©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Ligand Binding Sites in the I Domain of Integrin That Differentially Affect a Divalent Cation-dependent Conformation (*)

(Received for publication, May 31, 1995)

Sarah Lea McGuire Mary Lynn Bajt (§)

From the From Cell Biology and Inflammation Research, Upjohn Laboratories, Kalamazoo, Michigan 49001

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The I domains of the leukocyte beta(2) integrins have been shown to be essential for ligand recognition. Amino acid substitutions of Asp and Ser, which reside in a conserved cluster of oxygenated residues, abrogate divalent cation ligand binding function of alpha(M)beta(2). Presently, we evaluated the role of two I domain regions in alpha(M)beta(2) ligand recognition: 1) the conserved cluster of oxygenated residues (Asp, Asp, Ser, and Ser) and 2) a 7-amino acid region (Phe-Tyr), conserved in alpha(M) and alpha(X) but absent in alpha(L) of the beta(2) integrins. Recombinant alpha(M)beta(2) was expressed on COS-7 cells, and function was assessed by iC3b recognition. Alanine substitution at position Asp, Asp/Ser, Ser, or Ser produced a complete loss in the capacity of alpha(M)beta(2) to recognize iC3b and attenuated the binding of a divalent cation-dependent epitope recognized by monoclonal antibody 24. Moreover, alanine substitution at Asp or Tyr or deletion of Phe-Tyr 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 alpha(M)beta(2) ligand binding function and suggest that this region does not affect a divalent cation-dependent conformation of alpha(M)beta(2).


INTRODUCTION

The beta(2) leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses(1, 2, 3) . The beta(2) integrins consist of three glycoproteins, alpha(M)beta(2) (CD11b/CD18, MAC-1), alpha(L)beta(2) (CD11a/CD18, LFA-1), and alpha(x)beta(2) (CD11c/CD18, p150,95), containing a common beta subunit (beta(2)) noncovalently linked to three distinct but homologous alpha subunits (alpha(L), alpha(M), and alpha(X))(4) . All three alpha subunits contain an insertion of approximately 200 amino acids termed the ``I'' domain (5, 6, 7, 8, 9) . The I domain is absent in all other known integrin subunits except for alpha(1) of alpha(1)beta(1)(10) , alpha(2) of alpha(2)beta(1)(11), and alpha(E) of alpha(E)beta(7)(12) .

alpha(M)beta(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, alpha(M)beta(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 alpha(M)beta(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 alpha(M)beta(2). We have previously identified a cluster of oxygenated residues within beta(2) that are essential for ligand binding of alpha(M)beta(2) and alpha(L)beta(2)(21) . In addition, the I domain has been shown to be essential for alpha(M)beta(2) ligand binding by the localization of blocking antibody epitopes and by direct ligand binding to isolated I domain(22, 23, 24) . The alpha(M) I domain also binds cations(25) . Mutations of the highly conserved residues Asp, Ser, and Asp in alpha(M) I domain, which reside in a cluster of oxygenated residues, abolish divalent-cation binding and alpha(M)beta(2) ligand recognition of iC3b(25) . To date, the location and structure of additional ligand binding sites within the alpha(M) I domain is entirely speculative. In the present study, we have identified a novel alpha(M) I domain ligand binding site (Phe-Tyr), which is conserved in alpha(M) and alpha(X) but absent in alpha(L) (see Fig. 1). In addition, we have further characterized a cluster of highly conserved oxygenated residues (Asp, Asp, Ser, and Ser) in the alpha(M) I domain. Our results further implicate Asp and Ser and designate analogous importance to Ser, Asp, and Tyr in the ligand binding function of alpha(M)beta(2). Furthermore, these two identified ligand binding domains differentially alter the interaction of bound divalent cations to alpha(M)beta(2).


Figure 1: Amino acid alignment of the I domain of alpha with other integrin alpha subunit I domains. The deduced sequence of alpha(M) (CD11b, (5, 6, 7) ) was aligned with human alpha(X) (CD11c, (8) ), alpha(L) (CD11a, (9) ), alpha(1)(10) , alpha(2)(11) , and alpha(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 alpha(M) critical for ligand recognition(25) .




MATERIALS AND METHODS

Monoclonal Antibodies

Murine monoclonal anti-alpha(M) (mAb (^1)24) was used as purified IgG, and characterization was described previously(26, 27) . Murine monoclonal anti-alpha(M) (LPM19c) was used as a dilution of ascites(28) . The following murine anti-human antibodies were used as culture supernatants: 904 (anti-alpha(M))(25) , M1/70 (anti-alpha(M))(13) , LM2/1 (anti-alpha(M))(29) , and TS1/18 (anti-beta(2))(30) . Murine blocking anti-human alpha(M) (mAb 3H5) and anti-human beta(2) (mAb 8H1) were prepared in our laboratory as described previously by immunizing BALB/c mice with affinity-purified alpha(M)beta(2) from polymorphonuclear leukocytes obtained from healthy donors(21) . Both mAbs 3H5 and 8H1 were able to block alpha(M)beta(2)-dependent neutrophil adhesion to iC3b, fibrinogen, and purified ICAM-1.

Mutagenesis and Transfection

The full-length wild-type alpha(M) cDNA (6) was cloned into the expression vector pCDM8 (Invitrogen, San Diego, CA). A fragment containing the first 1347 nucleotides of alpha(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 alpha(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 beta(2)(32) subcloned into pCDNA1/neo (Invitrogen) and either wild-type or mutant alpha(M) constructs. Mock transfected cells were transfected 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-µl aliquot of cells (1 times 10^7 cells/ml) was pelleted in V-bottom 96-well plates (Costar Corp., Cambridge, MA). Cells were resuspended in 50 µl of anti-beta(2) or mAb 24 (20 µg/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 µl of TBS containing the appropriate divalent cations; with anti-beta(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 µl of fluorescein-conjugated goat F(ab`)(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 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 times g for 30 min at 4 °C. Cell extracts were immunoprecipitated with anti-beta(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.

Adhesion Assay

Transfected cell lines were detached from culture plates with nonenzymatic cell dissociation solution (Sigma) and 0.01% L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington Biochemical). Cells were washed in the presence of 0.1% soybean trypsin inhibitor (Sigma) and resuspended in modified Tyrode's buffer, pH 7.4 (137.5 mM NaCl, 2.6 mM KCl, 12 mM NaHCO(3), 1 mM CaCl(2), 1 mM MgCl(2), 1 mg/ml bovine serum albumin, 1 mg/ml dextrose). Transfected cell lines were fluorescently labeled with 2`,7`-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein (Molecular Probes, Inc., Eugene, OR) and adhered to immobilized iC3b as described previously(21) .

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 times 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 µl 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 µl 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 times 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 times 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-beta(2) mAb 8H1 (50 µg/ml). 100-µl aliquots of iC3b-E (2 times 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 (>10 erythrocytes/COS-7 cell, >50 cells examined) were examined by light microscopy at 200times magnification.


RESULTS

Generation and Expression of Mutant alphabeta(2)Receptors

The role of two distinct alpha(M) I domain regions in alpha(M)beta(2) ligand recognition function was investigated (Fig. 1). First, to examine the contribution that the cluster of highly conserved oxygenated residues in alpha(M) I domain play in ligand binding, alanine point mutations were introduced at Asp, Asp, Ser, Asp Ala/Ser Ala, or Ser into the wild-type full-length alpha(M) cDNA by site directed mutagenesis. Second, to investigate the contribution of the seven-amino acid region, which is conserved in alpha(M) and alpha(X) but absent in alpha(L), in alpha(M)beta(2) ligand binding function, deletion of these seven residues (DeltaPhe-Tyr) as well as single point mutations to alanine at Phe, Gly, Asp, Pro, Leu, or Tyr were inserted into alpha(M). We were unable to obtain alanine substitution of residue Gly. As a control, an alanine mutation outside of this region, Asn, was also introduced.

COS-7 cells were transiently co-transfected with the wild-type or mutant alpha(M) together with the wild-type beta(2). Cell surface expression and heterodimer association of the integrins were evaluated by immunoprecipitation of detergent-lysed surface-labeled cells (Fig. 2). Anti-beta(2) specific antibody (TS1/18) immunoprecipitated both the alpha and beta subunits from cells co-transfected with beta(2) and the wild-type alpha(M) or any of the mutant alpha(M), except for alpha(M)(D134A), indicating that both subunits were associated on the cell surface. Similar results were obtained utilizing anti-alpha(M) antibody (LM2/1) (data not shown). In contrast, alanine substitution of alpha(M) at D134A was not expressed when co-transfected with beta(2). Anti-beta(2) specific antibody did not precipitate alphabeta heterodimers. Moreover, immunoprecipitation of surface-labeled alpha(M)(Delta246-252)beta(2) consistently showed moderate cell surface expression in comparison with the wild-type or other mutant alpha(M)beta(2) receptors.


Figure 2: SDS-polyacrylamide gel electrophoresis of wild-type or mutant alphabeta(2)(s) transiently expressed on COS-7 cells. COS-7 cells co-transfected with alpha(M)beta(2) or alpha(M)beta(2) mutants were surface-iodinated, lysed, and immunoprecipitated with mAb TS1/18 (anti-beta(2)). The precipitated proteins were resolved by electrophoresis on 7.5% SDS-polyacrylamide gels under reduced conditions and detected by autoradiography. The co-transfected wild-type or mutant alpha(M)(s) in each cell line are listed above each lane. The position of the alpha and beta subunits are shown on the right, and molecular mass standards are indicated on the left in kDa.



Effects of alpha(M)Mutations on mAb Recognition

Surface expression of the wild-type or mutant alpha(M)beta(2) was confirmed by flow cytometry utilizing a panel of anti-alpha and anti-beta antibodies (Table 1). All co-transfected wild-type or mutant alpha(M)beta(2)(s) COS-7 cells exhibited strong immunostaining with anti-beta(2) antibodies TS1/18 and 8H1. Similarly, co-transfected wild-type or mutant alpha(M)beta(2)(s) COS-7 cells were strongly positive for four of the anti-alpha(M) antibodies: LM2/1, LPM19c, 904, and M1/70. However, recognition of the alpha(M)(Delta246-248)beta(2) by mAbs LM2/1, LPM19c, and 904 was attenuated. The epitopes of these inhibitory anti-alpha(M) antibodies have been localized to the I domain(22, 25) . In contrast, anti-alpha(M) blocking antibody, 3H5, failed to recognize recombinant alpha(M)(D248A)beta(2), alpha(M)(Y252A)beta(2), or alpha(M)(Delta246-248)beta(2) (Fig. 3). Therefore, mAb 3H5 recognizes an epitope close to or within residues Asp-Tyr. Since mAb 3H5 blocks the interaction of alpha(M)beta(2) to iC3b(21) , these residues may be involved in alpha(M)beta(2) ligand recognition. Fluorescence-activated cell sorting profiles of the recombinant wild-type or mutant alpha(M)beta(2)(s) heterodimers were very similar and were expressed on the cell surface of 40-50% of the cells with the exception of alpha(M)(Delta246-248)beta(2), which demonstrated moderate cell surface expression, 20-25% of the cells ( Fig. 3and results not shown). In agreement with the immunoprecipitation analysis, surface expression of recombinant alpha(M)(D134A)beta(2) was not detected on COS cells (results not shown).




Figure 3: Expression of 3H5 epitope on recombinant wild-type alphabeta(2) or representative mutant alphabeta(2) receptors as determined by flow cytometry. The binding of alpha(M)-specific antibodies LPM19c (a) and 3H5 (b) to cells bearing the wild-type alpha(M)beta(2) or alpha(M)beta(2) mutants was examined by flow cytometry. Results are depicted as histograms with the log of the fluorescence intensity on the abscissa and the cell number on the ordinate. Transfected cells were incubated in the presence of mAbs LPM19c or 3H5 for 30 min at room temperature. Cells were washed, stained with fluorescein-conjugated goat anti-mouse F(ab`)(2) for 30 min, and analyzed on a FACSan.



Interaction of Recombinant alpha(M)beta(2)Mutants with iC3b

To determine whether the mutations in the alpha(M) I domain were essential for ligand recognition, we tested the ability of the transfected COS-7 cells to bind to immobilized iC3b (Fig. 4). COS-7 cell transfectants expressing the wild-type alpha(M)beta(2) adhered to microtiter wells coated with iC3b. Adhesion was specific since it was completely abolished by anti-beta(2) blocking antibody 8H1. Similarly, cells expressing alpha(M)(F246A)beta(2), alpha(M)(G247A)beta(2), alpha(M)(P249A)beta(2), alpha(M)(L250A)beta(2), or alpha(M)(N224A)beta(2) showed no difference in adhesion to iC3b than cells expressing wild-type alpha(M)beta(2) and were blocked by mAb 8H1. In contrast, COS cell transfectants expressing alpha(M)(D140A)beta(2), alpha(M)(S142A)beta(2), alpha(M)(D140GS/A140GA)beta(2), alpha(M)(S144A)beta(2), alpha(M)(D248A)beta(2), alpha(M)(Y252A)beta(2), or alpha(M)(Delta246-248)beta(2) failed to bind to immobilized iC3b.


Figure 4: Adhesion of cells expressing wild-type or mutant alphabeta(2) receptors to immobilized iC3b. Fluorescently labeled cells were allowed to attach to microtiter wells coated with iC3b in the absence (open bars) or presence (hatched bars) of blocking mAb 8H1 (anti-beta(2)) for 30 min at 37 °C. Unbound cells were removed, and adherent cells were quantitated by florescence using a Pandex fluorescence concentration analyzer. The data are expressed as the percentage bound where 100% equals the total number of cells that bound to the mAb 8H1-coated wells to correct for the levels of integrin expressed by the different transfectants. Results are representative of three separate experiments. Bars represent the mean ± S.E. of three determinations.



To further substantiate the importance of these I domain alpha(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 2, Fig. 5). COS-7 cells expressing the wild-type alpha(M)beta(2) or those expressing alpha(M) mutations F246A, G247A, P249A, L250A, or N224A were able to rosette iC3b-E, and binding was blocked by anti-beta(2) antibody, 8H1. In contrast, cells expressing recombinant alpha(M)beta(2) with alpha(M) mutations D140A, S142A, D140GS/A140GA, S144A, D248A, Y252A, or Delta246-248 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 and Ser as playing an essential part in the ligand binding function of alpha(M)beta(2) and designate analogous importance to Ser, Asp, and Tyr in ligand recognition.




Figure 5: Binding of iC3b-E by cells expressing wild-type or representative mutant alpha(M)beta(2) receptors. COS-7 cells transiently transfected with wild-type alpha(M)beta(2) (A and B), alpha(M)(L250A)beta(2), or alpha(M)(D140A)beta(2) were detached and replated on 6-well tissue culture plates for 3 h at 37 °C. Cells were then incubated in the absence (A, C, and D) or presence (B) of blocking mAb 8H1 (anti-beta(2)) for 30 min at 37 °C. iC3b-E were added with transfectants for 60 min at 37 °C. Unbound erythrocytes were removed by washing, and rosettes were examined by light microscopy.



Expression of mAb 24 Epitope to Recombinant alpha(M)beta(2)Mutants

Double alanine substitutions at Asp and Ser of alpha(M) I domain abolish divalent cation-dependent binding to isolated I domain and block alpha(M)beta(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 alpha subunits of the beta(2) integrins(26) . mAb 24 does not recognize the I domain of alpha(L), suggesting that the epitope is not located in this domain(36) . Expression of mAb 24 is associated with the Mg- or Mn-occupied form of the beta(2) integrins(27) . In the presence of 0.5 mM Mn, cells expressing wild-type alpha(M)beta(2) stained brightly with mAb 24 (Fig. 6). Likewise, mAb 24 bound to cells expressing mutations at alpha(M)(F246A), alpha(M)(G247A), alpha(M)(P249A), alpha(M)(L250A), or alpha(M)(N224A). Surprisingly, cells expressing mutations at alpha(M)(D248A), alpha(M)(Y252A), or alpha(M)(Delta246-252), which did not recognize iC3b, bound mAb 24 similar to cells expressing the wild-type alpha(M)beta(2). No detectable binding of mAb 24 was observed in the absence of divalent cation (10 mM EDTA). In contrast, mAb 24 binding in the presence of 0.5 mM Mn to cells expressing alpha(M)(D140)beta(2), alpha(M)(S142)beta(2), or alpha(M)(D140GS/A140GA)beta(2) was markedly reduced, while cells expressing alpha(M)(S144A)beta(2) resulted in a modest attenuation of mAb 24 binding. These data suggest that the two identified ligand binding domains within the alpha(M) I domain differentially alter a divalent cation-dependent conformation of alpha(M)beta(2).


Figure 6: Expression of mAb 24 epitope on recombinant wild-type or mutant alpha(M)beta(2) receptors as determined by flow cytometry. The binding of cation-dependent antibody 24 to COS-7 cells transfected with wild-type or mutant alpha(M)beta(2) receptors was examined by flow cytometry. Transfected cells were incubated with mAb 24 in the presence of 0.5 mM MnCl(2) or 10 mM EDTA for 30 min at 37 °C. Cells were washed, stained with fluorescein-conjugated goat F(ab`)(2) anti-mouse immunoglobulins for 30 min, and analyzed. The binding of mAb 24 is expressed as the percent of mAb 24 binding in the presence of 0.5 mM MnCl(2) (mean linear fluorescence) to the total number of receptors/cell line as determined by the binding of anti-beta(2) antibody (mean linear fluorescence). Results represent the mean ± S.E. of three separate experiments.




DISCUSSION

These studies establish the following. 1) Individual alanine substitutions of alpha(M) I domain residues did not affect heterodimer formation or surface expression when co-transfected with beta(2) in COS-7 cells with the exception of residue Asp, which was not expressed. 2) Alanine substitution of the clustered oxygenated residues at position Asp, Ser, D140GS/A140GA, or Ser resulted in the complete loss of the capacity of alpha(M)beta(2) to recognize iC3b. 3) Alanine substitution of Asp, Tyr, or deletion of residues Phe-Tyr (Delta246-252) abolished the binding of alpha(M)beta(2) to iC3b as well as the recognition of the function blocking anti-alpha(M) antibody 3H5. 4) Substitution of residues Asp, Ser, D140GS/A140GA, or Ser with alanine attenuated the binding of the divalent cation-dependent antibody mAb 24. 5) Alanine substitution at Asp, Tyr, or Delta246-252 did not affect cation-dependent mAb 24 binding function. These results further implicate Asp and Ser and designate analogous importance to Ser, Asp, and Tyr in the ligand binding function of alpha(M)beta(2). Furthermore, these two identified ligand binding domains differentially alter the interaction of bound divalent cations to alpha(M)beta(2).

Since the disclosures of the contribution of the I domain in alpha(M)beta(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 and Ser (D140GS/A140GA), which reside in a cluster of oxygenated residues of the alpha(M) I domain, have been reported to abolish divalent cation-dependent binding of alpha(M)beta(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 in addition to Asp and Ser of alpha(M) I domain appear to be critical for ligand binding function of alpha(M)beta(2). This conclusion is based on the failure of cells expressing alpha(M)(D140A), alpha(M)(S142A), alpha(M)(D140GS/A140GA), or alpha(M)(S144) cotransfected with beta(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 attenuated binding of mAb 24 in the presence of Mn to cells expressing alpha(M)(D140)beta(2), alpha(M)(S142)beta(2), or alpha(M)(D140GS/ A140GA)beta(2) and to a lesser extent to cells expressing alpha(M)(S144A)beta(2). Expression of mAb 24 epitope is associated with the Mg or Mn bound form of the beta(2) integrins(27) . Therefore, the present work suggests that these I domain mutations perturb the interaction between bound divalent cation and alpha(M)beta(2). This is further supported by the identification of these residues as part of the three-dimensional metal coordination sites within the alpha(M) I domain by high resolution crystal structure: Ser, Ser, and Thr as well as secondary coordination sites between Asp to Ser and Asp to Ser(37) . Furthermore, alanine substitution of Asp and a synthetic peptide containing this residue inhibit iC3b binding to alpha(M)beta(2)(23) .

We have previously identified a conserved amino acid alignment of these alpha(M) I domain residues with a similar divalent cation-dependent ligand binding motif, DXSXS, in beta(2) and beta(3) subunits that contribute to ligand binding of alpha(M)beta(2), alpha(L)beta(2), and alphabeta(3), respectively (21, 38) . In addition, substitution of Asp in beta(3) disturbs binding of divalent cations(39) , and substitutions in the corresponding homologous residues in beta(1)(40) and beta(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 alpha(2) I domain are essential for the ligand binding function of alpha(2)beta(1)(42) . Alignment of these beta subunit sequences with the I domain has led to the proposal of an integrin beta subunit I domain(37) . Previous studies identifying natural beta(2) mutations (leukocyte adhesion deficiency) suggest that this region of beta(2) (residues 128-361) represents critical contact sites required for alphabeta heterodimer formation(43) . Interestingly, while alanine substitution of Asp, Ser, D140GS/A140GA, or Ser did not affect the capacity of alpha(M) to efficiently associate with beta(2) on COS cells, alanine substitution at Asp resulted in loss of alpha(M)beta(2) surface expression based on immunoprecipitation with anti-beta(2) antibody. These results suggest a role for the I domain not only in ligand recognition but in alphabeta heterodimer formation. It is particularly noteworthy that alpha(M) residue Asp aligns with the corresponding residue Asp of beta(2), which we previously identified as essential for surface expression of alpha(M)beta(2) and alpha(L)beta(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 beta(3) subunit(44) . It was proposed that residues 118-131 of beta(3) bind both divalent cation and ligand, resulting in the displacement of cation from this region of beta(3) and subsequently exposing secondary binding sites. In the present study we have identified a second region within alpha(M) I domain, which is important in alpha(M)beta(2) ligand recognition of iC3b. This conclusion is based on the inability of the cells expressing I domain mutations at Asp, Tyr or deletion of residues 246-252 to adhere to immobilized iC3b or to rosette iC3b-E. However, in contrast to mutations at Asp, Ser, or Ser, these mutations do not perturb the expression of the Mn-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 alpha(M)beta(2). This suggests that alanine substitution at Asp, Ser, or Ser perturb the interaction between bound cation and the integrin, while substitution at Asp, Tyr, or Delta246-252 do not. Therefore, an alternative hypothesis is that the binding of divalent cations to Asp, Ser, and Ser 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 and Tyr, if we assume that function blocking mAbs bind close to the ligand binding site. In contrast, substitution at Asp, Ser, or Ser did not affect the binding of any of the blocking anti-alpha(M) antibodies tested.

In conclusion, we have further characterized a cation ligand-interactive region in the alpha(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 alpha(M) I domain that does not affect cation-dependent conformation of alpha(M)beta(2). The mechanisms by which the multiple ligand binding domains in the alpha and beta 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-Tyr of the alpha(M) I domain. This region is unique to alpha(M) and alpha(X) but absent in alpha(L) of the beta(2) integrins. alpha(M)beta(2) and alpha(X)beta(2) share a common ligand, iC3b, that is not recognized by alpha(L)beta(2)(1, 2, 3) . We are presently addressing this issue.


FOOTNOTES

*
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§
To whom correspondence should be addressed: Upjohn Laboratories, Cell Biology and Inflammation Research, 7239-267-302, Kalamazoo, MI 49001.

(^1)
The abbreviations used are: mAb, monoclonal antibody; TBS, Tris-buffered saline.


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