in the competition assays were synthesized on an ABI 430 peptide synthesizer and were 95% homogeneous as judged by a reverse-phase C18 HPLC column (Vydac) by the Peptide Synthesis Core at The Scripps Research Institute. Masses of all synthetic peptides were confirmed by electrospray ionization mass spectrometry. The design and production of recombinant cytoplasmic domain model proteins have been described (13Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Briefly, polymerase chain reaction was used to generate anHindIII-BamHI fragment for each wild-type or mutant integrin cytoplasmic domain. Each polymerase chain reaction product was ligated into the pCR vector using a TA cloning kit (Invitrogen). After cDNA sequencing, each fragment was ligated intoHindIII-BamHI sites of the modified pET15b vector described before (13Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Each recombinant protein was expressed in BL21(DE3)pLysS cells (Novagen), isolated by Ni2+-charged resins, and further purified to >90% homogeneity using a reverse-phase C18 HPLC column (Vydac). Masses of all proteins were assessed by electrospray ionization mass spectrometry on an API-III quadrupole spectrometer (Sciex, Toronto, Canada) and varied by less than 0.1% from the predicted mass. Integrin tail affinity chromatography was performed as described (12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar,13Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Briefly, 1 mg of each recombinant integrin cytoplasmic domain dissolved in 5 ml of 20 mm Pipes, 50 mm NaCl, pH 6.8 (PN buffer), plus 1 ml of 100 mm sodium acetate was bound to 100 μl of Ni2+-charged His-Bind resins (Novagen) at 4 °C overnight. Resins were then washed twice with PN buffer and stored in an equal volume of PN buffer plus 0.1% NaN3. Jurkat T cells (or human platelets) were lysed on ice for 30 min with buffer A: 10 mm Pipes, 50 mm NaCl, 150 mm sucrose, 1 mmNa3VO4, 50 mm NaF, 40 mm sodium pyrophosphate, pH 6.8, plus 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm EDTA, 20 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride (PMSF) (plus 0.1 mm E-64, a calpain inhibitor, for platelets). After sonication, the cell lysate was clarified by centrifugation at 12,000 rpm for 20 min. 500–1000 μg of the clarified cell lysate was supplemented with 3 mmMgCl2 and then added to 50 μl of integrin tail-coated resins as described above. The mixture was incubated at 4 °C with rotation overnight. Resins were washed three times with buffer A. Bound proteins were extracted with 50 μl of reducing SDS sample buffer, were separated on 4–20% SDS-polyacrylamide gels (PAGE), and analyzed for total protein by Coomassie Blue staining or for specific proteins by immunoblotting. The expression and isolation of recombinant glutathione S-transferase (GST)-paxillin have been described (16Salgia R. Li J.L. Lo S.H. Brunkhorst B. Kansas G.S. Sobhany E.S. Sun Y. Pisick E. Hallek M. Ernst T. Tantravahi R. Chen L.B. Griffin J.D. J. Biol. Chem. 1995; 270: 5039-5047Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). For detection, an additional sequence, YPYDVPDYA (HA-tag), recognized by monoclonal antibody 12CA5, was joined to the C terminus of the paxillin. Aliquots of recombinant HA-tagged GST-paxillin were mixed with 300 μl of buffer A plus 20 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mm PMSF, 0.1% Triton X-100, 3 mm MgCl2, and 1 mg/ml of bovine serum albumin (BSA), added to 20 μl of model protein-loaded resins, and incubated at room temperature with rotation for 2 h. Resins were then washed three times with the same buffer. Bound proteins were extracted with reducing SDS sample buffer, separated on SDS-PAGE, and detected with antibody specific for HA-tag. For the peptide competition assay, binding assays were performed in the presence of competing peptide at concentrations indicated in each experiment. CHO cell lines were cell surface-labeled with sulfo-N-hydroxysuccinimide-biotin (Pierce) following the manufacturer's instructions. Cells were lysed on ice for 30 min in an immunoprecipitation buffer: Tris-HCl, 20 mm, pH 7.4; NaCl, 150 mm; EDTA, 10 mm; benzamidine HCl, 10 mm; sodium azide, 0.02%; Triton X-100, 1%; Tween 20, 0.05%; PMSF, 2 mm; aprotinin, 5 μg/ml; and leupeptin, 5 μg/ml (17Chen Y.-P. O'Toole T.E. Ylanne J. Rosa J.-P. Ginsberg M.H. Blood. 1994; 84: 1857-1865Crossref PubMed Google Scholar). After clarification by centrifuging at 12,000 rpm for 20 min at 4 °C, cell lysate was then incubated with protein G-Sepharose coated with antibody d-57 or an irrelevant mouse IgG overnight at 4 °C. The beads were washed with the immunoprecipitation buffer four times, and the precipitated polypeptides were extracted with SDS sample buffer. Precipitated cell surface biotin-labeled polypeptides were separated by SDS-PAGE under non-reducing conditions and detected with streptavidin-peroxidase followed by ECL (Amersham Pharmacia Biotech). In parallel, lysates of unmodified cells were precipitated with anti-αIIbβ3, d-57, and co-precipitated paxillin was detected by immunoblotting the reduced immunoprecipitates with biotin-labeled anti-paxillin antibody (clone 349). Assays of cell adhesion and spreading on fibrinogen (Fg) or fibronectin (FN) were performed as described previously (18Ylanne J. Chen Y.-P. O'Toole T.E. Loftus J.C. Takada Y. Ginsberg M.H. J. Cell Biol. 1993; 122: 223-233Crossref PubMed Scopus (194) Google Scholar). Briefly, for cell adhesion assay, 24-well plates were coated with 10 μg/ml Fg in a coating buffer: NaCl, 150 mm; NaH2PO4, 50 mm; and Na2HPO4, 50 mm, pH 8.0, at 4 °C overnight and blocked with 1% heat-denatured BSA at 37 °C for more than 1 h. CHO cells were labeled with fluorescent dye (CellTracker Green CMFDA; Molecular Probes) following the manufacturer's instructions. Equal numbers of the labeled cells were then plated on the Fg-coated wells and incubated in a 37 °C incubator for 30 min. At the end of the experiment, unattached cells were washed away with phosphate-buffered saline (PBS). Fluorescence of attached cells was detected using a Cytofluor II fluorescence reader (Millipore). For cell spreading assays, coverslips in 24-well plates were incubated with 10 μg/ml of either Fg or FN resuspended in coating buffer at 4 °C overnight and blocked with 1% heat-denatured BSA at 37 °C for more than 1 h. Cells were detached, washed twice with Dulbecco's modified Eagle's medium plus 1 mg/ml BSA, and resuspended in the same medium at a concentration of 1–2 × 105 The cells × were to to the coverslips at 37 °C for 1 h. cells were washed away with cells were with for min at room washed twice with and examined by Cells that and the presence of under were as spreading cells. were with a with a assessed the of cells that in each experiment. To the amino acid within the α4cytoplasmic domain that are responsible for paxillin binding, we examined the binding of paxillin to an α4 The α4 tail bound paxillin and a from of Jurkat T cells 1 The is most the protein, M. E. R. J. N. Cell. Biol. 1994; Scopus Google J. M. A. J. Cell 1998; Google Scholar). We examined paxillin binding of a of truncation mutants of α4 1 The 5 amino acid residues of α4 were for paxillin or binding further in α4 and each is a reduced binding of both proteins To from other cellular we these using purified recombinant paxillin and results were obtained Thus, the amino acid residues of within the α4 tail are required for binding of paxillin and To the paxillin-binding site in the α4 we assessed the of synthetic α4 cytoplasmic domain peptides 2 to for the binding of purified paxillin to the α4 A peptide the α4 tail sequence blocked paxillin paxillin binding, full-length αIIb tail peptide The concentration of recombinant α4 tail model protein was and the peptide binding at a concentration of 2 Thus, the α4 tail peptide paxillin binding. We synthesized three that the α4 tail with 2 The for paxillin binding with an of 2 from the results with truncation the was at concentrations to the at this Thus, contains a minimal sequence for paxillin binding. Furthermore, a peptide with an to on paxillin binding at a concentration of Thus, peptide paxillin binding. peptides were synthesized to further the and the sequence by 4 or 2 to that of of reduced of to in a in the blocked paxillin binding with an of Thus, contains a minimal sequence required to paxillin binding. The in and the full-length α4 cytoplasmic domain suggests that additional sequences are required for of the of paxillin binding by α4 cytoplasmic HA-tagged GST-paxillin was added to Ni2+-charged resins with α4 model protein in the of presence of each competing synthetic peptide at Bound were and separated by SDS-PAGE under reducing to a and with Bound paxillin was by scanning of these using the is the concentration of peptide at binding of recombinant paxillin was were and results were in a HA-tagged GST-paxillin was added to Ni2+-charged resins with α4 model protein in the of presence of each competing synthetic peptide at Bound were and separated by SDS-PAGE under reducing to a and with Bound paxillin was by scanning of these using the is the concentration of peptide at binding of recombinant paxillin was were and results were To the role of amino acid residues within that are important for paxillin binding, we synthesized peptides that each a single alanine substitution within this region and examined their to for paxillin binding. Alanine substitution of and Glu983 markedly reduced the of peptide on paxillin binding In alanine substitution of or 3 and Alanine substitution of blocked the of Thus, of competition by synthetic peptides implicated 2 amino acid and as for paxillin binding. To that the of alanine substitution in in paxillin binding, we examined the of these alanine substitutions on the paxillin binding properties of the α4 full-length α4 tail model proteins alanine substitutions at and Tyr991 were for binding to recombinant paxillin. Alanine substitution of either Glu983 or Tyr991 reduced paxillin binding to In substitution at and substitution at less on paxillin binding 4 Furthermore, the truncation that and paxillin to the same site in the α4 Furthermore, the same point mutations that disrupted paxillin binding blocked binding 4 Thus, Tyr991 and Glu983 are important amino acid residues for paxillin and binding to the α4cytoplasmic domain. To the binding of the α4 tail mutants to we used Jurkat T cell lysate as a of the chromatography with model proteins or mutations markedly reduced paxillin and binding relative to wild-type α4 5 Thus, and mutations disrupted paxillin binding to the α4 To the of these mutations in the of an we joined the cytoplasmic domain of α4 to the transmembrane and extracellular domains of integrin αIIb to an chimeric α subunit was co-transfected with a chimeric β in CHO cells. To the of Y991A and E983A mutations on the of paxillin with an we these mutations into The and chimeric integrins were expressed in CHO cells and with antibodies against the extracellular domain of of recombinant integrin were precipitated from each cell 5 less paxillin was co-precipitated with either or than with wild-type chimeric integrin 5 Thus, and mutations disrupted the of an integrin with paxillin. The α4 cytoplasmic domain cell spreading and focal adhesion formation P.D. Alon R. Springer T.A. Hemler M. Mol. Biol. Cell. 1995; 6: 661-674Crossref PubMed Scopus (96) Google Scholar, 12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar). To the of these we adhesion and spreading of cells expressing these Cell expressing the Y991A and E983A mutant on a ligand for the αIIbβ3 extracellular domain In cell spreading was markedly in cells expressing the wild-type or a an α4 mutation that the to These were to a in cell adhesion to Fg and at 30 Furthermore, were to in cell spreading, because all cell lines on a ligand for hamster integrin Thus, mutations that disrupt the paxillin binding function of the α4 cytoplasmic domain in increased cell spreading. The present have a sequence motif required for paxillin binding to the α4 integrin cytoplasmic domain. We a paxillin-binding sequence by of truncation and of paxillin binding to the α4 tail by synthetic and residues were identified within this sequence A motif of conserved in the α4 in all this motif is present in is that the paxillin binding function of α4 is evolutionarily Furthermore, the of this motif from α that to paxillin an for the of paxillin binding to required to the specific role of each of the residues important in paxillin binding. The paxillin-binding site identified in the α4 tail is from that in other paxillin-binding For the paxillin-binding site of is of a sequence a region is in the paxillin-binding site of the α4 cytoplasmic domain. Furthermore, the identified paxillin-binding sites of focal adhesion kinase and are from that of the α4 tail In an of a recombinant protein from the focal adhesion sequence of contains its paxillin-binding site J.D. Schaller M.D. J. Cell Biol. 1993; PubMed Scopus Google Scholar, T. N. J. 1995; PubMed Scopus Google paxillin binding to the Liu and M. that α4 can to paxillin that is with or required to this The interaction of paxillin with α4 is of affinity and is required for of the biological of α4 integrins (12Liu S. Thomas S.M. Woodside D.G. Rose D.M. Kiosses W.B. Pfaff M. Ginsberg M.H. Nature. 1999; 402: 676-681Crossref PubMed Scopus (291) Google Scholar). paxillin-binding is present in synthetic peptides from the α4 tail and can by single point These features of the interaction that peptides or that disrupt the interaction used to cellular functions.

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