Structure-Function of the Putative I-domain within the Integrin beta 2 Subunit*

Yu-Mei Xiong and Li ZhangDagger

From the Department of Vascular Biology, American Red Cross Holland Laboratory, Rockville, Maryland 20855

Received for publication, September 29, 2000, and in revised form, February 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The central region (residues 125-385) of the integrin beta 2 subunit is postulated to adopt an I-domain-like fold (the beta 2I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta 2, a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta 2I-domain with their homologous counterparts of the beta 1I-domain, has been deployed. This approach is based on the premise that beta 1 and beta 2 are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta 2I-domain. When these mutant beta 2 subunits were transfected together with wild-type alpha M in human 293 cells, all 16 beta 2 mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta 2I-domain are not critically involved in heterodimer formation between the alpha M and beta 2 subunits. Using these mutant alpha Mbeta 2 receptors, we have mapped the epitopes of nine beta 2I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro192-Glu197, destroyed ligand binding of the alpha Mbeta 2 receptor, suggesting that these function-blocking mAbs inhibit alpha Mbeta 2 function allosterically. Given the recent reports implicating the segment equivalent to Pro192-Glu197 in ligand binding by beta 3 integrins, these data suggest that ligand binding by the beta 2 integrins occurs via a different mechanism than beta 3. Finally, both the conformation of the beta 2I-domain and C3bi binding activity of alpha Mbeta 2 were dependent on a high affinity Ca2+ binding site (Kd = 105 µM), which is most likely located within this region of beta 2.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha Mbeta 2 is a member of the beta 2 integrin subfamily, which includes alpha Lbeta 2 (LFA-1, CD11a/CD18), alpha Xbeta 2 (p150,95, CD11c/CD18), and alpha Dbeta 2. Like all integrins, the beta 2 subfamily members are expressed on cell surfaces as heterodimers, but their expression is restricted primarily to leukocytes. alpha Mbeta 2 plays a multifunctional role on leukocytes. As examples, this integrin is important in leukocyte adhesion and transmigration through endothelium, in activation of neutrophils and monocytes, in phagocytosis of foreign material, and in apoptosis (1, 2). A wide variety of protein and non-protein ligands have been identified that interact with alpha Mbeta 2, with representative examples including fibrinogen (Fg)1 (3), ICAM-1 (4), C3bi (5), zymosan (6), and neutrophil inhibitory factor (7). C3bi and Fg are two particularly important ligands of alpha Mbeta 2; C3bi is critical to phagocytosis of opsonized foreign particles, and Fg, which interacts with alpha Mbeta 2 via its gamma -module (8), is involved in leukocyte adhesion and migration.

Central to the ligand binding function of alpha Mbeta 2 is its I(A) domain. The alpha MI-domain is an inserted segment of ~200 amino acids and is highly homologous to several I-domains found in integrin alpha  subunits (9). The three-dimensional structures of several I-domains (alpha M, alpha L, alpha X, alpha 2, etc.) have been solved (10-13). These I-domains are composed of six or seven alpha -helices and six beta -sheets arranged in a Rossman-type fold. A cation binding site, termed the MIDAS motif, is located within the I-domain. In the MIDAS motif, cation coordination is provided by a DXSXS sequence and by other two distant (in terms of primary sequence) oxygenated residues (10). In addition to the alpha  subunits with their I-domains, the beta  subunits also contribute to ligand binding to integrins. Studies of the beta  subunits have been focused primarily on their central regions (residues ~125-385 in a typical beta  subunit of >700 amino acids). This region is predicted to contain a MIDAS motif, and candidate residues for cation coordination have been identified by mutagenesis (14-17). Protein sequence analysis suggests that this region may also fold into an I-domain-like structure (10, 18). However, due to the low homology between the I-domains of the alpha  and beta  subunits, it is uncertain whether this putative I-domain region does, indeed, fold into an I-domain, or merely contains a MIDAS motif. What is clear is that this region does play a critical role in mediating ligand binding to integrins. In beta 3, it was reported that bound RGD peptides can be cross-linked to this region (19, 20). Substituting this segment within the beta 1I- or beta 5I-domain with its homologous counterpart from beta 3 imparts beta 3 ligand specificity to the beta 1 or beta 5 integrin (21, 22). A natural mutation of Arg214 to Gln in beta 3 abolishes ligand binding of alpha IIbbeta 3, and a synthetic peptide containing the sequence of beta 3 (211) blocks Fg binding to purified alpha IIbbeta 3 (23). Similar observations implicate the beta 1I-domain in the ligand binding functions of the beta 1 integrins. For example, it was shown that both activating and inhibiting mAbs recognize a small stretch of beta 1 (residues 124-160 and 207-218) (24, 25). Recently, the D134XSXS sequence of the proposed MIDAS motif within beta 2 was implicated in the binding of Fg, C3bi, and ICAM-1 to alpha Mbeta 2 (26, 27). These data indicate that this putative I-domain is important to ligand binding functions of the beta 2 integrins as well.

Recently, we have deployed homolog-scanning mutagenesis (28) to identify several segments critical to Fg and C3bi binding within the alpha MI-domain (8, 29). This approach entails switching sequences within the alpha MI-domain to their homologous sequences within the alpha LI-domain. This approach is feasible because the alpha MI- and alpha LI-domains are highly homologous, but alpha Mbeta 2 and alpha Lbeta 2 recognize different ligands. In the study reported here, we have applied this same strategy to the putative beta 2I-domain region. Our data are consistent with folding of the region into an I-domain-like structure. However, our results suggest that ligand recognition by the region of the beta 2 subunit is achieved in a distinct fashion from that involved in ligand recognition by the beta 3 integrins. In addition, we show that the epitopes of several blocking mAbs map to this region but their inhibitory activity is likely to be achieved via an allosteric mechanism. Finally, we show that the conformation and ligand binding functions of the beta 2I-domain are enhanced selectively by Ca2+, suggesting a unique cation-specific effect on the beta 2I-domain. Taken together, these results provide insight into the structure-function relationships of alpha Mbeta 2, which may also extend to other integrins in general.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Human kidney 293 cells and the expression vector, pCIS2M, were gifts from Dr. F. J. Castellino (University of Notre Dame, Notre Dame, IN). The cDNAs of CD11b and CD18 were obtained from Dr. B. Karan-Tamir (Amgen, Thousand Oaks, CA). The recombinant gamma -module of Fg was provided by Dr. Medved (American Red Cross, Rockville, MD). The mAbs used in this study were obtained from the following sources. mAb 6.5E was provided by Dr. D. P. Andrew (Amgen Inc., Boulder, CO); mAb MHM23 was from Dako (Carpinteria, CA); IB4 and TS1/18 were from the ATCC (Rockville, MD); mAb 44 was from Sigma; CLB-LFA-1/1,54 (CLB54) was from RDI (Flanders, NJ); YFC118.3 and R3.3 were from Chemicon (Temecula, CA); H20A was from VMRD Inc. (Pullman, WA); 6.7 was from PharMingen (San Diego, CA); 685A5, MEM-48, and 7E4 were from Biodesign (Kennebunk, ME).

Site-directed Mutagenesis and Development of Stable Cell Lines-- The detailed procedures used for homolog-scanning mutagenesis and to establish stable cell lines expressing wild-type and mutant alpha Mbeta 2 receptors in human kidney 293 cells have been published (30). Similar methods were used to express the alpha Mbeta 2 heterodimer and the single beta 2 subunit on the surface of the Chinese hamster ovary cells. To obtain cell lines with similar expressions, each mutant cell line was subcloned by cell sorting using an alpha M-specific mAb (2LPM19c). Up to 20 colonies were selected and analyzed for integrin expression by FACS analysis. Cells with receptor expression levels similar to wild-type alpha Mbeta 2 were chosen, and five different subclones were used for the subsequent studies reported in this work. To exclude the possibility of subcloning artifacts, all studies were repeated using the original pool of each mutant receptor.

Surface Labeling and Immunoprecipitation-- Cells expressing wild-type and mutant alpha Mbeta 2 were washed once with DPBS, biotinylated with EZ-link Sulfo-NHS-LC-Biotin (sulfosuccinimidyl 6-biotinamidohexanoate, Pierce), and lysed with a solution containing 20 mM Tris-Cl, 150 mM NaCl, pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, 25 µg/ml soybean trypsin inhibitor, and 20 µg/ml leupeptin. The cell lysates were subjected to immunoprecipitation with an alpha M-specific mAb 44a and a beta 2-specific mAb 6.7. The immunoprecipitates were analyzed on 7% acrylamide gels, and the surface-expressed alpha Mbeta 2 was visualized by Western blotting using a horseradish peroxidase-avidin conjugate.

C3bi Binding and Adhesion to Fg-- The ligand binding activity of the beta 2 mutants was assessed using two classic alpha Mbeta 2 ligands, C3bi and Fg, according to our published methods (27). For adhesion of alpha Mbeta 2-expressing cells to Fg, the recombinant gamma -module (10 µg/ml) was deposited at the center of each well in a 24-well non-tissue culture polystyrene plate. After blocking with 400 µl of 0.05% polyvinylpyrrolidone in DPBS, a total of 2 × 106 cells in Hank's balanced salt solution containing 1 mM Ca2+ and 1 mM Mg2+ was added to each well and incubated at 37 °C for 20 min. The unbound cells were removed by three washes with DPBS, and the adherent cells were quantified by cell-associated acid phosphatase as described previously (27).

FACS Analysis-- A total of 106 cells expressing wild-type or mutant alpha Mbeta 2 in Hank's balanced salt solution containing 1 mM Mg2+ and 1 mM Ca2+ was incubated with 1 µg of mAb for 30 min at 4 °C. A subtype-matched mouse IgG served as a control. After washing with PBS, cells were mixed with FITC-conjugated goat-anti-mouse IgG(H+L) F(ab')2 fragment (1:20 dilution) (Zymed Laboratories Inc.), and kept at 4 °C for another 30 min. Cells were then washed with PBS and resuspended in 500 µl of DPBS. The FACS analyses were performed using FACScan (Becton-Dickinson), counting 10,000 events. Mean fluorescence intensities were quantified using the FACScan program, and these values were used to compare alpha Mbeta 2 expression levels or the reactivity of the different alpha Mbeta 2 mutants with specific mAbs.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Homolog-scanning Mutagenesis of the beta 2I-domain-- As shown in Fig. 1, the purported I-domain within integrin beta 2 shares considerable sequence homology with the corresponding region of the beta 1 subunit. The major sequence differences are confined to regions that are predicted to be hydrophilic and surface-oriented based on hydropathy plots and molecular modeling, and, thereby, are the segments that are likely to contribute to the unique functions of the beta 2 integrins. For example, the beta 2 subunit partners with an entirely separate set of alpha  subunits from beta 1, and the beta 2 integrins recognize a set of ligands very distinct from the beta 1 integrins (there is no known peptide sequence recognized by both beta 1 and beta 2 integrins). Based on the sequence homology between the beta 1I- and beta 2I-domains, we sought to systematically probe the function of the hydrophilic and unique segments of this region (residues 125-385) using homolog-scanning mutagenesis. Accordingly, we replaced 16 non-conserved segments of three to nine residues within the beta 2I-domain with the corresponding segments from the beta 1 subunit (Fig. 1). These 16 segments covered the entire hydrophilic region of the beta 2I-domain predicted from hydropathy plots and molecular modeling. The primers used for mutagenesis are listed in Table I. The DNA sequence of the entire I-domain was confirmed for each mutant before and after transfer back into the pCIS2M expression vector containing the cDNA of beta 2.


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Fig. 1.   Sequence alignment between the putative beta 1I- and beta 2I-domains. The amino acid residues are from 141 to 395 for the beta 1I-domain and from 125 to 380 for the beta 2I-domain (the numbering is based on the entire protein sequence including the signal peptide). The conserved residues are underlined, and the mutated segments are shown in brackets.

                              
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Table I
Primers used in the homolog-scanning mutagenesis of the putative beta 2I-domain

Surface Expression and Heterodimer Formation-- A large number of natural mutations occur within the beta 2I-domain, which abolish surface expression and/or heterodimer formation (31-37). Nevertheless, when the beta 2 mutants were co-transfected with wild-type alpha M in human kidney 293 cells, all 16 mutants were expressed on the cell surface as heterodimers and the subunits had appropriate molecular weights. As shown in Fig. 2, immunoprecipitation of surface-labeled cells with 44a, a mAb specific for the alpha M subunit, yielded two bands of ~165 kDa (alpha M) and 95 kDa (beta 2) on SDS-PAGE. The patterns were similar to those obtained for wild-type alpha Mbeta 2 (27). In addition, FACS analyses were conducted on these 16 mutants using a panel of beta 2-specific mAbs (Table II). All 16 beta 2 mutants were recognized by three different mAbs to the beta 2 subunit MEM48, 7E4, and 6.7, as well as by the alpha M-specific mAb 44. To exclude selection artifacts, we established at least five independent stable cell lines for each mutant beta 2 integrin that expressed similar levels of receptors on their cell surfaces, as judged by FACS analysis using mAb 44. Heterodimer formation, as well as other results described below, was similar for all five clones.


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Fig. 2.   Surface expression and heterodimer formation of the 16 beta 2I-domain mutants. The 16 beta 2 homolog-scanning mutants were co-transfected with wild-type alpha M into human 293 cells, and stable cell lines were established. A total of 5 × 106 cells expressing the wild-type (wt) or mutant alpha Mbeta 2 were surface-labeled by biotin and then lysed. The biotinylated alpha Mbeta 2 was immunoprecipitated with 5 µg of an alpha M-specific mAb (44a) and analyzed on 7% SDS-PAGE. Lane 1, alpha Mbeta 2(Arg144-LysK148); lane 2, alpha Mbeta 2(Leu154-Glu159); lane 3, alpha Mbeta 2(Glu162-Glu164); lane 4, alpha Mbeta 2(Asn181-Asp185); lane 5, alpha Mbeta 2(Pro192-Glu197); lane 6, alpha Mbeta 2(Gln199-Ala203); lane 7, alpha Mbeta 2(Asn213-Glu220); lane 8, alpha Mbeta 2(Pro247-Glu249); lane 9, alpha Mbeta 2(Ala262-Asp265); lane 10, alpha Mbeta 2(Asp290-Glu298); lane 11, alpha Mbeta 2(Gly305-His309); lane 12, alpha Mbeta 2(Ser324-Thr329); lane 13, alpha Mbeta 2(Thr334-Ile336); lane 14, alpha Mbeta 2(Glu344-Asp348); lane 15, alpha Mbeta 2(His354-Asn358); lane 16, alpha mbeta 2, His371-Lys379.

                              
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Table II
Reactivity of function-blocking monoclonal antibodies with the beta 2I-domain mutants
FACS analysis was performed using 1 µg of each mAb and 106 alpha Mbeta 2-expressing cells. A "+" indicates that the mean fluorescence intensity of the mAb is at least 10 times that of the IgG control. A "-" indicates that the mean fluorescence intensity of the mAb is no more than that of the IgG control.

Epitope Mapping of Function-blocking mAbs-- To help locate the functional sites within the beta 2I-domain, we sought to map the epitopes of several beta 2-specific function-blocking mAbs: MHM23, IB4, 6.5E, TS1/18, CLB54, YFC118.3, R3.3, H20A, 685A5, and 7E4. The ability of these mAbs to block beta 2 integrin functions, such as alpha Mbeta 2-mediated adhesion and C3bi binding and alpha Lbeta 2-mediated binding to ICAM-1, has been well documented (38-42). Representative FACS analyses using mAb IB4 with five of the alpha Mbeta 2 mutants are shown in Fig. 3A and a summary of the FACS analyses for all 16 mutants and 12 beta 2-specific mAbs is shown in Table II. Among these 12 mAbs, 3 (6.7, MEM-48, and 7E4) reacted well with all 16 mutants, but not the mock-transfected 293 cells. The other nine mAbs recognized the beta 2I-domain, and their epitopes consisted of at least two noncontiguous sequences. For example, mAb IB4 reacted well with wild-type alpha Mbeta 2, and mutants alpha Mbeta 2(Leu154-Glu159), alpha Mbeta 2(Asn213-Glu220) and alpha Mbeta 2(His354-Asn358), but its binding to the two mutants alpha Mbeta 2(Arg144-Lys148) and alpha Mbeta 2(Pro192-Glu197) was ablated (Fig. 3A), suggesting that these two segments (Arg144-Lys148 and Pro192-Glu197) contribute to the epitope of IB4. As shown in Table II, in addition to IB4, mAbs MHM23, H20A, R3.3, and perhaps 6.5E also depended on segments Arg144-Lys148 and Pro192-Glu197 for their interactions with alpha Mbeta 2. mAb 685A5 required segments Arg144-Lys148, Pro192-Glu197, and Asn213-Glu220; mAb TS1/18 required segments Leu154-Glu159 and Glu344-Asp348; mAb CLB54 required segments Leu154-Glu159 and His354-Asn358; and finally mAb YFC118.3 required segments Arg144-Lys148, Leu154-Glu159, and His354-Asn358. These epitopes can be roughly divided into two different groups (see Fig. 8). The first contains segments Leu154-Glu159, Glu344-Asp348, and His354-Asn358, and is important for alpha Mbeta 2 interaction with mAbs TS1/18, CLB54, and YFC118.3, and the second contains segments Arg144-Lys148, Pro192-Glu197, and Asn213-Glu220, and is important for alpha Mbeta 2 binding of mAbs MHM23, H20A, IB4, R3.3, and 685A5. To further support our epitope mapping results and this grouping of the mAbs, we performed two additional experiments. First, competition was performed between mAbs MHM23, IB4, and R3.3 from group 2, TS1/18 from group 1, and 7E4, which recognizes an epitope that is likely located outside of the beta 2I-domain. In these experiments, alpha Mbeta 2-expressing cells were incubated first with the competitor mAb, IB4, R3.3, TS1/18, or 7E4, and then the reporter mAb MHM23 was added. Binding of MHM23 was measured by FACS analysis, and the results are shown in Fig. 3B. As predicated, mAbs IB4 and R3.3, which belong to the same group as MHM23 (group 2), blocked more than 95% of the binding of mAb MHM23 to alpha Mbeta 2. In contrast, mAb TS1/18 (group 1) and mAb 7E4 had little effect on MHM23 binding. The specificity of these assays was confirmed by the ability of unlabeled MHM23 but not a control IgG to block the binding of the fluorescence-labeled MHM23 to the cells. Second, the ability of mAb IB4 to block adhesion of alpha Mbeta 2-expressing cells to a representative ligand, the gamma -module of fibrinogen, was assessed using wild-type and two different alpha Mbeta 2 mutants. As shown in Fig. 3C, cells expressing these three different alpha Mbeta 2 receptors all adhered well to the gamma -module in the presence of a control IgG. Addition of mAb IB4 completely inhibited adhesion of cells expressing the wild-type and one of the mutant receptors alpha Mbeta 2(Leu154-Glu159). However, mAb IB4 had no effect on adhesion by the second mutant alpha Mbeta 2(Arg144-Lys148). These results are consistent with the FACS data presented in Fig. 3A, which show that the epitope of mAb IB4 was destroyed in mutant alpha Mbeta 2(Arg144-Lys148) but not in mutant alpha Mbeta 2(Leu154-Glu159).


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Fig. 3.   Epitope mapping of the beta 2I-domain function-blocking mAbs. A, representative FACS analysis using mAb IB4. alpha Mbeta 2-expressing 293 cells (106) were incubated with 1 µg of mAb at 4 °C for 30 min. After three washes with PBS, the cells were stained with FITC-conjugated goat anti-mouse IgG and analyzed using FACScan. An isotype-matched IgG was used as a control. B, competition among different function-blocking mAbs for binding to alpha Mbeta 2. The alpha Mbeta 2-expressing cells were first incubated with an IgG control, or with mAbs IB4, R3.3, MHM23, TS1/18, and 7E4 individually for 30 min at 4 °C, and then incubated with a MHM23-FITC conjugate for additional 30 min. After washing with PBS, bound MHM23 was measured by FACS analysis. C, inhibition of cell adhesion to the gamma -module of Fg by mAb IB4. A total of 2 × 106 alpha Mbeta 2-expressing cells was incubated with either a control IgG (open bar) or mAb IB4 (filled bar) for 30 min at 4 °C, and then added to 24-well non-tissue culture polystyrene plates, which were pre-coated with recombinant gamma -module (10 µg/ml) and subsequently blocked with 0.05% polyvinylpyrrolidone in DPBS. After incubation at 37 °C for 20 min, the unbound cells were removed by three washes with DPBS and the adherent cells were quantified by cell-associated acid phosphatase. For each cell line, the number of adherent cells in the presence of the control IgG was taken as 100%. Asterisk (*) indicates a value of less than 1%. Data shown are the means ± S.D. of three independent experiments.

Role of the beta 2I-domain in Ligand Binding-- A short disulfide loop of 7-8 amino acids has been implicated in the ligand binding functions of the beta 3 integrins (21, 22, 43). This disulfide loop is conserved in the beta 2 subunit, corresponding to Pro192-Glu197 within the putative beta 2I-domain. Given the high degree of homology between the beta 2 and beta 3 subunits, we tested the hypothesis that segment Pro192-Glu197 is also important to the ligand binding function of alpha Mbeta 2. The gamma -module of Fg and C3bi were used as model alpha Mbeta 2 ligands, and we assessed their interactions with the mutant alpha Mbeta 2(Pro192-Glu197), in which this segment was replaced with its homologous counterpart of the beta 1 subunit. We expected that this mutant would be defective in Fg and C3bi binding, should Pro192-Glu197 constitute a part of the ligand binding site within alpha Mbeta 2. As shown in Fig. 4 (A and B), this mutant bound C3bi and interacted with the gamma -module similarly to wild-type alpha Mbeta 2, suggesting that this sequence is not directly involved in ligand binding by alpha Mbeta 2. The specificity of the C3bi binding assay was confirmed using mock-transfected 293 cells and by inhibition experiments with EDTA. In addition, the specificity was further verified by blocking experiments using the alpha M-specific mAb 44a; addition of 44a blocked more than 90% C3bi binding to both the wild-type and the mutant receptors. Similarly, the specificity of the adhesion to the gamma -module was confirmed by blocking experiments with EDTA (data not shown) and mAb 44a. Thus, the contribution of the beta 2 subunit to ligand binding is different from that of beta 3, suggesting that ligand binding to the beta 2 integrins has different requirements.


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Fig. 4.   Ligand binding to beta 2I-domain mutants containing the epitopes of function-blocking mAbs. A, C3bi binding. Biotinylated EC3bi (2 × 107) were added to 2 × 105 cells expressing alpha Mbeta 2, which had been pre-seeded onto polylysine-coated 24-well plates. After 60 min at 37 °C, the amount of bound EC3bi was determined using avidin-alkaline phosphatase and p-nitrophenylphosphate, measuring the absorbance at 405 nm. The value for wild-type alpha Mbeta 2 was taken as 100%. Specificity was demonstrated by addition of 1 mM EDTA (shown with asterisks) and further verified by blocking experiments with an alpha M-specific mAb 44a; addition of mAb 44a blocked more than 90% C3bi binding to wild-type and three representative mutants: alpha Mbeta 2(Arg144-Lys148), alpha Mbeta 2(Pro192-Glu197), and alpha Mbeta 2(Leu154-Glu159). Data are the means ± S.D. of three to six independent experiments. B, Fg adhesion. Adhesion of alpha Mbeta 2-expressing cells to the gamma -module of Fg was performed as described in Fig. 3C except that the number of adherent cells expressing wild-type alpha Mbeta 2 was taken as 100%. Specificity was verified using alpha M-specific function-blocking mAb 44a (filled bar). Data are the means ± S.D. of three to six independent experiments.

In addition to the segment Pro192-Glu197, five other segments were recognized by the nine beta 2-blocking mAbs (MHM23, IB4, 6.5E, TS1/18, CLB54, YFC118.3, R3.3, H20A, and 685A5). To determine if these segments contribute directly to the ligand binding function of alpha Mbeta 2, we repeated the above ligand binding experiments with the five beta 2 mutants: alpha Mbeta 2(Arg144-Lys148), alpha Mbeta 2(Leu154-Glu159), alpha Mbeta 2(Asn213-Glu220), alpha Mbeta 2(Glu344-Asp348), and alpha Mbeta 2(His354-Asn358). As shown in Fig. 4 (A and B), the five additional mutants also displayed significant ligand binding activities toward both C3bi and the Fg gamma -module. For C3bi binding, all five mutants interacted well with C3bi, similar to wild-type alpha Mbeta 2 (Fig. 4A); for adhesion to the Fg gamma -module, two mutants (alpha Mbeta 2(Leu154-Glu159) and alpha Mbeta 2(His354-Asn358)) behaved similarly to the wild-type receptor, whereas one mutant (alpha Mbeta 2(Arg144-Lys148)) adhered less (~50% of wild-type), and the other two mutants (alpha Mbeta 2(Asn213-Glu220) and alpha Mbeta 2(Glu344-Asp348)) adhered more strongly (~300% of wild-type) to the gamma -module. Thus, these results suggested that the epitopes of the above beta 2I-domain specific mAbs do not contribute significantly to ligand binding per se, and, therefore their blocking functions occur most likely via an allosteric mechanism.

The Influence of Ca2+ on the Conformation of the beta 2I-domain-- Ligand binding to integrins depends upon divalent cations, and specific cations can influence ligand binding specificity. For example, the alpha MI-domain adopts different conformations in the presence of Ca2+ versus Mn2+ (44, 45), and conformational changes are induced in the beta 1I-domain by Mg2+ and Ca2+ (46, 47). In the course of our studies, we observed that binding of mAbs YFC118.3 and TS1/18 to alpha Mbeta 2 was supported by Ca2+ but not by Mg2+ and that addition of EGTA/Mg2+ or EDTA reduced the binding of these mAbs by 4-fold (for YFC118.3) or 5-fold (for TS1/18) (Fig. 5A). As these two mAbs recognize different and non-contiguous regions within the beta 2I-domain (see Table II; TS1/18 recognizes Leu154-Glu159 and Glu344-Asp348, whereas YFC118.3 recognizes Arg144-Lys148, Leu154-Glu159, and His354-Asn358), these results suggested that the overall conformation of the beta 2I-domain is differentially influenced by cations and that the conformation induced by Ca2+ is required for optimal reactivity with these mAbs. To further characterize these observations, we tested the effect of Ca2+ concentrations on the binding of these two mAbs. A constant concentration of each mAb of 20 nM was selected for these analyses, which is below the concentrations of each required for 50% of its maximal binding to alpha Mbeta 2. The Ca2+ titration curve for mAb YFC118.3 is shown in Fig. 5B. Binding of the mAb increased with increasing Ca2+ and saturated above 500 µM added Ca2+. These data could be fitted to a single binding site model. The estimated Kd of this Ca2+ binding site was 105 ± 9 µM. A similar Kd for Ca2+ binding site was obtained with TS1/18. This similarity suggests that the same Ca2+ binding site is involved in the binding of these two mAbs to alpha Mbeta 2.


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Fig. 5.   Effects of Ca2+ on the binding of mAbs TS1/18 and YFC118.3. A, binding of mAbs TS1/18 and YFC118.3 to alpha Mbeta 2 depends on Ca2+. alpha Mbeta 2-expressing 293 cells (106) in the presence of 1 mM Ca2+ (thick line) or 2 mM Mg2+ plus 1 mM EGTA (thin line) was determined by FACS analysis. An isotype-matched IgG was used as a control for each mAb (dotted line). B, mAb YFC118.3 binding to alpha Mbeta 2 as a function of different concentrations of Ca2+. Binding of mAbYFC118.3 (1 µg) to wild-type alpha Mbeta 2-expressing 293 cells (106) in the presence of different concentrations of Ca2+ was determined by FACS analysis. The mean fluorescence intensity was determined using the FACScan program. The titration data can be fitted into a single binding site model with an apparent Kd value of 105 ± 9 µM by non-linear regression analysis, using an equation provided by SigmaPlot, F = F0 + Fmax*[Ca]/(Kd+[Ca]), where F is the total bound mAb measured by FACS, F0 is the mAb bound in the absence of Ca2+, and [Ca] is the Ca2+ concentration added. C, mAbYFC118.3 binding to alpha Mbeta 2(S136A)-expressing cells. Binding of mAbYFC118.3 to alpha Mbeta 2(S136A) in the presence of different concentrations of Ca2+ was determined as described for wild-type alpha Mbeta 2.

To explore the possibility that the Ca2+ binding site reported by these two mAbs is located within the MIDAS motif (D134XSXS) of the beta 2I-domain, we tested YFC118.3 binding to a mutant beta 2, in which Ser136, a putative cation coordination site, was replaced by Ala. As shown in Fig. 5C, Ca2+ bound to this mutant alpha Mbeta 2 with a significantly (p < 0.03) reduced affinity (Kd = 151 ± 10 µM), compared with wild-type alpha Mbeta 2, indicating that the cation binding site reported by YFC118.3 is likely located within the beta 2I-domain. To exclude the possibility that Ca2+ binding to the alpha M subunit may allosterically affect YFC118.3 and TS1/18 binding to beta 2, we expressed the beta 2 subunit alone on the surface of the Chinese hamster ovary cells. The presence of beta 2 and absence of alpha M on the cell surface was confirmed by FACS analyses using an alpha M-specific mAb 44 and beta 2-specific mAbs 6.7 (Fig. 6A), 7E4, and MEM-48 (data not shown). That the beta 2 subunit is expressed alone on the cell surface is further supported by surface labeling and immunoprecipitation experiments; for the alpha Mbeta 2-expressing cells, both mAbs 44a (against alpha M) and 6.7 (against beta 2) yielded two bands of ~95 and 165 kDa on SDS-PAGE, whereas for the beta 2-expressing cells, mAb 44a did not produce any detectable band and mAb 6.7 yielded only a single band of 95 kDa (beta 2) (Fig. 6B). These data demonstrate that the beta 2 subunit is present alone on the cell surface and not complexed with alpha M or any other integrin alpha  subunits. To see whether the single beta 2 subunit still contains a high affinity Ca2+ binding site, we repeated the above Ca2+ titration experiments with mAbs YFC118.3 and TS1/18. Fig. 6C shows that, similar to the alpha Mbeta 2 heterodimer, mAb YFC118.3 bound to single beta 2 in a cation-dependent manner, and the Ca2+ titration curve can be fitted to a single binding site model. The estimated Kd of this Ca2+ binding site is 83 ± 2 µM, which is very close to the Kd of 105 µM for the heterodimeric receptor. A similar Kd for Ca2+ binding to beta 2 was obtained with TS1/18. Taking these data together, we conclude that the proper conformation for mAb binding to the beta 2I-domain depends upon a Ca2+ binding site within beta 2, possibly composed of Ser136 within the proposed MIDAS motif of the beta 2I-domain.


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Fig. 6.   Ca2+ binding to the surface-expressed single beta 2 subunit. A, FACS analysis of surface-expressed single beta 2. The beta 2-expressing cells (106) were incubated with 1 µg of an alpha M-specific mAb 44 (gray line), a beta 2-specific mAb 6.7 (black line), or a control IgG (dashed line) at 4 °C for 30 min. After three washes with PBS, the cells were stained with FITC-conjugated goat anti-mouse IgG and analyzed using FACScan. B, surface labeling and immunoprecipitation. A total of 5 × 106 cells expressing the alpha Mbeta 2 heterodimer (lanes 1 and 2) or the beta 2 subunit alone (lanes 3 and 4) were surface-labeled by biotin and then lysed. The biotinylated alpha Mbeta 2 or single beta 2 was immunoprecipitated with 5 µg of an alpha M-specific mAb 44a (lanes 1 and 3) or a beta 2-specific mAb 6.7 (lanes 2 and 4), and analyzed on 7% SDS-PAGE. C, mAbYFC118.3 binding to the beta 2-expressing cells. Binding of mAbYFC118.3 to the single beta 2 subunit in the presence of different concentrations of Ca2+ was determined as described for wild-type alpha Mbeta 2.

Several studies have reported that ligand binding by the alpha MI-, alpha LI-, alpha 1I-, alpha 2I-, beta 1I-, and beta 3I-domains is supported by Mg2+ but not Ca2+ (47-50). In fact, Ca2+ can inhibit ligand binding to several of these integrins. As our data suggest that the beta 2I-domain contains a unique high affinity Ca2+ binding site, we next tested the effects of Mg2+ and Ca2+ on C3bi binding by alpha Mbeta 2. As shown in Fig. 7, C3bi binding is supported by 1 mM Ca2+. This interaction can be further increased by addition of Mg2+. However, alpha Mbeta 2 only exhibited minimal ligand binding in the presence of Mg2+ alone (EGTA was added to exclude possible contributions by Ca2+). As expected, addition of 1 mM EDTA completely abolished C3bi binding to alpha Mbeta 2, confirming the cation dependence of the C3bi/alpha Mbeta 2 interaction. Thus, in the case of alpha Mbeta 2, Ca2+ is not inhibitory but is required for ligand binding.


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Fig. 7.   Ca2+ dependence of C3bi binding to alpha Mbeta 2. C3bi binding to wild-type alpha Mbeta 2 was measured in the presence of 1 mM Ca2+ (hatched bar), 2 mM Mg2+ plus 1 mM EGTA (gray bar), 1 mM Ca2+ plus 1 mM Mg2+ (solid bar), or 1 mM EDTA (open bar) as described in Fig. 4. The value for C3bi binding in the presence of both Ca2+ and Mg2+ was taken as 100%. Specificity was verified using mock-transfected 293 cells in the presence of 1 mM Ca2+ and 1 mM Mg2+. Data shown are the means ± S.D. of three independent experiments. Asterisk (*) represents a value of less than 1%.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this work, we have probed the function of the hydrophilic surface of the putative beta 2I-domain (residues 125-385), using a homolog-scanning mutagenesis approach. Our major findings are as follows. 1) The majority of the hydrophilic surface of the beta 2I-domain is not critically involved in heterodimer formation between the alpha M and beta 2 subunits. 2) Although the epitopes of several function-blocking mAbs map to the putative beta 2I-domain, these epitopes are not involved directly in ligand binding to alpha Mbeta 2. 3) The positioning of these epitopes is consistent with an I-domain-like fold for this region of the beta 2 subunit, as proposed by several investigators (10, 15-18, 42). 4) Of particular note, segment Pro192-Glu197, which has been implicated in direct ligand contact by the beta 3 integrins (21, 22, 43), is not critical to ligand binding by alpha Mbeta 2, suggesting a fundamental difference between the ligand binding mechanism by beta 2 versus beta 3. 5) The optimal conformation of the beta 2I-domain for C3bi binding depends on a functional Ca2+ binding site within the beta 2 subunit.

A number of studies have demonstrated the importance of the central region (residues 125-385 in beta 2) of the integrin beta  subunits in alpha /beta association. This region of beta 1 (residues 121-329) forms a heterodimer with alpha 5 (160) (47), and the same region of beta 3 (residues 111-318) complexes with alpha IIb (1) (51). In addition, many naturally occurring point mutations within this region, Asp128, Leu149, Gly169, Pro178, Lys196, Gly273, Gly284, and Asn351 (31-37), preclude cell-surface expression of the beta 2 integrins. Furthermore, swapping residues V275GSDNH between human and avian beta 3 was found to change the specificity of alpha IIb/beta 3 association (51). Taken together, these data strongly implicate this central region of the beta  subunits in either heterodimer formation or in controlling the pairing specificity between the alpha  and beta  subunits. None of the known alpha  subunits complex with both beta 1 and beta 2, and thus we expected heterodimer formation would be perturbed in some of our homolog-scanning mutants, particularly the one involving segment Asp290-Glu298, which is homologous to V275GSDNH of beta 3 (51). Nevertheless, when all 16 nonconserved segments within the beta 2I-domain, including segment Asp290-Glu298, were replaced with their counterpart sequences within the beta 1I-domain, surface expression and heterodimer formation were not affected, as assessed by surface labeling and immunoprecipitation experiments. Therefore, we conclude that the majority of the hydrophilic residues of the beta 2I-domain do not make a significant contribution to the heterodimer formation and specificity pairing of the alpha M and beta 2 subunits. As most of the hydrophobic residues are identical between beta 1I- and beta 2I-domains, it is possible that the remaining few non-identical hydrophobic residues within the beta 2I-domain, most of which have been mutated and found not critical in this study (see Fig. 1), are responsible for the specific paring between alpha M and beta 2. Alternatively, as the C-terminal cysteine-rich region is not involved in heterodimer formation (52, 53), the N-terminal plexin-homologous region is a likely candidate for determining the specificity of the heterodimer formation. It should be noted that most of the naturally occurring point mutations that prevent cell surface expression occur at conserved sites in the beta  subunit, and these could affect the overall fold of the beta I-domains, leading to intracellular degradation (the exception to this is Lys196, which is not conserved, but when we substituted a Glu at this position, surface expression also was not affected).

It was proposed recently that the central region within the beta  subunits (residues 125-385 for beta 2) folds into an I-domain-like structure, similar to that present in several integrin alpha  subunits (10, 18). However, homology between the I-domains of the alpha  and beta  subunits is very low, particularly in the C-terminal portions, and conflicting views exist in the literature as to whether this region assumes an I-domain fold or merely contains a metal binding MIDAS motif (DXSXS), such as that found in I-domains (14-17). Using the 16 homolog-scanning mutants of the beta 2I-domain, we have mapped the epitopes of nine mAbs (MHM23, IB4, 6.5E, TS1/18, CLB54, YFC118.3, R3.3, H20A, and 685A5). All of these mAbs reactive with the putative beta 2I-domain recognized epitopes that are composed of at least two non-contiguous sequences. For discussion purposes, these epitopes can be divided into two groups (Fig. 8): the first group contains segments Leu154-Glu159, Glu344-Asp348, and His354-Asn358 as part of their epitopes. This group includes TS1/18 (Leu154-Glu159 and Glu344-Asp348), CLB54 (Leu154-Glu159and His354-Asn358), and YFC118.3 (Arg144-Lys148, Leu154-Glu159, and His354-Asn358). The second group includes segments Arg144-Lys148, Pro192-Glu197, and Asn213-Glu220 as part of their epitopes. This second group includes MHM23, H20A, IB4, and R3.3 ( Arg144-Lys148 and Pro192-Glu197), and 685A5 (Arg144-Lys148, Pro192-Glu197, and Asn213-Glu220). The spatial relationship of these segments is consistent with the I-domain fold such that protein folding will bring the distal segments Leu154-Glu159, Glu344-Asp348, and His354-Asn358 (group 1), or Arg144-Lys148, Pro192-Glu197, and Asn213-Glu220 (group 2) together into spatial proximity to form the overlapping mAb epitopes. Our mapping results are consistent with a very recent study, in which the epitopes of some mAbs from group 1 were mapped and used to construct a three-dimensional model for the beta 2I-domain (42). Although our results support this model, there is one major difference. The identification of mAbs (MHM23, H20A, IB4, R3.3, and 685A5) belonging to group 2 allows us to define more accurately the position of the disulfide loop, C191PNKEKEC198, within the three-dimensional framework. The positioning of this loop is particularly important, given the recent report implicating the segments equivalent to Pro192-Glu197 within this loop in ligand binding to beta 3 and beta 5 (22, 43). As shown in Table II, segments Arg144-Lys148, Pro192-Glu197 (within the disulfide loop), and Asn213-Glu220 form the epitope for mAb 685A5 and must, therefore, be located in the vicinity of each other. Thus, the disulfide loop region Pro192-Glu197 should be positioned in the lower portion of the beta 2I-domain, close to segment Arg144-Lys148 (helix 1) and segment Asn213-Glu220 (helix 2) (Fig. 8). Such an arrangement is different from the proposed location of this disulfide loop on the upper portion of the I-domain by Huang et al. (42).


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Fig. 8.   Epitopes of function-blocking mAbs in the beta 2I-domain. The structure of the beta 2I-domain is modeled according to the crystal coordinates of the alpha MI-domain (10) and a recently published model of the beta 2I-domain by Huang et al. (42). The model is further modified based on the epitope mapping data in Table II using the Biosym software. The backbone of the beta 2I-domain is shown with helix 1 in green, helix 2 in silver, helix 6 in cyan, beta -sheet 6 in purple, the disulfide loop in yellow, and the bound Ca2+ in blue. The epitopes identified in this study can be divided in two groups. Group 1 (yellow circle) includes mAbs TS1/18 (Leu154-Glu159 and Glu344-Asp348), CLB54 (Leu154-Glu159and His354-Asn358), and YFC118.3 (Arg144-Lys148, Leu154-Glu159, and His354-Asn358); and group 2 (cyan circle) includes mAbs MHM23, H20A, IB4, and R3.3 (Arg144-Lys148 and Pro192-Glu197), and 685A5 (Arg144-Lys148, Pro192-Glu197, and Asn213-Glu220).

Recent studies from several laboratories have implicated segment 179-183 of beta 3, which is homologous to Pro192-Glu197 of beta 2, in ligand binding (21, 22, 43). However, the alpha Mbeta 2(Pro192-Glu197) mutant interacted well with C3bi and the gamma -module of Fg, similar to the wild-type receptor, suggesting that this segment is not involved directly in ligand contact within the beta 2 integrins. Thus, there appears to be a fundamental difference between the ligand binding requirements of beta 3 and that of beta 2. Of note, the integrins alpha IIbbeta 3 and alpha Vbeta 3 that utilize this sequence in ligand binding lack I-domains within their alpha  subunits. Therefore, integrins with or without I-domains in their alpha  subunits may employ different mechanisms for ligand binding. Support for this hypothesis also can be derived from recent findings showing that the W2 and W3 repeats within the beta -propeller of the alpha  subunits are located in close proximity to the sequence corresponding to Pro192-Glu197 of beta 2 within their beta  subunits, and together contribute to formation of the ligand binding site (43). Since the I-domains within the alpha  subunits are predicted to insert between the W2 and W3 repeats (54), this geometry would be altered and not be available for ligand binding to the beta 2 integrins.

In this study, we mapped the epitopes of nine beta 2-blocking mAbs to specific regions within the beta 2I-domain. The epitopes were restricted to six segments (Arg144-Lys148, Leu154-Glu159, Pro192-Glu197, Glu344-Asp348, Asn213-Glu220, and His354-Asn358). To determine whether these segments also are involved in ligand binding, we examine their binding of C3bi and Fg, two classic ligands of alpha Mbeta 2. All six beta 2 mutants interacted well with C3bi and the gamma -module of Fg, in a manner similarly to wild-type alpha Mbeta 2, except mutant alpha Mbeta 2(Arg144-Lys148), which exhibited 50% adhesive activity of the wild-type receptor. These data suggest that none of these segments is critically involved in ligand binding of alpha Mbeta 2. Therefore, it is very likely that these beta 2I-domain specific mAbs, like the beta 1-specific function-blocking mAb described by Mold et al. (55), inhibit receptor functions allosterically. We cannot exclude the possibility that some of these mAbs sterically hinder ligand binding. However, several activating mAbs of the beta 1 integrins map to the homologous region within the beta 1I-domain (25), suggesting that this region is conformationally flexible, consistent with an allosteric mechanism. In further support of this model, we found that mutant alpha Mbeta 2(Asn213-Glu220), which interacted more avidly with both C3bi and the gamma -module (Fig. 4, A and B), exhibited an active conformation, judged by its reactivity toward an activation-dependent mAb 24.2 This mAb has been used in a number of studies to probe the activated state of several beta 2 integrin receptors (49, 56, 57). Investigation of the underlying mechanism of activation is currently under way.

It has been well established that integrin-ligand interactions are cation-dependent, but the nature and location of these cation-binding sites are currently unclear. Recently, a novel cation binding site, termed the MIDAS motif, was identified in the crystal structures within the I-domains of several alpha  subunits and was found to be central to ligand binding functions of these I-domains (10, 58-61). Evidence for the existence of MIDAS motifs in the I-domains of beta 1, beta 2, and beta 3 has also been developed (14, 16, 17, 47, 51, 62, 64). Although the I-domains of the alpha  and beta  subunits are predicted to have similar MIDAS folds, their cation binding properties appear to differ significantly. Using several different approaches including x-ray crystallography, circular dichroism, and fluorescence, it appears that cation binding to the I-domains of the alpha  subunits and the beta 1, beta 3, and beta 5 subunits can lead to changes in conformation and ligand binding activity (14, 44, 45, 47, 50, 63). Compared with the beta 1 and beta 3 subunits, the role of cation binding in controlling the conformation of the beta 2 subunit is not well understood. In this study, we report that the binding of two mAbs (TS1/18 and YFC118.3) recognizing non-contiguous regions within the beta 2I-domain depend on Ca2+ for optimal recognition of alpha Mbeta 2 (Fig. 5). A single Ca2+ binding site with a Kd value of ~105 µM was estimated for both mAbs. This Kd value is very similar to that determined for Ca2+ binding to the alpha LI-domain (50 µM) (45) and those obtained for Mg2+ binding to the I-domains of alpha 1, alpha 2, beta 1, and beta 5 (80-100 µM) (14, 47, 50), suggesting that the cation binding site that controls the conformation of the beta 2I-domain is most likely located within the beta 2I-domain itself. To test this hypothesis, we evaluated Ca2+ binding activity of alpha Mbeta 2(S136A), in which the predicted coordinating residue within the MIDAS motif was changed. Using mAb YFC118.3 and FACS analysis, we found that the Ca2+ binding affinity obtained for this mutant beta 2 was significantly lower than that of wild-type beta 2 (151 ± 10 µM for the mutant versus 105 ± 9 µM for wild-type beta 2, p < 0.03) (Fig. 5C). Our results are in agreement with the studies of Lin et al. (14), showing that mutations of the residues within the MIDAS motif of the beta 5I-domain changed the apparent affinity of Mg2+ for alpha vbeta 5 from 80-180 µM to 125-300 µM. To exclude the possibility that Ca2+ could affect YFC118.3 and TS1/18 binding to the beta 2I-domain allosterically by binding to alpha M (via the Ca2+ binding site within either the alpha MI-domain or the beta -propeller), we expressed single beta 2 on the cell surface. We found that the beta 2 subunit alone, in the absence of alpha M or any other alpha  subunits, still possessed a high affinity Ca2+ binding site, which is required for optimal binding of mAbs YFC118 and TS1/18 to the beta 2I-domain (Fig. 6). The calculated Kd is 83 µM, which is very close to that of the alpha Mbeta 2 heterodimer (105 µM). These data strongly suggest that the Ca2+ binding site that promotes YFC118.3 and TS1/18 binding to the beta 2I-domain is located within the beta 2 subunit, possibly composed of Ser136 of the MIDAS motif. However, since mutation of Ser136 did not completely abolish Ca2+ binding, residues outside the MIDAS motif may also be involved in Ca2+ coordination.

Given the specificity of the beta 2I-domain for Ca2+, we next tested whether this Ca2+ binding site plays a role in ligand binding by alpha Mbeta 2, and found that C3bi binding to alpha Mbeta 2 was supported more effectively by Ca2+ than Mg2+ (Fig. 7). In light of the report that Ca2+ does not support C3bi binding to the recombinant alpha MI-domain (48), the Ca2+ binding site that supports C3bi binding of alpha Mbeta 2 is likely located within the beta  subunit, most probably in the beta 2I-domain. A similar cation-binding site was reported in the beta 1 subunit that modulates both ligand binding and mAb 12G10 recognition by integrin alpha 5beta 1 (46, 65). This mAb (12G10) recognizes an epitope (Val211-Met287) within the beta 1I-domain similar to that of TS1/18 and YFC118.3, and its binding depends on a single high affinity cation binding site with a Kd of 70 µM for Ca2+ (46). Mold et al. (46) proposed that divalent cations induced conformational changes within the beta 1I-domain, leading to an unmasking of the ligand binding site within alpha 5beta 1. Given the similarity between the Ca2+ binding sites within the beta 1I- and beta 2I-domains, it is very possible that the same mechanism is involved in the modulation of alpha Mbeta 2 function by Ca2+.

In summary, using homolog-scanning mutagenesis, we have systematically probed the hydrophilic surface of the beta 2I-domain. Our data suggest that the majority of the hydrophilic regions of the beta 2I-domain are not critically involved in the specific association of beta 2 with alpha M. Additionally, we have mapped the epitopes of nine beta 2-specific mAbs into two separate groups within the beta 2I-domain and showed that the spatial arrangement of the residues that constitute these mAb epitopes is consistent with an I-domain-like fold in this region. Most importantly, our data strongly demonstrate that the ligand binding site within beta 2 is distinct when compared with that of beta 3. This fact leads us to hypothesize that integrins containing I-domains in their alpha  subunits may utilize different regions of the beta I-domains for ligand recognition than the integrins lacking I-domains in their alpha  subunits. In addition, our C3bi binding and Fg adhesion data showed that the epitopes of the nine beta 2I-domain specific function-blocking mAbs are not critically involved in ligand binding, implying that they block alpha Mbeta 2 functions by allosteric mechanisms. Finally, we have demonstrated that both the conformation of the beta 2I-domain and C3bi binding to alpha Mbeta 2 depend on a functional Ca2+ binding site, which is located within the beta 2 subunit and probably in the beta 2I-domain. As C3bi binding to the alpha MI-domain is supported by Mg2+, but not Ca2+ (48), our data suggest a role for the Ca2+ binding site within the beta 2I-domain in C3bi-alpha Mbeta 2 interactions. Given the high degree of homology between all integrin beta  subunits, these conclusions should extend to other integrins as well.

    ACKNOWLEDGEMENTS

We thank Dr. Edward Plow for critical reading and editing of this manuscript and Dr. Tom Haas for help with protein modeling. We are also grateful for the help of Teresa Hawley with cell sorting and to Drs. Andrew and Medved for providing valuable reagents.

    FOOTNOTES

* This work was supported in part by the American Heart Association and National Institutes of Health Grant HL61589.

Dagger To whom correspondence should be addressed: Dept. of Vascular Biology, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0657; Fax: 301-738-0465; E-mail: zhangl@usa.redcross.org.

Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M008903200

2 Y. Xiong and L. Zhang, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: Fg, fibrinogen; DPBS, Dulbecco's phosphate-buffered saline; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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