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The Journal of Immunology, 1998, 161: 1363-1370.
Copyright © 1998 by The American Association of Immunologists

The Leukocyte Function-Associated Antigen-1 (LFA-1)-Binding Site on ICAM-3 Comprises Residues on Both Faces of the First Immunoglobulin Domain1

Elaine D. Bell2,*, Andrew P. May{dagger} and David L. Simmons3,*

* ICRF Cell Adhesion Laboratory, Imperial Cancer Research Fund Laboratories, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DS, U.K., and {dagger} Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, Department of Biochemistry, University of Oxford, Oxford, OX1 3UQ, U.K.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ICAM-3 (CD50), a member of the Ig superfamily, is a major ligand for the leukocyte integrin LFA-1 (CD11a/CD18). This interaction represents one of several Ig superfamily/integrin ligand-receptor pairs that have been described to date. ICAM-3 is highly expressed on resting leukocytes and on APCs. In addition to an adhesive function, ICAM-3 can act as a signal-transducing molecule on T cells, providing a costimulatory signal for cell proliferation. Eighteen point mutations in ICAM-3 were generated, and residues important for binding of functional blocking Abs were identified. Mutation of seven of the residues reduced or abrogated adhesion to LFA-1, including three residues that are located on strand A of the ABED face of domain 1. In contrast, extensive mutagenesis analysis of ICAM-1 has shown that only residues on the GFC face interact with LFA-1. Our results provide evidence for a more extensive binding interface between ICAM-3 and LFA-1 than has previously been described. ICAM-3 appears to be unique among the ICAMs in utilizing residues on both faces of domain 1 for interaction with its ligand LFA-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The three intercellular adhesion molecules ICAM-1, ICAM-2, and ICAM-3 are members of the Ig superfamily (IgSF)4 composed of five, two, and five Ig domains, respectively (1, 2, 3, 4, 5, 6, 7, 8, 11, 12). All three are counterreceptors for the ß2 integrin LFA-1 (CD18/CD11a, {alpha}Lß2). In addition, ICAM-3 has recently been shown to interact with a newly identified member of the ß2 family, CD18/CD11d ({alpha}dß2) (9, 10).

ICAM-3 appears to be the dominant ligand for LFA-1 during the initiation of the immune response (13), given that ICAM-1 and ICAM-2 either are not expressed or are expressed at very low levels on resting leukocytes and APCs (14, 15). In addition to an adhesive function, ICAM-3 can provide a costimulatory signal for T cell proliferation and IL-2 production (16, 17). Cross-linking of ICAM-3 induces changes in calcium flux and tyrosine phosphorylation in a T cell line (18). A consequence of this ICAM-3 signaling is increased adhesion mediated by ß1 and ß2 integrins, a process referred to as crosstalk or adhesion amplification (14, 19).

The LFA-1-binding site on ICAM-3 has been mapped to domain 1 (20), an Ig I-set structure consisting of seven ß strands arranged on two surfaces, an ABED face and a GFC face. Site-directed mutagenesis in conjunction with modeling studies permitted the LFA-1-binding site to be mapped to the GFC face of domain 1. The two residues homologous to E34 and Q73 in ICAM-1 have also been defined as key residues for LFA-1 binding to ICAM-3. A large panel of anti-ICAM-3 Abs were generated, of which five Abs were able to block ICAM-3/LFA-1 interactions and were functionally effective in inducing homotypic T cell aggregation, induction of T cell proliferation, IL-2 production, and expression of cell surface activation markers on T cells (16). The five blocking Abs define three overlapping epitopes in domain 1, none of which was affected by our previous panel of mutations (20).

In recent years, several studies on integrin/ligand interactions have shown that many ligands use short sequences as recognition motifs for integrin binding (21). RGD, the prototypic peptide motif, provides adhesive activity for various extracellular matrix components including fibronectin, fibrinogen, vitronectin, and von Willebrand’s factor. Other studies on VCAM-1 and the ICAMs have identified a second linear motif with the following consensus sequence: L/I-D/E-S/T/V-P/S (22). The solution of the VCAM-1 D1-2 crystal structure (23) has revealed that like RGD in fibronectin (24), the VCAM-1 IDSP motif is located in a prominently exposed loop, which presumably favors interaction with integrin. In contrast, the recently solved crystal structure of ICAM-2 shows that the critical glutamic acid residue at position 37 is located in a ß strand and is surrounded by a relatively flat surface which may complement the relatively flat surface surrounding the Mg2+-binding site in the I domains of Mac-1 and LFA-1 (25). It has been proposed that these short linear sequences are essential common components of IgSF/integrin interactions (26). Indeed, the QIDS region in domain 1 of VCAM-1 can be replaced with the similar ICAM-1 sequence GIET without affecting interaction with VLA-4, indicating that this region is part of a general integrin binding motif (27). Despite having common structural binding motifs, integrins clearly have discrete receptor specificities presumably determined by residues without the common motifs. The identification of such "specificity" residues will permit the development of novel therapeutics targeted to particular interactions.

A second common theme that has emerged from structural studies of IgSF proteins is that only residues located on the GFC face of the Ig fold contribute to ligand binding. This holds true for the interaction of VCAM-1 with the integrin {alpha}4ß1, and also for the binding of the adhesive proteins CD2 (29), E cadherin (28), and members of the sialoadhesin family (30, 31) to their respective ligands. Notably, mutational analysis of most of the ABED face residues in domain 1 of ICAM-1 has shown that none of these residues participates in binding to LFA-1 (32, 33). Interestingly, a recent study has shown that residues N23 and S25, which comprise a potential N-linked glycosylation site located on the ABED face of ICAM-3, contribute to the interaction with LFA-1 (34).

In this study, we set out to define the epitopes of the functional anti-ICAM-3 blocking Abs and hence delineate additional ICAM-3/LFA-1 contact sites. A panel of 18 single point mutations were generated, targeting 7 residues in domain 1 and 3 residues in domain 2 of ICAM-3. This panel of mutations has enabled us to identify residues that contribute to the epitopes of the five functional blocking anti-ICAM-3 Abs. Mutation of seven of the residues reduced or abrogated adhesion to LFA-1, including three residues located on strand A of the ABED face of domain 1. Mutation of residues in domain 2 of ICAM-3 had some effect on the interaction with LFA-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Monoclonal antibodies and cell culture

The mAbs used in this study were as follows. The anti-ICAM-3 Abs CH3.1, CH3.2, CH3.3, CAL3.4, CAL3.10, CAL3.38, and CAL3.41 were generated in this laboratory as described previously (16). The anti-ICAM-3 Abs BY44 and CG106 were provided by Dr. D. Mason (Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, U.K.). The anti-ICAM-3 Ab KS128 was purchased from Dako, High Wycombe, Buckinghamshire, U.K. Anti-CD18 Abs KIM185 and KIM127 (36) were provided by Dr. M. Robinson (Celltech, Slough, U.K.), and anti-CD11a Ab 38 (35) was obtained from Dr. N. Hogg (Imperial Cancer Research Fund (ICRF), London, U.K.). Six Ab that were submitted to the CD50 (ICAM-3) panel of the Sixth International Workshop on Human Leukocyte Differentiation Antigens were also used in this study: AO19 (BU68); AO41 (186–269); AO64 (B-N2); AO69 (B-P12); AO70 (B-R1); and AO85 (AZN-ICM3.1). Ab AO76 (B-S9), an anti-ICAM-2 Ab that cross-reacts with domain 2 of ICAM-3, and the CD50 reference Ab HP2/19 (Ref. 25 in panel) were also included.

KL/4 cells (K562 cells stably transfected with CD11a and CD18) (36) were a generous gift from Dr. M. Robinson (Celltech, Slough, U.K.) and were maintained in RPMI 1640 medium, 10% FCS, and 1 mg/ml G418 (Life Technologies, Paisley, U.K.). COS-1 cells were provided by the cell bank (ICRF, Clare Hall, U.K.) and grown in DMEM supplemented with 10% FCS, 2 mM glutamine, and penicillin-streptomycin.

Generation of ICAM-3 mutants

ICAM-3(D1-2)Fc and CD31(D1-2)Fc consisting of the first two extracellular domains fused to human IgG1-Fc fragment have previously been described (7, 37). Single point mutations were introduced into ICAM-3(D1-2)Fc using a two-step PCR strategy (38, 39) with common forward amplification (5'-ATAT AAGCTT ATG GTA CCA TCC GTG TTG TGG CCC-3') and reverse amplification primers (5'-ATCT AGATCT ACTTACCTGT GCG CGG GGG GGT CAC GGG CAG-3') in addition to sequence-specific mutagenic primers (a list of primers is available on request). All PCR amplifications were performed using Pwo DNA polymerase (Boehringer Mannheim, Mannheim, Germany) with ICAM-3(D1-5)Fc in pcDNA3neo as template. A maximum of 10 cycles in the first round and 15 cycles in the second round of PCR were performed to reduce the rate of generating unwanted mutations. PCR products were digested with HindIII and BglII and cloned into the HindIII/BamHI-digested Fc expression vector pIG1 (7, 40). All mutants were verified by sequencing of the amplified region.

Recombinant chimeric Fc plasmids were transiently expressed in COS cells and tissue culture supernatants containing secreted protein harvested after 7 to 10 days. Recombinant protein production was initially assessed by ELISA using the Ab CH3.1 which bound well to all the mutants.

Adhesion assays

Immulon 3 96-well flat-bottom plates (Dynatech, Chantilly, VA) were precoated overnight at 4°C with 1 µg/well Fc-specific goat anti-human IgG (Sigma, Poole, Dorset, U.K.) in bicarbonate buffer (pH 9.6), blocked for 2 h at room temperature with 0.4% BSA (fraction V, Sigma) in PBS, and then coated with chimeric Fc proteins by addition of neat tissue culture supernatant to the wells for at least 2 h at room temperature. Before the adhesion assay, HSB2 or KL/4 cells were labeled with the fluorescent dye BCECF-AM (10 µg/107 cells, Molecular Probes, Eugene, OR) for 30 min at 37°C and washed twice in assay buffer (DMEM, 10 mM HEPES buffer, 0.2% BSA). LFA-1 is not constitutively avid for ICAM-3 but requires activation to mediate stable adhesion. Therefore, HSB2 cells were stimulated by addition of either Mn2+ ions (0.5 mM MnCl2) or KIM185 Ab at 5 to 10 µg/ml, or KIM185 Ab at 5 to 10 µg/ml plus 50 ng/ml PMA (Sigma) for 10 min at room temperature before plating, and KL/4 cells were similarly stimulated by addition of KIM127 Ab at 5 to 10 µg/ml. Labeled, stimulated cells were added to wells at 2 x 105 cells/well in a volume of 50 µl and allowed to adhere for 35 min at 37°C. Plates were washed with prewarmed assay buffer until the cells in the negative control wells were sufficiently removed as judged by visual inspection. Typically two to three washes were required. The percentage of input cells bound (percentage of cell adhesion) was quantified using a Cytofluor II fluorescent plate reader (Millipore, Watford, U.K.) by comparing the total input fluorescence and the fluorescence of bound cells after washing.

Enzyme-linked immunosorbent assay of ICAM-3 Fc proteins

Immulon 3 96-well flat-bottom plates were precoated overnight at 4°C with Fc-specific goat anti-human IgG in bicarbonate buffer (pH 9.6), blocked for 2 h at room temperature with 2% BSA in PBS, and then coated with Fc proteins by addition of neat tissue culture supernatant to the wells for at least 2 h at room temperature. Primary Ab were added to saturation as either neat tissue culture supernatant, or at 5 to 10 µg/ml for purified Ab, or at 1:500 dilution for the Abs from the workshop panel, with the exception of AO19 (BU 68) which was added at 1:100 dilution. Bound Ab were detected using peroxidase-conjugated sheep anti-mouse Ig (1:1000 dilution, Amersham Life Sciences, Amersham, Buckinghamshire, U.K.) and visualized with o-phenylenediamine dihydrochloride (Sigma). Each layer was incubated at room temperature for 30 min, followed by three washes with 0.25% BSA in PBS. The absorbance was measured at 450 nm.

Modeling of ICAM-3

A molecular model of the N-terminal domain of ICAM-3 was produced, based on the structure of ICAM-2 domain 1 (25). The sequence of ICAM-3 was substituted into the ICAM-2 structure using the program MUTATE (R. Esnouf, unpublished data). No insertions or deletions were required, and in all regions the ICAM-2 main chain conformation was retained. The figure was produced using the program MOLSCRIPT (42).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutagenesis strategy

Previous work has established that ICAM-3 domains 1 and 2 contain the LFA-1 binding site (7). Using a nested set of ICAM-3 Fc domain deletion chimeras, it was shown that binding was not significantly increased by the presence of domains 3 to 5 of ICAM-3 (20). In this latter study, 9 residues in domain 1 (6 on the GFC face and 3 on the ABED face) and 3 residues in domain 2 were selected for mutational analysis. The domain 1 residues E37, L66, S68, and Q75 were found to contribute significantly to LFA-1 binding. However, the five functional blocking Abs were able to bind to all of the conformationally intact domain 1 mutants, suggesting that further sites of interaction between LFA-1 and ICAM-3 remain to be defined.

In this study, we have used a two-step PCR strategy to mutate the 7 previously unmutated charged residues in domain 1 (4 on the GFC face and 3 on the ABED face) as well as a further three residues in the E-F loop of domain 2. Figure 1Go shows an alignment of the primary amino acid sequences of ICAM-1, ICAM-2, and ICAM-3 and indicates the location of residues targeted for mutagenesis.



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  		FIGURE 1. Sequence alignment of ICAM-1, ICAM-2, and ICAM-3. Sequence alignment of the two N-terminal domains of ICAM-1, ICAM-2, and ICAM-3. The predicted ß strands of the Ig domains are underlined and labeled according to the method of Casanovas et al. (25), as defined for ICAM-2. Asterisks denote those residues in ICAM-3 that were targeted for site-directed mutagenesis. Potential N-linked glycosylation sites are numbered (-1- etc.) above the alignment.

 
Mutant ICAM-3D1-2Fc proteins were produced as tissue culture supernatants from COS cells and were used undiluted in adhesion assays and ELISAs. To demonstrate that application of neat Fc supernatants produced saturating conditions for Ab and cell binding assays, a titration ELISA was performed (Fig. 2Go). The primary Ab chosen for this ELISA was CH3.2, which binds well to all the ICAM-3 mutants with the exception of R6A and R6E. The results indicate that use of neat Fc supernatants produced saturating conditions for functional assays. Thus, differences observed in LFA-1-mediated cell binding to the mutants cannot be attributed to differences in the amount of mutant Fc protein coated on the plates.



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  		FIGURE 2. Titration of mutant ICAM-3 Fc proteins to demonstrate that ELISA and adhesion assays were performed under saturating conditions. Mutant ICAM-3 Fc proteins were produced as supernatants from COS cells. Various dilutions were applied to plates precoated with Fc-specific goat anti-human IgG and blocked with BSA. Bound protein was quantitated by ELISA with the primary Ab CH3.2 applied at 1/5 dilution of tissue culture supernatant. Binding to wild-type ICAM-3D1-2Fc was used to normalize the data, giving 100% binding by definition. Data are representative of two independent experiments. The results are expressed as means of three wells. The SDs were consistently within 10% of mean values and are not shown for clarity. A, Data for mutants in domain 1 of ICAM-3 that localize to the ABED face; B, Data for mutants in domain 1 of ICAM-3 that localize to the GFC face. C, Data for mutants in domain 2 of ICAM-3.

 
Assessment of structural integrity of ICAM-3 mutants and identification of blocking Ab epitopes by ELISA

The structural integrity of the ICAM-3 Fc mutant proteins was assessed by ELISA profile using a panel of 13 Abs that map to domain 1 and 5 Abs which map to domain 2 of ICAM-3 (Table IGo). Mutation of the domain 2 residue D154 to both alanine and lysine appears to cause a severe disruption of the domain 2 structure as indicated by lack of binding of all five Abs that map to domain 2. These mutants were excluded from functional assays. The remaining three mutations in domain 2 appeared to have little or no effect on the overall structure of domain 2. The majority of the domain 1 Abs recognize the domain 1 mutants, the main exception being mutation of those residues that contribute to the epitopes of blocking and partial blocking Abs. In particular, residues E32 and K33 seem to be crucial components of the blocking Ab epitopes, with residue K42 also making a significant contribution. The binding of the domain 1 Ab CH3.2 is significantly reduced by mutation of R6 to R6E.


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  		Table I. Assessment of structural integrity of ICAM-3 mutants and identification of blocking Ab epitopes by ELISA1

 
Effect of ICAM-3 mutants on interaction with LFA-1

To investigate the effect of the mutations on LFA-1 binding, adhesion assays were performed using two different cell lines that express LFA-1. Figure 3Go shows that HSB2 cells suboptimally activated with the anti-ß2-activating Ab KIM185 were unable to bind to the mutant K42D that maps to the D strand of domain 1 in a region close to the LETS motif. The mutants E32A, K33A, K33D, R64A, and R64E also resulted in a significant reduction in cell adhesion (p < 0.01 compared with wild type in each case). These residues are all located on the GFC face of domain 1. Interestingly, LFA-1-mediated cell binding to mutants E2K, R6E, E8A, and E8K, located on the ABED face of domain 1, was also significantly reduced (p < 0.02, p < 0.05, p < 0.01, and p < 0.01, respectively). The domain 2 mutants H155A and H155D had a significant effect on binding of HSB2 cells, with H155A resulting in decreased adhesion compared with wild type (p < 0.01) and H155D showing enhanced adhesion compared with wild type (p < 0.01). On the whole, residues mutated to opposite charges had greater effects on cell adhesion than those mutated to alanine, the exceptions being E32A and E32K.



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  		FIGURE 3. Adhesion of KIM185-stimulated HSB2 cells to the panel of ICAM-3 mutants. HSB2 cells were stimulated with the activating Ab KIM 185 at 10 µg/ml for 10 min at room temperature before addition to wells coated with ICAM-3 mutants, ICAM-3D1-2Fc wild type or the negative control protein CD31D1-2Fc. The percentage cell adhesion (percentage of input cells bound after two washes) was calculated, and results are expressed as mean ± SD (n = 3). Results are representative of three independent experiments. *, p < 0.01; **, p < 0.02; and ***, p < 0.05 compared with the wild-type control, as determined by Student’s t test. A, Data for mutants of residues H155, G156, E2, E8, E32, K33, and K42; B, Data for mutants of residues R6 and R64.

 
To determine the effect of the ICAM-3 mutants under optimal binding conditions, HSB2 cells were maximally activated by stimulation with KIM185 and the protein kinase C agonist PMA (Fig. 4Go). Under these conditions, the K42D mutant remained unable to bind HSB2 cells. The other domain 1 mutants produced a pattern of binding similar to that observed under suboptimal activating conditions. Maximal stimulation of HSB2 cells did not result in increased binding to E32A, K33A, K33D, or R64E. Although increased cell binding to E32K and R64A was observed, the adhesion was still significantly reduced compared with the wild-type control (p < 0.01 in each case). The domain 1 mutants located on the ABED face produced a pattern of binding similar to that observed for suboptimal activation of the cells. For the domain 2 mutants, adhesion to H155A remained significantly reduced (p < 0.01), although binding to H155D was not significantly different compared with the wild-type control.



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  		FIGURE 4. Adhesion of KIM185/PMA-stimulated HSB2 cells to the panel of ICAM-3 mutants. HSB2 cells were stimulated with the activating Ab KIM 185 at 10 µg/ml and the phorbol ester PMA at 10 ng/ml for 10 min at room temperature before addition to wells coated with ICAM-3 mutants, ICAM-3D1-2Fc wild type or the negative control protein CD31D1-2Fc. The percentage cell adhesion (percentage of input cells bound after two washes) was calculated, and results are expressed as mean ± SD (n = 3). Results are representative of three independent experiments. *, p < 0.01; **, p < 0.02; and ***, p < 0.05 compared with the wild-type control, as determined by Student’s t test. A, Data for mutants of residues H155, G156, E2, E8, E32, K33, and K42; B, Data for mutants of residues R6 and R64.

 
The effect of the mutations on LFA-1 binding were then examined in an alternative cell context, because it is known that cell background plays an important role in determining the ability of LFA-1 to recognize its ligands (43). When KL/4 cells were stimulated with KIM127, the cells bound to the panel of mutants in a pattern similar to that observed for adhesion of activated HSB2 cells; mutant K42D resulted in abrogation of adhesion, while mutants K33D and R64E profoundly reduced adhesion compared with the wild-type control (Fig. 5Go).



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  		FIGURE 5. Adhesion of KIM127-stimulated KL/4 cells to the panel of ICAM-3 mutants. KL/4 cells were stimulated with the activating Ab KIM127 at 10 µg/ml for 10 min at room temperature before addition to wells coated with ICAM-3 mutants, ICAM-3D1-2Fc wild type or the negative control protein CD31D1-2Fc. The percentage cell adhesion (percentage of input cells bound after two washes) was calculated, and results are expressed as mean ± SD (n = 3). Results are representative of three independent experiments. *, p < 0.01; **, p < 0.02; and ***, p < 0.05 compared with the wild-type control, as determined by Student’s t test. A, Data for mutants of residues H155, G156, E2, E8, E32, K33, and K42; B, Data for mutants of residues R6 and R64.

 
To map the location of the mutated residues, a model of domain 1 of ICAM-3 was generated based on the crystal structure of ICAM-2 (Fig. 6Go). The side chains of residues that, when mutated, resulted in reduced binding or no binding to LFA-1 positive cells are illustrated on the model. In this model, the critical glutamic acid residue at position 37 is located at the end of a ß strand and not in a loop region, and is surrounded by a relatively flat surface. Interestingly, residues identified in this study as being important for the interaction of ICAM-3 with LFA-1, are located on both faces of domain 1.



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  		FIGURE 6. Proposed model of domain 1 of ICAM-3 based on the crystal structure of ICAM-2. A side-on view of the domain is shown, with the GFC face on the left-hand side and the ABED face on the right-hand side. The side chains of residues that, when mutated, resulted in no binding (K42) or reduced binding (E2, R6, E8, E32, K33, and R64) to LFA-1 are illustrated. Residue E37, which has been defined as a key residue for ICAM-3 binding to LFA-1, is also shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have used mutant ICAM-3Fc proteins to build on previous work and further investigate the interaction between ICAM-3 and LFA-1. We have generated 18 single point mutants in an ICAM-3D1-2Fc backbone and used this panel of reagents to define residues that are crucial components of blocking Ab epitopes and residues that are components of the ICAM-3/LFA-1 interface.

The panel of ICAM-3 mutants was assessed by ELISA using a series of 18 Abs, 13 of which map to domain 1 and 5 that map to domain 2 of ICAM-3. Residues E32 and K33 were identified as crucial components of the epitopes of the 10 blocking and partial blocking Abs, with residue K42 also making a significant contribution. Residues Q75 and S68 are also important for binding of the Abs AO19 and AO85, which are both partial blocking Abs. Similarly, Sadhu et al. (44) have defined the epitopes of six other blocking Abs. Residues E32, K33, and E37 were identified as being important for Ab binding, while mutation of Y70 and Q75 also had some effect. Our model of ICAM-3 based on the ICAM-2 crystal structure indicates that these residues cluster in two distinct locations on the GFC face of domain 1: the B-C and F-G loops and adjacent residues located at the ends of ß strands in the top half of domain 1; and the C-D loop and adjacent residues in the lower half of domain 1. Interestingly, anti-ICAM-1 Abs that block the interaction with LFA-1 also have epitopes that map to these regions on the domain 1 structure (33, 45). The anti-ICAM-1 Abs RR1, WEHI-CAM-1, 7.5C2, 8.4A6, and R6.5D6 map to the F-G loop at the top of domain 1, and the epitopes of the Abs 84H10, BBIG-11, 1B9, and 4D3 include the residues K29, L30, K39, K40, K41, and Y66 (at equivalent positions to residues E32, K33, K42, none, E43, and S68, respectively, in ICAM-3).

We have investigated the role of 9 residues in both domain 1 and domain 2 of ICAM-3 in the interaction with LFA-1. We generated a panel of 18 single point mutations, which produced a similar pattern of cell adhesion when analyzed using two different LFA-1-positive cell types and various activation regimens. A lysine residue at position 42, which bound the nonblocking mAbs as wild type, was identified as a key residue for LFA-1 binding. In an alternative assay system, where COS cells were transfected with mutant ICAM-3 constructs and assayed for adhesion to purified LFA-1, the mutant K42A displayed wild-type activity and bound to a panel of Abs as wild type (34). In the structure of ICAM-2, the side chain of K42 forms a hydrogen bond with the main chain carbonyl of L36 (25). In our model of ICAM-3, K42 adopts a similar position, and it is possible that this interaction may be important in stabilizing the conformation of the C-D loop region. In previous studies, functional analysis of the equivalent residue in ICAM-1 (K39) has yielded conflicting results. The triple mutant K39KE/ERQ was found to bind as wild type to LFA-1 (32), whereas the single point mutants K39A, K39E, and K39M resulted in little or no adhesion to LFA-1 (33).

We have identified mutants of several residues that result in intermediate effects on LFA-1-mediated adhesion. Mutants of three residues on the GFC face of domain 1, E32, K33, and R64, significantly decreased LFA-1 adhesion compared with wild type even under optimal activation conditions. Residues E32 and K33 map to the B-C loop and the C strand, respectively, while R64 lies on the F strand in the lower region of domain 1. Using a similar system, Sadhu et al. (44) found that the double mutant E32K/AS resulted in an approximately sixfold decrease in binding to LFA-1, in complete agreement with our data for the single mutants. In an alternative assay system, where COS cells were transfected with mutant ICAM-3 constructs and assessed for binding to purified LFA-1, Klickstein et al. (34) found that the mutant E32A behaved as wild type. Interestingly, residues in ICAM-1 at positions equivalent to E32 and K33 (K29 and L30, respectively) have been mutated (K29A, K29M, K29Q, and L30A) and found to bind to LFA-1 equally as well as the wild type construct (33). The residue R64 has not previously been targeted for mutation and appears to have an effect on LFA-1 adhesion by virtue of its location in the main LETS motif region at the bottom of the F strand.

In addition to residues E32, K33, and R64 on the GFC face of domain 1, we have also identified mutants of ABED face residues that have statistically significant effects on LFA-1 adhesion. The mutants E2K, R6E, E8A, and E8K reduced adhesion compared with wild-type ICAM-3 (with p < 0.05 or less), although in general the magnitude of the reduction in adhesion was less than that observed for GFC face mutants. In a previous study, LFA-1-transfected COS cells that were suboptimally activated with PMA were observed to bind to the mutants P12A and F21A at ~50% of wild-type levels (20). When the LFA-1-positive COS cells were maximally activated using the anti-ß2-activating mAb KIM185 in conjunction with PMA, these two mutants were able to bind at 80% of wild type. In a recent study, the mutants N23 and S25, which both disrupt a potential N-linked glycosylation site on the ABED face, were found to profoundly disrupt binding to LFA-1 (24 and 19% binding compared with wild type at 100%) (34). The two residues at positions equivalent to R6 and E8 in ICAM-3 have been analyzed in ICAM-1 (residues S3 and S5, respectively) and found to bind to LFA-1 as wild type (33). Residues in ICAM-1 at positions equivalent to P12 and F21 in ICAM-3 have not been targeted for mutation in any studies of ICAM-1 to date. The residue in ICAM-1 that aligns with N23 is T20, which was changed to alanine in the mutant T20CS/ACT and had no significant effect on binding to LFA-1 (32). Mutation of S22 to alanine in ICAM-1, which corresponds to S25 of ICAM-3, similarly had no effect on adhesion to LFA-1 (33). Taken together, our data and the data from these recent studies seem to indicate that in contrast to ICAM-1, ABED face residues on domain 1 of ICAM-3 may contribute to the interaction with LFA-1.

Domain 2 mutants were found to have some effect on adhesion to LFA-1. Whereas non-domain 2 mutants generally resulted in statistically significant decreases in adhesion in all three assay systems, the data for the domain 2 mutants were somewhat variable in terms of statistical significance. It is therefore difficult to conclude whether these domain 2 residues play a major role in the interaction with LFA-1, although it would appear possible that they may make some contribution to the interaction.

In this study, we have provided evidence for a more extensive binding interface between ICAM-3 and LFA-1 than has previously been described. Notably, residues on the ABED face of ICAM-3 domain 1, as well as on the GFC face, appear to contribute to the interaction with LFA-1, although GFC face residues predominate. We have also compared the residues in ICAM-3 and ICAM-1 that mediate interaction with LFA-1 and show that although some residues, such as E37 in ICAM-3 and E34 in ICAM-1, are identical and located at equivalent positions in both molecules, other residues and their locations are very different. These findings complement several studies that show that LFA-1 can bind selectively to its ligands ICAM-1 and ICAM-3 and that there are distinct but overlapping binding sites in the I domain of LFA-1 for ICAM-1 and ICAM-3 (35, 36, 46, 47, 48). It has recently been shown that prior exposure to ICAM-1 increases the binding of LFA-1 to ICAM-3 (43), which further suggests that the binding sites on LFA-1 for these two ligands are distinct. In this article, we show that ICAM-3 is clearly very different from ICAM-1 in utilizing residues on both faces of domain 1 to interact with LFA-1, which may reflect a distinct stoichiometry of interaction. Although this study extends our knowledge of the interaction face between ICAM-3 and LFA-1, cocrystallization studies will be required to determine the precise interactions between LFA-1 and its ICAM ligands.


   Acknowledgments
 
We thank Dr. D. Mason (Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, U.K.) for the BY44 and CG106 Abs, and in particular Dr. M. K. Robinson (Celltech, Slough, U.K.) for the gift of KL/4 cells and KIM Abs. We also thank Justin Newton and Dr. Chris Buckley for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the Imperial Cancer Research Fund (E.D.B. and D.L.S.) and a BBSRC CASE award with the Yamanouchi Research Institute (A.P.M.). Back

2 Current address: The Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3 Canada. Back

3 Address correspondence and reprint requests to Dr. David L. Simmons, Cell Adhesion Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, U.K. E-mail address: Back

4 Abbreviations used in this paper: IgSF, Ig superfamily; ICRF, Imperial Cancer Research Fund. Back

Received for publication June 18, 1997. Accepted for publication March 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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