Distinct Ligand Binding Sites in the I Domain of Integrin αMβ2 That Differentially Affect a Divalent Cation-dependent Conformation

The I domains of the leukocyte β2 integrins have been shown to be essential for ligand recognition. Amino acid substitutions of Asp140 and Ser142, which reside in a conserved cluster of oxygenated residues, abrogate divalent cation ligand binding function of αMβ2. Presently, we evaluated the role of two I domain regions in αMβ2 ligand recognition: 1) the conserved cluster of oxygenated residues (Asp134, Asp140, Ser142, and Ser144) and 2) a 7-amino acid region (Phe246-Tyr252), conserved in αM and α but absent in αX of the β2 integrins. Recombinant αMβ2 was expressed on COS-7 cells, and function was assessed by iC3b recognition. Alanine substitution at position Asp140, Asp140/Ser142, Ser142, or Ser144 produced a complete loss in the capacity of αMβ2 to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp248 or Tyr252 or deletion of Phe246-Tyr252 abolished iC3b ligand recognition as well as the binding of a blocking antibody. In contrast, these mutations did not affect the binding of the cation-dependent epitope. These data implicate a second region within the I domain important for αMβ2 ligand binding function and suggest that this region does not affect a divalent cation-dependent conformation of αMβ2.

␣ M ␤ 2 is the major leukocyte integrin expressed on neutrophils and mediates phagocytosis of opsonized particles (13), adherence to the endothelium (14 -15), neutrophil homotypic aggregation, and chemotaxis (16). Like other integrins, ␣ M ␤ 2 is promiscuous and recognizes a multiplicity of protein ligands, including complement C3 fragment iC3b (13,17), ICAM-1 (18), fibrinogen (19), and Factor X (20). The identification of regions of ␣ M ␤ 2 that contribute to multifunctional ligand recognition have not been completely characterized. The current state of our knowledge indicates the presence of multiple ligand contact points in ␣ M ␤ 2 . We have previously identified a cluster of oxygenated residues within ␤ 2 that are essential for ligand binding of ␣ M ␤ 2 and ␣ L ␤ 2 (21). In addition, the I domain has been shown to be essential for ␣ M ␤ 2 ligand binding by the localization of blocking antibody epitopes and by direct ligand binding to isolated I domain (22)(23)(24). The ␣ M I domain also binds cations (25). Mutations of the highly conserved residues Asp 140 , Ser 142 , and Asp 242 in ␣ M I domain, which reside in a cluster of oxygenated residues, abolish divalent-cation binding and ␣ M ␤ 2 ligand recognition of iC3b (25). To date, the location and structure of additional ligand binding sites within the ␣ M I domain is entirely speculative. In the present study, we have identified a novel ␣ M I domain ligand binding site (Phe 246 -Tyr 252 ), which is conserved in ␣ M and ␣ X but absent in ␣ L (see Fig. 1). In addition, we have further characterized a cluster of highly conserved oxygenated residues (Asp 134 , Asp 140 , Ser 142 , and Ser 144 ) in the ␣ M I domain. Our results further implicate Asp 140 and Ser 142 and designate analogous importance to Ser 144 , Asp 248 , and Tyr 252 in the ligand binding function of ␣ M ␤ 2 . Furthermore, these two identified ligand binding domains differentially alter the interaction of bound divalent cations to ␣ M ␤ 2 .
Mutagenesis and Transfection-The full-length wild-type ␣ M cDNA (6) was cloned into the expression vector pCDM8 (Invitrogen, San Diego, CA). A fragment containing the first 1347 nucleotides of ␣ M was isolated by sequential digestion with HindIII and EcoRV and subcloned into the HindIII and EcoRV sites of pBluescript KS (Stratagene, La Jolla, CA). Nucleotide base substitutions or deletions were then incorporated by oligonucleotide-directed mutagenesis (31). Following transformation, mutant clones were confirmed by nucleotide sequencing of the mutated region, which was then ligated into the HindIII-and EcoRV-digested ␣ M cDNA in pCDM8. COS-7 cells (a monkey kidney fibroblastoid cell line from the ATCC) were maintained in Dulbecco's modified Eagle's medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 1% glutamine (Irvine Scientific), 1% penicillin and streptomycin (Irvine Scientific), and 1% nonessential amino acids (Sigma). COS-7 cells were co-transfected by electroporation with wild-type ␤ 2 (32) subcloned into pCDNA1/neo (Invitrogen) and either wild-type or mutant ␣ M constructs. Mock transfected cells were trans-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Upjohn Laboratories, Cell Biology and Inflammation Research, 7239-267-302, Kalamazoo, MI 49001. fected with pCDM8 vector without insert. Cells were evaluated for surface expression and function 48 h after electroporation.
Flow Cytometric Analysis-Flow cytometric analysis was carried out as described previously (33) or with modification for mAb 24 epitope expression. Briefly, cells were harvested with 3.5 mM EDTA and 0.01% L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin (Worthington) in chelex-treated Tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Cells were washed in the presence of 0.05% soybean trypsin inhibitor (Sigma) and resuspended in TBS. A 50-l aliquot of cells (1 ϫ 10 7 cells/ml) was pelleted in V-bottom 96-well plates (Costar Corp., Cambridge, MA). Cells were resuspended in 50 l of anti-␤ 2 or mAb 24 (20 g/ml) in TBS containing 0.5 mM MnCl 2 or 10 mM EDTA. Following incubation for 30 min at 37°C, cells were pelleted by centrifugation and washed with 100 l of TBS containing the appropriate divalent cations; with anti-␤ 2 antibody, cells were washed with TBS containing 1 mM CaCl 2 and 1 mM MgCl 2 . Cells were pelleted by centrifugation and resuspended in 50 l of fluorescein-conjugated goat F(abЈ) 2 anti-mouse immunoglobulins, heavy and light chains (Biosource International, Camarillo, CA) for 30 min at 4°C. Cells were pelleted by centrifugation and resuspended in TBS containing the appropriate divalent cations. Antibody binding to the transfected cell lines was analyzed by flow cytometry on a FACScan (Beckman Instruments).
Surface Iodination, Immunoprecipitation, and SDS-PAGE-Transiently transfected cell lines were harvested with 3.5 mM EDTA in phosphate-buffered saline (Irvine Scientific, Santa Ana, CA). Cells were washed 2 times with phosphate-buffered saline and then resuspended in TBS containing 1 mM MgCl 2 and 1 mM CaCl 2 . Cells were surface labeled with 125 I (Amersham Corp.) by the lactoperoxidase-glucose oxidase method and then solubilized in lysis buffer (100 mM Tris-HCL (pH 8.0), 0.15 M NaCl, 2 mM MgCl 2 , 1% Triton X-100, 0.025% NaN 3 , 2 mM phenylmethylsulfonyl fluoride, and 2 mM aprotinin). The insoluble material was removed from the lysate by centrifugation at 14,000 ϫ g for 30 min at 4°C. Cell extracts were immunoprecipitated with anti-␤ 2 antibody (mAb TS1/18). mAb TS1/18 was attached to preswollen protein A-Sepharose beads (Pharmacia Biotech Inc.) as described previously (34). Precleared detergent lysates from the surface-labeled cells were incubated with the antibody-conjugated Sepharose beads overnight with shaking at 4°C. The Sepharose beads containing the antibody-antigen complex were washed 2 times with lysis buffer followed by two washes with lysis buffer containing 0.3 M NaCl. Beads were resuspended in sample buffer (35), boiled for 3 min, and centrifuged, and the precipitated proteins were resolved by SDS-polyacrylamide gel electrophoresis (reducing 7.5% acrylamide gels). The gels were dried and visualized by autoradiography.
iC3b-E Preparation and Rosette Assay-Sheep erythrocytes (Colorado Serum Co., Denver, CO) were coated with iC3b (iC3b-E) as described previously with modification (22). Briefly, 6 ϫ 10 8 sheep erythrocytes were washed with Hanks' balanced salt solution containing 10 mM Hepes (pH 7.3), 1 mM MgCl 2 , 1 mM CaCl 2 , and 0.1% bovine serum albumin (HBSS buffer). Cells were resuspended in 10 ml of HBSS and incubated with 400 l of anti-sheep erythrocyte antibody (MAS 012b) (Sera-lab, United Kingdom) for 25 min at room temperature with gentle shaking. Cells were washed extensively with HBSS buffer and resuspended in 4 ml of HBSS buffer. 300 l of C5 depleted normal human serum (Quidel, San Diego, CA) was added to the IgM-coated erythrocyte (IgM-E) for 1 h at 37°C with gentle shaking. Cells (iC3b-E) were washed extensively with HBSS buffer and resuspended to a final concentration of 2 ϫ 10 8 cells/ml and stored at 4°C.
Forty-eight hours post-transfection, COS-7 cells were harvested with tissue culture trypsin/EDTA solution (Sigma), and 2 ϫ 10 5 cells were seeded in duplicate onto 6-well tissue culture plates in supplemented Dulbecco's modified Eagle's medium as described above. Cells were allowed to adhere for 3 h at 37°C. Adherent monolayers were washed once with HBSS buffer and incubated for 30 min at 37°C in 0.5 ml of HBSS in the presence or absence of blocking anti-␤ 2 mAb 8H1 (50 g/ml). 100-l aliquots of iC3b-E (2 ϫ 10 8 cells/ml) were added, and the plates were further incubated for 1 h at 37°C. Adherent monolayers were gently washed with HBSS buffer to remove nonadherent erythrocytes. Rosettes (Ͼ10 erythrocytes/COS-7 cell, Ͼ50 cells examined) were examined by light microscopy at 200ϫ magnification. (Fig. 1). First, to examine the contribution that the cluster of highly conserved oxygenated residues in ␣ M I domain play in ligand binding, alanine point mutations were introduced at Asp 134 , Asp 140 , Ser 142 , Asp 140 3 Ala/Ser 142 3 Ala, or Ser 144 into the wild-type full-length ␣ M cDNA by site directed mutagenesis. Second, to investigate the contribution of the seven-amino acid region, which is conserved in ␣ M and ␣ X but absent in ␣ L , in ␣ M ␤ 2 ligand binding function, deletion of these seven residues (⌬Phe 246 -Tyr 252 ) as well as single point mutations to alanine at Phe 246 , Gly 247 , Asp 248 , Pro 249 , Leu 250 , or Tyr 252 were inserted into ␣ M . We were unable to obtain alanine substitution of residue Gly 251 . As a control, an alanine mutation outside of this region, Asn 224 , was also introduced. COS-7 cells were transiently co-transfected with the wildtype or mutant ␣ M together with the wild-type ␤ 2 . Cell surface expression and heterodimer association of the integrins were evaluated by immunoprecipitation of detergent-lysed surfacelabeled cells (Fig. 2). Anti-␤ 2 specific antibody (TS1/18) immunoprecipitated both the ␣ and ␤ subunits from cells co-transfected with ␤ 2 and the wild-type ␣ M or any of the mutant ␣ M , except for ␣ M (D134A), indicating that both subunits were associated on the cell surface. Similar results were obtained utilizing anti-␣ M antibody (LM2/1) (data not shown). In contrast, alanine substitution of ␣ M at D134A was not expressed when co-transfected with ␤ 2 . Anti-␤ 2 specific antibody did not precipitate ␣␤ heterodimers. Moreover, immunoprecipitation of surface-labeled ␣ M (⌬246 -252)␤ 2 consistently showed moderate cell surface expression in comparison with the wild-type or other mutant ␣ M ␤ 2 receptors.

Generation and Expression of Mutant
Effects of ␣ M Mutations on mAb Recognition-Surface expression of the wild-type or mutant ␣ M ␤ 2 was confirmed by flow cytometry utilizing a panel of anti-␣ and anti-␤ antibodies ( Table I) (11), and ␣ E (12) using single-letter code. Gaps were introduced to maximize alignment (dots). Boxed residues were mutated to alanine. Asterisks indicate previously identified residues in ␣ M critical for ligand recognition (25). cells exhibited strong immunostaining with anti-␤ 2 antibodies TS1/18 and 8H1. Similarly, co-transfected wild-type or mutant ␣ M ␤ 2 (s) COS-7 cells were strongly positive for four of the anti-␣ M antibodies: LM2/1, LPM19c, 904, and M1/70. However, recognition of the ␣ M (⌬246 -248)␤ 2 by mAbs LM2/1, LPM19c, and 904 was attenuated. The epitopes of these inhibitory anti-␣ M antibodies have been localized to the I domain (22,25). In contrast, anti-␣ M blocking antibody, 3H5, failed to recognize recombinant ␣ M (D248A)␤ 2 , ␣ M (Y252A)␤ 2 , or ␣ M (⌬246 -248)␤ 2 (Fig. 3). Therefore, mAb 3H5 recognizes an epitope close to or within residues Asp 248 -Tyr 252 . Since mAb 3H5 blocks the interaction of ␣ M ␤ 2 to iC3b (21), these residues may be involved in ␣ M ␤ 2 ligand recognition. Fluorescence-activated cell sorting profiles of the recombinant wild-type or mutant ␣ M ␤ 2 (s) heterodimers were very similar and were expressed on the cell surface of 40 -50% of the cells with the exception of ␣ M (⌬246 -248)␤ 2 , which demonstrated moderate cell surface expression, 20 -25% of the cells (Fig. 3 and results not shown). In agreement with the immunoprecipitation analysis, surface expression of recombinant ␣ M (D134A)␤ 2 was not detected on COS cells (results not shown).
To further substantiate the importance of these I domain ␣ M residues in iC3b ligand recognition, we tested the ability of the adherent COS-7 cell transfectants to bind to iC3b-coated erythrocytes (iC3b-E) ( Table II, Fig. 5). COS-7 cells expressing the wild-type ␣ M ␤ 2 or those expressing ␣ M mutations F246A, G247A, P249A, L250A, or N224A were able to rosette iC3b-E, and binding was blocked by anti-␤ 2 antibody, 8H1. In contrast, cells expressing recombinant ␣ M ␤ 2 with ␣ M mutations D140A, S142A, D140GS/A140GA, S144A, D248A, Y252A, or ⌬246 -248 were unable to rosette iC3b-E. None of the COS-7 transfectants were able to bind erythrocytes coated with IgM (results not shown). These results further implicate Asp 140 and Ser 142 as playing an essential part in the ligand binding function of ␣ M ␤ 2 and designate analogous importance to Ser 144 , Asp 248 , and Tyr 252 in ligand recognition.
Expression of mAb 24 Epitope to Recombinant ␣ M ␤ 2 Mutants-Double alanine substitutions at Asp 140 and Ser 142 of ␣ M I domain abolish divalent cation-dependent binding to isolated I domain and block ␣ M ␤ 2 cation-dependent ligand recognition of iC3b, suggesting that these residues may provide coordinating ligands for divalent cations (25). We hypothesized that these mutations may perturb the interaction between bound divalent cation and the integrin. To test our hypothesis, the expression of a cation-sensitive epitope recognized by mAb 24 was analyzed by flow cytometry to determine if mutations that disrupt ligand binding differentially alter mAb 24 expression. mAb 24 recognizes a common cation-dependent epitope on all three ␣ subunits of the ␤ 2 integrins (26). mAb 24 does not recognize the I domain of ␣ L , suggesting that the epitope is not located in this domain (36). Expression of mAb 24 is associated with the Mg 2ϩ -or Mn 2ϩ -occupied form of the ␤ 2 integrins (27). In the presence of 0.5 mM Mn 2ϩ , cells expressing wild-type ␣ M ␤ 2 stained brightly with mAb 24 (Fig. 6)

TABLE I Summary of reactivities of anti-␣ M or ␤ 2 antibodies to wild-type or mutant ␣ M ␤ 2 on COS-7 cells by flow cytometry
COS-7 cells co-transfected with wild-type or mutant ␣ M ␤ 2 receptors were incubated with mAbs for 30 min at room temperature. Cells were then washed and further incubated for 30 min with fluorescein isothiocyanate-conjugated goat anti-mouse F(abЈ) 2 fragments and analyzed on a FACScan. ϩ, positive staining similar to that shown in Fig. 3; Ϫ, staining not significantly different from the negative control (Mock); ϩ/Ϫ, staining marginally greater than the negative control. Since the disclosures of the contribution of the I domain in ␣ M ␤ 2 ligand recognition, the location and structure of the ligand binding sites within the I domain has been a subject of intense investigation. Double alanine substitutions of the highly conserved oxygenated residues Asp 140 and Ser 142 (D140GS/A140GA), which reside in a cluster of oxygenated residues of the ␣ M I domain, have been reported to abolish divalent cation-dependent binding of ␣ M ␤ 2 to iC3b (25). Our data are in agreement with these previous observations and specifically extend the functional role of this highly conserved cluster of oxygenated residues in this domain in ligand recognition. The present work suggests that Ser 144 in addition to Asp 140 and Ser 142 of ␣ M I domain appear to be critical for ligand binding function of ␣ M ␤ 2 . This conclusion is based on the failure of cells expressing ␣ M (D140A), ␣ M (S142A), ␣ M (D140GS/ A140GA), or ␣ M (S144) cotransfected with ␤ 2 to adhere to immobilized iC3b or to bind iC3b-E.
In addition to the effect on ligand binding, our results support a direct interaction between ligand binding and a divalent cation-dependent conformation. This conclusion is based on the  iC3b-coated erythrocytes COS-7 cells co-transfected with ␤ 2 and wild-type or ␣ M mutants were incubated in the presence or absence of blocking mAb 8H1 (anti-␤ 2 ) for 30 min at 37°C. At the end of the incubation, cells were gently washed to remove unbound erythrocytes and examined by light microscopy. ϩ, positive rosetting similar to that shown in Fig. 5 (Ͼ10 erythrocytes/ COS-7 cell, Ͼ50 cells examined); Ϫ, negative rosetting not significantly different from the negative control (Mock).
Ϫ Ϫ attenuated binding of mAb 24 in the presence of Mn 2ϩ to cells expressing ␣ M (D140)␤ 2 , ␣ M (S142)␤ 2 , or ␣ M (D140GS/ A140GA)␤ 2 and to a lesser extent to cells expressing ␣ M (S144A)␤ 2 . Expression of mAb 24 epitope is associated with the Mg 2ϩ or Mn 2ϩ bound form of the ␤ 2 integrins (27). Therefore, the present work suggests that these I domain mutations perturb the interaction between bound divalent cation and ␣ M ␤ 2 . This is further supported by the identification of these residues as part of the three-dimensional metal coordination sites within the ␣ M I domain by high resolution crystal structure: Ser 142 , Ser 144 , and Thr 209 as well as secondary coordination sites between Asp 140 to Ser 142 and Asp 242 to Ser 144 (37). Furthermore, alanine substitution of Asp 242 and a synthetic peptide containing this residue inhibit iC3b binding to ␣ M ␤ 2 (23).
We have previously identified a conserved amino acid alignment of these ␣ M I domain residues with a similar divalent cation-dependent ligand binding motif, DXSXS, in ␤ 2 and ␤ 3 subunits that contribute to ligand binding of ␣ M ␤ 2 , ␣ L ␤ 2 , and ␣ IIb ␤ 3 , respectively (21,38). In addition, substitution of Asp 119 in ␤ 3 disturbs binding of divalent cations (39), and substitutions in the corresponding homologous residues in ␤ 1 (40) and ␤ 6 (41) abolish the ligand binding function. Therefore, we hypothesized that this common binding motif participates in all integrin functions through the interaction of ligand with divalent cations occupying a divalent cation binding site in the integrins (21,38). This is further supported by the finding that alanine substitution of the corresponding residues in the ␣ 2 I domain are essential for the ligand binding function of ␣ 2 ␤ 1 (42). Alignment of these ␤ subunit sequences with the I domain has led to the proposal of an integrin ␤ subunit I domain (37). Previous studies identifying natural ␤ 2 mutations (leukocyte adhesion deficiency) suggest that this region of ␤ 2 (residues 128 -361) represents critical contact sites required for ␣␤ heterodimer formation (43). Interestingly, while alanine substitution of Asp 140 , Ser 142 , D140GS/A140GA, or Ser 144 did not affect the capacity of ␣ M to efficiently associate with ␤ 2 on COS cells, alanine substitution at Asp 134 resulted in loss of ␣ M ␤ 2 surface expression based on immunoprecipitation with anti-␤ 2 antibody. These results suggest a role for the I domain not only in ligand recognition but in ␣␤ heterodimer formation. It is particularly noteworthy that ␣ M residue Asp 134 aligns with the corresponding residue Asp 128 of ␤ 2 , which we previously identified as essential for surface expression of ␣ M ␤ 2 and ␣ L ␤ 2 (21).
These I domain sequences may participate directly in a complex between cation, ligand, and receptor as has been reported for the identified homologous ligand binding domain in the ␤ 3 subunit (44). It was proposed that residues 118 -131 of ␤ 3 bind both divalent cation and ligand, resulting in the displacement of cation from this region of ␤ 3 and subsequently exposing secondary binding sites. In the present study we have identified a second region within ␣ M I domain, which is important in ␣ M ␤ 2 ligand recognition of iC3b. This conclusion is based on the inability of the cells expressing I domain mutations at Asp 248 , Tyr 252 or deletion of residues 246 -252 to adhere to immobilized iC3b or to rosette iC3b-E. However, in contrast to mutations at Asp 140 , Ser 142 , or Ser 144 , these mutations do not perturb the expression of the Mn 2ϩ -induced mAb 24 epitope. Taken together, these results demonstrate that the two distinct I domain regions differentially alter the interaction of a cation-dependent conformation of ␣ M ␤ 2 . This suggests that alanine substitution at Asp 140 , Ser 142 , or Ser 144 perturb the interaction between bound cation and the integrin, while substitution at Asp 248 , Tyr 252 , or ⌬246 -252 do not. Therefore, an alternative hypothesis is that the binding of divalent cations to Asp 140 , Ser 142 , and Ser 144 may maintain an integrin conformational structure that allows access to distinct binding sites within the receptor. This is supported by the localization of the epitope recognized by receptor blocking mAb 3H5 to Asp 248 and Tyr 252 , if we assume that function blocking mAbs bind close to the ligand binding site. In contrast, substitution at Asp 140 , Ser 142 , or Ser 144 did not affect the binding of any of the blocking anti-␣ M antibodies tested.
In conclusion, we have further characterized a cation ligandinteractive region in the ␣ M I domain that further supports the proposal of a common ligand binding mechanism (21, 38) essential for all integrin receptor function. In addition, we have identified a second unique ligand binding domain in the ␣ M I domain that does not affect cation-dependent conformation of ␣ M ␤ 2 . The mechanisms by which the multiple ligand binding domains in the ␣ and ␤ subunits participate in ligand specificity is speculative. Differences in ligand recognition and specificity may be controlled by small sequence differences in specific regions such as residues Phe 246 -Tyr 252 of the ␣ M I domain. This region is unique to ␣ M and ␣ X but absent in ␣ L of the ␤ 2 integrins. ␣ M ␤ 2 and ␣ X ␤ 2 share a common ligand, iC3b, that is not recognized by ␣ L ␤ 2 (1-3). We are presently addressing this issue.