Eur J Biochem 271, 3729–3740 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04313.x Integrin receptor specificity for human red cell ICAM-4 ligand Critical residues for aIIbb3 and aVb3 binding Patricia Hermand1, Pierre Gane1, Isabelle Callebaut2, Nelly Kieffer3, Jean-Pierre Cartron1 and Pascal Bailly1 INSERM U76, Institut National de la Transfusion Sanguine, Paris, France; 2De´partement de Biologie Structurale, LMCP, CNRS UMR 7590, UP6/UP7, Paris, France; 3Laboratoire de Biologie et de Physiologie inte´gre´e (CNRS/GDRE-ITI), Universite´ du Luxembourg, Luxembourg The red cell intercellular adhesion molecule-4 (ICAM-4) binds to different members of the integrin receptor families To better define the ICAM-4 integrin receptor specificity, cell transfectants individually expressing various integrins were used to demonstrate that aLb2, aMb2, and aIIbb3 (activated) bind specifically and dose dependently to the recombinant ICAM-4-Fc protein We also show that cell surface ICAM-4 interacts with the cell surface aVb3 integrin In addition, using a a4b1 cell transfectant and b2 integrin-deficient LAD cells, we show here that ICAM-4 failed to interact with a4b1 even after a4b1 activation by phorbol ester or with the monoclonal antibody TS2/16 (+ Mn2+) ICAM-4 amino acids that are critical for aIIbb3 and aVb3 interaction were identified by domain deletion analysis, site-directed muta- The LW (Landsteiner–Wiener) blood group glycoprotein has been renamed ICAM-4 (CD242) based on sequence similarities with the family of intercellular adhesion molecules (ICAMs) [1], a group of molecules which plays a crucial role in cell–cell interactions in the immune system [2–4] ICAM genes are all clustered on chromosome 19 except ICAM-2 which is located on chromosome 17 All ICAM proteins exhibit several Ig-like domains, two for ICAM-4 and ICAM-2 They bind to ab-heterodimeric integrins and have a differential pattern of expression and cellular distribution [5,6] Data consistent with this first showed that ICAM-4 binds to the integrins aLb2 (LFA-1, CD11a/CD18) and aMb2 (Mac1, CD11b/CD18) [1,7] Based on a three-dimensional model of ICAM-4 deduced from the crystal structure of ICAM-2 [8] and using mutated chimeric ICAM-4-Fc proteins, we have previously shown that aLb2 binds to the first Ig-domain of ICAM-4, whereas the aMb2 binding site encompassed both Ig-domains [9] These interactions were also reported to Correspondence to J.-P Cartron, INSERM U76, INTS, rue Alexandre Cabanel, 75015 Paris, France Fax: +33 43 06 50 19, Tel.: +33 44 49 30 00, E-mail: cartron@idf.inserm.fr Abbreviations: aIIbb3 CHO*, activated aIIbb3 CHO cells; aIIbb3*, activated aIIbb3 integrin; LAD, leukocyte adhesion deficiency; L-cells, L929 mouse fibroblast cells; LW, Landsteiner–Wiener; mAb, monoclonal antibody; MAdCAM-1, mucosal addressin cell adhesion molecule 1; PMA, 4b-phorbol 12-myristate 13-acetate; RBC, red blood cell; VCAM-1, vascular cell adhesion molecule (Received July 2004, accepted 29 July 2004) genesis and synthetic peptide inhibition Our results provide evidence that the b3 integrin binding sites encompass the first and second Ig-like domains of ICAM-4 However, while the aIIbb3 contact site comprises the ABED face of domain D1 with an extension in the C¢-E loop of domain D2, the aVb3 contact site comprises residues on both faces of D1 and in the C¢-E loop of D2 These data, together with our previous results, demonstrate that different integrins bind to different but partly overlapping sites on ICAM-4, and that ICAM-4 may accommodate multiple integrin receptors present on leukocytes, platelets and endothelial cells Keywords: adhesion; ICAM-4; integrins; site-directed muta1 genesis; structure model be highly sensitive to the presence of divalent cations for high affinity binding to b2 integrins In parallel, it was established that ICAM-4 on red blood cells (RBCs) and transfected fibroblasts interacted specifically with recombinant and purified I-domains of aLb2 and aMb2 integrins [10] More recently, using ICAM-4 positive and negative RBCs, and normal or type-I Glanzmann’s thrombastenia platelets lacking b3 integrins, we identified the platelet fibrinogen receptor integrin aIIbb3 (GPIIb-IIIa, CD41/ CD61) in its high affinity state as the receptor for RBC ICAM-4, suggesting a potential physiological significance of the ICAM-4 mediated RBC–platelet interaction in hemostasis and thrombosis [11] Other studies, based on adhesion of hemopoietic and nonhemopoietic cells, reported that ICAM-4 might also interact with aVb1 (CD51/ CD29) and aVb5 integrins [12] and that the binding site for these integrins is distinct but adjacent to binding sites for b2 integrins [13] However, controversy still exists regarding the ligand specificity of a4b1, as ICAM-4 was considered as a ligand for this integrin by some authors [12] but not by others [7,9] As some of these results were obtained with erythroid and non-erythroid cell lines that display a large number of integrins, we have developed cell adhesion assays using stable recombinant cell lines expressing unique human integrins obtained by cotransfection with appropriate cDNA encoding the a- and b-subunits We also used a permanent cell line, established from a patient with leukocyte adhesion deficiency [14], lacking b2 integrins but strongly expressing the a4b1 integrin Moreover, using ICAM-4 mutants generated by site-directed mutagenesis of 3730 P Hermand et al (Eur J Biochem 271) surface exposed residues as well as synthetic peptides of ICAM-4, we have identified critical amino acids involved in the binding sites of aIIbb3 and aVb3 integrins, and shown by synthetic peptide inhibition that ICAM-4–b3 integrin interactions might be modulated, at least in vitro Experimental procedures Reagents Specific monoclonal antibodies (mAbs) used include clones Lia1/2, 7E4 and SZ21 specific for the b1-chain (CD29), b2-chain (CD18), and b3-chain (CD61), respectively; clones 25.3.1, BEAR-1, AMF7, HP2/1, SAM1 and SZ22 recognizing the aL-chain (CD11a), aM-chain (CD11b), aV-chain (CD51), a4-chain (CD49d), a5-chain (CD49e) and aIIb-chain (CD41) in the absence of the b3-chain, respectively, which were purchased from Coulter/Immunotech (Marseille, France) mAb AP-2 specific for an epitope of the complex aIIbb3 integrin came from GTI (Brookfield, WI, USA) Stimulatory antib1 (CD29) mAb TS2/16 came from Endogen (Woburn, MA, USA) Murine mAbs BS46/56 to ICAM-4/LWab were described previously [15] ImmunoPure mouse IgG and human Fc fragment came from Pierce (Rockford, IL, USA) and ImmunoReseach-Jackson (West Grove, PA, USA), respectively The human plasma fibronectin, and fibrinogen depleted of vWF and fibronectin were purchased from Life Technologies (Cergy Pontoise, France) and Enyme Research Laboratories Inc (South Bend, IN, USA), respectively Calcein AM was purchased from Molecular Probes (Eugene, OR, USA) Peptides Gly65–Val74 (residues 65–74 of ICAM-4, hereafter called G–V), and peptide Phe26–Ser40 (residues 26–40 of ICAM-4, hereafter called F–S) and corresponding random peptides were synthesized and purified by Neosystem (Strasbourg, France) Soluble recombinant Fc proteins Chimeric ICAM-pIgI constructs derived from intact or mutagenized ICAM-4 (LWa allele) carrying the two Ig-like domains D1 and D2 (residues 1–208) and deletion mutants D1-ICAM-4 (residues 1–101) or D2-ICAM-4 (residues 102– 208) were used to produce soluble Fc-fusion proteins in COS-7 cells as described [9] Similarly, ICAM-1- and ICAM-2-pIgI constructs (kindly provided by D Simmons and E Ferguson, University of Oxford, UK) were used to produce ICAM-1- and ICAM-2-Fc proteins Human vascular cell adhesion molecule (VCAM-1)-Fc was purchased from R & D Systems Europe (Abingdon, Oxfordshire, UK) Transfectants, cell lines and adhesion assays L cell transfectants expressing a4b1 or aVb3 integrins were generated by cotransfection into L929 mouse fibroblast cells (hereafter called L cells) of the CD49d (a4-chain)-pFNEO [16] plus the CD29 (b1-chain)-pECE [17], or the CD51 (aV-chain)-pcDNA I NEO [18] plus the CD61 (b3-chain)pcDNA3.1 expression constructs, respectively Stable expression of ICAM-4 in L cells was performed by Ó FEBS 2004 transfecting the cells with pcDNAI-ICAM-4 (LWa) [19] Transfectants resistant to G418 (geneticin) were selected by immunomagnetic separation for a4b1 or aVb3 integrin expression or ICAM-4 expression Stable clones were isolated and the antigen expression of CD49d, CD51 and ICAM-4 was analysed by flow cytometry, as reported [11] L cell transfectants expressing aLb2 or aMb2 integrins and the unactivated or dithiothreitol-activated aIIbb3 CHO cell transfectants (activated cells hereafter referred to as aIIbb3 CHO* and activated integrin as aIIbb3*) were also used in these studies [9,11] All transfectants were grown in Iscove’s modified Dulbecco medium with Glutamax-1 (Life Technologies, Inc.) supplemented with G418 600 lgỈmL)1 and 1% (v/v) amphotericin-B/penicillin/streptamycin and 7,8 10% (v/v) fetal bovine serum Briefly, for cell adhesion assays to immobilized proteins under static conditions, indicated amounts of purified wild type or mutant ICAM-Fc, VCAM-1-Fc, fibrinogen and fibronectin in 25 mM Tris, pH 8.0, 150 mM NaCl, mM MgCl2 plus mM CaCl2 were adsorbed overnight to flatbottom 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) The wells were then blocked for h at 22 °C with 1% (w/v) nonfat milk in the same buffer and washed once with binding buffer A (RPMI containing 10 mM Hepes, mM MgCl2 and mM CaCl2) Transfectants (1 · 105 per well) in 100 lL of binding buffer were added to washed protein coated wells for 45 at 22 °C before washing to remove nonadherent cells and carry out microscopic observation, as described previously [9,11] For cell–cell adhesion assays under static conditions, parental L cells and transfected aVb3 L cells (5 · 104 per well) resuspended in cell culture medium, were added to each well Twenty-four hours later, the wells were washed three times with binding buffer A before adding parental L cells and ICAM-4 L cell transfectants (1 · 105 per well in 100 lL of buffer A) labeled with calcein AM, as described previously [20] After 45 at 22 °C, nonadherent cells were removed as indicated above using NaCl/Pi solution containing MgCl2 and CaCl2 Labeled cells that adhered to the well walls were recovered by vigourous shaking and counted by flow cytometry as described [11] For blocking experiments, transfectants or protein coated wells were pretreated for 30 at room temperature with specific mAbs (25 lgỈmL)1) or peptides (31–250 lM final concentration) The Epstein–Barr virus-transformed B lymphoblastoid cell line established from a patient with leukocyte adhesion deficiency (LAD) (gift of C Figdor, Netherland Cancer Institute, Amsterdam, the Netherlands) [14] was grown in RPMI with 10% (v/v) fetal bovine serum LAD cells (1 · 105 per well) in 100 lL of binding buffer A supplemented with 5% (v/v) fetal bovine serum were used in cell adhesion assays as indicated above For blocking experiments, cell lines were pretreated with specific mAbs (25 lgỈmL)1) For phorbol ester activation, cells were pretreated for 15 at 37 °C with 80 lM 4b-phorbol 12-myristate 13-acetate (PMA; Sigma), as described [12] For mAb anti-b1 (TS2/16) activation [21], cells were washed twice in Hank’s balanced salts (Sigma–Aldrich), then resuspended in Hank’s balanced salts containing 1.0 mM Mn2+ and the TS2/16 mAb (final concentration 1.0 lgỈmL)1) for 30 at room temperature Ó FEBS 2004 ICAM-4–b3 integrin interaction (Eur J Biochem 271) 3731 Table Integrin profiles of cell lines Integrin subunit expression was quantified by analysis using flow cytometry Cell lines were stained with mAbs anti-CD49d (HP2/1), anti-CD49e (SAM1), anti-CD51 (AMF7), anti-CD41 (SZ22), anti-CD11a (25.3.1) and anti-CD11b (BEAR-1) followed by phycoerythrin-goat anti-mouse IgG (Fab¢2) fragments The estimated number of copies per cell, measured as specific antibody binding capacity per cell, was deduced from a calibration curve obtained with Qifikit calibration beads (Dako, Denmark) Transfectants a4b1 L cell aLb2 L cell aMb2 L cell aVb3 L cell aIIbb3 CHO LAD cells Integrin a-subunits expressed Copies per cell a4(CD49d) aL(CD11a) aM(CD11b) aV(CD51) aIIb(CD41) 10 32 46 62 18 a4(CD49d) aL(CD11a) aM(CD11b) a5(CD49e) aV(CD51) aIIb(CD41) Cell lines 40 000 < 300 < 300 1100 1300 < 100 200 000 000 300 600 Results Characterization of the integrin transfectants and cell lines We first established a stable transfectant expressing human a4b1 or aVb3 that was obtained by cotransfection of CD49d (a4-chain) plus CD29 (b1-chain) or CD51 (aVchain) plus CD61 (b3-chain), respectively, into L cells Then, all transfectants, including those established earlier and expressing aLb2, aMb2 and aIIbb3 [9], as well as the LAD cell line, were characterized by flow cytometry analysis with specific mAbs Each transfectant reacted strongly with the respective mAbs against the transfected Fig Specific adhesion of b-integrin transfectants to ICAM-4-Fc (A) Dose-dependent cell adhesion aIIbb3-CHO* transfectant ( ), L cell transfectants expressing a4b1 (d), aMb2 (,), aLb2 (n) or aVb3 (e) Controls (Ctrl.) including parental L cell (h), parental CHO cell 24 dithiothreitol-treated or not, and native aIIbb3-CHO transfectant (s) to ICAM-4-Fc coated to plastic wells at varying concentrations The results are expressed as mean percentage of bound cells ± SEM of three experiments (B) Effect of mAbs on adhesion of L cell transfectants expressing aLb2, aMb2, aVb3 and aIIbb3 CHO*-transfectant (dithiothreitol-activated) to coated ICAM-4-Fc (500 ngỈwell)1) Cell transfectants were pretreated or not with saturating concentrations of indicated mAbs specific for the a- or b-chains of each integrin (see text for specificities) and ICAM-4 (BS46/56) Controls included unrelated mouse IgG antibody (ctrl.IgG) and wells without Fc-protein The results are expressed as the relative percentage of bound transfectants, where 100% is calculated from the total number of transfectants bound to ICAM-4-Fc without mAb The mean ± SEM from three experiments is shown By Student’s t-test analysis, ***P < 0.001, and *P < 0.05 human a-chains, and the estimated copy number for each cell type was determined (Table 1) Parental L cells and CHO cells were nonreactive with the mAbs used (not shown) As expected, LAD cells that lack the b2-chain (CD18) had no detectable surface expression of aL and aM integrins (Table 1) LAD cells were negative for aIIband aV-chains but had a high copy number of a4-chain, and the b1 integrin was also strongly expressed (not shown) These findings indicate that a4b1 integrin is the predominant ab heterodimer expressed on LAD cells Ó FEBS 2004 3732 P Hermand et al (Eur J Biochem 271) Adhesion of integrin transfectants to ICAM-4-Fc Cell adhesion assays of parental and transfected L or CHO cells to the chimeric ICAM-4-Fc protein coated on plastic were performed in the presence of Mg2+ and Ca2+ (Fig 1A) Moreover, dithiothreitol activation of the aIIbb3 integrin (aIIbb3*) was performed as reported [11] All transfectants dose-dependently adhered to coated ICAM4-Fc, except a4b1 L cells, nonactivated aIIbb3 CHO cells and all parental cells whether treated or not with dithiothreitol (Fig 1A) For all transfectants that bound to ICAM-4-Fc, a plateau was reached at 500 ngỈwell)1 For all cell clones tested, the extent of adhesion correlated with the level of integrin expression and the relative binding: aVb3 > aIIbb3* > aMb2 > aLb2 In contrast, despite their high copy number of a4-chains, a4b1-transfected L cells did not bind to ICAM-4 (Fig 1A) In order to control the specificity of the ICAM-4–integrin interactions, the effect of different mAbs on the adhesion of transfectants to coated ICAM-4-Fc was investigated (Fig 1B) mAbs against either the a- or b-chains, or against ICAM-4 blocked approximately by 50–70% the binding of aLb2, and aMb2 L cells Binding of aIIbb3 CHO* cells was blocked (70%) by mAb AP2 against a complex-specific epitope of the aIIbb3 integrin and by mAbs BS46/56 (50%) against ICAM-4, but was not blocked by other mAbs, as reported previously [11] In contrast, mAb SZ21, specific for the b3-chain alone, did not block binding of aIIbb3 CHO* cells, but efficiently inhibited (60%) binding of aVb3 L cells to ICAM-4 Similarly, mAbs AMF7 and BS46/56 against aV-chain and ICAM-4, respectively, inhibited binding of aIIbb3 CHO* cells These data strongly support a specific interaction between ICAM-4 and aVb3 As expected, control mouse IgG had no effect As a specific interaction between red cell ICAM-4 and platelet aIIbb3 was demonstrated previously [11], we provide further evidence that cell surface ICAM-4 might interact with cell surface aVb3 Accordingly, a cell–cell adhesion assay using ICAM-4 and aVb3 L cell transfectants, was performed Figure shows that ICAM-4 L cells did not bind to immobilized parental L cells, but did bind to immobilized aVb3 L cells This cell–cell adhesion was efficiently blocked ( 57%) by BS56 mAb specific for ICAM-4 while control mouse IgG had no effect As another control, parental L cells did not bind significantly to immobilized aVb3 L cells, as expected Altogether, our findings indicate that ICAM-4 interacts specifically with the four integrins aLb2, aMb2, aIIbb3* and aVb3 but not with the a4b1 integrin This raised the possibility that a4b1 might need some activation inducing a favorable conformational change to interact with ICAM-4 To test this hypothesis, the effect of PMA stimulation [22] and of antibody activation on a4b1mediated cell adhesion [21,23] was investigated As shown in Fig 3A, after activation with either PMA or mAb TS2/16 (+ Mn2+), a4b1 L cells still failed to bind to coated ICAM-4-Fc, while both native and stimulated a4b1 L cells showed adhesion to coated VCAM-1-Fc (from 10 to 32%) and fibronectin (from 27 to 39%), two well known ligands of a4b1 [16,24,25] PMA pretreatment had a 1.4-fold stimulatory effect on adhesion to fibronectin, but no significant enhancement on adhesion to VCAM-1-Fc By contrast, stimulation by mAb TS2/16 (+Mn2+) enhanced adhesion to VCAM-1-Fc and fibronectin by 3.2- and 1.2-fold, respectively We also found that even under experimental conditions (buffer composition and cations) similar to those described by Mankelow et al [13], a4b1 L cells did not react with ICAM-4-Fc (not shown) As a control for these experiments, no binding of native or treated parental L cells to coated ICAM-4-Fc, VCAM-1-Fc and fibronectin was observed Adhesion of LAD cells to ICAM-4-Fc Fig Adhesion of ICAM-4 L cell transfectant to immobilized aVb3 L cell transfectant ICAM-4 L cells (black bars) or parental L cells (white bar) (105 cellsỈwell)1) labeled with the fluorescent dye calcein (#) were deposited onto confluent aVb3 L cell or parental L cell monolayers ICAM-4 L cells, either native or pretreated with saturating concentrations of mAbs to ICAM-4 (BS56) or with unrelated mouse IgG antibody (ctrl.IgG), were used At the end of the incubation period and after washings, adherent calcein-labeled cells were collected and counted by flow cytometry analysis The results are expressed as mean percentage of bound cells ± SEM of three experiments By Student’s t-test analysis, *P < 0.05 To obtain further evidence that ICAM-4 may or may not interact with a4b1, we examined the binding of LAD cells, which strongly express a4b1, but not b2 and b3 integrins (Table 1), to ICAM-4, VCAM-1 and fibronectin Figure 3B shows that whatever the experimental conditions, including activation by PMA or mAb TS2/16, LAD cells did not bind to ICAM-4-Fc, but efficiently bound to VCAM-1-Fc (39–79%) and fibronectin (11–25%) In the presence of mAb TS2/16, LAD cell adhesion to VCAM-1 was stimulated twofold, whereas PMA-stimulated cell adhesion to fibronectin was only 2.2-fold LAD cells carry a4-, b1-, a5and aV-chains (Table 1) and therefore probably express the three integrins a4b1, a5b1 and aVb1 Figure 3B shows that binding of LAD cells to VCAM-1 was almost completely blocked by mAbs specific for a4-chain (clone HP2/1) or Ó FEBS 2004 ICAM-4–b3 integrin interaction (Eur J Biochem 271) 3733 Fig Adhesion of a4b1 L cell transfectant and LAD cells to CAM-Fc and fibronectin (A) Adhesion of a4b1 L cells (gray bars) and parental L cell (black bars) to coated ICAM4-Fc, VCAM-1-Fc and fibronectin (FN) (500 ngỈwell)1) in binding buffer A containing mM MgCl2 plus mM CaCl2 Cells were untreated (left), pretreated with 4b-phorbol 12-myristate 13-acetate (PMA) (middle) or used under optimal binding conditions in the presence of mAbTS2/16 and 1.0 mM Mn2+ (right) ***P < 0.001 vs parental L cell (B) Adhesion of LAD cells to coated ICAM-4-Fc (black bars), VCAM-1-Fc (hatched bars) and fibronectin (FN, white bars) in binding buffer A containing mM MgCl2 plus mM CaCl2 Cells were pretreated or not with saturating concentrations of indicated mAbs specific for a4-chain (HP2/1), b1-chain (Lia1/ 2) (left), pretreated with PMA (middle) or used under optimal binding conditions as indicated (right) Control including unrelated mouse IgG antibody and Hank’s balanced salts binding buffer without cations and stimulatory mAb The results are expressed as mean percentage of bound cells ± SEM of three experiments b1-chain (clone Lia1/2), whereas binding to fibronectin was only weakly affected As a control, mouse IgG had no effect These data provide clear evidence that, native and stimulated L cell transfectants or LAD cells expressing an active form of a4b1 integrin are unable to interact with ICAM-4, although they interact with VCAM-1 and fibronectin Identical results were obtained when the same buffer and cation composition described by Mankelow et al [13] was used (not shown) b3 Integrin binding sites on ICAM-4 Previous studies from our laboratory have shown that the b2 integrins aLb2 and aMb2, interact with ICAM-4 through distinct contact sites [9] The fact that mAb SZ21 did not block aIIbb3* binding to ICAM-4 but inhibited aVb3 binding suggested that these integrins may also establish distinct contact sites with ICAM-4 To determine which ICAM-4 Ig domains contain aIIbb3 and aVb3 binding sites, ICAM-4 domain deletion mutants lacking either domain D1 or D2 were used in cell adhesion assays The binding of aIIbb3 CHO* and aVb3 L cells required the presence of both extracellular Ig domains because the absence of either domain D1 or D2 was associated with a 55% decrease in adhesion (Fig 4) To better define the binding sites of both b3 integrins, we tested 32 ICAM-4-Fc mutant proteins, obtained by site-directed mutagenesis of the most prominent surface exposed residues, which could participate in molecular interactions The mutated residues are mainly located on the ABED and CFG faces of domain D1 and on the C¢-E loop of domain D2, and have previously been used to define critical amino acids involved in the interaction between ICAM-4 and b2 integrins [9] As shown in Fig 4A, aIIbb3 CHO* cell adhesion to six ICAM-4-Fc mutants, namely the mutants Gln30 fi Ala, Gly32 fi Ala, Lys33 fi Ala, Gln36 fi Ala, Trp77 fi Ala and Glu151 fi Ala, was reduced by more than 55% as compared to wild type ICAM-4 Five out of the six mutated residues were located in domain D1, in the A-B loop (Gln30 fi Ala, Gly32 fi Ala), at the beginning of the B strand (Lys33 fi Ala, Gln36 fi Ala) and in the middle of the E-F loop (Trp77 fi Ala), while the sixth residue (Glu151 fi Ala) was located in domain D2, at the top of the C¢-E loop Based on these results, we conclude that the six amino acids critical for ICAM-4 binding to aIIbb3* integrin are spatially close and located at the bottom of the ABED face of domain D1 with an extension in the domain D2 through residue Glu151 (Fig 5, left) Ten mutations located within domain D1 in the B and E strands at positions Lys33, Trp66, Tyr69 and Asp73, in the C, F and G strands at positions Arg52, Leu80 and Arg97, in the E-F loop at position Trp77 and in the C¢-E loop of domain D2 at positions Glu151 and Thr154 caused a reduced adhesion of aVb3 L cells by at least 40% (Figs 4B 10 and 5, right) These data suggest that the aVb3 binding site on ICAM-4 comprises residues on both the ABED and CFG faces of domain D1 and in the C¢-E loop of domain D2 (Fig 5, right) In addition, the three-dimensional Ó FEBS 2004 3734 P Hermand et al (Eur J Biochem 271) Fig Adhesion of aIIbb3 CHO* and aVb3 L-cell transfectants to ICAM-4 mutants Equivalent amounts of wild type ICAM-4-Fc, ICAM-4-Fc domain deletion mutants (D1 or D2) and 32 ICAM-4-Fc mutants (indicated using one letter amino acid code), were coated on plastic well (500 ngỈwell)1) and used in a cell adhesion assay with aIIbb3 CHO* (dithiothreitol-activated) (A) and aVb3 L cells (B) Negative control (Ctrl.) was carried out in the absence of ICAM-4-Fc protein The results are expressed as in Fig 1B The mean ± SEM from three determinations is shown ICAM-4 model reveals that six of 10 critical residues are clustered between the CFG face of D1 and the C¢-E loop of 11 D2 which are spatially close (Fig 5, far-right at 90°) Regarding residues 97 and 151, we found that substitution of a charged residue (Glu or Arg, respectively) by the opposite charge had little or no effect on cell adhesion, whereas substitution by an apolar amino acid (Ala) inhibited adhesion Moreover, replacement of the aromatic residue (Trp) at position 77 by a small apolar (Ala), but not by another aromatic residue (Phe), reduced adhesion of aIIbb3 CHO* cells, although this may not be true for avb3 L cell adhesion (Fig 4) The replacement of the apolar residue (Leu) at position 80 by another apolar residue (Gly) had no effect, whereas substitution by an aromatic residue (Phe) severely reduced avb3 L cell adhesion These observations suggest the importance of the surface exposed residues over their electric charge or hydrophobicity in ICAM-4/b3 integrin interaction, as already found for b2 integrin interaction [9] Inhibition of ICAM-4–b3 integrin interaction by ICAM-4 peptides To confirm the results of site-directed mutagenesis, blocking experiments with ICAM-4 synthetic peptides (31–250 lM) covering the binding regions involved in b3 integrin interaction were performed We used the Phe26–Ser40 peptide (F–S) covering residues Gln30, Gly32, Lys33 and Gln36 involved in aIIbb3* interaction, and the Gly65–Val74 peptide (G–V) covering residues Trp66, Tyr69 and Asp73, involved in aVb3 interaction Figure 6A shows that adhesion of aIIbb3 CHO* cells to ICAM-4 was efficiently inhibited by 63% and 78% with the F–S and G–V peptides (125 lM), respectively, whereas corresponding random peptides used as controls had no effect The G–V peptide inhibited adhesion of aIIbb3 CHO* cells to fibrinogen by 64%, whereas the F–S peptide did not Because the G–V peptide harbors a Gln-X-X-Asp-Val motif involved in the aIIbb3*–fibrinogen interaction [26] and because mutations at position 69 and 73 did not affect adhesion of aIIbb3 CHO* cells to ICAM-4 (Fig 4A), we conclude, contrary to our previous speculation [11], that the aIIbb3* binding site resides along the A-B strand (peptide F–S) but not on the E strand (peptide G–V) in domain D1 of ICAM-4 In addition, F–S peptide inhibited the binding of aIIbb3 CHO* cells to ICAM-4 in a concentrationdependant manner (Fig 6, bottom) Adhesion of aVb3 L cells to ICAM-4 was efficiently inhibited by 55% and 90% with 125 and 250 lM of the G–V peptide, respectively, whereas the F–S peptide and corresponding random peptides had no inhibitory effect (Fig 6B) These data are consistent with those of site-directed mutagenesis (Fig 4B) and strongly suggest that the aVb3 binding site partly resides along the E strand Discussion Current experimental evidence suggests that red cell ICAM-4 is a ligand for b2 integrins, which is of potential significance in a variety of physiological processes, including erythropoiesis and red cell destruction [7,9,11,27] The physiological relevance of these molecular interactions is also supported by the fact that, under depressed venous shear rates, adhesion of RBCs to activated adherent neutrophils is mediated through aMb2–ICAM-4 interaction [28] More recently, we found that ICAM-4 interacts with the activated platelet fibrinogen receptor, integrin aIIbb3* [11], suggesting that this interaction might be responsible for platelet–red cell aggregate formation in patients with sickle cell anemia, further contributing to the vaso-occlusion events characteristic of this disease [29,30] Here, we have confirmed and further extended these findings by providing the first demonstration that human aVb3 integrin expressed in L cells functions as a receptor for recombinant ICAM-4 protein and ICAM-4 expressed in L cells, thus suggesting a potential role in the physiology and pathology implicating endothelial cells and platelets Indeed, preliminary data indicate that ICAM-4 might be the sickle red cell protein that mediates binding to endothelial aVb3 12 integrin through a protein kinase A-dependent signaling pathway [31] and that mAbs specific to aVb3 inhibit sickle red blood cell–endothelium interaction induced by plateletactivating factor [32] Our current results extend the data from Spring et al [12] who showed that ICAM-4 is a novel ligand for the aV integrin family, although their studies only identified aVb1 and aVb5, but not aVb3, as ICAM-4 receptors Ó FEBS 2004 ICAM-4–b3 integrin interaction (Eur J Biochem 271) 3735 Fig Critical residues of ICAM-4 involved in interaction with b3 integrins Three-dimensional representation of an ICAM-4 model constructed on the basis of ICAM-2 structure A ribbon representation of the secondary structures of the ICAM-4 model is shown inside its transparent solventaccessible surface Each domain (D1 and D2) shows two faces formed by the ABED strands of one antiparallel b-sheet and the CFG strands of the second antiparallel b-sheet Each strand is labeled by an italic capital letter Amino acids are designated by a one letter code and residue numbers The solvent-accessible surfaces of critical amino acid residues involved in the ICAM-4–aIIbb3 (left side) and –aVb3 interactions (right side, two views at 90° to each other) are colored (red, acidic residues Q30, Q36, D73, E151; blue, basic residues K33, R52, R97; pink/purple, aromatic residues W66, Y69, W77; green, hydrophobic residue L80; yellow/orange, other residues G32, T154 Residues involved in both b3 integrin binding sites are underlined This figure has been prepared using the SWISSPDB VIEWER software [45] Our findings and those reported above clearly indicate that ICAM-4 exhibits the unusual property of accommodating a broad repertoire of integrin receptors involved in different cellular functions [7,9,11–13] However, controversial results have been published with respect to a4b1 interaction with ICAM-4 [9,12] Therefore, in order to further delineate the integrin receptor specificity of ICAM4, we have developed cell adhesion assays using various cell lines that express recombinant a4b1 such as L cells, or LAD cells that express a4b1, but lack b2 integrins as well as aIIband aV-chains Our results show that although both cell lines strongly express a functional a4b1 integrin, as assessed by VCAM-1 and fibronectin binding after activation, they not bind to immobilized ICAM-4-Fc Thus, we conclude that ICAM-4 is not a ligand for a4b1 integrin On a structural basis, although a minimal adhesion motif LDV is present on ICAM-4, it may not be sufficient for integrin binding [33,34] Moreover, the critical motif IDSP required for a4b1 integrin binding to its known ligands [VCAM-1, 13 mucosal addressin cell adhesion molecule (MAdCAM-1)] is missing on ICAM-4 [35] Next, to complement previous studies defining the critical residues of ICAM-4 involved in b2 integrin interaction we performed an analysis of ICAM-4–b3 integrin interactions at a molecular level with essentially the same panel of ICAM-4 mutants [9] The mutated residues were chosen according to their significant solvent accessibility observed on the modeled structure of ICAM-4 Mutations were thus expected to affect the function and interaction potential, rather than the structure itself However, an effect on the structure could not be excluded especially for the Tyr69 mutant, which exposes only its phenol hydroxyl to solvent These studies revealed that distinct but partly overlapping sites spread over the domains D1 and D2 of ICAM-4 mediate interactions with aIIbb3* and aVb3, as both integrin binding sites share some residues (Lys33, Trp77 and Glu151) The aIIbb3* binding site comprises at least six residues clustered at the bottom of the ABED face (Gln30, Gly32, Lys33, Gln36) and at the top of the C¢-E loop of domain D1 (Trp77) and D2 (Glu151), respectively (Fig 5) Sequence alignments revealed that these residues are not conserved within human ICAM proteins [5], explaining why aIIbb3* integrin has not been found to interact with other members of the ICAM family The aVb3 binding site comprises at least 10 residues, including eight distributed on the ABED face (Lys33, Trp66, Tyr69, Asp73) and the CFG face (Arg52, Trp77, Leu80 and Arg97) of domain D1 and two (Glu151 and Thr154) in the C¢-E loop of domain D2 (Fig 5) These data provide evidence for a more extensive binding area between 3736 P Hermand et al (Eur J Biochem 271) Ó FEBS 2004 Fig Effects of ICAM-4 peptides on adhesion of b3 transfectants to ICAM-4 and fibrinogen Adhesion of aIIbb3 CHO* cells (dithiothreitol-activated) (A) and aVb3 L cell transfectant (B) to coated ICAM-4-Fc and fibrinogen (500 ngỈwell)1) and effects of ICAM-4 peptides on cells binding Cell transfectants were pretreated or not with the peptides Phe26–Ser40 (F–S, black bars) and Gly65–Val74 (G–V, hatched bars) derived from ICAM-4 at different final concentrations (31–250 lM) After 30 incubation, the cells were tested for binding to coated ICAM4-Fc or fibrinogen For each peptide tested, the corresponding random (rd, white bars) peptide was used as control The results are expressed as in Fig 1B 100% relative percentages are in gray bars for each histogram The mean ± SEM from three experiments is shown.Student’st-testanalysis,***P < 0.001 ICAM-4 and aVb3 than between ICAM-4 and other integrins Accordingly, we conclude that the aVb3 site comprises residues on both faces of domain D1 for interaction with ICAM-4, as previously reported for ICAM-3 binding to aLb2, and it was postulated that this 14 might reflect a distinct stoichiometry of interaction [36] However, the critical residues involved in aVb3 interaction appear to cluster on the edges of both b sheets, defining a potential contact area (Fig 5) Protein sequence examination suggests that aVb3 does not interact with other human ICAM molecules as the critical residues identified here are not conserved among other family members Different sites on immunoglobulin super15 family proteins are known to mediate binding to integrins (for review see [6]), defining for each protein unique structural features which can be associated with its ligand specifity: a flat surface on the D1 domain of ICAM-1 and ICAM-2, with a glutamic acid playing a key role (see below), a protruding CD loop on the D1 domain of VCAM-1 and MAdCAM-1 including a critical aspartic acid Interestingly, an unusually long D strand in domain D2 of MAdCAM-1 appears to contribute to integrinbinding [37] These two separate integrin-recognition motifs encompassing both IgSF domains of MAdCAM-1 might thus be compared to those defined here on ICAM-4 for aIIbb3* and aVb binding, probably forming a cleft for ligand binding Binding sites involving two consecutive IgSF domains were already observed in other IgSF proteins, not involved in integrin binding, such as the cytokine receptor family (hormone-binding site I) [38] Importantly, except for positions 32 (aIIbb3), 66 and 80 (aVb3), most mutations affecting ICAM-4 interaction with b3 integrins involved amino acid residues different from those implicated in the binding of the monoclonal antibody BS46/56 that includes positions 19, 32, 56, 66, 70, 75, 80 and 91 in domain D1 [9] Therefore, it cannot be excluded that mutant L80G did not react correctly with aVb3 due to protein misfolding, as previously noted for interaction with b2 integrins [9] However, as discussed below, residues 32 and 66 belong to peptide regions involving amino acids 26–40 (peptide F–S) and 65–74 (peptide G–V), respectively, which are clearly involved in ICAM-4–aIIbb3 and ICAM-4–aVb3 interactions Inhibition studies with the synthetic peptides F–S (Phe26–Ser40) and G–V (Gly65–Val74) located on A-B loop/B strand and E strand, respectively, provided additional support to the b3 binding sites on ICAM-4 identified by site-directed mutagenesis The results point to the critical role of F–S peptide in the ICAM-4–aIIbb3* interaction and of G–V peptide in the ICAM-4–aVb3 interaction However, the G–V peptide also inhibits ICAM-4–aIIbb3* interaction Ó FEBS 2004 ICAM-4–b3 integrin interaction (Eur J Biochem 271) 3737 Fig Compilation of amino-acid residues which are involved in ICAM-4/b integrin interactions (A) Schematic two-dimensional representation of the D1 domain of ICAM-4 showing the the two antiparallel b-sheets with ABED and CFG strands, and the loops between strands (B) Compilation and location of relevant amino acids on ICAM-4 which caused reduced interaction with aLb2, aMb2, aIIbb3*, aVb3, aVb1 and aVb5 integrins Right column illustrates predicted localization of residues on ICAM-4 Strands are illustrated by vertical black bars and loops by open rectangles It is worth noting that these two peptides are adjacent on the three-dimensional level (Fig 5) As the G–V peptide harbors a Gln-X-X-Asp-Val motif involved in aIIbb3*– fibrinogen interaction, it might occupy the fibrinogen binding site of aIIbb3*, thus preventing the docking of ICAM-4 This is consistent with the observation that the peptide G–V, but not F–S, also prevents aIIbb3*–fibrinogen interaction (Fig 6A) Therefore, it is assumed that these peptides might be of interest to modulate ICAM-4–b3 integrin interactions occurring in some physiological as well as pathological situations Comparison of the present findings with our previous results regarding ICAM-4–b2 integrin interaction [9] and with very recent data on ICAM-4 interaction with aVb1 and 16 aVb5 [13] are summarized in Fig and lead to the following conclusions: (a) the binding sites of all integrins encompass the two Ig-like domains of ICAM-4, except that for aLb2 which is restricted to domain D1, (b) the b2 and b3 (aIIb and aV) binding sites, which were defined with the same panel of ICAM-4 mutants, appear clearly distinct although they all share Trp77, thus suggesting that there is no consensus binding motif and that ICAM-4 binding might be modulated by I/A domain interactions (see below), (c) the E-F loop of domain D1 and the C¢-E loop of domain D2 which are spatially close (Fig 5) bear residues Trp77 and Glu151, respectively, that are crucial for binding to aMb2 and b3 (aIIb and aV) integrins and (d) the aVb3 binding site defined here is distinct from the reported binding site for aVb1 and aVb5, although all share at least two residues (Trp66, Arg97), although this should be considered with caution as analyses were carried out with different sets of ICAM-4 mutants [13] With this restriction in mind, it appears that amino acids 3738 P Hermand et al (Eur J Biochem 271) Ó FEBS 2004 critical for binding to b2 and aV (b1 and b5) integrins are 19 membrane complex [44], but whether it is present as a monomer or as a dimer is currently unknown According to mainly located in the A and G strands of domain D1 and the interface observed for the ICAM-1 dimer [42], which is share a common residue (Arg97) suggested to mediate physiologic dimerization on the cell I/A domains of integrins, including a metal-coordination surface, ICAM-4 dimerization might also largely involve the site, play a key role in ligand recognition Aspartate or ABED sheet of D1 domain However, in the cell adhesion glutamate residues of ligands form part of the coordination assay used in our studies, the recombinant ICAM-4-Fc sphere and consequently are central to the integrin recogfusion protein is present as an obligate dimer and therefore nition site [39] I/A domains are present on top of all the b impairment of interaction with different integrins for subunits, whereas they are present only in some a-subunit mutants in this region, as seen in cell adhesion assays, (in aL and aM, but not in aIIb and aV), inserted in the loop of might reflect either an effect on the oligomerization or a a b propeller structure In the aVb3 experimental structure, direct effect on ligand binding Different stoichiometry, the propeller, lacking an I/A domain, is in complex with the however, cannot be excluded, as already suggested by Bell b I/A domain to form the ligand binding head of the integrin [40] Binding of a cyclic peptide presenting an RGD 20 and colleagues [36] In conclusion, because of its broad repertoire of integrin sequence involves major interface between the aV- and receptors, ICAM-4 is likely to play a role in the normal b3-subunits and extensive contacts with both, including physiology through interaction with macrophages, platelets formation of salt bridges between the arginine side chain and leukocytes that express a variety of integrins, and and aspartic residues on the integrin [41] A similar situation through abnormal interactions with blood cells and/or might occur for the interaction of ICAM-4 with aVb3 (with vascular endothelium in pathologic situations, as discussed a central role played by Arg97) and also with aIIbb3, both of above However, more studies are required to better which lack I/A domains in the a-chains and for which we document such interactions and their potential modulation have shown here that a relatively large surface of ICAM-4 by synthetic peptides derived from ICAM-4 as additional might be involved specific tools for future therapeutic strategies in situations In contrast to integrins lacking an a I/A domain, only the were these interactions may occur I/A domain inserted into the a-subunit appears to be sufficient for integrin binding [39] Recombinant a I/A domains can indeed bind ligand with the same affinity and Acknowledgements specificity as intact integrins Accordingly, ICAM-4 binding This investigation was supported in part by the Institut National de la to aLb2 and aMb2 integrins solely involves the a I/A domain ´ ´ Sante et de la Recherche Medicale (INSERM) and the Institut National [10], similarly to what occurs for the ICAM-1–aLb2 or de la Transfusion Sanguine (INTS) –aMb2 interaction [42] However, the interface between We would like to thank Mrs M Huet (INTS, Paris) for technical ICAM-4 and a I/A domain probably differs drastically assistance We also thank Dr P.J Newman (Blood Center of southfrom that observed for ICAM-1 [42], due to the presence of eastern Wisconsin, Milwaukee, WI), Dr D Cheresh (Scripps Reseach an arginine (Arg52) in ICAM-4, instead of the critical Institute, La Jolla, CA), Dr M Hemler (Dana Faber, Cancer Institute, ICAM-1 glutamic acid (Glu34) which coordinated the I/A Boston, MA) and Dr E Ruoslahti (Burnham Institute, La Jolla, CA) 2+ domain Mg Regardless of the connection of I/A for the supply of the b3-chain-pcDNA3.1, aV-chain-pcDNAI-NEO, 17 domains to a (aLb2/aMb2) or b (aIIbb3/aVb3) chains, the a4-chain-pF-NEO and b1-chain-pECE constructs, respectively exact nature of the different interfaces of ICAM-4 with these domains remains to be determined Although it is References possible that an acidic residue of ICAM-4 in another position might participate in the metal-binding site of I/A 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112, 99–111 43 Bloy, C., Hermand, P., Blanchard, D., Cherif-Zahar, B., Goossens, D & Cartron, J.P (1990) Surface orientation and antigen Ó FEBS 2004 properties of Rh and LW polypeptides of the human erythrocyte membrane J Biol Chem 265, 21482–21487 44 Cartron, J.P (1999) RH blood group system and molecular basis of Rh-deficiency Baillieres Best Pract Res Clin Haematol 12, 655–689 45 Guex, N & Peitsch, M.C (1997) SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling Electrophoresis 18, 2714–2723  ... using ICAM-4 and aVb3 L cell transfectants, was performed Figure shows that ICAM-4 L cells did not bind to immobilized parental L cells, but did bind to immobilized aVb3 L cells This cell? ? ?cell. .. Adhesion of LAD cells to ICAM-4- Fc Fig Adhesion of ICAM-4 L cell transfectant to immobilized aVb3 L cell transfectant ICAM-4 L cells (black bars) or parental L cells (white bar) (105 cellsỈwell)1)... between red cell ICAM-4 and platelet aIIbb3 was demonstrated previously [11], we provide further evidence that cell surface ICAM-4 might interact with cell surface aVb3 Accordingly, a cell? ??cell