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Related Collections Hemostasis, Thrombosis, and Vascular Biology Cell Adhesion and Motility Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 January 2001, Vol. 97, No. 1, pp. 175-182 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Ligand binding to integrin v3 requires tyrosine 178 in the v subunit Shigenori Honda, Yoshiaki Tomiyama, Nisar Pampori, Hirokazu Kashiwagi, Teruo Kiyoi, Satoru Kosugi, Seiji Tadokoro, Yoshiyuki Kurata, Sanford J. Shattil, and Yuji Matsuzawa From the Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, and the Department of Blood Transfusion, Osaka University Hospital, Osaka, Japan, and the Departments of Vascular Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Integrin v3 has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in v, a non-I-domain  subunit, remain to be identified. Recently in IIb, the other partner of the 3 subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of v in ligand binding. Wild-type v3 was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl2) or a mutation of 3 threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of v abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Av also failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-v3 antibody, and the expression of 3 ligand-induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with IIb, none of the mutations in the predicted W3 4-1 loop in v impaired ligand binding. These results implicate v Y178 in ligand binding to v3, and they suggest that there are key structural differences in the adhesive ligand-binding sites of v3 and IIb3. (Blood. 2001;97:175-182) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Integrins are a large family of heterodimeric adhesion receptors that is often subdivided into groups based on 8 known integrin  subunits.1 The 3-integrin subfamily is composed of v3, originally identified as the vitronectin receptor, and IIb3, a platelet-specific receptor for fibrinogen and von Willebrand factor. IIb3 plays a crucial role in platelet aggregation, normal hemostasis, and pathological thrombus formation.2 On the other hand, v3 is expressed in a number of tissues, including platelets, endothelial cells, vascular smooth muscle cells, and osteoclasts, and it plays a key role in angiogenesis and bone resorption.3,4 The IIb and v subunits are homologous and 36% identical in primary amino acid sequence,5 and most ligands that bind to IIb3, including fibrinogen, von Willebrand factor, and vitronectin, also bind to v3. However, there are some distinctive features between these 2 integrins.3 First, the IIb subunit has been found only in combination with 3, whereas v can associate with at least 5  subunits (1, 3, 5, 6, and 8).6 Second, some ligands, such as osteopontin, matrix metalloproteinase-2, and adenovirus penton base, bind to v3 but not to IIb3. Third, treatment of IIb3 with ethylenediamine tetraacetic acid (EDTA) at 37°C dissociates the complex into its individual subunits; v3 remains a heterodimer. Finally, the ligand-binding function of v3, but not IIb3, is suppressed by calcium (Ca2+).7 Generally, all integrins require divalent cations for ligand recognition, and multiple residues important for ligand binding have been identified on both  and  subunits.8 The N-terminal region of integrin  subunits has 7 repeats of homologous sequences of about 60 amino acid residues. Some integrin  subunits (eg, 2, L, and M) contain an inserted domain of about 200 amino acids residues (the I-domain) between the second and the third repeats in the  subunit, which is critically involved in ligand binding.9,10 The crystal structure of the I-domain has been determined, and the metal ion-dependent adhesion site (MIDAS) motif that contributes to cation binding, as well as ligand binding, has been clarified.11 Interestingly, a MIDAS-like motif essential for the ligand-binding function was also identified in integrin  subunits.12,13 On the other hand, integrin  subunits, such as v, IIb, and 4, do not have the I-domain. The structural basis for the interaction between non-I-domain  subunits and their ligands remains elusive. For the IIb subunit, peptide cross-linking studies have shown that the histamine-histamine-leucine-glycine-glycine-alanine-lysine-glutamine-alanine-glycine-aspartic acid-valine (HHLGGAKQAGDV) sequence derived from the COOH terminus of the -chain of fibrinogen interacts with residues 294-314, encompassing the second putative calcium-binding domain of IIb.14 However, recent characterization of molecular defects in Glanzmann thrombasthenia and mutagenesis studies demonstrated several discontinuous residues important for ligand binding: proline (P)145, D163, L183, G184, tyrosine (Y)189, Y190, phenylalanine (F)191, G193, and D224.15-19 Springer has proposed that the 7 N-terminal sequence repeats of integrin  subunits are folded into a -propeller domain.20 The proposed domains contain seven 4-stranded -sheets (W1-W7) arranged in a torus around a pseudosymmetry axis. Interestingly, the discontinuous residues identified in IIb as important for ligand binding were located in the regions predicted to adopt a -turn structure on the upper face of the -propeller model: P145 within the W3 4-1 loop; L183, G184, Y189, Y190, F191, and G193 within the predicted W3 2-3 loop; and D224 within the predicted W4 4-1 loop. Furthermore, the analysis of a Japanese variant of Glanzmann thrombasthenia, KO, and alanine-scanning mutagenesis have shown that D163 in the W3 4-1 loop (cystein [C]146-C167) is essential for ligand binding.16 In contrast, the single available peptide cross-linking study of v3 showed that 2 distinct linear regions in v, residues 139-167 and 312-349, cross-link to an arganine-glycine-aspartic acid (RGD) peptide.21 In this study, we took advantage of the data regarding ligand-binding sites in IIb and investigated the role in ligand binding of the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop) in v. By performing alanine-scanning mutagenesis of recombinant v3 expressed in 293 cells, we demonstrate a critical role for Y178 within the predicted W3 2-3 loop of v in ligand binding. In contrast to IIb, however, no mutations in the W3 4-1 loop affect ligand binding to v3, thereby implying key structural differences in the adhesive ligand-binding sites of the 2 3 integrins.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Monoclonal antibodies and peptides The following monoclonal antibodies (mAbs) were used in the study: LM609,22 a murine mAb specific for v3, and LM142,23 a mAb specific for v (gift from Dr David Cheresh, The Scripps Research Institute, La Jolla, CA); AP5,24 a mAb specific for 3 (gift from Dr Thomas Kunicki, The Scripps Research Institute); anti-LIBS1 (anti-ligand-induced binding site 1) mAb (specific for 3)25 and anti-LIBS6 mAb (specific for 3)26 (Dr Mark Ginsberg, The Scripps Research Institute); and AP3 (specific for 3)27 (Dr Peter Newman, The Blood Center of Southeastern Wisconsin, Milwaukee, WI). WOW-1 Fab,28 a monovalent ligand-mimetic mAb specific for activated v3, was created by replacing the heavy chain hypervariable region 3 (H-CDR3) of PAC-1 Fab, a ligand-mimetic mAb specific for activated IIb3,29 with a single integrin-binding domain of adenovirus penton base. We also used the RGDW peptide (gift from Dr Jiro Seki, Fujisawa Pharmaceutical Co., Osaka, Japan).30 Construction of v expression vectors and cell transfection Wild-type (WT) v complementary DNA (cDNA) (gift from Dr David Cheresh, The Scripps Research Institute) and WT 3 cDNA (gift from Dr Gilbert White, University of North Carolina, Chapel Hill, NC) were cloned into mammalian expression vector pcDNA3 (Invitrogen Corp, San Diego, CA). To introduce single alanine substitutions into v, overlap extension polymerase chain reaction (PCR) was carried out as previously described.16 For example, to generate the Y178A (Y178A) v mutant, we synthesized mismatched sense primer v178A-s, 5'-GGTCCTGGTAGCTTTGCATGGCAAGGTCAGC-3' (sense, nucleotides [nt] 648-678; mismatched sequences underlined) and antisense primer v178A-as, 5'-GCTGACCTTGCCATGCAAAGCTACCAGGACC-3' (antisense, nt 678-648; mismatched sequences underlined), which were constructed based on the published sequence.31 PCR was performed by using v cDNA as a template and primers v258-s, 5'-GCAAACACCACCCAGCC-3' (sense, nt 258-274) and v178A-as, or primers v178A-s and v952-as, 5'-GCAGCCATCTGCTCGCCAG-3' (antisense, nt 952-934). The 2 individually amplified PCR products were mixed and used as a template for PCR using primers v258-s and v952-as. The amplified PCR products were digested with PpuMI and AflIII. The fragments digested with AflIII and XbaI were isolated from the full-length v cDNA cloned into pcDNA3. These 2 fragments were introduced together into the pcDNA3 that had been digested with PpuMI and XbaI. The nucleotide sequences of the inserts were confirmed by sequence analysis. In a selected experiment, the C142-C155 loop in v was swapped with the corresponding sequence of IIb C146-C167. The WT or mutant v construct was cotransfected into 293 cells with WT 3 construct by the calcium-phosphate method as previously described.32 The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) and analyzed 2 days after transfection. Immunoprecipitation Immunoprecipitation was performed as previously described with slight modification.33 In brief, cells were surface-labeled with sulfo-NHS-biotin (Pierce, Rockford, IL) and lysed in a buffer containing 1% Triton X-100, 25 mM Tris-HCl (tris[hydroxymethyl] aminomethane-hydrochloride), 100 mM sodium chloride (NaCl) (pH 7.4), 0.1 mg/mL leupeptin, 4 µg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 10 mM benzamide. Then 200 µg protein from each sample was immunoprecipitated with the mAb LM609, and precipitated bands were identified on Western blots using peroxidase-conjugated avidin. Ligand-binding studies Fibrinogen (Kabi, Stockholm, Sweden) was labeled with fluorescein isothiocyanate (FITC) as previously described34 and stored at 80°C until use. FITC-fibrinogen binding to 293 cells was assessed by flow cytometry as described.33 Briefly, 50-µL aliquots of 1.5 × 105 washed cells in Ca2+-free-Tyrode-HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer containing 1 mM magnesium dichloride (MgCl2) were incubated with mAb LM142, specific for v (5 µg/mL), for 30 minutes on ice. After washing, 0.5 mM manganese dichloride (MnCl2) was added into the cell suspension to induce a high-affinity state of v3. Cells were then incubated with 150 µg/mL FITC-fibrinogen in the presence or absence of 1 mM RGDW peptide and phycoerythrin (PE)-conjugated antimouse immunoglobulin G (IgG) (1:5 dilution) for 25 minutes at 22°C and then incubated with propidium iodine (PI) (Sigma Chemical Co., St Louis, MO) for 5 minutes at 22°C. After washing, fibrinogen binding (FL1) was analyzed on the gated subset of single high v3-expressing (FL2) live cells (PI-negative, FL3). Specific fibrinogen binding was defined as that inhibited by 1 mM RGDW peptide. For WOW-1 Fab binding to v3, cells in Ca2+-free-Tyrode-HEPES buffer containing 1 mM MgCl2 were incubated with 5 µg/mL WOW-1 Fab in the presence of 0.5 mM MnCl2 for 30 minutes at 22°C. After washing, cells were incubated with 5 µg/mL Alexa-conjugated goat antimouse IgG F(ab')2 (Molecular Probes, Eugene, OR) for 25 minutes on ice and then incubated with PI for 5 minutes at 22°C. After washing, WOW-1 binding (FL1) was analyzed on the gated subset of single, living cells. WOW-1 binding to untransfected 293 cells was routinely taken as a measure of nonspecific binding because this value was similar to that obtained for WOW-1 binding to v3 transfected cells in the presence of 1 mM RGDW. For the induction of LIBS on v3, washed cells in Tyrode-HEPES buffer containing 1 mM MgCl2 and 1 mM calcium dichloride (CaCl2) were incubated with 1 mM RGDW for 30 minutes at 22°C. The cells were then incubated for 30 minutes with AP5, anti-LIBS1, or anti-LIBS6 at a final concentration of 5 µg/mL. After washing, cells were incubated with FITC-conjugated goat F(ab')2 antimouse IgG for 25 minutes. The mixtures were incubated with PI for an additional 5 minutes and washed, and mAb binding was analyzed on the gated subset of single, living cells. Adhesion assays Adhesion assays were performed as described by Faull et al.35 Wells of 96-well microtiter plates were coated with up to 1 µg fibrinogen per well in 100 µL phosphate-buffered saline (PBS) and incubated at 4°C overnight. After washing with PBS, wells were blocked with PBS containing 1% bovine serum albumin (BSA) (Sigma) for 90 minutes at 22°C. To determine background adhesion, control wells were coated with 1% BSA. Cells were washed twice with PBS and resuspended in DMEM containing 0.1% BSA at a concentration of 1 × 106 cells per mL. Then, 100-µL aliquots of cell suspension were added to wells in triplicate. The plate was incubated in a humidified 37°C incubator for 60 minutes. After washing with PBS, the adherent cells were checked by visual inspection, and adhesion was quantified by measuring endogenous cellular acid phosphatase activity in an enzyme-linked immunosorbent assay.     Results Top Abstract Introduction Materials and methods Results Discussion References Expression of v3 mutants in 293 cells Recent studies have demonstrated that several residues within the C146-C167 loop and the G184-G193 loop in the IIb subunit, which correspond to the W3 4-1 loop and W3 2-3 loop in the proposed -propeller model, respectively, play a critical role in ligand binding. Figure 1 shows the corresponding regions (C142-C155 and G172-G181) in the v subunit and the residues we replaced with alanine. The WT or mutant v construct was transiently cotransfected into 293 cells with the WT 3 construct, and v3 surface expression was examined by flow cytometry using the v3 complex-specific mAb LM609. As shown in Figure 2A, the surface expression of mutant v3 was 70% to approximately 123% of WT v3. Similar results were obtained when mAb LM142 was used to quantify v and mAb AP3 was used to quantify 3 (data not shown). View larger version (32K): [in this window] [in a new window]   Figure 1. Amino acid sequences of the predicted W3 4-1 loop between the N-terminal repeats 2 and 3 and the predicted W3 2-3 loop within repeat 3 in the  subunits of 3 integrins. Both loops are located on the upper face of the -propeller model.20 The asterisks indicate that the residues were substituted by alanine in this study. This figure is adapted from a -propeller model proposed by Springer,20 and the arrows indicate  strands. View larger version (41K): [in this window] [in a new window]   Figure 2. Surface expression of v3 mutants in transiently transfected 293 cells. (A) The surface expression of transfected v3 was analyzed 2 days after transfection by flow cytometry. Cells expressing WT or mutant v3 were incubated with 5 µg/mL v3 complex-specific mAb LM609 for 30 minutes on ice and then washed once. Bound antibodies were detected by FITC-conjugated goat F(ab')2 antimouse IgG. Relative amounts of the binding were normalized to a 100% value for LM609 binding to cells expressing WT v3. The results are representative of 3 separate experiments. (B) Immunoprecipitation showing the surface expression of transfected v3. Transiently transfected cells were surface-labeled with sulfo-NHS-biotin and lysed in the lysing buffer containing 1% Triton X-100. WT or mutant v3 was precipitated with LM609 (specific for the v3 complex) and separated on a 6% sodium dodecyl sulfate polyacrylamide gel under reducing conditions. After transfer, a membrane was incubated with peroxidase-conjugated avidin and developed with chemiluminescence. The results are representative of 3 separate experiments. Because 293 cells normally express v1 but not v3,36 we were concerned that endogenous v might associate with transfected 3 and contribute to the total v3 expressed on these cells. However, by comparing v3 transfectants to 3 transfectants, we found that endogenous v contributed no more than approximately 17% to the total v3 expressed. Immunoprecipitation experiments employing LM609 further showed that the expression of endogenous v associated with transfected 3 in 293 cells is low, and that the surface expression levels of mutant F177A, Y178A, and W179Av3 are comparable to those of WT v3 (Figure 2B). These results indicate that transfected v, not endogenous v, was the major contributor toward v3 expression in 293 cells. Moreover, none of the alanine-scanning mutants of v adversely affected surface expression of v3. The Y178A mutation in v abolishes soluble ligand binding to v3 To analyze the ligand-binding function of each mutant v3, we initially examined the binding of FITC-conjugated soluble fibrinogen to v3. Because v3 expressed on 293 cells is present in a low-affinity state and does not bind soluble ligands, cells were incubated with 0.5 mM MnCl2, which induces a high-affinity state of integrins by a direct effect on the extracellular domain (Figure 3A).37 To avoid even a minimal contribution of endogenous v in 293 cells to fibrinogen binding, we selectively analyzed the subset of transfectants expressing high levels of exogenous v3 monitored by LM142. As shown in Figure 3B, a D119Y mutation within the ligand-binding site of 3 abolished fibrinogen binding to v3 as expected,38 confirming the specificity of the ligand-binding assay in this system. Moreover, a Y178A mutation within the predicted G172-G181 loop of v abolished fibrinogen binding, and both F177A and W179A mutations adjacent to the Y178 also moderately impaired binding. However, none of the mutations within the C142-C155 loop of v (S144A, Q145A, D146A, D148A, D150A, Q152A, and G153A) disturbed fibrinogen binding to the receptor. View larger version (33K): [in this window] [in a new window]   Figure 3. Ligand-binding function of v3 mutants. (A) The binding of soluble fibrinogen and a ligand-mimetic mAb, WOW-1 Fab, to WT or mutant v3 were examined in the presence or absence of 0.5 mM MnCl2 by flow cytometry. For fibrinogen binding, washed cells were first incubated with 5 µg/mL mAb LM142 (specific for v) for 30 minutes on ice. After washing, cells with 0.5 mM MnCl2 were incubated with 150 µg/mL FITC-conjugated fibrinogen and PE-conjugated antimouse IgG (1:5 dilution) for 25 minutes at 22°C and then incubated with PI for 5 minutes at 22°C. After washing, fibrinogen binding (FL1) was analyzed on the gated subset of single, high v3 expression (FL2) and live cells (PI-negative, FL3) as indicated. (B) Relative amounts of fibrinogen binding are normalized to a 100% value for the binding to cells expressing WT v3 (% of WT). Fibrinogen binding in the presence of 1 mM RGDW was used as a negative control. Data represent the mean ± SE of 3 experiments. (C) WOW-1 Fab binding. For WOW-1 binding, cells with 0.5 mM MnCl2 were first incubated with 5 µg/ml WOW-1 Fab for 30 minutes at 22°C. After washing, cells were incubated with 5 µg/mL Alexa-conjugated antimouse IgG for 25 minutes on ice and then incubated with PI for 5 minutes at 22°C. After washing, bound antibodies were analyzed. WOW-1 binding to 293 cells was used as a negative control. Relative amounts of WOW-1 Fab binding are expressed by the following formula: % binding of WOW-1 Fab to WT v3/% binding of LM609 to WT v3. The vIIb mutant represents a chimera in which the C142-C155 loop in v was swapped with the corresponding sequence of IIb C146-C167. Data represent the mean ± SE of 3 experiments. To further examine the ligand-binding function of mutant v3, we next examined the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-v3 antibody. The Y178A mutation in v, as well as the D119Y mutation in 3, markedly inhibited WOW-1 binding to MnCl2-treated cells. On the other hand, swapping of the C142-C155 region of v with the corresponding region of IIb (C146-C167) did not show a marked inhibition of WOW-1 Fab binding. Because WOW-1 Fab is sensitive to changes in v3 affinity rather than avidity,28 these results suggest that the Y178A mutation disrupts the conformation of the ligand-binding pocket in v3. Because the integrin activator MnCl2 may have additional effects on cells, we cotransfected the WT or the mutant v with the T562N3 mutant, which constitutively activates 3 integrins and eliminates the need for MnCl2.33 As shown in Figure 4, T562N3 augmented fibrinogen binding when complexed with the WT v, but it failed to induce fibrinogen binding when complexed with the Y178Av. These results indicate that Y178 in the v subunit is critical for soluble ligand binding to v3. View larger version (19K): [in this window] [in a new window]   Figure 4. Effects of 3-activating mutant (T562N) on fibrinogen binding. WT () or Y178Av () construct was transiently cotransfected with WT or T562N3 construct into 293 cells. Fibrinogen binding to transfected v3 was determined by flow cytometry. Cells were first incubated with 5 µg/mL LM142 (specific for v) for 30 minutes on ice. After washing, cells were incubated with 150 µg/mL FITC-conjugated fibrinogen and PE-conjugated antimouse IgG for 25 minutes at 22°C in the presence or absence of 1 mM RGDW and then incubated with PI for 5 minutes at 22°C. After washing, cells expressing high levels of transfected v3 were analyzed. In these experiments, all were performed in the absence of MnCl2. The results are representative of 3 separate experiments. The Y178A mutation in v prevents the induction of LIBS epitopes by RGDW peptide To further clarify the effects of the Y178A mutation on the structure of v3, the reactivities of several mAbs (AP5, LIBS1, and LIBS6) specific for LIBS on 3 were examined in the absence of ligands. These LIBS epitopes are believed to be outside the ligand-binding pocket of the receptor. As shown in Figure 5A, there was no apparent difference in the reactivities of these mAbs between WT v3 and Y178Av3, suggesting that this mutation does not grossly alter the conformation of v3. The binding of activation-independent ligands, such as RGD peptides to v3, has been shown to induce LIBS expression on the receptor.39 Indeed, 1 mM RGDW increased the binding of all 3 LIBS mAbs to WT v3. However, it failed to induce LIBS expression on the Y178Av3 mutant. These data suggest that the Y178A mutation in v disturbs the binding of small and macromolecular RGD ligands to v3. View larger version (25K): [in this window] [in a new window]   Figure 5. LIBS expression on Y178Av3 mutant. WT or Y178Av construct was transiently cotransfected with WT 3 construct into 293 cells. Three different mAbs specific for 3 LIBS (AP5, anti-LIBS1, and anti-LIBS6) were employed to assess the LIBS expression, and LM609 (specific for v3) was employed to monitor the surface expression of transfected v3. (A) LIBS expression in the absence of RGDW peptide. Closed and open histograms represent the binding of anti-LIBS antibodies and control mouse IgG1, respectively. The results are representative of 2 separate experiments. (B) LIBS expression in the presence () or absence () of 1 mM RGDW peptide. Closed and open histograms represent the binding of anti-LIBS antibodies in the presence and absence of RGDW, respectively. The results are representative of 2 separate experiments. The Y178A mutation of v inhibits cell adhesion to immobilized fibrinogen Because immobilized vitronectin and fibrinogen are activation-independent ligands for v3, we further examined cell adhesion to immobilized ligands in the absence of integrin activation. Parent 293 cells showed marked adhesion to vitronectin probably via endogenous v1 (data not shown),36 whereas they showed only modest adhesion to fibrinogen even at a concentration of 10 µg/mL. Therefore, we examined the effect of the Y178Av3 on the cell adhesion to immobilized fibrinogen but not to vitronectin. Transfection of both WT v and 3 markedly increased the adhesion of the transfectants; transfection of WT 3 alone induced only a modest increase in adhesion at concentrations of 2.5 and 5 µg/mL (Figure 6). As compared with the WT 3 transfectant, Y178Av3 failed to increase cell adhesion to immobilized fibrinogen. However, the adhesion of F177A and W179Av3 mutants was only slightly impaired, especially at a relatively low concentration of fibrinogen (1.25 µg/mL). Thus, the effect of Y178A on binding of v3 to soluble fibrinogen is also observed with immobilized fibrinogen. View larger version (25K): [in this window] [in a new window]   Figure 6. Adhesion of v3 mutants to immobilized fibrinogen. WT or mutant v construct was transiently cotransfected with WT 3 construct into 293 cells. In WT 3 cells (), only WT 3 construct was transfected into cells. WT () or mutant v3-transfected cells were incubated for 60 minutes at 37°C with immobilized fibrinogen at serial concentrations. After washing with PBS, the adherent cells were quantified with a colorimetric reaction using endogenous cellular acid phosphatase activity. , Phe177Ala; , Tyr178Ala; , Trp179Ala; , untransfected cells. Data represent the mean ± SE of triplicate measures of optical density at 415 nm.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The aim of this study is to reveal the structural basis for the interaction between v3 and its ligands, especially ligand-binding sites in the v subunit of v3. In non-I-domain integrin  subunits, particularly the IIb subunit, several ligand-binding sites have been identified by the characterization of naturally occurring mutations in patients with Glanzmann thrombasthenia as well as mutagenesis analyses.15-19 However, ligand-binding regions of v remain elusive. To clarify the critical regions for ligand binding in the v subunit, we focused on the predicted W3 4-1 loop (C142-C155) and W3 2-3 loop (G172-G181) and investigated their role in ligand binding by alanine-scanning mutagenesis. The results demonstrate that Y178 in the W3 2-3 loop in v is one of the critical residues for ligand binding. In sharp contrast to IIb, none of the mutations in the predicted W3 4-1 loop impaired ligand binding, which suggests that there are key structural differences in the adhesive ligand-binding sites of v3 and IIb3. The differences in the locations of the ligand-binding sites between v and IIb in the -propeller model are summarized and illustrated in Figure 7. View larger version (38K): [in this window] [in a new window]   Figure 7. Critical residues for ligand binding in the  subunits of 3 integrins. (A) Comparison of critical residues for ligand binding in v with those in IIb. In IIb multiple residues (underlined) critical for ligand binding have been identified in both the W3 4-1 and W3 2-3 loops. In sharp contrast, in v only Y178 within the W3 2-3 loop is critical for ligand binding. The figures in panels B and C are adapted from a -propeller model proposed by Springer,20 and they show the location of these critical residues. The view is shown from the top (B) and from the side (C). There is mounting evidence that the predicted loops between N-terminal repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop) are important for ligand binding in non-I-domain subunits.18,40-43 Previous studies have shown that the N-terminal one-third of the  subunit regulates ligand-recognition specificity of 3 integrins44 and that residues 139-167 in v corresponding to the W3 4-1 loop are a cross-linking site for RGD peptides.21 However, the v Y178 identified in this study is located in the predicted W3 2-3 loop, and none of the alanine substitutions within the W3 4-1 loop examined impaired ligand binding. The inhibition of ligand binding by alanine substitutions at the residues adjacent to Y178 (F177 and W179) and the failure of the induction of 3 LIBS with RGDW, even at high concentrations, provide further support for the critical role of Y178 in ligand binding. Although the W3 4-1 loop and W3 2-3 loop are separated in the primary structure, the proposed propeller model suggests that these regions are close to each other in 3-dimensional space and may explain the apparent discrepancy between the previous cross-linking study and the present study (Figure 7). Interestingly, many critical residues identified in non-I-domain  subunits have aromatic side chains, suggesting that in addition to oxygenated residues such as D163 in IIb,16 aromatic residues are important for ligand binding.45 The corresponding residues to the v Y178 in IIb (Y190),18 4 (Y187),42,43 and 5 (F187),43 and the adjacent residues Y186 and W188 in 340 have been demonstrated to be critical for ligand binding, although the role of W3 2-3 loop in 3 is still controversial.41 In contrast, the role of W3 4-1 loop appears to be different between integrin  subunits. D163 in IIb and threonine (T)162 and G163 in 3, which are located in the predicted W3 4-1 loop, appear critical for ligand binding, whereas the W3 4-1 loop in 4 and in v (this study) do not. More recently it has been demonstrated that the replacement of the W3 4-1 loop in v with the corresponding loop in 5 did not disturb ligand binding but changed ligand-recognition specificity.46 Our swapping mutagenesis of the W3 4-1 loop of v with the corresponding region of IIb did not abolish WOW-1 Fab binding, suggesting that the W3 4-1 loop is not critical for ligand-recognition specificity between v3 and IIb3. WOW-1, a monovalent ligand-mimetic antibody, was created by replacing the H-CDR3 of PAC-1 Fab with a single integrin-binding domain of the adenovirus penton base, a viral coat protein that consists of 5 subunits, each containing an integrin-binding RGD motif. Penton base is known to facilitate adenovirus internalization through v integrins, particularly v3 and v5.47 WOW-1-like penton base recognizes that activated state of v3 and v5. Using mAb B5-IVF2 specific for 5, we have determined that 293 cells express 5 as well as v (S.H. and Y.T., unpublished data, June 1999). Thus, WOW-1 Fab might bind to v3-transfected 293 cells through v5 as well as v3. Nonetheless, the bulk of WOW-1 Fab binding to transfected cells appeared to be through v3 because antibody binding was largely abolished by the Y178A substitution in v or the D119Y substitution in 3. Integrin activation encompasses at least 2 events: (1) modulation of receptor affinity through conformational changes and (2) modulation of receptor avidity through facilitation of lateral diffusion and/or clustering of heterodimers. The binding of monovalent ligand WOW-1 Fab to v3 likely reflects affinity modulation, whereas the binding of multivalent ligand, such as fibrinogen, likely reflects both affinity and avidity modulation.28 Our data with WOW-1 Fab suggest that the Y178A substitution disturbs integrin affinity modulation rather than avidity modulation. In addition to ligand binding, cell adhesion to immobilized ligand can be strongly influenced by post-ligand-binding events. Indeed, in spite of the moderate inhibition of soluble fibrinogen binding by F177A as well as W179A substitution, these substitutions induced only a modest inhibition of cell adhesion to immobilized fibrinogen. One could argue the possibility that the Y178A substitution in v may disturb conformational changes from resting to activated states of integrin because of the failure of the induction of 3 LIBS with RGDW.48 However, this possibility is unlikely because the Y178A substitution in v completely abolished the interaction with immobilized fibrinogen, an activation-independent ligand. Thus, this residue is likely involved in direct contact with the ligand. It would be interesting to know whether the Y178A substitution may affect divalent cation binding. Recently, employing human-to-mouse chimeras, Puzon-McLaughlin et al49 localized binding sites for ligand-mimetic murine mAbs against IIb3 and demonstrated that the involvement of several discontinuous sites in both IIb and 3 is unique to ligand-mimetic antibodies. Involvement of certain residues in both v (Y178) and 3 (D119) subunits in WOW-1 binding is consistent with their data, and these residues may participate in a ligand-binding pocket in v3. The v3 is expressed in a number of cell types: endothelial cells, arterial smooth muscle cells, platelets, subpopulation of leukocytes, osteoclasts, and tumor cells. The v3 is involved in cell adhesion, proliferation, and migration and has been shown to play a crucial role in tumor angiogenesis, intimal hyperplasia after arterial injury, wound healing, and osteoporosis in the adult organism. Recently, human clinical trials are in progress to evaluate the effects of the humanized anti-v3 mAb in patients with late-stage cancer.50 The present results provide new information concerning the interaction between RGD ligands and the non-I-domain v subunit as well as key structural differences between v3 and IIb3 with regard to ligand binding. This information may facilitate the development of novel antagonists specific for v3.     Acknowledgments We thank Dr David Cheresh for mAbs LM142 and LM609 and the vector containing WT v cDNA; Dr Mark Ginsberg for mAbs anti-LIBS1 and anti-LIBS6; Dr Thomas Kunicki for a mAb AP5; Dr Peter Newman for a mAb AP3; Dr Gilbert White for the vector containing WT 3 cDNA; Dr Martin Hemler for a mAb B5-IVF2; and Dr Jiro Seki for RGDW peptide.     Footnotes Supported by a grant from the Ministry of Education, Science and Culture, Tokyo, Japan; a grant from the Japan Society for the Promotion of Science, Tokyo, Japan; a grant from the Senri Life Science Foundation, Osaka, Japan; a grant from the Yamanouchi Foundation for Research on Metabolic Disorders, Tokyo, Japan; Welfide Medical Research Foundation, Osaka, Japan; and grant HL56595 from the National Institutes of Health, Bethesda, MD. Submitted July 6, 2000; accepted September 14, 2000. 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