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J. Biol. Chem., Vol. 275, Issue 30, 22736-22742, July 28, 2000

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Paxillin Binding to a Conserved Sequence Motif in the ₄ Integrin Cytoplasmic Domain^*

Shouchun Liu and Mark H. Ginsberg^§

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

Received for publication, January 19, 2000, and in revised form, April 14, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

₄₁ integrin-mediated cell adhesion results in increased cell migration, reduced cell spreading, and focal adhesion formation relative to other ₁ integrins. Paxillin, a signaling adapter protein, binds tightly to the ₄ cytoplasmic domain and is implicated in ₄ integrin signaling. We now report the mapping of a paxillin-binding site in the ₄ cytoplasmic domain and an assessment of its role in the ₄ tail-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 ₄ 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 ₄ tail sequence. Y991A or E983A substitution disrupted the interaction of ₄ integrins with paxillin. These same two point mutations reversed the effects of the ₄ tail on cell spreading. The key features of the identified paxillin-binding sequence are present in all ₄ 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 ₄ integrins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Integrin adhesion receptors are heterodimers of  and  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 (1, 2). Integrins mediate cell adhesion and play roles in cell migration and cytoskeletal organization (1, 3). The ₄₁ integrin is expressed on leukocytes and their precursors, neural crest cells, and in developing skeletal muscle (4-6). It plays important roles in embryogenesis, hematopoiesis, myogenesis, and immune responses (5, 7). The ₄ integrin subunit is indispensable for these biological processes, possibly because ₄ regulates cell migration, cytoskeletal organization, and gene expression differently from other integrin  subunits (8). ₄ integrins increase cell migration and oppose cell spreading and focal adhesion formation. These unusual biological properties depend on the ₄ cytoplasmic domain (8, 9). Indeed, this region of ₄ markedly stimulates cell migration and opposes cell spreading and focal adhesion formation when joined to other integrin  subunits (10-12).

To investigate the unusual biological properties of ₄ integrins, we previously analyzed the binding of cellular proteins to the ₄ cytoplasmic domain using ₄ tail affinity chromatography (12). We reported that the ₄ cytoplasmic tail binds tightly to the signaling adaptor protein, paxillin. In the current study, we have characterized the ₄ cytoplasmic domain sequences required for paxillin binding and examined effects of mutations that disrupt paxillin binding on an ₄ tail-specific function. Here we report that a region of 9 amino acid residues (Glu⁹⁸³-Tyr⁹⁹¹) within the ₄ cytoplasmic domain is sufficient for paxillin binding. An alanine substitution at either Glu⁹⁸³ or Tyr⁹⁹¹ within this region can disrupt the ₄ tail-paxillin association. Furthermore, introduction of E983A or Y991A point mutation into the ₄ tail can abolish the effects of the ₄ 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 ₄ integrins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cell Lines, Cell Culture, and Reagents-- 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 (12, 13). CHO cells expressing chimeric _IIb3 integrins were created and cultured as described (12, 14). Briefly, the extracellular and transmembrane domains of the _IIb integrin subunit were joined to the cytoplasmic domain of ₄, ₄(Y991A), ₄(E983A), or ₄(R985A), respectively. The extracellular and transmembrane domains of the ₃ integrin subunit were joined to the cytoplasmic domain of the ₁A integrin subunit. The _IIb and ₃ chimeric subunits were co-transfected into CHO cells. CHO cells expressing the chimeric _IIb3 integrin were selected with neomycin, and clonal lines were isolated by single cell fluorescence-activated cell sorting using _IIb3-specific antibody D-57 (15). _IIb6A₃₁A-expressing CHO cells have been described previously (14). 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 _IIb3 (D-57) has been described previously (15). 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.

Integrin Cytoplasmic Domain Model Proteins and Affinity Chromatography-- The design and production of recombinant cytoplasmic domain model proteins have been described (13). Briefly, polymerase chain reaction was used to generate an HindIII-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 into HindIII-BamHI sites of the modified pET15b vector described before (13). 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 (12, 13). 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 mM Na₃VO₄, 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 mM MgCl₂ 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.

Binding of Recombinant Paxillin to Model Proteins and the ₄ Peptide Competition Assay-- The expression and isolation of recombinant glutathione S-transferase (GST)-paxillin have been described (16). 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.

Immunoprecipitation and Western Blot Analysis-- 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 (17). 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-_IIb3, D-57, and co-precipitated paxillin was detected by immunoblotting the reduced immunoprecipitates with biotin-labeled anti-paxillin antibody (clone 349).

Cell Adhesion and Spreading Assays-- Assays of cell adhesion and spreading on fibrinogen (Fg) or fibronectin (FN) were performed as described previously (18). 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 cells/ml. The cells (5-10 × 10⁴ cells/well) were permitted to attach to the coverslips at 37 °C for 1 h. Unattached cells were washed away with PBS. Attached cells were fixed with 3.7% paraformaldehyde for 15 min at room temperature, washed twice with PBS, and examined by phase microscopy. Cells that exhibited flattening, and the presence of lamellipodia under microscope examination were scored as spreading cells. Photographic images were acquired with a Nikon Diaphot microscope equipped with a Sensys-cooled charge-coupled device video camera. Two independent observers assessed the percentage of cells that exhibited spread morphology in each experiment.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Identification of a Minimal Paxillin Binding Sequence-- To identify the amino acid residue(s) within the ₄ cytoplasmic domain that are responsible for paxillin binding, we first examined the binding of native paxillin to an immobilized ₄ tail. The ₄ tail bound paxillin and a faint 55-kDa paxillin-related polypeptide from extracts of Jurkat T cells (Fig. 1B). The 55-kDa polypeptide is most likely the paxillin-related protein, Hic-5 (19, 20). We next examined paxillin binding of a series of C-terminal truncation mutants of ₄ (Fig. 1A). The C-terminal 5 amino acid residues of ₄ were dispensable for paxillin or Hic-5 binding (Fig. 1). However, further N-terminal deletions in ₄ (990X, 985X, and 982X; each "X" is a stop codon) dramatically reduced binding of both proteins (Fig. 1B). To minimize possible contributions from other cellular proteins, we repeated these experiments using purified recombinant paxillin and similar results were obtained (data not shown). Thus, the amino acid residues N-terminal of Ser⁹⁹⁴ within the ₄ tail are required for binding of paxillin and Hic-5.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Paxillin binding to 4 tail deletion mutants. A, amino acid sequences of full-length and C-terminal deletion mutants of the 4 cytoplasmic domain. B, Ni2+-charged resins were loaded with full-length or mutant 4 tail proteins. Bound proteins from a Jurkat T cell lysate (Ly) were separated on 4-20% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and immunoblotted with antibody (clone 349) reactive with Hic-5 and paxillin. Loading of each tail was assessed by Coomassie Blue staining (Loading). Depicted is a representative result from three experiments.

To localize the paxillin-binding site in the ₄ tail, we assessed the capacity of synthetic ₄ cytoplasmic domain peptides (Fig. 2A) to compete for the binding of purified paxillin to the ₄ tail. A peptide containing the entire ₄ tail sequence completely blocked paxillin (100 nM paxillin added) binding, whereas full-length _IIb tail peptide had no inhibitory effect (data not shown). The estimated concentration of immobilized recombinant ₄ tail model protein was ~70 µM, and the soluble peptide inhibited binding completely at a concentration of 54 µM (Fig. 2B). Thus, the soluble ₄ tail peptide specifically inhibited paxillin binding.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Inhibition of paxillin binding by 4 peptides. A, amino acid sequences of synthetic peptides derived from the 4 cytoplasmic domain. B, recombinant HA-tagged GST-paxillin was added to Ni2+-charged resins loaded with 4 tail protein in the absence (None) or presence of the competing synthetic peptide indicated. The concentration of the full-length 4 tail was 54 µM; other peptides were present at a concentration of 540 µM. Bound fractions were collected and separated by SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and stained with HA-tag-specific antibody, 12CA5 (upper panel). Bound paxillin was quantified by scanning densitometry of these immunoblots using NIH "Image" program. These values were used to calculate percentage inhibition = 100 × [(A0 A)/A0], where A = intensity in the presence of competitor, and A0 = intensity in the absence of competitor (lower panel). Depicted is a representative result from three experiments.

We next synthesized three pentadecapeptides that spanned the ₄ tail with 5-8 residue overlaps (Fig. 2A). The middle peptide, ₄(Ser⁹⁷⁸-Ile⁹⁹²), competed for paxillin binding with an IC₅₀ of ~200 µM (Fig. 2B). As expected from the previous results with C-terminal truncation mutants, the N-terminal pentadecapeptide, ₄(Lys⁹⁶⁸-Glu⁹⁸²), was without effect (Fig. 2) at concentrations up to 540 µM. Similarly, the C-terminal peptide, ₄(Arg⁹⁸⁵-Asp⁹⁹⁹), also manifested little activity at this concentration. Thus, ₄(Ser⁹⁷⁸-Ile⁹⁹²) contains a minimal recognition sequence for paxillin binding. Furthermore, a scrambled peptide with an identical composition to ₄(Ser⁹⁷⁸-Ile⁹⁹²) (WRSLDYENIEQISSR) had no inhibitory effect on paxillin binding at a concentration of 540 µM (data not shown). Thus, ₄(Ser⁹⁷⁸-Ile⁹⁹²) peptide specifically blocks paxillin binding.

Additional peptides were synthesized to further define the recognition specificity. Peptides Arg⁹⁷⁴-Ile⁹⁹² and Ser⁹⁷⁸-Ser⁹⁹⁴, which extended the ₄(Ser⁹⁷⁸-Ile⁹⁹²) sequence by 4 or 2 residues, respectively, showed similar activity to that of ₄(Ser⁹⁷⁸-Ile⁹⁹²) (Table I). However, deletion of Ile⁹⁹² (Ser⁹⁷⁸-Y⁹⁹¹) reduced activity. Similarly, deletion of Ser⁹⁷⁸ to Glu⁹⁸² resulted in a 3-fold reduction in activity. Nonetheless, the nonapeptide, Glu⁹⁸³-Ile⁹⁹², still blocked paxillin binding with an IC₅₀ of 670 µM. Thus, Glu⁹⁸³-Ile⁹⁹² contains a minimal sequence required to block paxillin binding. The >10-fold difference in activity between ₄(Glu⁹⁸³-Ile⁹⁹²) and the full-length ₄ cytoplasmic domain suggests that additional flanking sequences are required for maximal activity.

View this table: [in this window] [in a new window]   Table I Quantification of the inhibition of paxillin binding by 4 peptides Recombinant HA-tagged GST-paxillin was added to Ni2+-charged resins loaded with 4 model protein in the absence of presence of each competing synthetic peptide at different concentrations. Bound fractions were collected and separated by SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and stained with HA-tagged-specific antibody, 12CA5. Bound paxillin was quantified by scanning densitometry of these immunoblots using the NIH Image program. IC50 is the concentration of peptide at which 50% binding of recombinant paxillin was inhibited. Two experiments were performed, and similar results were obtained.

Amino Acid Residues Essential for Paxillin Binding-- To assess the role of individual amino acid residues within ₄(Glu⁹⁸³-Ile⁹⁹²) that are important for paxillin binding, we synthesized ₄(Ser⁹⁷⁸-Ile⁹⁹²) peptides that each contained a single alanine substitution within this region (Fig. 3) and examined their capacity to compete for paxillin binding. Alanine substitution of Tyr⁹⁹¹ abolished (IC₅₀ > 1100 µM) and Glu⁹⁸³ markedly reduced (IC₅₀ ~ 800 µM) the inhibitory effect of ₄(Ser⁹⁷⁸-Ile⁹⁹²) peptide on paxillin binding (Fig. 3). In contrast, alanine substitution of Arg⁹⁸⁵, Glu⁹⁸², or Trp⁹⁸⁹ had little effect (Fig. 3 and data not shown). Alanine substitution of Arg⁹⁸⁶ partially blocked the inhibitory capacity of ₄(Ser⁹⁷⁸-Ile⁹⁹²) (IC₅₀ ~ 450 µM, Fig. 3). Thus, analysis of competition by synthetic peptides implicated 2 amino acid residues, Glu⁹⁸³, and Tyr⁹⁹¹, as being essential for paxillin binding.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Competition for paxillin binding to 4 cytoplasmic domain by alanine-substituted 4 peptides. Upper panel, recombinant HA-tagged GST-paxillin was added to Ni2+-charged resins loaded with 4 tail protein in the presence of the synthetic peptides indicated. Bound fractions were collected and separated on 4-20% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and stained with antibody specific for HA-tag, 12CA5. Bound paxillin was quantified as described in Fig. 2B. Lower panel, amino acid sequences of the 4 peptides with the single alanine substitutions indicated. Depicted are results of one of three experiments performed with similar results.

To confirm that the effect of alanine substitution resulted in differences in paxillin binding, we examined the effect of these alanine substitutions on the paxillin binding properties of the intact ₄ tail. Recombinant full-length ₄ tail model proteins containing alanine substitutions at Glu⁹⁸³, Arg⁹⁸⁵, Arg⁹⁸⁶, and Tyr⁹⁹¹ (Fig. 4A) were assayed for binding to recombinant paxillin. Alanine substitution of either Glu⁹⁸³ or Tyr⁹⁹¹ reduced paxillin binding (Fig. 4B) to near background levels. In contrast, substitution at Arg⁹⁸⁵ had no effect and substitution at Arg⁹⁸⁶ had much less effect on paxillin binding (Fig. 4B). Furthermore, the previous truncation experiment (Fig. 1) suggested that Hic-5 and paxillin bind to the same site in the ₄ tail. Furthermore, the same point mutations that disrupted paxillin binding also blocked Hic-5 binding (Fig. 4C). Thus, Tyr⁹⁹¹ and Glu⁹⁸³ are important amino acid residues for paxillin and Hic-5 binding to the ₄ cytoplasmic domain.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Paxillin binding to 4 tail mutants. A, amino acid sequences of wild-type and mutant recombinant 4 cytoplasmic domains are depicted. Note that Ala969 was changed to Leu to preserve a HindIII restriction site. B, recombinant HA-tagged GST-paxillin was added to Ni2+-charged resins loaded with wild-type or mutant4, or IIb model proteins. Bound fractions were collected and separated on 4-20% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and stained with antibody specific for the HA-tag, 12CA5. The quantity of bound paxillin was estimated by scanning densitometry using the NIH Image program. Depicted is a representative result from three experiments. C, Ni2+-charged resins with wide-type or mutant 4 tail as well as IIb tail proteins. Bound proteins from a human platelet lysate (Ly) were separated on 4-20% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and immunoblotted with antibody (clone 349) reactive with Hic-5 and paxillin. Loading of each tail was assessed by Coomassie Blue staining (Loading). Depicted are results of one of two experiments performed with similar results.

To examine the binding of the ₄ tail mutants to native paxillin, we used Jurkat T cell lysate as a source of the protein. Affinity chromatography with model proteins containing ₄(Y991A) or ₄(E983A) mutations exhibited markedly reduced paxillin and Hic-5 binding relative to wild-type ₄ (Fig. 5A, upper panel). Thus, ₄(Y991A) and ₄(E983A) mutations also disrupted native paxillin binding to the ₄ tail. To examine the effect of these mutations in the context of an intact integrin, we joined the cytoplasmic domain of ₄ to the transmembrane and extracellular domains of integrin _IIb to form an _IIb4 chimera. This chimeric  subunit was co-transfected with a chimeric  subunit, ₃₁A, in CHO cells.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   E983A and Y991A mutations disrupt native paxillin binding to the 4 cytoplasmic domain and its physical association with intact integrins. A, Jurkat T cell lysate (Ly) was added to Ni2+-charged resins coated with 4 tail proteins as indicated. Bound proteins were collected and fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with antibody specific for paxillin (upper panel). Similar loading of each tail was assessed by Coomassie Blue staining (lower panel). Depicted are results of one of three experiments performed with similar results. B, upper panel, cell lysates from CHO cells expressing IIb431A, IIb4(Y991A)31A, or IIb4(E983A)31A chimeras were precipitated with IIb3-specific antibody, D-57. Immunoprecipitated proteins were separated by 4-20% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane, and reacted with biotin-labeled anti-paxillin antibody, and bound antibody was detected with streptavidin peroxidase followed by ECL. Two paxillin bands detected by the antibody probably represent the  and  isoforms of paxillin (23). Lower panel, in parallel, these cells were surface-labeled with biotin and subjected to immunoprecipitation with D-57. Precipitated surface proteins were separated on 4-20% SDS-PAGE under non-reducing conditions and detected with streptavidin peroxidase and ECL. Immunoprecipitation with an irrelevant mouse IgG did not result in detectable paxillin co-precipitation (data not shown). Depicted are results of one of three experiments performed with similar results.

To examine the effect of Y991A and E983A mutations on the association of paxillin with an intact integrin, we introduced these mutations into _IIb4 chimera. The _IIb431A, _IIb4(Y991A)₃₁A, and _IIb4(E983A)₃₁A chimeric integrins were expressed in CHO cells and immunoprecipitated with antibodies against the extracellular domain of _IIb3. Similar quantities of recombinant integrin were precipitated from each cell line (Fig. 5B, lower panel). Approximately 12-fold less paxillin was co-precipitated with either _IIb4(Y991A)₃₁A or _IIb4(E983A)₃₁A than with wild-type _IIb431A chimeric integrin (Fig. 5B, upper panel). Thus, ₄(Y991A) and ₄(E983A) mutations also disrupted the association of an intact integrin with paxillin.

Suppression of Cell Spreading Requires Integrity of the Paxillin-binding site-- The ₄ cytoplasmic domain inhibits cell spreading and focal adhesion formation (11, 12). To examine the functional effect of these mutants, we assayed adhesion and spreading of cells expressing these chimeras. Cell expressing the Y991A and E983A mutant chimeras spread extensively on Fg, a ligand for the _IIb3 extracellular domain (Fig. 6A). In contrast, cell spreading was markedly retarded in cells expressing the wild-type chimera or a chimera containing an ₄ mutation that retains the capacity to bind paxillin, ₄(R985A). These differences were not due to a change in _IIb3-dependent cell adhesion to Fg (_IIb431A, 60 ± 18%; _IIb4(Y991A)₃₁A, 57 ± 3%; _IIb4(E983A)₃₁A, 69 ± 13%; and _IIb4(R985A)₃₁A, 52 ± 6% adherence at 30 min). Furthermore, they were not due to generalized defects in cell spreading, because all cell lines spread well on fibronectin, a ligand for endogenous hamster integrin 5₁ (data not shown). Thus, mutations that disrupt the paxillin binding function of the ₄ cytoplasmic domain resulted in increased cell spreading.

View larger version (45K): [in this window] [in a new window]   Fig. 6.   Effect of 4 cytoplasmic domain mutations on cell spreading. A, cells were added to Fg-coated coverslips in 24-well plates and incubated in a 37 °C incubator for 1 h. The plates were washed with PBS twice, and the cells were fixed and examined by phase microscopy as described under "Materials and Methods." Magnification, ×200. Three independent clones from both IIb431A and IIb4(Y991A)31A-CHO cells and two independent clones from both IIb4(E983A)31A and IIb4(R985A)31A-CHO cells were examined, and similar results were obtained (data not shown). B, spread cells were enumerated as described under "Materials and Methods." The data represent the mean ± S.D. of triplicate determinations.

Evolutionary Conservation of the Paxillin Binding Motif-- The present studies have defined a sequence motif required for paxillin binding to the ₄ integrin cytoplasmic domain. We defined a core paxillin-binding sequence by analysis of truncation mutants, and inhibition of paxillin binding to the ₄ tail by synthetic peptides. Critical Glu, Arg, and Tyr residues were identified within this core sequence (Fig. 7A). A core motif of E(X)_2-3R(X)₄Y is conserved in the ₄ tails in all species so far sequenced. Because this motif is present in Xenopus, it is likely that the paxillin binding function of ₄ is evolutionarily conserved. Furthermore, the absence of this motif from  tails that fail to bind paxillin (Fig. 7B) provides an explanation for the specificity of paxillin binding to ₄. High resolution structural studies will be required to define the specific functional role of each of the residues important in paxillin binding.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Sequence comparison of the 4 cytoplasmic domain from different species and of different human  cytoplasmic domains. A, sequences of the 4 cytoplasmic domain from human, mouse, and Xenopus are depicted. The amino acid residues that are critical for paxillin binding are bold and underlined. The italicized amino acid residues are within the paxillin-binding domain but were not critical for paxillin binding. B, amino acid sequences of human 3A, 6A, 4, IIb, and 5 integrin cytoplasmic domains and a consensus sequence (plurality of 3) are depicted. Amino acid residues that are conserved among different  integrins are capitalized. The paxillin-binding motif that is unique to the 4 cytoplasmic domain is boxed. C, sequences of the paxillin-binding sites from PTP-PEST (24, 25), FAK (22), vinculin (22, 26), and the 4 cytoplasmic domain are depicted.

The paxillin-binding site identified in the ₄ tail is distinct from that in other paxillin-binding proteins. For example, the paxillin-binding site of PTP-PEST is composed of a sequence containing a proline-rich region (Fig. 7C), which is absent in the paxillin-binding site of the ₄ cytoplasmic domain. Furthermore, the identified paxillin-binding sites of focal adhesion kinase (FAK) and vinculin are also distinct from that of the ₄ tail (Fig. 7C). In addition, an excess of a recombinant protein derived from the focal adhesion targeting sequence of FAK, which contains its paxillin-binding site (21, 22), did not inhibit paxillin binding to the ₄ tail.² Consequently, our data suggest that ₄ can bind simultaneously to paxillin that is associated with PTP-PEST or FAK. Future studies will be required to test this possibility.

The interaction of paxillin with ₄ is of high affinity and is required for many of the biological functions of ₄ integrins (12). As shown here, paxillin-binding activity is present in small synthetic peptides derived from the ₄ tail and can be ablated by single point mutations. These features of the ₄-paxillin interaction suggest that small peptides or peptidomimetics that disrupt the interaction could be used to inhibit ₄-dependent cellular functions.

	FOOTNOTES

^* This work was supported in part by grants from the National Institutes of Health. This is publication number 13010VB from the Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by National Service Research Award IF32HL09922-01.

^§ To whom correspondence should be addressed: Dept. of Vascular Biology, VB-2, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7143; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M000388200

² S. Liu and M. H. Ginsberg, unpublished data.

	ABBREVIATIONS

The abbreviations used are: CHO cells, Chinese hamster ovary cells; GST, glutathione S-transferase; BSA, bovine serum albumin; Fg, fibrinogen; FN, fibronectin; Pipes, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; FAK, focal adhesion kinase.

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