©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of the Binding Site on Intercellular Adhesion Molecule 3 for the Leukocyte Integrin Lymphocyte Function-associated Antigen 1 (*)

(Received for publication, August 29, 1994; and in revised form, November 8, 1994)

Claire L. Holness (1) Paul A. Bates (3) Amanda J. Littler (1) Christopher D. Buckley (1) Alison McDowall (2) David Bossy (1) Nancy Hogg (2) David L. Simmons (1)(§)

From the  (1)Cell Adhesion Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU and the (2)Leukocyte Adhesion & (3)Biomolecular Modelling Laboratories, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Intercellular adhesion molecule 3 (ICAM-3, CD50) is a member of the immunoglobulin superfamily and is a constitutively expressed ligand for the leukocyte integrin LFA-1 (CD11a/CD18). ICAM-3 is expressed at high levels by all resting leukocyte populations and antigen presenting cells and is a major ligand for LFA-1 in the resting immune system. ICAM-3 is a signal transducer and may play a key role in initiating immune responses. Mutant ICAM-3 Fc-chimeric proteins were quantitatively analyzed for their ability to bind COS cells expressing human LFA-1. The LFA-1-binding site on ICAM-3 is located in the N-terminal 2 Ig domains. Domains 3-5 do not significantly contribute to adhesion. The binding site has been further resolved by rational targeting of 14 point mutations throughout domains 1 and 2, coupled with modeling studies. Within domain 1 a cluster of residues (Glu, Leu, Ser, and Gln), that are predicted to lie on the CC`FG face of the Ig fold, play a dominant role in LFA-1 binding.


INTRODUCTION

Cell adhesion molecules (CAMs) (^1)play a key role in stabilizing and strengthening cell-matrix and cell-cell interactions. Leukocyte CAMs provide antigen-nonspecific recognition and are involved in a wide range of intercellular interactions, including those between helper T cells and antigen presenting cells, cytotoxic T cells, and natural killer cells and their targets (Springer 1990). In addition to these interleukocyte activities, CAMs are also involved in mediating leukocyte adhesion to and transmigration across vascular endothelium.

A key receptor-ligand interaction that contributes significant adhesion to all of these areas is the binding of the ICAMs (intercellular adhesion molecules) to their integrin ligands, most notably LFA-1 and Mac-1 (Pardi et al., 1992). The three known ICAMs (ICAM-1, -2, and -3) are all members of the immunoglobulin superfamily and have five, two, and five Ig domains, respectively (Rothlein et al., 1986; Simmons et al., 1988; Staunton et al., 1988, 1989; de Fougerolles et al., 1991, and 1993; Fawcett et al., 1992. Vazeux et al., 1992). ICAMs 1 and 3 are the most closely related. The ICAMs all bind to the leukocyte integrin LFA-1 (CD11a/CD18; alpha(L)beta(2),), and ICAM-1 has been shown to bind an additional integrin Mac-1 (CD11b/CD18; alpha(M)beta(2),) (Diamond et al., 1991). While the integrins are only expressed on hematopoietic cells, the ICAMs have very different expression profiles. ICAM-1 is expressed at low levels under normal conditions and is strongly up-regulated by cytokines on many cell types, including all leukocytes, endothelial cells, keratinocytes, and fibroblasts. ICAM-2 is constitutively expressed by all leukocytes, and endothelial cells, but at low levels. ICAM-3 is constitutively highly expressed by leukocytes and appears to be inducible on vascular endothelium in certain disease states, especially in lymphomas and myelomas (Doussis-Anagnostopoulou et al., 1993).

The different expression profiles support the notion that the ICAMs serve different functions and are not functionally redundant. ICAM-3 is expressed by antigen presenting cells, specifically by Langerhans cells in the skin (Acevedo et al., 1993). ICAMs 1 and 2 are either absent or expressed at very low levels on these cells. In addition, on resting T cells, ICAM-3 is the dominant ligand for LFA-1 (Campanero et al., 1993) and provides a costimulatory signal for cell proliferation (de Fougerolles et al., 1994). Recent work shows ICAM-3 to be a signal transducer. Cross-linking ICAM-3 produces a significant Ca flux and association with tyrosine kinases (Juan et al., 1994). A consequence of signaling through ICAM-3 is increased cell adhesion via integrin beta1 and beta2 pathways (Campanero et al., 1993; Cid et al., 1994). Thus, ICAM-3 appears to be the major ligand for LFA-1 in the primary or initiating phases of immune responses (Hernandez-Caselles et al., 1993) and plays a role in cell adhesion and signal transduction.

The interaction of ICAM-1 with LFA-1 is a multistep process, involving conformational changes in the integrin, partly controlled by engagement of ligand ICAM-1 (Cabanas and Hogg 1993). LFA-1 seems to have an intrinsically low affinity for its ICAM ligands, and that affinity is increased by a series of events that can be mimicked by a number of different stimuli including: changes in bound divalent cations (Dransfield and Hogg 1989; Dransfield et al., 1992), signal transduction agonists such as phorbol esters (Dustin and Springer 1989); and a small number of mAbs that induce or stabilize conformation changes (van Kooyk et al., 1991; Landis et al., 1993). However, apart from ligand itself, the normal cellular regulators of this affinity change are not yet known.

The LFA-1-binding site in ICAM-1 has been mapped to the N-terminal immunoglobulin (Ig) domain, and 2 residues, namely Glu and Gln, form a key part of that site (Staunton et al., 1990). Domain 1 is an Ig C2 fold, consisting of seven beta strands arranged on two surfaces, an ABE face and a CC`FG face. A model of the structure of the domains 1 and 2 of ICAM-1 has been produced which places these key residues on the CC`FG face; Glu is in the C-C` loop, and Gln is placed in the F to G loop (Berendt et al., 1992). These residues are conserved in ICAM-3 (Fawcett et al., 1992; Vazeux et al., 1992; de Fougerolles et al., 1993). We have previously shown that the N-terminal two Ig domains of ICAM-3 are competent to bind LFA-1 (Fawcett et al., 1992) and now report a detailed analysis of the LFA-1-binding site in ICAM-3. In addition to those residues predicted to be critical for interaction with LFA-1, we identify 2 amino acids on the F strand of D1 that are also essential components of the LFA-1-binding site. Based on this mutagenesis screen, we propose a model for domains 1 and 2 of ICAM-3 and discuss the critical area for interaction with ligand LFA-1.


MATERIALS AND METHODS

Monoclonal Antibodies and Cell Culture

Anti-LFA-1 alpha subunit monoclonal mAb 38 has been described (Dransfield et al., 1989). Anti ICAM-3 mAbs CH3.1, CH3.2, and CH3.3 were obtained by the fusion of spleen cells from a BALB/c mouse immunized with ICAM-3(D1-D2)-Fc. Anti-ICAM-3 mAbs CAL3.1, CAL3.4, CAL3.10, CAL3.16, CAL3.38, and CAL3.41 were obtained by the fusion of spleen cells from a BALB/c mouse immunized with ICAM-3(D1-D5)-Fc (Bossy et al., 1994). Other mAbs were provided as follows: anti-CD11a MHM24, anti-CD18 MHM23, and anti-ICAM-3 mAbs KS128, BY44, and CG106 by Dr. D. Mason (Nuffield Department of Pathology, John Radcliffe Hospital, Oxford) and anti-CD18 mAb KIM185 by Dr. M. Robinson (Celltech, Slough, United Kingdom). In adhesion assays, purified mAbs KIM185 and 38 were used at 5-10 µg/ml and other mAbs as 1/2 dilution of tissue culture supernatant final concentration.

COS-7 cells were obtained from the ICRF cell bank and grown in Dulbecco's modified Eagle's medium, 5% fetal calf serum. Activated T cells were expanded from peripheral blood mononuclear cells as described (Cabanas and Hogg, 1993).

Fc-chimeras and Mutants

ICAM-1(D1-D2)-Fc and ICAM-3(D1-D2)-Fc consisting of the first two extracellular domains fused to human IgG1-Fc fragment have previously been described (Fawcett et al., 1992). ICAM-3(D1-D3)-Fc, ICAM-3(D1-D4)-Fc, and ICAM-3(D1-D5)-Fc chimeras were generated by polymerase chain reaction (PCR) amplification of ICAM-3 cDNA and cloning into Fc expression vector pIG1 (Fawcett et al., 1992; Simmons, 1993). Primers used for PCR amplification were CDM8 forward sequencing primer 5`-CTAGAGAACCCACTGCTTAAC-3` and 5`-AC AGATCT ACTTACCTGT GAA GCT AAA GAC CGT CAA GTT-3` for ICAM-3(D1-D3)-Fc; CDM8 forward sequencing primer and 5`-AC AGATCT ACTTACCTGT TTT CCA TTT CAA GTG CTG GGG-3` for ICAM-3(D1-D4)-Fc; CDM8 forward sequencing primer and 5`-AC AGATCT ACTTACCTGT GTG GGA GCT CCC AGC CTC AAT-3` for ICAM-3(D1-D5)-Fc. The mouse-human chimera was made by PCR ligation of domain 1 of mouse ICAM-1 to human ICAM-3 domains 2-5 as an Fc chimera. Plasmid encoding ICAM-1(D1-D5)-Fc was a gift from Dr. A. Craig, Institute of Molecular Medicine, Oxford. The NCAM-Fc consisted of the entire extracellular domain (five Ig related domains and two type III fibronectin-related domains) of neural cell adhesion molecule fused to human IgG1-Fc fragment. Mutant forms of ICAM-3(D1-D2)Fc were made by a two-step PCR strategy (Higuchi, 1990). A list of mutagenic primers can be provided on request. All constructs and mutants were sequenced in their entirety.

Recombinant chimeric-Fc plasmids were transiently expressed in COS cells and secreted protein purified from tissue culture supernatants with protein A-coupled Sepharose (Simmons, 1993).

Transfection of COS Cells with LFA-1 and Cytofluorometric Analysis

CD11a and CD18 cDNAs cloned into pcDNA/AMP (Invitrogen) were transiently expressed in COS cells. The cDNAs (at 5 µg/ml each) were transfected in the presence of 1 mg/ml DEAE-dextran and 100 µM chloroquine for 2-4 h at 37 °C followed by a 2-min shock in PBS, 10% Me(2)SO. For cytofluorometric analysis, COS transfectants were lifted 72 h post-transfection in PBS, 2 mM EDTA, washed in PBS, 2 mM EDTA, 2 mg/ml bovine serum albumin at 4 °C, stained with primary mAbs (1:10 dilution of tissue culture supernatants of anti-CD11a mAb MHM24 or anti-CD18 mAb MHM23), washed, stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG, washed three times, fixed in PBS, 2% formaldehyde, and analyzed on a Becton Dickinson FACScan. COS cell transfected with LFA-1 were routinely 15-40% positive by FACScan analysis.

Adhesion Assays

Immulon-3 96-well plates (Dynatech Laboratories Inc., Chantilly, VA) were precoated with 1 µg/well goat-anti-human-Fc Ig (Sigma, Poole, Dorset U.K.) overnight at 4 °C, blocked with PBS, 0.4% bovine serum albumin (Fraction V, Sigma) for 2 h at room temperature, and then coated with chimeric proteins in PBS for at least 2 h at room temperature. Prior to adhesion assay at 72-h post-transfection, COS cells were labeled for 12-24 h with ^3[H]thymidine at 10 µCi/2 times 10^7 cells and harvested prior to the assay with PBS, 2 mM EDTA. Cells were washed twice in assay medium, consisting of RPMI, 2.5% fetal calf serum. Stimulation with phorbol 12-myristate 13-acetate (PMA, Sigma) was performed by addition of PMA to 50 ng/ml (80 nM), incubation for 30 min at 37 °C, and then removal by washing in assay buffer. Cells were added to ICAM-Fc plates to give 3 times 10^4/well in a final volume of 50 µl. Cells were preincubated on ice for 20 min in the presence of antibodies as appropriate, followed by a further 30-min incubation at 37 °C. Cells that remained bound after two washes with prewarmed RPMI were lysed in 1% SDS, scintillant added (Ready Safe, Beckman), and incorporated radioactivity counted using a Beckman LS 5000 CE counter. The percent of LFA-1-positive cells was calculated from the incorporated radioactivity by taking into account the total cell input and the transfection efficiency.

Enzyme-linked Immunosorbent Assay of ICAM-3 Fc Proteins

Immulon-3 96-well plates were precoated with 1 µg/well goat-anti human-Fc Ig overnight at 4 °C, blocked with PBS, 0.4% bovine serum albumin (Fraction V, Sigma) for 2 h at room temperature, and then coated with chimeric proteins (5 µg/ml) in PBS for at least 2 h at room temperature. Antibody was added in saturating amounts as either neat supernatant or appropriately diluted ascitic fluid, followed by peroxidase-conjugated goat anti-mouse Ig (1:500 dilution, Dako, Glostrup, Denmark). Each layer was incubated for 30 min at room temperature and followed by six washes. The assay was visualized with O-phenylenediamine dihydrochloride (Sigma) and absorbance read at 450 nm.


RESULTS

Regulation of LFA-1 Activation State in Nonhematopoietic Cells

In order to dissect the interaction of ICAM-3 with the leukocyte integrin LFA-1, the experimental system involved quantitative binding assays of LFA-1 cells to soluble recombinant fusion proteins. Previous work has established that LFA-1 leukocytes bind to an ICAM-3-Fc chimeric protein containing only the N-terminal two domains of ICAM-3 (Fawcett et al., 1992). Recent work has shown that ligand itself may influence the binding avidity of T cell LFA-1 (Cabanas and Hogg, 1993). As leukocytes express LFA-1, ICAM-1, and ICAM-3, potential problems might occur when trying to dissect the nature of LFA-1 binding to ICAM-3, due to the influence of intercellular LFA-1/ICAM-1 or ICAM-3 interaction. In order to avoid these possible complications, COS cells were used as the recipient cells for LFA-1. DEAE-dextran transfection of COS cells with pCD18 and pCD11a results in transient expression of LFA-1 on 15-40% of cells as detected by flow cytometry with mAbs MHM24 (CD11a) or MHM23 (CD18). In contrast, no surface expression of CD11a or CD18 was detected when either pCD11a or pCD18 were transfected alone (data not shown).

In order to assess the activation state of LFA-1 expressed by COS cells, binding assays were performed onto ICAM-1 and ICAM-3 in the presence or absence of activating stimuli. LFA-1 COS were allowed to adhere to soluble recombinant fusion proteins containing the two N-terminal domains of ICAM-1 or ICAM-3 (Fawcett et al., 1992). The activation state of COS cell LFA-1 could be regulated both by the protein kinase C agonist PMA and by an ``activating'' anti-LFA-1 monoclonal antibody (Fig. 1). The anti-CD18 mAb KIM185 is known to induce a change in the conformation of CD18 and promote LFA-1-dependent adhesion (Andrew et al., 1993). Adhesion of LFA-1 COS to ICAM-1 and ICAM-3 is also enhanced by the addition of KIM185 (Fig. 1).


Figure 1: Regulation of LFA-1 activation state in COS cells. COS cells transiently expressing LFA-1 (40% COS cells LFA-1) were allowed to adhere to ICAM-1(D1-D2)-Fc or ICAM-3(D1-D2)-Fc. Where indicated, cell binding occurred in the presence of monoclonal antibodies: mAb38 is an anti-CD11a mAb which blocks LFA-1/ICAM interactions and KIM185 is an anti-CD18 mAb which induces LFA-1-dependent adhesion. Where indicated, COS cells were treated with 80 nM PMA. Results are expressed as means ± 1 S.D. unit (n = 5).



ICAM-3 Domains 1 and 2 Are Necessary and Sufficient for LFA-1 Binding

Previous work has established that ICAM-3 domains 1 and 2 contain the LFA-1-binding site (Fawcett et al., 1992). However, the possibility that there might be additional contributions to this binding from domains 3, 4, and 5 has not been addressed. To investigate this a series of ICAM-3-Fc chimeric proteins, comprising a nested set of ICAM-3 domain deletion mutants, was generated (Fig. 2). Fusion protein ICAM-3(D1-D5)-Fc is the wild-type molecule containing all five immunoglobulin domains whereas proteins ICAM-3(D1-D4)-Fc, ICAM-3(D1-D3)-Fc, and ICAM-3(D1-D2)-Fc have sequential, C-terminal, domain deletions. Cell binding assays were performed with chimeric proteins immobilized onto plastic via anti-Fc polyclonal antibodies which allowed normalization of the amount of wild-type and mutant proteins presented to the cells.


Figure 2: The domain deletion series of ICAM-3-Fc chimeric proteins. Proteins were purified as described under ``Materials and Methods.'' 5 µg of each protein were resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions and visualized with Coomassie stain. Molecular masses deduced from electrophoretic mobilities are ICAM-3(D1-2)-Fc 80 kDa (lane 1), ICAM-3(D1-3)-Fc 107 kDa (lane 2), ICAM-3(D1-4)-Fc 130 kDa (lane 3), ICAM-3(D1-5)-Fc 136 kDa (lane 4), and NCAM-Fc (negative control) 153 kDa (lane 5).



In order to assess the effect of the C-terminal domains of ICAM-3 on the interaction of LFA-1, the set of domain deletion mutants was used in a binding assay with LFA-1 COS that had been activated with PMA. Binding was not significantly increased by the presence of domains 3-5 of ICAM-3 (Fig. 3). This binding is LFA-1 specific as it is blocked by the anti-CD11a mAb 38.


Figure 3: The first two amino-terminal domains of ICAM-3 (D1-2) contain the LFA-1-binding site. LFA-1 COS cells were assayed for binding to the domain deletion series of ICAM-3-Fc chimeric proteins coated onto plastic via anti-Fc polyclonal antibodies. LFA-1 COS (15% COS cells LFA-1) were activated with 80 nM PMA. NCAM-Fc is included as a negative control having 5 C2-set IgSF domains. Results are expressed as means ± 1 S.D. unit (n = 10).



Further Definition of the LFA-1-binding Site on ICAM-3 by Directed Mutagenesis

Having established that the LFA-1-binding site is contained within domain 1 and 2, a mutagenesis screen of these domains was undertaken. We aligned the primary amino acid sequences of ICAM-1 and ICAM-3, and, based on a molecular model for ICAM-1 (Berendt et al., 1992), predicted which ICAM-3 residues contributed to the beta strands of the immunoglobulin structure (Fig. 4). The key residues for ICAM-1 binding to LFA-1 (Glu and Gln) are present on the CC`FG face of domain 1. To determine the importance of ICAM-3 domain 1 for interaction with LFA-1, six specific mutations were targeted onto the CC`FG face and three onto the opposite ABE face. The CC`FG face mutations included substitution of residues homologous to ICAM-1 Glu and Gln. In addition, five mutations were targeted onto domain 2, particularly in connecting loops between beta strands, to assess its contribution toward the LFA-1-binding site. We selected residues predicted to be solvent accessible and exposed at the surface of ICAM-3. For the most part charged amino acids were substituted with alanine (Cunningham and Wells, 1989) by site-directed mutagenesis of pICAM-3(D1-D2)-Fc, and the location of the mutations is indicated in Fig. 4. Mutant ICAM-3(D1-D2)-Fc fusion proteins produced by expression of ICAM-3(D1-D2)Fc plasmids in COS cells were checked by SDS-polyacrylamide gel electrophoresis analysis and found to migrate at the predicted size of 80 kDa (Bossy et al., 1994).


Figure 4: Sequence alignment of ICAM-1 and ICAM-3. Sequence alignment of the first two domains of ICAM-1 and ICAM-3. The predicted location of the beta sheets of the immunoglobulin domains is indicated below the alignment. Asterisks denote those residues that were targeted for site-directed mutagenesis. Potential N-linked glycosylation sites are numbered (-1-, etc.)



Effect of ICAM-3 Mutations on Interaction with LFA-1

To address the effect of the mutations on LFA-1 binding, adhesion assays were performed onto a concentration range of mutant and wild-type ICAM-3 protein using LFA-1 COS cells as the ligand bearing cells (Fig. 5). When compared to an equivalent concentration of wild-type ICAM-3, no binding was exhibited to mutants E37A, L66K, S68K, Q75H, and Q75A. These residues all map onto the CC`FG face of domain 1 and are critical for interaction with LFA-1. Glu is also located in this region of ICAM-3, and LFA-1 binding to E43A is reduced to 30-50% of wild-type. In contrast, substitution of Asp to alanine does not affect interaction with LFA-1. Compared with those residues critical for LFA-1 binding, Asp is located on the opposite face of domain 1. The domain 1 mutants F21A and P12A are also located on this ABE face, and binding of LFA-1 to these proteins is reduced to 50% of wild-type.


Figure 5: Effect of ICAM-3 mutations on LFA-1 binding. COS cells transiently expressing LFA-1 (15% COS cells LFA-1) were treated with PMA and then allowed to adhere to a concentration range (1, 5, 10, and 20 µg/ml) of ICAM-3(D1-D2)-Fc mutant and wild-type chimeric proteins. The concentration range was achieved by precoating with 1 µg/well anti-human-Fc Ig and then coating with chimeric protein at the indicated concentration. Results are expressed as means ± 1 S.D. unit (n = 6).



COS cells expressing LFA-1 bound to the domain 2 mutants (Fig. 5). Interaction of LFA-1 with P158A and E143A was not affected whereas binding to D166A, R127A, and R127WE/AAA was perturbed. This indicates that domain 2 of ICAM-3 may have a role in the interaction with ligand LFA-1.

In order to distinguish the most important binding area, LFA-1 expressed in COS cells was fully activated with a combination of PMA and anti-beta2 activating mAb KIM185 and allowed to bind to mutant and wild-type ICAM-3 chimeric proteins (Fig. 6). Under these conditions of enhanced LFA-1 activation, there is still no binding to mutants E37A, L66K, S68K, Q75H, and Q75A. This emphasizes the critical nature of these residues for interaction with ligand. In contrast, under these optimal conditions binding to mutants on the ABE face of domain 1 and within domain 2 is largely restored and so may be less important for interaction with LFA-1.


Figure 6: Effect of ICAM-3 mutations on binding of fully activated LFA-1. COS cells transiently expressing LFA-1 (25% COS cells LFA-1) were treated with PMA plus mAb KIM185. Cells were allowed to adhere to ICAM-3(D1-D2)-Fc mutant and wild-type proteins or NCAM-Fc as a negative control. Results are expressed as means ± 1 S.D. (n = 6).



Effect of ICAM-3 Mutations on Interaction with Anti-ICAM-3 mAbs

Eight anti-ICAM-3 mAbs were mapped to domain 1 by enzyme-linked immunosorbent assay onto ICAM-3(D1-D2)-Fc and a mouse-human chimeric protein composed of D1 of mouse ICAM-1 linked to domains 2-5 of human ICAM-3. MAbs CH3.1, CH3.2, CH3.3, BY44, CG106, CAL3.10, CAL3.38, and CAL3.41 recognized ICAM-3(D1-D2)-Fc but not the mouse-human ICAM-1/ICAM-3 chimera, indicating that these mAbs have epitopes in domain 1 or possibly at the interface between domains 1 and 2.

To define relationships between the epitopes of the mAbs, competitive inhibition assays were performed (data not shown). CH3.1, CH3.2, and CH3.3 recognize overlapping regions of ICAM-3 that are distinct from the epitopes of BY44 and CG106. In addition, CAL3.10, CAL3.38, and CAL3.41 are defined within a separate cluster. Mabs BY44, CG106, CAL3.10, CAL3.38, and CAL3.41 are also functionally distinct since they block the interaction of COS LFA-1 with ICAM-3(D1-D5)-Fc (Bossy et al., 1994). All of the domain 1 mAbs recognize the domain 1 mutants indicating the overall conformation of this Ig domain to be preserved (Table 1). As the blocking mAbs BY44, CG106, CAL3.10, CAL3.38, and CAL3.41 bind to all of the mutant proteins, this suggests that there may be further regions to be discovered that are important for the interaction of ICAM-3 with LFA-1.



KS128 bound to the mouse-human chimeric protein, although binding was 50% of that to ICAM-3(D1-D5)-Fc. This suggests that domain 2 and domain 1 of ICAM-3 contribute to the epitope of KS128. In addition, when screened on the mutant panel, KS128 bound poorly to both the domain 2 mutants R127A, R127WE/AAA, and D166A and the domain 1 mutants P12A and E43A. Thus the epitope for KS128 appears to span both N-terminal domains of ICAM-3 and this is consistent with another report (Klickstein et al., 1993). Three further mAbs, CAL3.1, CAL3.4, and CAL3.16, also appear to be influenced by domain 1 and domain 2. Like KS128, the binding of these mAbs is decreased by mutations in both domain 1 and domain 2. An explanation for this is that Pro, Glu, Arg, and Asp are in close association, a prediction that is supported by modeling data (see below).

The mAbs CAL3.1, CAL3.4, CAL3.16, and KS128 do not recognize all of the domain 2 mutants (Table 1). Since binding of each domain 1/domain 2 mAb is perturbed to R127A, R127WE/AAA, and D166A, it must be considered that these mutations are destabilizing. In contrast, the majority of the domain 1/domain 2 mAbs recognize E143A and P158A suggesting that these mutants maintain structural integrity. In addition, each domain 2 mutant is recognized by all of the domain 1 mAbs, and this confirms the conformation of the first domain in each case.

Molecular Model for ICAM-3

In order to map the relative position of the mutations, a model of the two N-terminal domains of ICAM-3 was constructed (Fig. 7). The protocol for replacement of side chains and protein fragments (usually loop regions) was initiated by a critical comparison of the primary protein sequence alignment of ICAM-1 and ICAM-3 (Fig. 4) and then followed by methodologies previously described for the ICAM-1 model (Berendt et al., 1992). An important feature of the molecular model is the relative position of the two domains. For both ICAM-1 and ICAM-3, a close association between the domains is predicted with domain 2 rotated at approximately 160 ° with respect to domain 1. Key conserved hydrophobic patches at the base of domain 1 and at the top of domain 2 support this arrangement (data not shown) as does the epitope mapping in this present study.


Figure 7: Model for domains 1 and 2 of ICAM-3. The beta strands are labeled in the normal convention for immunoglobulin folds, A to G, with the two beta sheets CC`FG and ABE, indicated by shading differences. Disulfide bonds are denoted by dark zig-zag lines. The locations of site-directed mutations are indicated by residue number and side chain, and potential N-linked glycosylation sites are numbered (1-7) and shown as bullet. This diagram was produced with the aid of the display program MOLSCRIPT (Kraulis, 1991).



The location of the 14 mutations is indicated on the molecular model (Fig. 7). The residues critical for the association of ICAM-3 with LFA-1 (Glu, Leu, Ser, and Gln) are aligned on the CC`FG face of domain 1. The residue Asp, within the F-G loop of domain 2, also contributes to the binding site. The model shows the CC`FG face of domain 1 and the F-G loop of domain 2 to be associated along the same side of ICAM-3 and this supports the participation of these two regions in the binding site for LFA-1.

There are seven potential N-linked glycosylation sites in the two N-terminal domains of ICAM-3, and their position is relevant when considering the LFA-1-binding site. In a previously solved x-ray crystal structure of an Fc antibody fragment, an N-linked oligosaccharide covered hydrophobic residues on one face of the Ig fold (Diesenhofer, 1981). The oligosaccharide from this Fc fragment was clipped and placed on each of the potential glycosylation sites on the molecular model of ICAM-3. Freely rotatable bonds of the Asn-oligosaccharide complexes were then adjusted to remove steric clashes and to cover exposed hydrophobic residues. The general conclusion drawn from this exercise is that the CC`FG face on the first domain is relatively free from oligosaccharide cover, and the oligosaccharides at positions four and five impinge only on the edges of this region (Fig. 7).


DISCUSSION

To study the binding site for LFA-1 on ICAM-3, we measured the adhesion of COS cells expressing LFA-1 to a series of mutant ICAM-3 proteins. This model system had two significant benefits. First, by using COS cells to present LFA-1, we avoided the influence of interleukocyte LFA-1/ICAM binding events that may affect the binding avidity of LFA-1 on leukocytes (Cabanas and Hogg, 1993). Therefore, COS cells expressing LFA-1 permitted a direct analysis of ICAM-3 binding. Second, all adhesion assays were normalized for the amount of wild-type or mutant protein presented to the LFA-1/COS cells. All ICAM-3 site-directed mutants were produced as recombinant D1-D2-Fc fusion proteins, and this allowed even small differences in the binding properties between the mutants to be quantitatively assessed. This system overcomes the problems of expressing equivalent amounts of mutant proteins in COS cells and eliminates the possibility of accessory cell-cell interactions which may contribute to background binding in a two-cell system.

Here we show that LFA-1 expressed by a non-leukocytic cell type, COS cells, can exist in a variety of activation states with different affinities for ligand. The avidity state of COS cell LFA-1 can be regulated both by ``outside-in'' signals (mediated by mAb KIM185) and ``inside-out'' signals (mediated by the protein kinase C agonist, PMA). In a separate study, COS cell LFA-1 was activated with anti-CD11a mAb MEM83 and binding to ICAM-1 was enhanced (Landis et al., 1994). This ability to regulate COS cell LFA-1, differs from a previous report, in which LFA-1 expressed in COS cells was shown to be constitutively active (Larson et al., 1990). This discrepancy may reflect differences in the level of expression of LFA-1 on the COS cells. Larson et al.(1990) achieved a higher transfection efficiency (50% of LFA-1 cells in the COS cell population) and higher expression of LFA-1/COS cell. These factors might affect avidity by altering the distribution of LFA-1 on the COS cell surface.

Domains 1 and 2 of ICAM-3 are necessary and sufficient for interaction with LFA-1. There are no protein recognition sequences for LFA-1 within domains 3-5. These domains may, however, have a role in presentation of the binding site to LFA-1 when ICAM-3 is expressed at the cell surface in the context of the cell glycocalyx. This appears to be true for the interaction of ICAM-1 with its receptor on Plasmodium falciparum infected erythrocytes (Berendt et al., 1992). The ICAM-1-binding site for malaria-infected erythrocytes maps to the first two domains, yet adhesion to a shortened version of ICAM-1 containing only these domains is dramatically reduced presumably due to steric hindrance. In the present work, domain-deleted and wild-type chimeric proteins were presented equally to LFA-1 via anti-Fc antibodies so eliminating the issue of binding site accessibility.

Having established the importance of domains 1 and 2 of ICAM-3 for interaction with LFA-1, site-directed mutants were generated to dissect the binding site. Mutations were targeted onto the CC`FG face and ABE face of the first domain of ICAM-3. The CC`FG face of ICAM-3 was of particular interest since the equivalent region of ICAM-1 contributes to LFA-1 binding (Berendt et al., 1992). We show that glutamine at position 37 of ICAM-3 is essential for LFA-1 binding. ICAM-3 E37 is equivalent to ICAM-1 Glu which is also critical for association with LFA-1 ligand (Staunton et al., 1990). To extend the study of ICAM-3 E37, we made substitutions of the amino acids to which Glu is predicted to hydrogen bond. Modeling data associates Glu in an interaction triad with Leu and Ser. By generating mutants L66K and S68K, binding of ICAM-3 to LFA-1 was prevented, presumably by disrupting the inter-residue contacts within this triad.

A second key residue for the ICAM-3/LFA-1 interaction is Gln. The homologous residue is Gln in ICAM-1 and is essential for LFA-1 binding. Staunton et al. (1990) found that mutating ICAM-1 Gln to histidine decreased LFA-1 binding 10-fold, and substitution to threonine decreases binding 2-fold. In contrast, we find that mutation of ICAM-3 Q75 to either a histidine or an alanine eliminates LFA-1 binding. Therefore, ICAM-3 residue Gln distinguishes the binding site for LFA-1 on ICAM-3 from that on ICAM-1.

The fact that ICAM-3 binding was eliminated by mutations at Leu and Ser on the F strand in the middle of the CC`FG face indicates that those residues important for interaction with LFA-1 are aligned along one face of the molecule. This region of ICAM-3 is predicted to be free from oligosaccharide cover and therefore accessible for ligand binding. Indeed, a recent study (Landis et al., 1994) shows that the glycosylation of ICAM-3 domain 1 does not have a role in ligand binding. Although our assignment of oligosaccharide chains to the periphery of the CC`FG face is speculative, a recent study of the glycan structures of human CD2 suggests a similar situation with regard to the ligand-binding site (Withka et al., 1993). The three-dimensional structure of the fully glycosylated form of domain 1 of human CD2, determined by nuclear magnetic resonance spectroscopy, places the oligosaccharide at the top of the connecting loops between the beta strands, at the perimeter of the CD58 ligand-binding site.

The ICAM-3 D1 ABE face mutations had less dramatic effects than most of the CC`FG face substitutions. The mutant D27A bound ligand to the same extent as wild-type ICAM-3, and this residue is equivalent to ICAM-1 S24 which also is not involved in LFA-1 binding (Staunton et al., 1990). The two other ABE face mutations, Pro and Phe, did perturb ligand binding. This raises the possibility that there are regions of ICAM-3 D1, in addition to the CC`FG face, that have a role in ligand binding. Residue Pro is located on the A strand of ICAM-3 D1. Interestingly, mutation of the A strand of ICAM-1 D1 does not affect binding to LFA-1 (Staunton et al., 1990). Since LFA-1 binds selectively to ICAM-1 and ICAM-3 (Landis et al., 1994), the binding interfaces are likely to be distinctive. For both ICAM-1 and ICAM-3, the CC`FG face of domain 1 appears to have a dominant role in interaction with ligand, but the binding sites may be defined by other areas within domain 1.

Alignment of ICAM-1 with ICAM-3 reveals that the highest degree of sequence homology occurs in the second domain (77% amino acid identity), suggesting functional importance for this region of the ICAMs. Because of lack of reagents (mAbs and mutants), only limited analysis of ICAM-1 domain 2 has been reported. To date, only the F-G loop of ICAM-1 D2 polypeptide is known to contribute to LFA-1 binding (Berendt et al., 1992; Staunton et al., 1992). To determine the extent of interaction of LFA-1 with D2 of ICAM-3, domain 1 proximal and distal mutations were made. Mutation of Arg and Asp reduced binding to LFA-1 but may also destabilize the Ig structure. Thus, the second domain of ICAM-3 may contribute to the LFA-1-binding site, but the extent of the interaction face has yet to be determined. There may be regions within domain 2 that are critical for LFA-1 binding, or it is possible that this domain has only a supportive role in association with ligand LFA-1.

LFA-1 interacts with key residues on the CC`FG face of domain 1. For members of the immunoglobulin superfamily, the faces of the beta strands of the Ig-related domains may be as important for ligand interaction as the connecting loop regions. Indeed, the binding site for LFA-3 (CD58) is located along one face (in fact the CC`FG face) of CD2 (Somoza et al., 1993; Arulanandam et al., 1993). A molecular model of the first two domains of ICAM-3 indicates that the domains are closely associated, with the second domain rotated at approximately 160 °. Although molecular modeling cannot predict domain packing with certainty, solved structures for CD4 and CD2 support this orientation (Wang et al., 1990; Ryu et al., 1990: Jones et al., 1992; Brady et al., 1992). Thus, the LFA-1 binding surface on ICAM-3 encompasses the CC`FG face of domain 1 and may extend into domain 2. Although it is possible that, within domains 1 and 2, there are other important binding areas to be defined.

Amino acids Glu and Gln within ICAM-3 domain 1 may represent an essential, common motif in the ligand-binding site of all ICAMs. Homologous residues are found in all members of the ICAM family, ICAM-1 (Simmons et al., 1988; Staunton et al., 1988), ICAM-2 (Staunton et al., 1989), and ICAM-3 (Fawcett et al., 1992; Vazeux et al., 1992; de Fougerolles et al., 1993), and are critical for binding of LFA-1 to ICAM-1 and ICAM-3. These key residues are also conserved across species and are present in chimpanzee ICAM-1 (Hammond and McClelland 1993), rat ICAM-1 (Kita et al., 1992), mouse ICAM-1 (Horley et al., 1989), and mouse ICAM-2 (Xu et al., 1992). This motif may be generally important for the interaction of integrins with members of the immunoglobulin superfamily (IgSF). To date, there are three examples of IgSF/integrin interactions: ICAMs with LFA-1 and/or Mac-1 (Simmons et al., 1988; Staunton et al., 1989; Diamond et al., 1990), VCAM with VLA-4 (alpha4beta1) and alpha4beta7 integrins (Elices et al., 1990; Ruegg et al., 1992), and MadCAM-1 with the alpha4beta7 integrin (Berlin et al., 1993). Acidic residues at equivalent locations on VCAM-1 domains 1 and 4 (i.e. within the C-C` region) are also important for binding to its cognate integrin VLA-4 (alpha4beta1) (Osborn et al., 1994; Vonderheide et al., 1994; Renz et al., 1994).

In conclusion, we have identified the CC`FG face of ICAM-3 to be an important binding region for LFA-1. Residues critical for interaction with LFA-1 are present on this face of domain 1. Some of these essential residues may form the basis of a common motif for all IgSF-integrin interactions.


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 correspondence should be addressed: Cell Adhesion Laboratory, ICRF, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. Tel.: 44-865-222355; Fax: 44-865-222431.

(^1)
The abbreviations used are: CAMs, cell adhesion molecules; ICAM, intercellular adhesion molecule; LFA-1, lymphocyte function-associated antigen 1; mAb, monoclonal antibody; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We thank Martyn Robinson (Celltech) for the generous gift of mAb KIM185, Alister Craig (Institute of Molecular Medicine, Oxford) for ICAM-1(D1-D5)-Fc; Paul Crocker (IMM, Oxford) for NCAM-Fc protein; David Mason (Nuffield Department of Pathology) for gifts of ICAM-3 and CD11a/CD18 mAbs; Michael J. E. Sternberg, Alister Craig, Andy Gearing, and John Clements (British Bio-technology, Oxford) for discussions; and Paul Crocker and Anthony Berendt for comments on the manuscript.


REFERENCES

  1. Acevedo, A., del Pozo, M. A., Arroyo, A. G., Sanchez-Mateos, P., Gonzalez-Amaro, R., and Sanchez-Madrid, F. (1993) Am. J. Pathol. 143, 774-782 [Abstract]
  2. Andrew, D., Shock. A., Ball, E., Ortlepp, S., Bell, J., and Robinson, M. (1993) Eur. J. Immunol. 23, 2217-2222 [Medline] [Order article via Infotrieve]
  3. Arulanandam, A. R. N., Withka, J. M., Wyss, D. F., Wagner, G., Kister, A., Pallai, P., Recny, M. A., and Reinherz, E. L. (1993). Proc. Natl. Acad. Sci. U. S. A. 90, 11613-11617 [Abstract]
  4. Berendt, A. R., McDowall, A., Craig, A. G., Bates, P., Sternberg, M. J. E., Marsh, K., Newbold, C. I., and Hogg, N. (1992) Cell 68, 71-81 [Medline] [Order article via Infotrieve]
  5. Berlin, C., Berg, E. L., Briskin, M. J., Andrew, D. P., Kilshaw, P. J., Holzmann, B., Weissman, I. L., Hamann, A., and Butcher, E. C. (1993) Cell 74, 185-195 [Medline] [Order article via Infotrieve]
  6. Bossy, D., Buckley, C. D., Holness, C. L., Littler, A. J., Murray, N., Collins, I., Simmons, D. L. (1994) Eur. J. Immunol ., in press
  7. Brady, R. L., Dodson, E. J., Dodson, G. G., Lange, G., Davis, S. J., Williams, A. F., and Barclay, A. N. (1993) Science 260, 979-983 [Medline] [Order article via Infotrieve]
  8. Cabanas, C., and Hogg, N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5838-5842 [Abstract]
  9. Campanero, M. R., del Pozo, M. A., Arroyo, A. G., Sanchez-Mateos, P., Hernandez-Caselles, T., Craig, A., Pulido, R., and Sanchez-Madrid, F. (1993) J. Cell Biol. 123, 1007-1016 [Abstract]
  10. Cid, M. A., Esparza, J., Juan, M., Miralles, A., Ordi, J., Vilella, R., Urbano-Marquez, A., Gaya, A., Vives, J., and Yague, J. (1994) Eur. J. Immunol. 24, 1377-1382 [Medline] [Order article via Infotrieve]
  11. Cunningham, B. C., and Wells, J. A. (1989) Science 244, 1081-1085 [Medline] [Order article via Infotrieve]
  12. de Fougerolles, A. R., Stacker, S. A., Schwarting, R., and Springer, T. A. (1991) J. Exp. Med. 174, 253-267 [Abstract]
  13. de Fougerolles, A. R., Klickstein, L. B., and Springer, T. A. (1993) J. Exp. Med. 177, 1187-1192 [Abstract]
  14. de Fougerolles, A. R., Qin, X., and Springer, T. A. (1994) J. Exp. Med. 179, 619-629 [Abstract]
  15. Deisenhofer, J. (1981). Biochemistry 20, 2361-2369 [Medline] [Order article via Infotrieve]
  16. Diamond, M. S., Staunton, D. E., de Fougerolles, A. R., Stacker, S. A., Garcia-Aguilar, J., Hibbs, J., and Springer T. A. (1990) J. Cell Biol. 111, 3129-3139 [Abstract]
  17. Diamond, M. S., Staunton, D. E., Marlin, S. D., and Springer T. A. (1991) Cell 65, 961-971 [Medline] [Order article via Infotrieve]
  18. Doussis-Anagnostopoulou, I., Kaklamanis, L., Cordell, J., Jones, M., Turley, H., Pulford, K., Simmons, D., Mason, D., and Gatter, K. (1993) Am. J. Pathol. 143, 1040-1043 [Abstract]
  19. Dransfield, I., and Hogg, N. (1989) EMBO J. 12, 3759-3765
  20. Dransfield, I., Cabanas, C., Craig, A., and Hogg, N. (1992) J. Cell Biol. 116, 219-226 [Abstract]
  21. Dustin, M. L., and Springer, T. A. (1989) Nature 341, 619-624 [CrossRef][Medline] [Order article via Infotrieve]
  22. Elices, M. J. L., Osborn, Y., Takada, C., Crouse, C., Luhowskyj, S., Hemler, M. E., and Lobb, R. R. (1990) Cell 60, 577-584 [Medline] [Order article via Infotrieve]
  23. Fawcett, J., Holness, C. L. L., Needham, L. A., Turley, H., Gatter, K. C., Mason, D. Y., and Simmons, D. L. (1992) Nature 360, 481-484 [CrossRef][Medline] [Order article via Infotrieve]
  24. Garrett, T. P. J., Wang, J., Yan, Y., Liu, J., and Harrison, S. C. (1993) J. Mol. Biol. 234, 763-778 [CrossRef][Medline] [Order article via Infotrieve]
  25. Hammond, L., and McClelland, A. (1993) Genbank Release 80.0 , National Center for Biotechnology Information, National Library of Medicine, NIH, Bethesda, MD
  26. Hernandez-Caselles, T., Rubio, G., Campanero, M. R., del Pozo, M. A., Muro, M., Sanchez-Madrid, F., and Aparicio, P. (1993) Eur. J. Immunol. 23, 2799-2806 [Medline] [Order article via Infotrieve]
  27. Higuchi R. (1990) in PCR Protocols : A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninisky, J. J., and White, T. J., eds) pp. 177-183, Academic Press, San Diego
  28. Horley, K. J., Carpenito, C. Baker, B., and Takei, F. (1989) EMBO J. 8, 2889-2896 [Abstract]
  29. Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K., and Stuart, D. I. (1992) Nature 360, 232-239 [CrossRef][Medline] [Order article via Infotrieve]
  30. Juan, M., Vinas, O., Pino-Otin, M. R., Places, L., Martinez-Caceres, E., Barcelo, J. J., Miralles, A., Vilella, R., de la Fuente, M. A., Yague, J., and Gaya, A. (1994) J. Exp. Med. 179, 1747-1756 [Abstract]
  31. Kita, Y., Takashi, T., Iigo, Y., Tamatari, T., Miyasaka, M., and Horiuchi, T. (1992) Biochim. Biophys. Acta 1131, 108-110 [Medline] [Order article via Infotrieve]
  32. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950 [CrossRef]
  33. Klickstein, L. B., deFougerolles, A. R., York, M. R., and Springer, T. A. (1993) Tissue Antigens 42, 270
  34. Landis, R. C., Bennett, R. I., and Hogg, N. (1993) J. Cell Biol. 120, 1519-1527 [Abstract]
  35. Landis, R. C., McDowall, A., Holness, C. L., Littler, A. J., Simmons, D. L., and Hogg, N. (1994) J. Cell Biol. 126, 529-537 [Abstract]
  36. Larson, R. S., Hibbs, M. L., and Springer, T. A. (1990) Cell Regul. 1, 359-367 [Medline] [Order article via Infotrieve]
  37. Moebius, U., Pallai, P., Harrison, S. C., and Reinherz E. L. (1993). Proc. Natl. Acad. Sci. U. S. A. 90, 8259-8263 [Abstract/Free Full Text]
  38. Osborn, L., Vassallo, C., Browning, B. G., Tizard, R., Haskard, D. O., Benjamin, C. D., Dougas, I., and Kirchhausen, T. (1994) J. Cell Biol. 124, 601-608 [Abstract]
  39. Pardi, R., Inverardi, L., Rugarli, C., and Bender, J. R. (1992) J. Cell Biol. 116, 1211-1220 [Abstract]
  40. Renz, M. E., Chiu, H. H., Jones, S., Fox, J., Kim, K. J., Presta, L. G., and Fong, S. (1994) J. Cell Biol. 125, 1395-1406 [Abstract]
  41. Rothlein, R., Dustin, M. L., Marlin, S. D., and Springer, T. A. (1986) J. Immunol. 137, 1270-1274 [Abstract/Free Full Text]
  42. Ruegg, C., Postigo., A. A., Sikorski, E. E., Butcher, E. C., Pytela, R., and Erle, D. J. (1992) J. Cell Biol. 117, 179-189 [Abstract]
  43. Ryu, S.-E., Kwong, P. D., Truneh, A., Porter, T. G., Arthos, J., Rosenberg, M., Dai, X., Xuong, N.-h., Axel, R., Sweet, R. W., and Hendrickson, W. A. (1990) Nature 348, 419-426 [CrossRef][Medline] [Order article via Infotrieve]
  44. Simmons, D. L. (1993) in Cellular Interactions in Development : a Practical Approach (Hartley, D. A., ed) pp. 93-128, IRL Press Oxford
  45. Simmons, D. L., Makgoba, M. W., and Seed, B. (1988) Nature 331, 624-627 [CrossRef][Medline] [Order article via Infotrieve]
  46. Somoza, C., Driscoll, P. C., Cyster, J. G., and Williams, A. F. (1993) J. Exp. Med. 178, 549-558 [Abstract]
  47. Springer, T. A. (1990) Nature 346, 425-434 [CrossRef][Medline] [Order article via Infotrieve]
  48. Staunton, D. E., Marlin, S. D., Stratowa, C., Dustin, M. L., and Springer, T. A. (1988) Cell 52, 925-933 [Medline] [Order article via Infotrieve]
  49. Staunton, D. E., Dustin, M. L., and Springer, T. A. (1989) Nature 339, 361-364
  50. Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) Cell 61, 243-254 [Medline] [Order article via Infotrieve]
  51. Van Kooyk, Y., Wiel van Kememade, P., Weder, P., Kuijpers, T. W., and Figdor, C. G. (1991) Nature 342, 811-813
  52. Vazeux, R., Hoffman, P. A., Tomita, J. K., Dickinson, E. S., Jasman, R. L., St. John, T., and Gallatin, W. M. (1992) Nature 360, 485-488 [CrossRef][Medline] [Order article via Infotrieve]
  53. Vonderheide, R. H., Tedder, T. F., Springer, T. A., and Staunton, D. E. (1994) J. Cell Biol. 125, 215-222 [Abstract]
  54. Wang, J., Yan, Y., Garrett, T. P. J., Liu, J., Rodgers, D. W., Garlick, R. L., Tarr, G. E., Husain, Y., Reinherz, E. L., and Harrison, S. C. (1990) Nature 348, 411-418 [CrossRef][Medline] [Order article via Infotrieve]
  55. Withka, J. M., Wyss, D. F., Wagner, G., Arulanandam, A. R. N., Reinherz, E. L., and Recny, M. A. (1993) Structure 1, 69-81 [Medline] [Order article via Infotrieve]
  56. Xu, H., Tong, I. L., de Fougerolles, A. R., and Springer, T. A. (1992) J. Immunol. 149, 2650-2655 [Abstract/Free Full Text]

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