Changing Ligand Specificities of alpha vbeta 1 and alpha vbeta 3 Integrins by Swapping a Short Diverse Sequence of the beta  Subunit*

(Received for publication, April 22, 1997)

Junichi Takagi , Tetsuji Kamata , Jere Meredith , Wilma Puzon-McLaughlin and Yoshikazu Takada §

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin alpha vbeta 3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while alpha vbeta 1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the beta 1 and beta 3 subunits. When the sequence CTSEQNC (residues 187-193) of beta 1 is replaced with the corresponding CYDMKTTC sequence of beta 3, the ligand specificity of alpha vbeta 1 is altered. The mutant (alpha vbeta 1-3-1), like alpha vbeta 3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The alpha vbeta 1-3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal beta 3-1-3 mutation blocks binding of alpha vbeta 3 to these multiple ligands and to LM609, a function-blocking anti-alpha vbeta 3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of beta , in which the sequence is located in the ligand binding site.


INTRODUCTION

Integrins are a family of alpha /beta heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions (1-5). Integrin-ligand interactions are critically involved in the pathogenesis of many diseases in human and animal models. Although integrin-ligand interaction is a therapeutic target, we poorly understand at the molecular level how integrins recognize multiple ligands. Evidence suggests that the I or A domain, a set of inserted sequences consisting of about 200 amino acid residues, of several integrin alpha  subunits (alpha M, alpha L, alpha 1, alpha 2) is important in ligand binding and receptor activation (reviewed in Ref. 6 and references therein). The presence of an I domain-like structure within the beta  subunit has been suggested based on the similarity in hydropathy profiles between the I domain and part of the beta  subunit (7). Interestingly, this region of beta  has been reported to be critical for ligand binding and its regulation (reviewed in Ref. 8) (Fig. 1). The Asp-119 (beta 3) (9) and Asp-130 (beta 1) (10, 11) and the corresponding residues in beta 2 and beta 6 are critical for ligand binding (12, 13). A synthetic peptide of beta 3 (MDLSYSMKDDLWSI, residues 118-131) has been shown to produce a ternary complex with cations and ligand (14). Also, the sequence DDLW (residues 126-129 of beta 3) was shown to be critical for interaction with the RGD sequence using a phage display system (15). A synthetic peptide of beta 3, DAPEGGFDAIMQATV (residues 217-231 of beta 3), has been shown to bind to immobilized fibrinogen (Fg),1 von Willebrand's factor (vWf), and fibronectin (Fn) (16, 17). A synthetic peptide of beta 3, SVSRNRDAPEG (residues 211-221 of beta 3), has been reported to block binding of Fg to alpha IIbbeta 3 (18, 19). We identified a small region of beta 1 (residues 207-218, a regulatory epitope) that is recognized by both activating and inhibiting anti-beta 1 antibodies (20). These antibodies probably induce high or low affinity states, respectively, by changing the conformation of the beta 1 subunit through binding to the non-ligand binding site (20).


Fig. 1. The diverse sequence within the putative I domain-like structure of the beta  subunit. This region of the beta  subunit contains eight predicted beta strands and five predicted alpha -helices (6). Eight critical oxygenated residues (Asp-130, Ser-132, Asn-224, Asp-226, Glu-229, Asp-233, Asp-267, and Asp-295 in beta 1) are located in several separate predicted loop structures (or at the boundary between a predicted alpha -helix and a beta -strand), which probably constitute multiple ligand/cation binding sites within the I domain-like structure of the beta  subunit. Although most of the sequences of the predicted turn structures containing the critical oxygenated residues are conserved among integrin beta  subunits, a large predicted loop region (residues 176-199) is not conserved among the beta  subunits. Particularly, the sequence and size of the disulfide-linked short sequence (e.g. residues 187-193 in beta 1, boxed area) is diverse. We hypothesized that the predicted loop region of the beta  subunit is involved in ligand binding specificity.
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We and researchers at other laboratories have recently identified residues critical for ligand binding in the putative I domain-like structure of beta 1 (6), beta 2 (21), and beta 3 (22). In beta 1, eight critical oxygenated residues are located in several separate predicted loop structures, which probably constitute multiple ligand/cation binding sites within the I domain-like structure of the beta  subunit. These critical oxygenic residues are conserved among integrin beta  subunits, indicating that these residues are ubiquitously involved in ligand binding regardless of ligand and integrin species. We observed that a large predicted loop region (residues 176-199 of beta 1) is diverse among the beta  subunits (Fig. 1). Furthermore, a recent structural model (23) and our preliminary model (not shown) of the I domain-like structure of beta  suggest that the sequence is also on the same side of the domain as residues critical for ligand binding. We hypothesized that the predicted loop (especially the disulfide-linked short sequences, e.g. residues 187-193 of beta 1) is involved in ligand specificity of integrins. alpha vbeta 3 has been shown to recognize a wide variety of ligands, including Fn, Fg, vWf, and vitronectin (Vn); alpha vbeta 1 is specific to Fn. We designed experiments, using alpha vbeta 1 and alpha vbeta 3 integrins, to determine whether a diverse sequence in the predicted loop (e.g. residues 176-199 in beta 1, residues 166-190 in beta 3) is involved in ligand specificity of integrins.


EXPERIMENTAL PROCEDURES

Antibodies

mAb 4B4 (to human beta 1) (24) was kindly provided by C. Morimoto (Dana-Farber Cancer Institute, Boston, MA); 8A2 (to human beta 1) (25) by N. Kovach and J. Harlan (University of Washington, Seattle, WA); A1A5 (to human beta 1) (26) by M. Hemler (Dana-Farber Cancer Institute, Boston, MA); LM142 (to human alpha v), LM609 (to alpha vbeta 3) (27), and P3G2 (to alpha vbeta 5) (28) by D. Cheresh (Scripps); 15 (to human beta 3) (29) by M. H. Ginsberg (Scripps); and 15/7 (to human beta 1) (30) by T. Yednock (Athena Neurosciences, San Francisco, CA). P5D2 (to human beta 1) and polyclonal anti-alpha v cytoplasmic peptide antibody were purchased from Chemicon (Temecula, CA).

Adhesive Peptides

A Fn 110-kDa fragment was prepared from bovine plasma Fn (Life Technologies, Inc.) as described (31). Bovine Fg was purchased from Daiichi Chemical (Tokyo, Japan). Purified vWf was provided by Z. Ruggeri (Scripps). Bovine Vn was purified according to Yatohgo et al. (32). Fg, Vn, and Fn 110-kDa fragment were coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instructions. The ligand concentration was 4.0, 1.2, and 2.3 mg/ml gel for Fg-, Vn-, and Fn 110-kDa fragment-Sepharose, respectively. The GRGDS peptide (6 mg/ml gel, Peptide Institute, Osaka, Japan) was coupled at the 6-carbon spacing arm of CH-Sepharose (Sigma) according to the manufacturer's instructions.

Transfection of Mammalian Cells

Human alpha v and beta 3 cDNAs were provided by J. Loftus (Scripps). Ten µg of wild-type human alpha v cDNA in pBJ-1 vector (33, 34) was transfected into parental CHO-K1 cells (8 × 106 cells) together with 1 µg of pFneo plasmid containing a neomycin-resistant gene by electroporation as described (35). After they were selected for G418 resistance, cells expressing alpha v were cloned by cell sorting in FACStar cell sorter (Becton-Dickinson) with mAb LM142 (the cloned cells are designated alpha v-CHO cells). Human beta 1 or beta 3 (WT/mutant) cDNA in pBJ-1 vector was transfected into alpha v-CHO cells together with 1 µg of pCD-hygro plasmid with a hygromycin-resistant gene or into parent CHO cells together with 1 µg of pFneo; cells were then selected with hygromycin (500 µg/ml; Calbiochem) or G418 essentially as described above. Cells expressing human beta 1 or beta 3 were cloned by sorting with mAb A1A5 or 15 as described above. The flow cytometric analysis was carried out using FACScan (Becton-Dickinson).

Adhesion Assays

Wells of 96-well Immulon-2 microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) containing Fg, vWf, Fn, and Vn at a concentration of 10 µg/ml overnight at 4 °C. The remaining protein binding sites were blocked by incubating with 1% bovine serum albumin (Calbiochem) for 1 h at room temperature. Cells (105 cells/well) in 100 µl of Dulbecco's modified Eagle's medium containing 0.5 mg/ml bovine serum albumin were added to the wells and incubated at 37 °C for 1 h. After gently rinsing the wells three times with PBS to remove unbound cells, bound cells were quantified using endogenous phosphatase activity (36).

Affinity Chromatography

Cells were harvested with 3.5 mM EDTA in PBS and washed with PBS. Cells (about 5 × 106) were then surface-labeled with 125I by using IODO-GEN (Pierce) (37), washed three times with PBS, and solubilized in 1 ml of 100 mM octyl glucoside in 10 mM Tris-HCl, 0.15 M NaCl, pH 7.4 (TBS), containing 2.5 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride (Sigma) at 4 °C for 15 min. The insoluble materials were removed by centrifugation at 15,000 × g for 10 min. The supernatant was then incubated with a small amount of underivatized Sepharose 4B at 4 °C for 15 min to remove nonspecific binding material. The supernatant was incubated at 4 °C for 1 h with 200-500 µl of packed Fg-, Vn-, Fn 110-kDa fragment-, or GRGDS-Sepharose that had been equilibrated with TBS containing 2.5 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 25 mM octyl glucoside (washing buffer). The unbound materials were washed with a 20 × column volume of washing buffer, and the bound materials were eluted with 20 mM EDTA instead of 1 mM MnCl2 in washing buffer; and then 0.5-ml fractions were collected. Twenty-µl aliquots from each fraction was analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel followed by autoradiography.

Immunostaining

Glass coverslips (Fisher) were treated with 10% KOH in methanol for 1 h at room temperature, washed three times with distilled H2O, and stored in ethanol. Etched coverslips were then coated with 50 µg/ml Fg, 50 µg/ml Fn, or 22 µg/ml Vn in PBS overnight at 4 °C and then blocked with 10 mg/ml heat-denatured bovine serum albumin (Calbiochem) in PBS for 10 min at room temperature. For plating experiments, cells were washed and then detached with 2.5 mM EDTA/PBS. Detached cells were isolated, washed, resuspended in Dulbecco's modified Eagle's medium, and then replated on coated coverslips. Cells were allowed to attach and spread for 2 h. Prior to fixation, cells were chilled on ice for 5 min, washed with cold PBS, and then extracted with cold PIPES buffer (0.1 M PIPES, pH 6.8, 1 mM MgCl2, and 1 mM EGTA) containing 1% glycerol and 0.5% Nonidet P-40 for 1-2 min. Extracted cells were washed with cold PIPES buffer and then fixed with 3.7% methanol-free formaldehyde (Polysciences) in PIPES buffer for 20 min at room temperature. Following fixation, cells were washed with PBS and then blocked with 10% normal goat serum (Life Technologies, Inc.)/PBS for 20 min at 37 °C. Human integrins were detected using either the anti-human beta 1 antibody P5D2 or the anti-human beta 3 antibody 15. Cells were immunostained for 1 h at 37 °C, washed, and then stained with a fluorescein isothiocyanate-conjugated sheep anti-mouse IgG secondary antibody (Molecular Probes) for 30 min at 37 °C; cells were also labeled with rhodamine phalloidin (Molecular Probes) to detect actin stress fibers. Stained cells were mounted in Fluoromount-G (Fisher) and photographed using a Nikon Diaphot inverted microscope.

Other Methods

Site-directed mutagenesis of the beta 1 and beta 3 cDNA in a pBJ-1 vector was carried out using unique restriction site elimination (38). The presence of mutations was confirmed by DNA sequencing. Immunoprecipitation was carried out as described previously (20).


RESULTS

Swapping the CTSEQNC Sequence of beta 1 (residues 187-193) with the Corresponding CYDMKTTC Sequence of beta 3 Induces Adhesion of alpha vbeta 1 to Fg and vWf

To determine whether a diverse sequence in the predicted loop (residues 176-199 in beta 1 and residues 166-190 in beta 3) is involved in ligand specificity of integrins, we replaced the CTSEQNC sequence of beta 1 with the corresponding CYDMKTTC sequence of beta 3 by site-directed mutagenesis. The CYDMKTTC sequence of beta 3 has been reported to be disulfide-linked (39). The resulting mutant beta 1-3-1, wild-type beta 1, or wild-type beta 3 cDNA constructs were transfected into either parental CHO cells or CHO cells expressing wild-type human alpha v (alpha v-CHO). Parent CHO cells have been reported to express endogenous hamster alpha v (40) but not beta 3 (41). Consistent with these findings, we found that CHO cells express alpha vbeta 5 using mAb P3G2 (data not shown). The cloned cells expressing WT or mutant beta 1 in association with exogenous human alpha v are designated alpha vbeta 1-, alpha vbeta 1-3-1-, alpha vbeta 3-CHO cells, and those with only endogenous hamster alpha v are designated as beta 1-, beta 1-3-1-, beta 3-CHO cells.

alpha vbeta 3 recognizes multiple ligands, including Fn, Fg, vWf, and Vn; alpha vbeta 1 is specific to Fn on CHO cells. Therefore, we tested the ligand specificity of the beta 1-3-1 mutant. As shown in Fig. 2A, we found that cells expressing alpha vbeta 3 or alpha vbeta 1-3-1, but not alpha vbeta 1, adhered to both Fg and vWf. Adhesion of the alpha vbeta 1-3-1- but not alpha vbeta 3-CHO cells was blocked by the inhibitory anti-human beta 1 mAb 4B4 (Fig. 2B), indicating that adhesion of the alpha vbeta 1-3-1-expressing cells to Fg and vWf is mediated by human beta 1 sequences. Similar results were obtained with the beta 1-, beta 3-, and beta 1-3-1-CHO cells (Fig. 2, A and B). These results suggest that the region spanning residues 187-193 of beta 1 or 177-184 of beta 3 is involved in the regulation of ligand specificity.


Fig. 2. Adhesion of WT and mutant alpha v integrins to vWf and Fg. Wells of 96-well microtiter plates were coated with vWf (black column), Fg (white column), or bovine serum albumin (hatched column). Adhesion assay was performed with no added antibody (A) and with 4B4 (inhibiting anti-beta 1 mAb) (B). Antibodies were added at a 1000 × dilution of ascites. All of the cell lines used adhered well (more than 70%) to Fn and Vn under the same assay conditions, probably due to endogenous fibronectin receptors alpha vbeta 1 and alpha 5beta 1 and vitronectin receptor, alpha vbeta 5, respectively (data not shown). The data indicated that the beta 1-3-1 mutation induces adhesion of alpha vbeta 1 to Fg and vWf. Mean fluorescence intensities for human beta 1 are 170 (in beta 1-CHO), 313 (in beta 1-3-1-CHO), 766 (in alpha vbeta 1-CHO), 640 (in alpha vbeta 1-3-1-CHO), and 3 (in parent CHO cells, negative control).
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Binding Specificity of alpha vbeta 1-3-1 to Fg, Vn, and Fn 110-kDa Fragment Immobilized to Sepharose

The specificity of the interaction between alpha vbeta 1-3-1 and ligands was further analyzed by affinity chromatography. Lysates from surface 125I-labeled alpha vbeta 1-3-1-CHO cells (as well as control alpha vbeta 1- and alpha vbeta 3-CHO cells) were incubated with immobilized Fg or Fn 110-kDa fragments, and bound materials were eluted with EDTA. As shown in Fig. 3A, bands corresponding to alpha v and beta 1 in size were eluted from Fg-Sepharose using a lysate of alpha vbeta 1-3-1-CHO cells, while bands corresponding to human alpha v and beta 3 were eluted from Fg-Sepharose with a lysate of alpha vbeta 3-CHO cells. Immunoprecipitation of the eluate from alpha vbeta 1-3-1 cells using anti-beta 1 mAb A1A5 (Fig. 3C, lane 5) and anti-alpha v mAb LM142 (Fig. 3D, lane 5) confirmed that these two bands are human alpha v and beta 1 (beta 1-3-1). In contrast, very low levels of alpha v and beta 1 were detected in the Fg-Sepharose eluate with lysate of alpha vbeta 1-CHO cells. These results suggest that alpha vbeta 1-3-1 exhibits a much higher affinity for Fg than alpha vbeta 1. Similar results were obtained with Vn-Sepharose (data not shown), suggesting that alpha vbeta 1-3-1 shows a much higher affinity to Vn as well. In experiments done in parallel, we have detected bands corresponding to alpha vbeta 1, alpha vbeta 1-3-1, and alpha vbeta 3 in the eluate from Fn 110-kDa fragment-Sepharose with lysates from alpha vbeta 1-, alpha vbeta 1-3-1, and alpha vbeta 3-CHO cells, respectively (Fig. 3B). Immunoprecipitation confirmed that the major beta  subunits in the eluates are beta 1, beta 1-3-1, and beta 3, respectively (Fig. 3C). These results suggest that the alpha vbeta 1-3-1 mutant, like alpha vbeta 3, binds to Fg, Vn, and Fn 110-kDa fragments in a solubilized form.


Fig. 3. Affinity chromatography on Fg- and Fn 110-kDa fragment-Sepharose. Lysates of surface 125I-labeled cells were incubated with Fg-Sepharose (A) or Fn 110-kDa fragment-Sepharose that had been equilibrated with buffer containing 2.5 mM MnCl2 (B). The bound materials were eluted with 20 mM EDTA. Twenty-µl aliquots from the first four 0.5-ml fractions were analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel under nonreduced conditions. C, immunoprecipitation of whole lysates (lanes 1-3) and eluted materials from Fg-Sepharose (lanes 4-6) and from Fn 110-kDa fragment-Sepharose (lanes 7-9) with alpha vbeta 1-CHO (lanes 1, 4, and 7), alpha vbeta 1-3-1-CHO cells (lanes 2, 5, and 8), and alpha vbeta 3-CHO cells (lanes 3, 6, and 9). Antibodies used were anti-beta 1 mAb A1A5 (lanes 1, 2, 4, 5, 7, and 8) and anti-beta 3 mAb 15 (lanes 3, 6, and 9). Control antibody (nonimmune rabbit serum) did not precipitate anything from any samples (data not shown). D, immunoprecipitation of the whole lysate (lanes 1-3) and the eluted materials (lanes 4-6) with alpha vbeta 1-3-1-CHO cells using anti-beta 1 mAb A1A5 (lanes 1 and 4), anti-alpha v mAb LM142 (lanes 2 and 5), and control antibody (lanes 3 and 6). The data suggest that alpha v and beta 1-3-1 are major integrin subunits in the eluted material.
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The alpha vbeta 1-3-1 Mutant Is Recruited to Focal Contacts and Transduces Signals on Adhesion to Fg and Vn

Next we determined if the altered ligand specificity of the alpha vbeta 1-3-1 chimera affected intracellular signaling. Cells were plated on Fn, Vn, or Fg, and localization of the human integrin was determined by immunostaining with anti-human beta 1 (alpha vbeta 1 and alpha vbeta 1-3-1) or anti-human beta 3 (alpha vbeta 3). While all three receptors localized to focal adhesions in cells plated on Fn (Fig. 4, A, C, and F), only beta 1-3-1 and beta 3 localized to focal adhesions in cells on Vn; alpha vbeta 1-CHO cells did attach and spread on Vn due to endogenous alpha vbeta 5. However, alpha vbeta 1 exhibited a diffuse staining pattern. This result is consistent with the binding data and indicates that the alpha vbeta 1-3-1 chimera is able to generate intracellular signals. In addition, we found that the alpha vbeta 1-3-1 chimera, like alpha vbeta 3, induced cell spreading and focal adhesion formation in cells plated on Fg; alpha vbeta 1 cells did not adhere to Fg. Similar results were obtained with the beta 1-, beta 1-3-1-, and beta 3-CHO cells that express lower levels of the transfected integrins (data not shown). These results indicate that the alpha vbeta 1-3-1 chimera is a functional receptor and has the same signaling properties as alpha vbeta 3.


Fig. 4. Localization of WT alpha vbeta 1, WT alpha vbeta 3, and alpha vbeta 1-3-1 mutant integrins on Fg, Vn, and Fn. alpha vbeta 1-CHO (A and B), alpha vbeta 3-CHO (C, D, and E) and alpha vbeta 1-3-1-CHO (F, G, and H) cells were plated on Fn (A, C, and F), Vn (B, D, and G) and Fg (E and H). Cells were allowed to spread for 2 h. Human integrins were detected using either the anti-human beta 1 mAb P5D2 (for alpha vbeta 1 and alpha vbeta 1-3-1 CHO cells) or the anti-human beta 3 mAb 15 (for alpha vbeta 3-CHO cells) and fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody. alpha vbeta 1-CHO cells did not attach to a Fg-coated glass plate.
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The Reciprocal beta 3-1-3 Swapping Mutation Blocks Binding of alpha vbeta 3 to Fg, vWf, and Vn and to LM609, a Function-blocking Anti-alpha vbeta 3 mAb

To determine whether the reciprocal swapping mutation has any effect on the ligand specificity of alpha vbeta 3, we replaced the CYDMKTTC sequence of beta 3 (residues 177-184) with the corresponding CTSEQNC sequence of beta 1 (the beta 3-1-3 mutation). The resulting mutant beta 3-1-3 and WT beta 3 cDNA constructs were transfected into alpha v-CHO cells, and cells stably expressing alpha vbeta 3-1-3 or alpha vbeta 3 were cloned by sorting (alpha vbeta 3-1-3-CHO and alpha vbeta 3-CHO cells, respectively). The levels of alpha v and beta 3 expression were comparable in clonal WT alpha vbeta 3-CHO and alpha vbeta 3-1-3-CHO cells used. alpha vbeta 3-1-3-CHO cells showed significantly lower adhesion activity than alpha vbeta 3-CHO cells to both Fg and vWf; alpha vbeta 3-1-3-CHO cells required higher ligand concentrations for adhesion than WT alpha vbeta 3-CHO cells (Fig. 5, A and B). In addition, solubilized alpha vbeta 3-1-3 did not bind to either Fg or Vn immobilized to Sepharose, although solubilized WT alpha vbeta 3 did (Fig. 5C). These results suggest that the beta 3-1-3 mutation significantly reduces binding of alpha vbeta 3 to Fg, Vn, and vWf. Although we observed that alpha vbeta 3-1-3 mutant binds to Fn 110-kDa fragments and to the GRGDS peptide on affinity chromatography (data not shown), we could not determine whether the beta 3-1-3 mutation changes the binding affinity of alpha vbeta 3 to Fn 110-kDa fragment or the GRGDSP peptide (because of the presence of other fibronectin receptors, endogenous alpha vbeta 1 and alpha 5beta 1).


Fig. 5. The beta 3-1-3 mutation blocks binding of alpha vbeta 3 to Fg, vWf, and Vn. Adhesion of alpha vbeta 3-1-3-CHO cells to Fg (A) and vWf (B) is shown. Wells of 96-well microtiter plates were coated with varying concentrations of Fg and vWf. Cells (105 cells/well) in 100 µl of Dulbecco's modified Eagle's medium were added to the wells and incubated at 37 °C for 1 h. After gently rinsing the wells three times with PBS to remove unbound cells, bound cells were quantified. Mean fluorescence intensities are 246 for human alpha v and 214 for human beta 3 in WT alpha vbeta 3-CHO cells and 331 for human alpha v and 262 for human beta 3 in alpha vbeta 3-1-3-CHO cells. C, affinity chromatography of alpha vbeta 3-1-3- and alpha vbeta 3-CHO cells on Vn and Fg. Lysates of surface 125I-labeled cells were incubated with Vn-Sepharose or Fg-Sepharose that had been equilibrated with buffer containing 2.5 mM MnCl2. The bound materials were eluted with 20 mM EDTA. Twenty-µl aliquots from the first four 0.5-ml fractions were analyzed by SDS-polyacrylamide gel electrophoresis using 7% polyacrylamide gel under nonreduced conditions.
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The immunoprecipitation of whole lysate of alpha vbeta 3-1-3-CHO cells using anti-alpha v and anti-beta 3 mAbs showed that anti-alpha v and anti-beta 3 co-precipitated beta 3-1-3 and alpha v subunits, respectively, suggesting that the beta 3-1-3 mutation does not affect the alpha -beta association. However, the alpha vbeta 3-1-3 mutant did not react with LM609, a function-blocking anti-alpha vbeta 3 mAb, upon immunoprecipitation (Fig. 6) and flow cytometric analysis (data not shown), suggesting that the beta 3-1-3 mutation destroyed the LM609 epitope and that the CYDMKTTC sequence of beta 3 is closely located to ligand binding sites of alpha vbeta 3.


Fig. 6. The beta 3-1-3 mutation blocks binding of alpha vbeta 3 to LM609, a function-blocking anti-alpha vbeta 3 antibody. Immunoprecipitation of alpha vbeta 3 and alpha vbeta 3-1-3 is shown, LM142 (to human alpha v), polyclonal anti-alpha v cytoplasmic peptide antibody, 15 (to human beta 3), LM609 (to alpha vbeta 3).
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DISCUSSION

We established that swapping the CTSEQNC sequence of beta 1 with the corresponding CYDMKTTC sequence of beta 3 induces significant changes in ligand specificity of alpha vbeta 1. The beta 1-3-1 mutation markedly increases affinity of alpha vbeta 1 to Fg, vWf, and Vn (a gain-of-function effect). Since the alpha vbeta 1-3-1 mutant is functional in cultured cells and transduces signals on adhesion to the ligands, the swapping did not induce a detectable adverse effect on the other receptor functions (e.g. alpha -beta association and signal transduction). In reciprocal experiments, swapping a disulfide-linked CYDMKTTC sequence of beta 3 with the corresponding CTSEQNC sequence of beta 1 blocks the binding function of alpha vbeta 3 to Fg, vWf, and Vn. Taken together, the present study suggests that a small disulfide-linked CYMKTTC sequence of beta 3 (and the CTSEQNC sequence of beta 1 as well) defines a novel site of integrin beta  critical for ligand specificity. Sequence diversity among beta  subunits and localization within an I domain-like structure of beta , close to putative ligand binding sites (see Introduction) is consistent with the proposed function of the sequence. In a preliminary study, we introduced mutations into the corresponding predicted loop of the beta 2 subunit. We found that these mutations showed profound effects on the ligand binding function of alpha Lbeta 2 integrin,2 indicating that the diverse predicted loops of the beta  subunits are ubiquitously involved in the regulation of ligand binding functions.

Mechanisms by which the disulfide-linked sequences in a predicted loop within the I domain-like structure of the beta  subunits define ligand specificity of integrins have yet to be studied. In preliminary studies, we did not obtain evidence that the beta 1-3-1 mutation induces constitutive activation of beta 1 integrins or induces drastic conformational changes. We determined the reactivity of the beta 1-3-1 mutant to an activation-dependent anti-beta 1 mAb 15/7, which recognizes the highly activated form of beta 1 integrin (30). The binding profiles of 15/7 to the beta 1-3-1 mutant and wild-type beta 1 were identical; binding of 15/7 was dependent on activation in both cases (data not shown). The epitope for 15/7 has been localized within the residues 354-425 of beta 1 (in the non-ligand binding region outside the I domain-like region) (42). Therefore, there is a possibility that the effect of the beta 1-3-1 mutation on conformation remains local (e.g. within the I domain-like structure of beta 1) and 15/7 does not detect it. The amino acid residues surrounding the tripeptide RGD of the ligands have been reported to be critical for receptor specificity of snake venom disintegrins (43-45). One possible mechanism is that the predicted loop structures of beta 3 or beta 1 interact with the residues surrounding the tripeptide RGD of ligands, if we assume that the predicted loop structure of beta  is close to the ligand binding site of alpha vbeta 3 or alpha vbeta 1. Another possibility is that the predicted loops regulate the access of a group of ligands (in the case of alpha vbeta 3, Fg, Vn, and vWf) to the ligand binding site.

The CTSEQNC sequence of beta 1 (or the CYDMKTTC sequence of beta 3) is located within a predicted beta -turn in the putative I domain-like structure of the beta  subunit (6). A recent model of the beta  I domain-like structure (23), the folding diagram, appears to be consistent with our previous and present mutagenesis data (Fig. 7), and this model is similar to our preliminary model (not shown). All of the residues critical for ligand binding (e.g. Asp-130 and Glu-229 of beta 1) (6, 10) are located in the upper face of the model (predicted as the ligand binding site). Also, the regulatory epitope (residues 207-218 of beta 1) (20), which is recognized by both activating and inhibiting anti-beta 1 mAbs, is located in the non-ligand binding site (in the lower face) of the domain. Interestingly, a diverse sequence in the predicted loop (e.g. residues 176-199 in beta 1, residues 166-190 in beta 3), which is involved in ligand specificity of integrins in the present study, is located in the upper face of the domain in this model. The finding that the beta 3-1-3 mutation blocked binding of the function-blocking anti-alpha vbeta 3 antibody LM609 supports the idea that the predicted loop structure is close to the ligand binding site of alpha vbeta 3. Taken together, the present and previous mutagenesis data strongly support this model. Recently, Collins Tozer et al. (22) published an interesting atomic model of the putative I domain of beta 3, which is based on the crystal structure of the alpha M I domain (7). However, our mutagenesis data do not fit in very well with their model, since 1) the sequence CYDMKTTC of beta 3, which is critically involved in ligand binding to alpha vbeta 3, is not close to the MIDAS site (apparently in a non-ligand binding site) in their model, and 2) although Thr-197 of beta 3 is located in the MIDAS site of beta 3 in this model, the corresponding residue of beta 1 (Thr-206) is very close to the regulatory epitope. This epitope is probably located in a non-ligand binding site of beta 1 because 1) binding of some mAbs actually activates, instead of inactivating, the beta 1 integrins, and 2) this epitope has recently been shown to be an allosteric effector site of beta 1 (46), since the binding of an inhibitory anti-beta 1 mAb 13 to the regulatory epitope is also dramatically attenuated by ligands (Fn fragments or the GRGDS peptide). Further biochemical and structural characterization of this region of the beta  subunit may be required to substantiate these models.


Fig. 7. Positions of residues critical for ligand specificity in a hypothetical model of the I domain-like structure of beta . This hypothetical model was taken from Ref. 23 and modified. Arrows indicate beta -sheets, and columns indicate alpha -helices. Closed circles show residues critical for ligand binding in beta 1 (6). In this model, the diverse disulfide-linked sequence critical for ligand specificity (residues 187-193 of beta 1 and 177-184 of beta 3) is located in the predicted loop, surrounded by conserved oxygenated residues critical for ligand binding (e.g. Asp-130 in beta 1) in the upper face of the I domain-like structure of beta . The upper face of this domain is predicted as a ligand binding site, based on the homology to the I domains of alpha M and alpha L (23). The small region that is recognized by activating or inhibiting antibodies (residues 207-218, a regulatory epitope) (20) is located in a predicted loop on the other side of the domain (a non-ligand binding site).
[View Larger Version of this Image (25K GIF file)]

alpha vbeta 3 has been shown to be involved in the progression of melanoma and induction of neovascularization by tumor cells. alpha vbeta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels (47, 48). We identified a critical region for ligand binding and specificity of integrins using a gain of function mutant of the beta  subunit. The predicted loop sequence of the integrin beta 3 subunit is a new potential target for designing inhibitors of ligand binding functions of alpha vbeta 3.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants GM47157 and GM49899.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.

This is publication 10153-VB from The Scripps Research Institute.


   Visiting from the Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226, Japan.
§   To whom correspondence should be addressed: Dept. of Vascular Biology, VB-1, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7122; Fax: 619-784-7323; E-mail: takada{at}scripps.edu.
1   The abbreviations used are: Fg, fibrinogen; CHO, Chinese hamster ovary; Fn, fibronectin; Vn, vitronectin; vWf, von Willebrand's factor; mAb, monoclonal antibody; WT, wild type; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid.
2   T. Kamata and Y. Takada, unpublished results.

ACKNOWLEDGEMENTS

We thank Drs. D. Cheresh, M. H. Ginsberg, M. E. Hemler, R. L. Juliano, J. Loftus, C. Morimoto, and Z. Ruggeri for valuable reagents.


REFERENCES

  1. Springer, T. A. (1994) Cell 76, 301-314 [Medline] [Order article via Infotrieve]
  2. Ruoslahti, E. (1991) J. Clin. Invest. 87, 1-7 [Medline] [Order article via Infotrieve]
  3. Hynes, R. O. (1992) Cell 69, 11-25 [Medline] [Order article via Infotrieve]
  4. Hemler, M. E. (1990) Annu. Rev. Immunol. 8, 365-400 [CrossRef][Medline] [Order article via Infotrieve]
  5. Yamada, K. M. (1991) J. Biol. Chem. 266, 12809-12812 [Free Full Text]
  6. Puzon-McLaughlin, W., and Takada, Y. (1996) J. Biol. Chem. 271, 20438-20443 [Abstract/Free Full Text]
  7. Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638 [Medline] [Order article via Infotrieve]
  8. Loftus, J. C., Smith, J. W., and Ginsberg, M. H. (1994) J. Biol. Chem. 269, 25235-25238 [Free Full Text]
  9. Loftus, J. C., O'Toole, T. E., Plow, E. F., Glass, A., Frelinger, A. L., and Ginsberg, M. H. (1990) Science 249, 915-918 [Medline] [Order article via Infotrieve]
  10. Takada, Y., Ylanne, J., Mandelman, D., Puzon, W., and Ginsberg, M. (1992) J. Cell Biol. 119, 913-921 [Abstract]
  11. Kamata, T., Puzon, W., and Takada, Y. (1995) Biochem. J. 305, 945-951 [Medline] [Order article via Infotrieve]
  12. Bajt, M., and Loftus, J. (1994) J. Biol. Chem. 269, 20913-20919 [Abstract/Free Full Text]
  13. Huang, X., Chen, A., Agrez, M., and Sheppard, D. (1995) Am. J. Resp. Cell Mol. Biol. 13, 245-251 [Abstract]
  14. D'Souza, S. E., Haas, T. A., Piotrowicz, R. S., Byers-Ward, V., McGrath, E., Soule, H. R., Cierniewski, C., Plow, E. F., and Smith, J. W. (1994) Cell 79, 659-667 [Medline] [Order article via Infotrieve]
  15. Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1995) J. Cell Biol. 130, 1189-1196 [Abstract]
  16. Cook, J., Trybulec, M., Lasz, E., Khan, S., and Niewiarowski, S. (1992) Biochim. Biophys. A. 1119, 312-321
  17. Lasz, E., McLane, M., Trybulec, M., Kowalska, M., Khan, S., Budzynski, A., and Niewiarowski, S. (1993) Biochem. Biophys. Res. Commun. 190, 118-124 [CrossRef][Medline] [Order article via Infotrieve]
  18. Charo, I. F., Nannizzi, L., Phillips, D. R., Hsu, M. A., and Scarborough, R. M. (1991) J. Biol. Chem. 266, 1415-1421 [Abstract/Free Full Text]
  19. Steiner, B., Trzeciak, A., Pfenninger, G., and Kouns, W. (1993) J. Biol. Chem. 268, 6870-6873 [Abstract/Free Full Text]
  20. Takada, Y., and Puzon, W. (1993) J. Biol. Chem. 268, 17597-17601 [Abstract/Free Full Text]
  21. Goodman, T., and Bajt, M. (1996) J. Biol. Chem. 271, 23729-23736 [Abstract/Free Full Text]
  22. Tozer, E., Liddington, R., Sutcliffe, M., Smeeton, A., and Loftus, J. (1996) J. Biol. Chem. 271, 21978-21984 [Abstract/Free Full Text]
  23. Tuckwell, D., and Humphries, M. (1997) FEBS Lett. 400, 297-303 [CrossRef][Medline] [Order article via Infotrieve]
  24. Morimoto, C., Letvin, N. L., Boyd, A. W., Hagan, M., Brown, H., Kormacki, M., and Schlossman, S. F. (1985) J. Immunol. 134, 3762-3769 [Abstract/Free Full Text]
  25. Kovach, N. L., Carlos, T. M., Yee, E., and Harlan, J. M. (1992) J. Cell Biol. 116, 499-509 [Abstract]
  26. Hemler, M. E., Sanchez-Madrid, F., Flotte, T. J., Krensky, A. M., Burakoff, S. J., Bhan, A. K., Springer, T. A., and Strominger, J. L. (1984) J. Immunol. 132, 3011-3018 [Abstract/Free Full Text]
  27. Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69 [Medline] [Order article via Infotrieve]
  28. Wayner, E. A., Orlando, R. A., and Cheresh, D. A. (1991) J. Cell Biol. 113, 919-929 [Abstract]
  29. Frelinger, A., III, Cohen, I., Plow, E., Smith, M., Roberts, J., Lam, S., and Ginsberg, M. (1990) J. Biol. Chem. 265, 6346-6352 [Abstract/Free Full Text]
  30. Yednock, T. A., Cannon, C., Vandevert, C., Goldbach, E. G., Shaw, G., Ellis, D. K., Liaw, C., Fritz, L. C., and Tanner, L. I. (1995) J. Biol. Chem. 270, 28740-28750 [Abstract/Free Full Text]
  31. Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597 [Abstract/Free Full Text]
  32. Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M. (1988) Cell Struct. Funct. 13, 282-292
  33. Takebe, Y., Seiki, M., Fujisawa, J.-I., Hoy, P., Yokota, K., Arai, K.-I., Yoshida, M., and Arai, N. (1988) Mol. Cell. Biol. 8, 466-472 [Medline] [Order article via Infotrieve]
  34. Lin, A. Y., Devaux, B., Green, A., Sagerstrom, C., Elliott, J. F., and Davis, M. (1990) Science 249, 677-679 [Medline] [Order article via Infotrieve]
  35. Irie, A., Kamata, T., Puzon-McLaughlin, W., and Takada, Y. (1995) EMBO J. 14, 5542-5549 [Abstract]
  36. Prater, C. A., Plotkin, J., Jaye, D., and Frazier, W. A. (1991) J. Cell Biol. 112, 1031-1040 [Abstract]
  37. Braciale, T. J., Henkel, T. J., Lukacher, A., and Braciale, V. L. (1986) J. Immunol. 137, 995-1002 [Abstract/Free Full Text]
  38. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200, 81-88 [Medline] [Order article via Infotrieve]
  39. Calvete, J. J., Henschen, A., and Gonzalez-Rodriguez, J. (1991) Biochem. J. 274, 63-71 [Medline] [Order article via Infotrieve]
  40. Ferrer, M., Fernandez-Pinel, M., Gonzalez-Manchon, C., Gonzalez, J., Ayuso, M., and Parrilla, R. (1996) Thromb. Haemostasis 76, 292-301 [Medline] [Order article via Infotrieve]
  41. Ylanne, J., Chen, Y., O'Toole, T., Loftus, J. C., Takada, Y., and Ginsberg, M. H. (1993) J. Cell Biol. 122, 223-233 [Abstract]
  42. Puzon-McLaughlin, W., Yednock, T., and Takada, Y. (1996) J. Biol. Chem. 271, 16580-16585 [Abstract/Free Full Text]
  43. Lu, X., Williams, J. A., Deadman, J. J., Salmon, G. P., Kakkar, V. V., Wilkinson, J. M., Baruch, D., Authi, K. S., and Rahman, S. (1994) Biochem. J. 304, 929-936 [Medline] [Order article via Infotrieve]
  44. Scarborough, R. M., Rose, J. W., Naughton, M. A., Phillips, D. R., Nannizzi, L., Arfsten, A., Campbell, A. M., and Charo, I. F. (1993) J. Biol. Chem. 268, 1058-1065 [Abstract/Free Full Text]
  45. Lu, X., Rahman, S., Kakkar, V., and Authi, K. (1996) J. Biol. Chem. 271, 289-294 [Abstract/Free Full Text]
  46. Mould, A., Akiyama, S., and Humphries, M. (1996) J. Biol. Chem. 271, 20365-20374 [Abstract/Free Full Text]
  47. Brooks, P., Montgomery, A., Rosenfeld, M., Reisfeld, R., Hu, T., Klier, G., and Cheresh, D. (1994) Cell 79, 1157-1164 [Medline] [Order article via Infotrieve]
  48. Montgomery, A., Reisfeld, R., and Cheresh, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8856-8860 [Abstract]

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