(Received for publication, August 29, 1994; and in revised form, November 8, 1994)
From the
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.
Cell adhesion molecules (CAMs) ()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;
,), and ICAM-1 has been shown to bind
an additional integrin Mac-1 (CD11b/CD18;
,) (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
1 and
2 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
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.
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).
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).
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).
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).
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 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.)
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-2 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).
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.
Figure 7:
Model
for domains 1 and 2 of ICAM-3. The strands are labeled in the
normal convention for immunoglobulin folds, A to G, with the two
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
. 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).
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
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 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 (
4
1) and
4
7
integrins (Elices et al., 1990; Ruegg et al., 1992),
and MadCAM-1 with the
4
7 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 (
4
1) (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.