©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Amino Acids in the CD11a I-domain Important for Binding of the Leukocyte Function-associated Antigen-1 (LFA-1) to Intercellular Adhesion Molecule-1 (ICAM-1) (*)

Caroline P. Edwards (§) , Mark Champe , Tania Gonzalez (1), Mary Ellen Wessinger (2), Steven A. Spencer (2), Leonard G. Presta (1), Phillip W. Berman , Sarah C. Bodary

From the (1) Department of Immunology, Department of Protein Engineering, and (2) Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of the LFA-1 subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I-domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I-domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.


INTRODUCTION

Leukocyte function antigen-1 (LFA-1),() like Mac-1 and p150/95, is a member of the integrin family. It is expressed exclusively on leukocytes and mediates a range of adhesive interactions including priming of T cells by antigen presenting cells, migration of leukocytes across the endothelium, and the binding of effector cells (e.g. cytotoxic T cells) to their targets (1, 2, 3) . Like all integrins, LFA-1 is a noncovalent heterodimer consisting of an unique chain (, CD11a) of approximately 180 kDa and a common chain (, CD18) of approximately 95 kDa. The CD11a subunit, as well as CD11b, CD11c, and the integrin-associated chains, and , contains an approximately 200-amino acid domain located amino-terminal to the divalent cation binding domain which is know as the ``inserted/interactive'' domain (I-domain) (1) . This conserved motif is thought to participate in ligand binding of all I-domain containing integrins (3) .

LFA-1 binds to three distinct ligands: ICAM-1, -2, and -3 (1, 4, 5, 6) . LFA-1/ICAM-1 interactions are the best understood and data from a number of groups indicates that the I-domain of CD11a plays a critical role in this interaction. For example, we and others have shown that a number of monoclonal antibodies (mAbs) that block LFA-1 binding to ICAM-1 map to the CD11a I-domain (7, 8) . In addition, CD11a I-domain-IgG chimeras bind specifically to and can inhibit T-cell interactions with ICAM-1 (8, 9) . To date, the amino acids within the intact or isolated I-domain, which are critical to ICAM-1 binding, have not been defined.

In previous studies, using site-directed mutagenesis, we localized the epitopes recognized by eight mAbs to murine and human CD11a (MHM-24, 50G1, CLB LFA 1/2, 32E 6.2, 25.3, TS 1/22, M17, I 21/7) that blocked the binding of LFA-1 to its ligand ICAM-1. Residues critical for the binding of all eight mAbs were localized to an 80-amino acid region of the I-domain. Within this region three discrete epitopes were localized and specific amino acids residues essential for the binding of all eight mAbs were identified (7) . These epitopes may define residues within the I-domain that contact ICAM-1, since Lollo et al.(10) suggest that at least one of the antibodies, M17, blocks CD11a function by competitive inhibition of ligand binding.

Other investigators (11) have identified a novel cation binding site in the I-domain of the closely related integrin Mac-1 (CD11b/CD18) that is essential for the binding of divalent cations as well as iC3b. Mac-1 also binds ICAM-1 and this interaction also appears to be mediated by the I-domain of CD11b (12) . It is not known whether CD11b possesses a distinct binding site for each ligand or whether the different ligands recognize the same or overlapping sites. In the present study, we analyzed the ligand binding properties of two sets of CD11a variants. One set, in which part or all of the human I-domain was replaced with murine sequences, suggested that the human ICAM-1 binding site of CD11a is not contained within the I-domain epitopes recognized by mAbs that block LFA-1 function. A second set was generated in which amino acids conserved throughout CD11a, -b, and -c I-domains were mutagenized. With these constructs, two aspartic acids (Asp-137 and Asp-239) were identified as important for binding of LFA-1 to ICAM-1. A third residue, Pro-192, appears to play a role in maintaining the I-domain conformation.


EXPERIMENTAL PROCEDURES

Generation of the Mutants

The expression vector used to produce membrane-associated CD11a (pRKLFAm) closely resembles that described previously for the expression of membrane-associated Mac-1 (13). To generate the pRKCD11a.mu3 (mu3) construct, the entire murine CD11a I-domain was synthesized de novo by oligonucleotide-directed mutagenesis using uracil containing human CD11a I-domain cDNA as a template (14) . Briefly, the human CD11a I-domain was generated by polymerase chain reaction from a plasmid containing the entire extracellular domain of CD11a and subcloned into a vector containing the M13 origin of replication (pLFAhuID). The fidelity of the polymerase chain reaction was confirmed by sequencing using Sequenase version 2.0 (U. S. Biochemical Corp.). A series of I-domain oligonucleotides were generated to incorporate the murine-specific I-domain amino acids into the human CD11a I-domain. The oligonucleotides were pooled and the murine I-domain (pLFA.IDmu3) was generated by oligonucleotide-directed mutagenesis in two steps. Additionally, the first PflM 1 site 3` to the I-domain was abolished. The clones generated at each step were sequenced to confirm that the oligonucleotides had been incorporated correctly. The murine I-domain from pLFA.IDmu3 was isolated by digestion with Nar 1 and PflM 1 and the fragment was inserted into the human CD11a plasmid (pRKLFA) at the comparable Nar 1 and PflM 1 sites. Correct insertion of the murine I-domain was confirmed by sequencing.

The generation of the H/M chimeras has been described previously (7) . The H11a.D137A (D137A), H11a.D137K (D137K), H11a.S139A (S139A), H11a.P192A (P192A), H11a.D239A (D239A), H11a.D239K (D239K) constructs contain the indicated point mutations in the human CD11a I-domain and were generated by oligonucleotide-directed mutagenesis using pRKLFA as the template (14) .

Transfections

Human 293 cells were transfected by a standard calcium phosphate method as described previously (15) . Briefly, 1 day prior to transfection, 293 cells were seeded at 30-40% confluence in 10-cm plates and incubated overnight. For each condition, 10 µg of pRKCD18, 1 µg of pAdvRNA, and 10 µg of the plasmid containing the specified CD11a constructs were precipitated in CaCl and HEPES buffer and added to the plates. Mock transfected cells were transfected with the pRKCD18 and pAdvRNA constructs. CD18 transfected in the absence of a CD11a construct was not expressed at the cell surface (data not shown). After 3 days transfectants were tested for adhesion to full-length recombinant human ICAM-1 (rICAM-1) immobilized on plastic or for expression of the CD11a/CD18 complexes on the cell surface. Transfection efficiencies ranged from 15 to 90%.

Adhesion Assays

Full-length human ICAM-1 was purified from a stably transfected 293 cell line expressing rICAM-1. Detergent extracted rICAM-1 was purified on a lentil-lectin column followed by gel filtration chromatography on a Superdex 200 column (Pharmacia Biotech Inc.). Details of the purification will be presented elsewhere. The purity and concentration of the isolated protein were determined by SDS-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (T Cell Diagnostics), respectively. 350 ng of rICAM-1 in PBS was added to each well of Nunc Maxisorb 96-well plates and incubated overnight at 4 °C. Plates were washed and blocked with a 2% bovine serum albumin/PBS solution for 2 h at 37 °C. The plates were washed with PBS and 100 µl of cells were added per well in triplicate determinations for each condition. Each experiment was carried out at least twice.

Transfected cells were detached with 5 mM EDTA/PBS and resuspended at a concentration of 200,000 cells/100 µl of adhesion buffer (140 mM NaCl, 20 mM HEPES, pH 7.5, 0.2% glucose) containing the specified concentration of divalent cations and antibodies. Antibodies were bound in buffer containing 1 mM each of MgCl and CaCl. (FACS analysis determined that the I-domain mutants bound the anti-CD18 activating antibody, MEM-48 (16), at levels comparable to wild-type CD11a/CD18 under these conditions (data not shown)). The cells were added to the plates and incubated at 37 °C for 1.5 h. Nonadherent cells were removed by three washes with PBS. Cell attachment was measured using the p-nitrophenyl n-acetyl--D-glucosaminide method of Landegren (17). Mean OD of triplicate determinations is presented. For comparison 30,000 cells gave an average OD of 1.

Antibody Staining and FACS Analysis

Three days post-transfection cells were detached and resuspended in 100 µl of Dulbecco's modified Eagle's medium, 10% fetal bovine serum with antibodies against CD11a or CD18. Purified mAbs to CD11a were used at 1 µg/ml (MHM-24 (18) , M17 (ATCC, Rockville, MD), 25.3 (Amac, Inc., Westbrook, ME); L16 (19) , 32E6 (7) ) or at 1/100 dilution of ascites (TS 1/22 (ATCC, Rockville, MD); 3D6, 50G1 (7) ). MAbs to CD18 were used at 5 µg/ml (MEM-48 (R& Systems, Abingdon, United Kingdom), H52 (20) ) or 1 µg/ml (MHM-23 (18) ). Cells were incubated with antibodies on ice for 1 h, washed, and fluorescein isothiocyanate-conjugated secondary antibodies (goat anti-mouse (Cappell) and goat anti-rat (Caltag)) were added at 1/500 and 1/100 dilutions, respectively. Cells were washed, fixed in 2% paraformaldehyde, and analyzed by FACS analysis using the FACScan Research software (Becton Dickinson).


RESULTS AND DISCUSSION

I-domain Epitopes Recognized by Antibodies That Block LFA-1 Function Are Distinct from Critical ICAM-1 Binding Site(s) in CD11a

Previously, a panel of human/murine (H/M) chimeras of the CD11a I-domain were used to localize the epitopes recognized by mAbs that block the function of human and murine CD11a. The antibodies recognized three discrete epitopes (IdeA, IdeB, and IdeC). Fig. 1 shows the location of the epitopes and the chimeric proteins used to localize them. The chimeric genes H/M 48, H/M 49, H/M 50, H/M 51, H/M 52, H/M 53, and H/M 54 all incorporated mutations in which murine sequences were substituted into human CD11a. Of the seven constructs tested, three (H/M 52, H/M 53, H/M 54) were effective in blocking antibody binding. Given that the mAbs studied all inhibit LFA-1 mediated adhesion to ICAM-1, we set out to test whether the amino acids that disrupted antibody binding were also important in LFA-1 binding to ICAM-1.


Figure 1: Amino acid sequences of the murine and human CD11a I-domains and the human/murine chimeras and point mutants. A, schematic representation of CD11a with the respective locations of the I-domain and the metal binding (EF-hand) domains. B, the residues of the human and murine I-domains, as well as the chimeras and point mutations, are illustrated. In the chimeras and point mutants a dash represents the human residue. Where the sequences differ from the human sequence the residue is shown. The mu3 construct depicts the amino acid differences between the human and murine I-domains. MAbs and their epitopes (IdeA, -B, -C) are indicated.



Previous studies have demonstrated that murine LFA-1 does not bind to human ICAM-1 and that this species specificity is encoded in the CD11a subunit (21) . Since the I-domains of LFA-1, Mac-1, , and integrins are known to be necessary for binding of their respective ligands (ICAM-1, iC3b, fibrinogen, collagen, and laminin) (12, 22, 23, 24) , we hypothesized that this domain was responsible for the species specificity of murine LFA-1. A cDNA fragment encoding the entire I-domain of murine CD11a (amino acids 125-308) was synthesized de novo using human CD11a I-domain cDNA as a template and M13-based mutagenesis (14) . It was inserted into the cDNA for the full-length human CD11a at the corresponding position (amino acids 126-308), to generate the mu3 variant. The mu3 cDNA was co-transfected with the cDNA for human CD18 into 293 cells and transfectants were analyzed for cell surface expression, antibody binding, and adhesion to human ICAM-1. shows the average values from the FACS analysis of mu3/CD18 expression. The chimeric mu3/CD18 construct was expressed at the cell surface at approximately the same levels as wild-type human CD11a/CD18. All mAbs to human LFA-1 that mapped to the I-domain (32E6, TS 1/22, MHM-24, 25.3, and 50G1) failed to bind mu3. Other mAbs known to bind outside of the I-domain (L16, 3D6), bound approximately the same percentage of cells and with approximately the same mean fluorescence intensity (data not shown) as human CD11a/C18 expressing cells. M17, a rat mAb to murine CD11a, bound mu3/CD18 transfectants about 50 times better than human CD11a/CD18 transfectants. MAbs to human CD18 bound the mu3/CD18 transfectants at levels comparable to human CD11a/CD18. In addition MHM-23, which recognizes CD18 when paired with an chain (20),() also bound the mu3 transfectants. This demonstrates that mu3 formed heterodimers with CD18. When transfectants were tested for their ability to bind to human ICAM-1-coated plates, adhesion was dependent on the presence of CD11a/CD18 at the cell surface, as mock transfected 293 cells showed background levels of binding (Fig. 2). Adhesion to ICAM-1 required the presence of divalent cations. Under conditions which promoted robust binding of wild-type CD11a/CD18, the mu3/CD18 transfectants failed to adhere to ICAM-1 (Fig. 2A). This data suggests that replacement of the human CD11a I-domain with murine CD11a sequences yields a heterodimeric CD11a/CD18 complex that is properly folded and exported to the cell surface, but is unable to bind to human ICAM-1.


Figure 2: Attachment of the human/murine I-domain chimeras to human ICAM-1. A, cells transfected with CD18 alone, mock (), or together with wild-type CD11a () or mu3 () were tested for their ability to adhere to human ICAM-1 in the absence and presence of 500 µM MnCl, 5 mM MgCl, 10 µg/ml irrelevant IgG1 or 10 µg/ml of the activating antibody, MEM-48, in the presence of 1 mM MgCl and 1 mM CaCl. B, 293 cells transfected with the indicated constructs were tested for their ability to bind to ICAM-1 in the absence () and presence () of 200 µM MnCl. Staining with MHM-23 determined that the CD11a, mu3, and H/M 48-H/M 54 transfectants expressed comparable levels of LFA-1 heterodimers on the cell surface (data not shown).



One mechanism by which antibodies to LFA-1, such as M17, may block function is by competitive inhibition of ICAM-1 binding to LFA-1 (10) . Since mAbs 25.3 and TS 1/22 bind to homologous residues as M17 (7) , it is likely that they inhibit LFA-1/ICAM-1 interactions by binding to the same site on CD11a as ICAM-1. Hence, it might be predicted that the chimera H/M 53, which contains the M17 epitope, would fail to bind to human ICAM-1 when complexed with CD18. Therefore, we tested whether the H/M chimeras disrupt the binding of ICAM-1. The H/M constructs were transiently co-transfected with the human CD18 expression plasmid into 293 cells and assayed for cell surface expression and ICAM-1 binding. FACS analysis of the binding of a panel of antibodies to these chimeras was consistent with the epitope mapping studies (7) (, Fig. 1). Mutants which contain murine sequences at positions corresponding to I-domain antibody epitopes did not bind the relevant antibodies, but bound other I-domain antibodies at levels comparable to wild-type CD11a/CD18. All chimeras bound MHM-23 with values comparable to wild-type, indicating the formation of heterodimers with CD18. Antibodies to CD18 and CD11a that map outside the I-domain bound at levels comparable to wild-type. When the transfectants were tested for adhesion to human ICAM-1 in the presence of 200 µM MnCl, all of the H/M chimeras bound to human ICAM-1 at levels comparable (within 2.5-fold) to wild-type CD11a/CD18 (Fig. 2B). These results indicate that the residues within these mAb epitopes are not strictly required for human ICAM-1 binding. It cannot, however, be ruled out that these epitopes may correspond to only part of the ICAM-1 binding domain which is composed of multiple residues at discontinuous locations within the I-domain. If this were the case, mutating residue(s) in a single region would not necessarily destroy ICAM-1 binding. Preliminary evidence supporting this hypothesis suggests that substitution of murine residues for human residues is required at multiple locations within the human CD11a I-domain in order to abolish ICAM-1 binding (data not shown).

Mutating Asp-137, Pro-192, and Asp-239 in the I-domain of CD11a Abolishes Binding of LFA-1 to ICAM-1

As described previously, the epitopes mapped by the H/M mutants were specific for CD11a and were not conserved in the closely related integrin, Mac-1, that also binds ICAM-1 (7) . Assuming that ICAM-1 binds similar sequences in CD11a and CD11b, we hypothesized that conserved sequences located in the region of these epitopes might be important for ligand binding. The sequence between IdeA and IdeB is highly conserved between these two molecules and has been found to be important for ligand binding of Mac-1 by Michishita et al.(11) . These investigators showed that mutating D140GS of CD11b to AGA abolished Mac-1 adhesion to iC3b. Binding of isolated I-domain to Mn was also abolished by this mutation. To further localize residues in this region of CD11a involved in the LFA-1/ICAM-1 interaction, we generated variants with the following point mutations in the CD11a I-domain: D137A, D137K, and S139A. These were assayed for their ability to promote binding of LFA-1 to ICAM-1 in the presence of varying concentrations of MnCl (Fig. 3A), MgCl (Fig. 3B), and MEM-48 (Fig. 3C), an antibody to CD18, which activates LFA-1 (16) . (We consistently found higher levels of binding of wild-type LFA-1 transfectants to ICAM-1 in the presence of MnCl than MgCl.) We found that the substitution of Ala or Lys for Asp at position 137 abrogated the ability of LFA-1 to bind to ICAM-1 under any of the conditions tested. However, substitution of Ala for Ser at position 139 had no significant effect on LFA-1 binding to ICAM-1 (Fig. 3, A-C).


Figure 3: Attachment of I-domain point mutants to human ICAM-1. 293 cells were transfected with the following CD11a/CD18 constructs and tested for ability to adhere to ICAM-1 in increasing concentrations of MnCl (A) or MgCl (B): mock (), wild-type CD11a (), D137A (), D137K (), S139A (), P192A (), D239A (), D239K (▾). C, the indicated transfectants were assayed for ability to adhere to ICAM-1 in the absence () or presence of 1 mM CaCl, 1 mM MgCl (), 10 µg/ml MEM-48 (), or 500 µM MnCl (). Staining with MHM-23 determined that the transfectants expressed comparable levels of LFA-1 heterodimers on the cell surface (data not shown).



FACS analysis demonstrated that these mutants formed heterodimers with CD18 at the cell surface, as the mAb MHM-23 bound at levels comparable to wild-type CD11a/CD18 (). Subtle changes in the conformation of the I-domain of these mutants were suggested by the decrease in binding of mAb TS 1/22 to the D137A variant, and in the moderate reduction in mAbs 32E6, 50G1, and TS 1/22 binding to D137K. No decrease in antibody binding was observed in the S139A variant. The gross conformation of these variants appeared to be intact since the antibodies to CD18 and most of the antibodies to CD11a bound these variants at levels comparable to wild-type CD11a/CD18 (). These results suggest that amino acids common to Mac-1 and LFA-1 are critical for the binding of these integrins to distinct ligands (iC3b and ICAM-1).

We next examined amino acids Pro-192 and Asp-239 in CD11a, which are conserved in CD11 I-domains and have been shown to be important for Mac-1 binding to Mn and iC3b (11) . An Ala was substituted for Pro at position 192 and an Ala or Lys was substituted for the Asp at position 239. Mutating Asp-239 in the CD11a I-domain to either Ala or Lys completely abolished LFA-1 binding to ICAM-1 under the conditions tested (Fig. 3, A-C). Binding of MHM-23 confirmed that these mutants were expressed at the cell surface as heterodimers with CD18. It appears that these substitutions had little or no effect on the gross conformation of LFA-1 as judged by antibody binding (). No reduction in binding of any of the antibodies was observed for the D239A variant and substituting Lys at this position reduced the binding of only TS 1/22. This is noteworthy given the considerable distance along the primary sequence between Asp-239 and the TS 1/22 epitope (IdeA) (Fig. 1) (7) . It will be of interest to determine whether Asp-239, as well as Asp-137 whose TS 1/22 binding is also perturbed, are located proximal to IdeA in the three-dimensional structure of this receptor.

Mutating Pro-192 to Ala also completely destroyed the ability of LFA-1 to bind to ICAM-1 (Fig. 3, A-C). No obvious changes in the gross morphology of this mutant were observed since the antibodies to CD18 and the antibodies to CD11a that map outside the I-domain bound at levels comparable to wild-type CD11a/CD18 (). Mutation of residue 192 did appear to alter the conformation of the I-domain as mAbs 32E6, 50G1, and TS 1/22 showed a 28-70% reduction in binding. Immunoprecipitations of the P192A variant also showed dramatic reduction in binding of all the I-domain antibodies, which was not the case for any of the other mutants (data not shown). These data suggest that the loss of ICAM-1 binding by this mutant is due to conformational changes within the I-domain. Mutating the analogous Pro in CD11b resulted in less drastic changes in Mac-1 binding to iC3b, but did reduce the binding of isolated CD11b I-domain to Mn and shifted the optimal cation concentration required for adhesion of Mac-1 expressing cells to iC3b (11) . In CD11a, this mutant was unable to bind to ICAM-1 even at high concentrations of divalent cations (500 µM MnCl or 2 mM MgCl) (Fig. 3, A and B). This suggests that either the Pro-192 mutation in the CD11a I-domain exerts a more profound effect on the I-domain structure or that the LFA-1/ICAM-1 interaction is more sensitive to structural constraints than the Mac-1/iC3b interaction.

Our results demonstrate the importance of Pro-192 and the aspartic acids, Asp-137 and Asp-239, for LFA-1 binding to ICAM-1. Pro-192 appears to play a structural role, as evidenced by the significant changes in I-domain mAb binding to the P192A mutant. While we cannot rule out a structural function for Asp-137 and Asp-239, substitution of alanine at either of these positions resulted in moderate or no changes in mAb binding. This suggests that loss of ICAM-1 binding in the D137A and D239A mutants is not a consequence of gross conformational changes in the I-domain. Recently, the significance of these aspartic acids in other I-domain containing integrins, and , has been determined. Mutation of Asp-253 in the I-domain abrogated binding to collagen IV and significantly reduced binding to laminin (23) . Mutation of Asp-151 and Asp-254 in the I-domain abolished binding to collagen I (22) . Interestingly, the data for both Mac-1 and suggests that these residues are not ligand contact points, but rather cation binding sites. In Mac-1, these aspartic acids appear to bind cations and mutation of these residues destroyed iC3b binding of the full-length receptor as well as the isolated I-domain (11, 25) . However, a 14-amino acid peptide derived from the CD11b I-domain that includes Asp-242 bound iC3b in a cation independent manner. Substitution of Ala for Asp at position 242 of this peptide did not affect binding to iC3b (25) . For , Kamata et al.(22) demonstrated that mutation of the analogous aspartic acids in full-length abrogated collagen binding. However the isolated I-domain bound to collagen I in a cation-independent manner (26). Moreover, in the isolated I-domain, mutation of these residues did not completely abrogate collagen binding, suggesting that these aspartic acids are not critical contact points in adhesion to collagen.

Taken together, these data suggest that by isolating all or part of these I-domains away from their full-length receptors (Mac-1, ), it is possible to promote receptor-ligand interactions in the absence of cations. Our preliminary data suggests that the isolated CD11a I-domain also binds ICAM-1 in a cation-independent manner (data not shown). Hence, it appears that the ligand binding sites in these full-length integrins are masked in the absence of cations, and cation binding is required to expose these sites. Conceivably the binding of cations to the above-defined aspartic acids regulates ligand accessibility of these integrins, which bind ligands as diverse as collagen, laminin, iC3b, and ICAM-1. The role of these residues in direct binding of divalent cations and ICAM(s) to the CD11a I-domain is currently being investigated. A full understanding of these interactions is likely to require a combination of three-dimensional structural and mutational analysis of both ICAM-1 and LFA-1. Determination of the domains of LFA-1 that confer ligand specificity, as well as understanding the role of amino acids that are involved in cation binding or activation, will be important to the development of highly specific anti-inflammatory therapeutics.

  
Table: 0p4in NA, not applicable.(119)


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Genentech, Inc., MS 34, 460 Point San Bruno Blvd., S. San Francisco, CA 94080. Tel.: 415-225-8036; Fax: 415-225-8221.

The abbreviations used are: LFA-1, leukocyte function-associated antigen-1; ICAM-1, intercellular adhesion molecule-1; rICAM-1, recombinant ICAM-1; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; H/M, human/murine.

G. R. Nakamura and P. W. Berman, unpublished observation.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Yvette van Kyook for providing us with the mAb L16; Dr. James Hildreth for providing us with the mAbs MHM-23, MHM-24, and H52; and Dr. Bradley McIntyre for providing us with the mAbs 3D6, 32E6, and 50G1. We also thank the Genentech oligonucleotide synthesis group and the Genentech flow cytometry laboratory.


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