(Received for publication, March 18, 1997, and in revised form, May 12, 1997)
From the Wellcome Trust Centre for Cell-Matrix
Research, School of Biological Sciences, University of Manchester,
2.205 Stopford Building, Oxford Road, Manchester, M13 9PT, United
Kingdom, ¶ Zeneca Pharmaceuticals, Alderley Park, Macclesfield,
Cheshire, SK10 4TF, United Kingdom, the
British Biotech Ltd.,
Watlington Road, Cowley, Oxford OX4 5LY, United Kingdom, and the
** Laboratory of Molecular Biophysics, Oxford Centre for Molecular
Sciences, Department of Biochemistry, University of Oxford, South Parks
Road, Oxford OX1 3QU, United Kingdom
Integrins are a family of heterodimeric adhesion
receptors that mediate cellular interactions with a range of matrix
components and cell surface proteins. Vascular cell adhesion molecule-1
(VCAM-1) is an endothelial cell ligand for two leukocyte integrins
(4
1 and
4
7). A related CAM, mucosal addressin cell adhesion
molecule-1 (MAdCAM-1) is recognized by
4
7 but is a poor ligand
for
4
1. Previous studies have revealed that all
4
integrin-ligand interactions are dependent on a key acidic ligand motif
centered on the CAM domain 1 C-D loop region. By generating
VCAM-1/MAdCAM-1 chimeras and testing recombinant proteins in cell
adhesion assays we have found that
4
1 binds to the MAdCAM-1
adhesion motif when present in VCAM-1, but not when the VCAM-1 motif
was present in MAdCAM-1, suggesting that this region does not contain
all of the information necessary to determine integrin binding
specificity. To characterize integrin-CAM specificity further we
measured
4
1 and
4
7 binding to a comprehensive set of mutant
VCAM-1 constructs containing amino acid substitutions within the
predicted integrin adhesion face. These data revealed the presence of
key "regulatory residues" adjacent to integrin contact sites and an
important difference in the "footprint" of
4
1 and
4
7
that was associated with an accessory binding site located in VCAM-1 Ig
domain 2. The analogous region in MAdCAM-1 is markedly different in
size and sequence and when mutated abolishes integrin binding
activity.
Under normal conditions, leukocytes exhibit a weakly adhesive phenotype, however, at sites of inflammation or at specialized lymphoid tissues, leukocyte adhesion receptor activity is modulated and cells become capable of interacting with ligands expressed on the lumenal surface of the vasculature. Leukocyte integrin receptors and endothelial immunoglobulin superfamily cell adhesion molecules (IgCAMs) make key contributions to the events that facilitate leukocyte emigration into the tissues (1, 2).
Integrins are /
heterodimeric cell surface adhesion receptors
that recognize a wide variety of extracellular ligands (3). For most
integrins, the mechanism of ligand recognition appears to be critically
dependent upon one of two short acidic peptide motifs: RGD and LDV (4,
5). Several extracellular matrix molecules express the RGD motif in an
invariant form and are ligands for a number of integrins
(e.g.
5
1,
IIb
3,
v
1,
v
3; Refs. 6-10). In contrast, the LDV motif exhibits sequence variation. For
example, the leukocyte integrins
4
1 and
4
7 recognize an LDVP motif in fibronectin (11-15), an IDSP sequence in the N-terminal domain of IgCAM vascular cell adhesion molecule-1
(VCAM-1)1 (16-22), and an LDTS sequence in
a second IgCAM, mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
(23-26).
Integrin-ligand interactions can be perturbed by short peptides based
on RGD or LDV. Interestingly LDV-containing peptides (and small peptide
mimetics) competitively inhibit 4
1 interactions with both
fibronectin and VCAM-1 indicating that the integrin ligand-binding
sites for both of these ligands are either overlapping or identical
(16, 27, 28).2
The x-ray crystal structure of the two N-terminal Ig domains of human
VCAM-1 has recently been solved. Both domains adopt a -
sandwich
topology, composed of an anti-parallel array of
-strands (28, 29).
As predicted by homology modeling studies the IDSP motif is located on
a projected loop between
-strands C and D, atypical of previously
characterized Ig domains (16, 30, 31). Other homology modeling and
sequence analysis studies have suggested that three
2 integrin IgCAM
ligands (intercellular adhesion molecules 1, 2 and -3, (ICAM-1, -2, -3)) also have an Ig structure analogous to that of VCAM-1 (32-36).
Since ICAM-1, -2, and -3 are also dependent on an LDV-like motif for
adhesive activity (IETP, LETS, and LETS, respectively) this suggests
that integrin-binding IgCAMs have a common active site (33, 37-39). Extensive mutational analysis of IgCAMs has provided invaluable information regarding integrin-CAM interactions, and certain residues that lie outside the LDV motif of VCAM-1 (16, 18, 19, 22), ICAM-1 (33),
and ICAM-3 (38, 39) have been shown to play an important role in
integrin recognition.
Thus, integrin-IgCAM interactions appear to share several common features: a characteristic Ig-fold and a dominant acidic peptide motif flanked by non-contiguous accessory residues. Key questions that remain are: what are the principal features of IgCAM structures that govern receptor selection and specifically does the dominant LDV-like motif encode integrin receptor specificity or are additional residues required to discriminate between integrin receptors?
Using the VCAM-1 crystal structure (29) as a basis for a mutagenesis
study we have generated IgCAM chimeras and investigated the link
between IgCAM LDV motif degeneracy and integrin binding. Using this
data we have further characterized the interaction of 4
1 and
4
7 with VCAM-1 and have identified additional key binding/regulatory residues both within and outside the C-D loop of Ig
domain 1. Our results provide evidence of distinct but overlapping
4
1/
4
7 adhesion footprints that extend over both of the most membrane distal domains of VCAM-1 and provide an insight into the mode
of integrin-IgCAM specificity.
A375-SM cells, a human metastatic melanoma cell line (provided by I. J. Fidler, M. D. Anderson Hospital and University of Texas, Houston, TX) were cultured as described (40) in Eagle's minimal essential medium containing 10% (v/v) fetal calf serum, minimal essential medium vitamins, non-essential amino acids, 1 mM sodium pyruvate, and 2 mM glutamine (all from Life Technologies, Inc., Paisley, United Kingdom). COS-1 cells were cultured in Dulbecco's minimal essential medium, 0.11g/liter of sodium pyruvate, 10% fetal calf serum, 2 mM glutamine as described (27). JY cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine. Anti-VCAM-1 monoclonal antibodies (mAbs) 4B2 (anti-domain 1), 1E10 (anti-domain 1), and 19C3 (anti-domain 2) were gifts of J. Clements (British Biotech, Cowley, Oxford, UK).
CAM-Fc Cloning and ExpressionTruncated CAM-Fc chimeras
consisting of Ig domains 1 and 2 of human VCAM-1 or murine MAdCAM-1
fused to the hinge and Fc region of human IgG1 were constructed as
follows. pCDM8-VCAM (a gift of J. Clements) and pCDM8-MAdCAM (a gift of
Dr. Peter Altevogt, German Cancer Research Center, Heidelberg, Germany)
were used as templates for the polymerase chain reaction amplification
of DNA encoding 5 and 3
restriction sites, the 5
-untranslated region, Ig domains 1 and 2 of VCAM-1 or MAdCAM-1 and a 3
splice donor
consensus sequence (35). Amplified DNA was cloned into the phagemid
vector pUC118 (VCAM) or pUC119 (MAdCAM) and sequenced. CAM cDNA
constructs were then subcloned using EcoRI (VCAM) or EcoRI/BamHI (MAdCAM) into the pIg mammalian
expression vector (a gift of D. Simmons, Institute of Molecular
Medicine, Oxford, UK) encoding a consensus splice acceptor sequence and
the hinge and Fc portion of human IgG1 downstream of the multiple
cloning site.
Mutations were introduced into CAM cDNAs by the method of Kunkel et al. (41) using the Mutagene phagemid mutagenesis kit (Bio-Rad, Hemel Hemstead, Herts, UK). Mutants were confirmed by sequencing. A list of mutagenic primers (size range 20-74-mer) is available on request.
COS-1 cells in exponential phase were trypsinized and washed twice with divalent cation-free phosphate-buffered saline and resuspended in cation-free phosphate-buffered saline to a concentration of 6-8 × 106 cells/750-µl aliquot. Triplicate aliquots were mixed with 10-20 µg of pIg-CAM DNA, prepared by alkaline lysis and anion-exchange resin (Qiagen, Dorking, Surrey, UK), and chilled on ice for 15-20 min in 0.4-cm gap electroporation cuvettes. Cells were electroporated in a Gene Pulser II (Bio-Rad) electroporator (parameters: 950 microfarads, 0.25 kV). Triplicate transfectants were pooled and resuspended in culture medium and seeded into 3 × 225-cm2 flasks each containing 35 ml of culture medium. Cells were cultured for 16 h in a humidified atmosphere of 5% (v/v) CO2 at 37 °C. Culture supernatants were then replaced with Dulbecco's minimal essential medium, 2 mM glutamine, 1% fetal calf serum (precleared of immunoglobulins with protein G-Sepharose, Pharmacia, St. Albans, Herts, UK) and cells cultured for 7-10 days. Expressed proteins were isolated from conditioned media by mixing with protein A-Sepharose (Pharmacia) for 16 h and eluting bound proteins with 0.1 M citric acid, pH 3.5, into a neutralizing volume of 1 M Tris, pH 9.0. CAM-Fc proteins were dialyzed against 25 mM HEPES, 150 mM NaCl, pH 7.4 (HEPES-buffered saline, HBS).
Enzyme-linked Immunosorbent Assay of CAM-Fc ProteinsAssays were carried out in 96-well plates (Costar, Northumbria Biologicals, Cramlington, UK). Wells were coated for 60 min at room temperature with 50 µl of CAM-Fc (10 µg/ml) and nonspecific sites blocked with 5% (w/v) bovine serum albumin, 150 mM NaCl, 10 mM Tris, pH 7.4, for 60 min. Plates were washed and 50-µl aliquots of primary mAbs (10 µg/ml) in HBS were added to triplicate wells and incubated for 60 min at room temperature. Bound antibody was detected with rabbit anti-mouse IgG (Fc-specific) alkaline phosphatase-conjugated secondary antibody (1:1000 dilution in HBS; Sigma, Poole, Dorset, UK) and chromogenic substrate, p-nitrophenyl phosphate (Sigma).
Cell Spreading AssayCell spreading assays were performed as described previously (42, 43). Assays were carried out in 96-well plates. Wells were coated for 60 min at room temperature with 50 µl of CAM-Fc diluted with HBS. Wells were blocked for 60 min at room temperature with 100 µl of 10 mg/ml heat-denatured bovine serum albumin (42). A375-SM cells were detached with 0.05% (w/v) trypsin, 0.02% (w/v) EDTA, resuspended to 1 × 105/ml in Dulbecco's minimal essential medium, 25 mM HEPES and allowed to recover for 10 min at 37 °C. 100-µl aliquots of cells were added to each well and plates incubated in a humidified atmosphere of 7% (v/v) CO2 for 60 min at 37 °C. Cells were fixed by the addition of 20 µl of 50% (v/v) glutaraldehyde and monitored for the degree of spreading using phase-contrast microscopy as described (43). Each data point was obtained by counting at least 300 cells/well from a number of randomly selected fields. No cell spreading was observed on wells coated only with heat-denatured bovine serum albumin.
Cell Attachment Assay96-well plates were coated for 60 min at room temperature with 50 µl of CAM-Fc diluted with HBS and blocked with heat-denatured bovine serum albumin (see above). JY cells were centrifuged at 1400 × g for 3 min, washed in Dulbecco's minimal essential medium, 25 mM HEPES (assay medium), resuspended at 1 × 106/ml in assay medium and allowed to recover at 37 °C for 10 min. 50-µl aliquots of cells were added to CAM-Fc-coated wells containing 50 µl of HBS, 0.2 mM MnCl2 and incubated in a humidified atmosphere of 7% (v/v) CO2 for 20 min at 37 °C. Cells attached to untreated wells were fixed directly with 20 µl of 50% (v/v) glutaraldehyde to enable quantitation of cell number. Nonspecifically attached cells were aspirated from experimental wells and the residual cells fixed with 5% (v/v) glutaraldehyde. Fixed cells were then washed three times with H2O and stained for 60 min with 0.1% (w/v) crystal violet in 0.2 M MES, pH 6. Excess stain was removed with three water washes before cells were lysed with 10% (v/v) acetic acid and the released stain measured spectrophotometrically at A560 nm.
The predicted C-D loop sequences of VCAM-1, MAdCAM-1, and ICAMs
1-3 have all been implicated as sites for integrin binding and,
without exception, possess an acidic residue essential for receptor
interaction. Alignment of these sequences reveals a consensus integrin-binding motif (Fig. 1). Despite only subtle
variations within this consensus and the use of a relatively conserved
Ig domain structure, integrins are capable of discriminating between IgCAM ligands. Although the two related integrins, 4
1 and
4
7, can both bind VCAM-1 and MAdCAM-1, there is a ligand
preference between the two integrins;
4
1 is primarily a receptor
for VCAM-1 whereas
4
7 is primarily a receptor for MAdCAM-1. The
C-D loops of VCAM-1 and MAdCAM-1 differ in two major respects: (i) the
VCAM-1 loop is composed of 8 amino acids, the MAdCAM-1 loop only 6, and (ii) two positions flanking the active site motif differ in MAdCAM-1, Gly for Gln and Ser for Pro (see Fig. 1). We therefore tested the
hypothesis that IgCAM selection by these two integrins is regulated by C-D loop sequence differences.
For this purpose truncated VCAM-1/MAdCAM-1 proteins containing the two
N-terminal Ig domains were synthesized as fusion proteins joined to the
Fc region of human IgG1. Expression of IgCAM protein in a
soluble form provides a major advantage over cell-surface expressed
molecules since it is possible to examine the dose dependence of
adhesive activity in a quantitative manner and determine relative activities. Since VCAM-1 and MAdCAM-1 Ig domain 1 C-D loops are predicted to differ in size, two VCAM-1/MAdCAM-1 chimeras and two
MAdCAM-1/VCAM-1 chimeras were constructed, each chimera differing with
respect to their parent molecule and C-D loop size (see Fig. 2). CAM-Fc fusion proteins were purified and tested for
their ability to support 4
1-dependent or
4
7-dependent cell adhesion and structural integrity
monitored using a panel of mAbs (Fig. 3, Table
I). VCAM-1-Fc supported
4
1-dependent
cell spreading, but MAdCAM-1 was a poor ligand confirming previous
reports (23, 44). When VCAM/MAdCAM C-D loop chimeras were tested for
4
1 binding activity, one chimera (VCAM-Fc chimera B) demonstrated a super-adhesive phenotype compared with the native VCAM-Fc construct (Fig. 3). In contrast, the VCAM-Fc chimera A failed to support cell
adhesion as did both MAdCAM-Fc chimeras. Similarly
4
7-dependent assays revealed that both MAdCAM-Fc
chimeras demonstrated very low adhesive activities. However, again
VCAM-Fc chimera B supported increased
4
7 binding compared with
native VCAM-Fc (Fig. 3). Although local structural perturbations may be
responsible for the reduced binding activities of some of the chimeras,
as a whole these data suggest that the VCAM-1 C-D loop is not entirely
responsible for preferential recognition of VCAM-1 by
4
1 compared
with MAdCAM-1. However, interestingly, they do suggest that the
MAdCAM-1 C-D adhesion loop represents a more ideal
4 integrin
binding structure.
|
We next attempted to identify those residues of the VCAM-1 C-D loop
that were responsible for 4
1/
4
7 binding activity and thus
provide a molecular explanation for the super-adhesive activity of the
MAdCAM-1 adhesion loop. The x-ray crystal structure of VCAM-1 domain 1 C-D loop (29) was analyzed and those residues that were both conserved
between species (human, mouse, and rat) and were solvent-accessible
were targeted for mutagenesis. Residues Arg, at position 36 of the
mature protein (Arg36), Gln38,
Ile39, Asp40, Pro42, and
Leu43 met these criteria and VCAM-Fc proteins, mutated at
each of these positions, were expressed, purified, and tested for their
ability to promote
4
1/
4
7-dependent cell
adhesion and for their structural integrity (Figs. 4, 5, 6,
Table I). The D40A mutation (alanine substituted for aspartate at
position 40) has been previously shown to abolish VCAM-1-
4
1
binding and was used as a control (16, 18, 19, 21, 22). In contrast to
published data (19), substitution of glutamate for aspartate (D40E) at
this position markedly reduced
4
1-dependent cell
spreading, but did not abolish it (Fig. 4). Mutation R36A was found to
have no effect on
4
1-dependent cell adhesion,
however, R36E markedly reduced activity. Mutations at position 38 that
introduced either a positive or negative charge (Glu or Arg) also
reduced cell adhesion. Mutations at positions 39 and 43 where aliphatic
side chains were replaced with charged residues (Arg or Lys) also had
negative effects on VCAM-1 activity and, in the case of mutant L43K,
abolished
4
1 binding (Fig. 4).
We further investigated the role of amino acids Glu38 and
Ile39 in 4
1-VCAM-1 interactions by making further
amino acid alterations at these positions. Gln38 mutated to
either a glycine or a leucine demonstrated a super-adhesive phenotype
with significant changes in the concentration of protein required for
half-maximal cell spreading (2-4-fold decrease, Fig. 5). Thus, removing a hydrophilic side chain at this
position, or replacing it with an aliphatic group, conferred not a
negative effect but instead augmented
4
1-VCAM-1 binding. These
data are consistent with the effects observed with one of the
VCAM-1/MAdCAM-1 chimeras (VCAM-Fc chimera B), where the equivalent
amino acid to VCAM-1 Gln38 in the MAdCAM-1 C-D loop is
glycine. The effect of increasing hydrophobicity at position 38 was
further tested by the introduction of a large aromatic residue,
phenylalanine. Intriguingly,
4
1-dependent cell
adhesion was still supported with only a 2-fold increase in the
concentration for half-maximal spreading (Fig. 5). Two further
mutations at position 39 were performed to test the effects of subtle
changes in the aliphatic side chain characteristics, I39A and I39V.
Remarkably, the smaller alanine side chain severely affected
4
1
binding ability with a marked reduction in maximal spreading; however,
a valine at this position only slightly affected activity (Fig. 5).
4
7 adhesion data largely mirrored that of
4
1, however, no
super-adhesive phenotype was obtained with mutations at position Gln38. In addition, D40E abolished
4
7-dependent attachment and mutants Q38F and I39V
markedly reduced
4
7 binding (Fig. 6). MAdCAM-1 C-D
loop residues were also tested for their contribution to
4
1 and
4
7 binding. Residues Gly39, Asp41, and
Leu44 were mutated and expressed as MAdCAM-Fc fusion
proteins.
4
1-dependent cell adhesion was abolished
and
4
7-dependent cell attachment severely affected by
all four mutations, reinforcing the role of this region of the molecule
in integrin binding (Fig. 7 and data not shown). Of
note, however, is the finding that the MAdCAM-D41E-Fc mutant had a very
low
4
7 binding activity, in marked contrast to the equivalent
VCAM-1 mutation (D40E), which could support a significant level of
4
1-mediated cell spreading.
The results described above clearly suggest that residues outside the
VCAM-1 C-D loop play a role in integrin selection. We therefore
attempted to map the adhesion "footprints" of 4
1 and
4
7
in an effort to identify a difference in binding requirements and an
explanation for the preference for VCAM-1 by
4
1. Since the VCAM-1
C-D loop appears to be central to
4 integrin binding we reasoned it
was likely that residues that comprise this "face" of the Ig domain
1 might be capable of participating directly in integrin binding. In
addition, since VCAM-1 Ig domain 2 has been predicted to play a role in
4
1 binding from studies with ICAM-1 chimeras, this region was
also targeted for mutagenesis (17). Those residues that constitute the
predicted integrin-binding face that are both conserved between species
and solvent-accessible (as revealed by the x-ray crystal structure,
Ref. 29) are highlighted in Fig. 8. These residues were
mutated to either alanine or another amino acid (often to swap or
introduce charge) and expressed as VCAM-Fc fusion proteins. All mutants
were examined for structural perturbations using anti-VCAM-1 mAbs in
enzyme-linked immunosorbent assay (Table I). Only mutation V47K
affected binding of both domain 1 mAbs. VCAM-Fc mutants were then
tested for their ability to support
4
1-dependent
spreading or
4
7-dependent attachment (Figs.
9 and 10, respectively).
For clarity and visual presentation the effects of mutations were
classified according to the maximal level of adhesion attained and the
concentration required to reach half-maximum adhesion. "Severe"
effects produced <50% of the maximal level of spreading on native
VCAM-Fc and the protein concentration required for half-maximal spreading was either not reached or was >7-fold higher than native VCAM-Fc. "Marked" effects were classified as those that reached 60-70% maximum native adhesion with a 3-4-fold increase in
concentration required for half-maximal concentration. "Slight"
effects produced 80-100% of maximum spreading with only a 2-fold
increase in concentration required for half-maximal adhesion. In
addition, if adhesion was augmented (maximum adhesion unchanged but
protein concentration required for half-maximum adhesion decreased
two-fold or more) then a mutant VCAM-Fc was described as
"super-adhesive" (see Fig. 11).
Only two residues outside the C-D loop, when mutated, were found to
affect 4
1-VCAM-Fc binding severely, Leu70 and
Glu87. Several residues, however (Thr72,
Glu81, Asp143, Ser148, and
Glu150), markedly affected
4
1 interactions. Mutations
of amino acids at six further positions resulted in a slight
perturbation of
4
1-dependent adhesion
(Lys46, Val47, Ser68,
Ser77, Glu155, and Thr157).
Interestingly, three amino acids outside the C-D loop
(Thr74, Leu80, and Lys147), when
mutated to alanine, conferred a super-adhesive phenotype on VCAM-Fc. In
contrast,
4
7 appeared to differ in its sensitivity to mutation of
residues outside the C-D loop region. Only one residue in Ig domain 1 severely affected
4
7 binding (Glu87) and mutations of
Leu70 had no effect on
4
7 binding (Fig. 10).
Furthermore, Ig domain 1 mutations that markedly affected
4
1
binding (Thr72 and Glu81) had no effect on
4
7-mediated cell attachment. One of the main differences between
4
1- and
4
7-VCAM-1 interactions appeared to lie in Ig domain
2; mutation of three residues at positions 143, 148, and 150 severely
affected
4
7-dependent cell adhesion. In addition,
residues that markedly affected
4
7 binding were also located in
domain 2 (Lys152 and Glu155). A further
difference was the super-adhesive activity of T151A for
4
7. The
key integrin-binding residues identified in VCAM-1 domain 2 correspond
to the C
-E loop region, a region in MAdCAM-1 domain 2 highlighted to
be of potential functional importance (29). We therefore directly
tested the role of this region of MAdCAM-1 in
4 integrin binding by
engineering a deletion mutant in which the central acidic region
(143-150) of the highly extended Ig domain 2 C
-E loop had been
deleted. The resultant mutant (MAdCAM-
C
-E-Fc) was then assayed for
4
1 and
4
7 binding activity (Fig. 12). In
both experiments, MAdCAM-
C
-E-Fc failed to support
4
1 or
4
7-mediated cell adhesion, confirming the importance of this region for both
4 integrin interactions.
Using IgCAM chimeras and a structure-guided site-directed
mutagenesis strategy, we have analyzed the molecular basis of 4 integrin binding to the IgSF members VCAM-1 and MAdCAM-1. All VCAM-1
and MAdCAM-1 mutants were produced as recombinant IgG Fc fusion
proteins permitting quantitative analysis of purified mutant binding
properties.
The main findings reported here are that the C-D loops of these IgCAMs
are only partially responsible for integrin specificity, and that
4
1/
4
7 integrin binding footprints on VCAM-1 differ in their
relative dependence on residues within VCAM-1 Ig domains 1 and 2. In
addition, we report that several amino acids within VCAM-1 modulate
integrin interactions with residues at predicted integrin contact
sites. These findings for the first time demonstrate a direct role for
VCAM-1 Ig domain 2 and suggest a mechanism for integrin selection by
IgCAMs.
Integrin-IgCAM interactions are dependent on a relatively conserved
acidic peptide motif presented as a surface-exposed structure supported
on a conserved Ig-domain scaffold. Despite only slight variations in
this binding motif, integrin-CAM specificities are exquisite. This
suggests two possibilities, either slight variations within this motif
are sufficient to discriminate integrin receptors or this motif might
represent a general binding structure and integrin selection may be
governed by distal sites. We investigated the role of IgCAM C-D loop
motifs in regulating integrin specificity by making chimeras between
VCAM-1 and MAdCAM-1 (the former is primarily a ligand for 4
1, the
latter for
4
7).
4
1-dependent cell spreading
assays revealed that MAdCAM-1 chimera Fc fusion proteins expressing
either of two VCAM-1 C-D adhesion loop variants failed to demonstrate
increased binding to
4
1 over native MAdCAM-1, indicating that the
VCAM-1 C-D adhesion loop alone was insufficient to confer full
4
1
binding activity. Intuitively, complementary VCAM-1 (MAdCAM C-D loop)
chimeras might therefore be expected to possess lower
4
1 binding
activity compared with native VCAM-Fc. However, one chimera (VCAM Fc
chimera B) demonstrated super-adhesive
4
1 binding activity. When
both sets of chimeras were tested for
4
7 binding activity,
essentially identical results were obtained; MAdCAM-1 chimeras with the
VCAM-1 C-D adhesion loop supported reduced levels of cell attachment
whereas the super-adhesive VCAM-Fc chimera B again demonstrated a
slight increase in the ability to bind integrin relative to native
VCAM-Fc. These data reinforce the importance of CAM C-D loops in
integrin binding and suggest that
4 integrins have a distinct
preference for the C-D loop structure presented by MAdCAM-1 Ig domain
1. Furthermore, since
4
1 has a greater affinity for VCAM-1
compared with MAdCAM-1 this would suggest that
4
1-VCAM-1 binding
is enhanced (or MAdCAM-1 binding is attenuated) by sites outside the
C-D loop.
Despite several studies on 4
1-VCAM-1 interactions,
4
1
contact sites within the VCAM-1 C-D loop are not fully understood. In
an effort to enhance our understanding of integrin-IgCAM interactions we made a detailed analysis of the VCAM-1 and MAdCAM-1 C-D loops. To
aid our study we analyzed the x-ray crystal structure of VCAM-1 (29)
and identified those C-D loop residues that were both conserved between
species (mouse, rat, and human) and surface-exposed, reasoning that
functionally important residues would be conserved and accessible to
integrin.
4 integrin-dependent adhesion data obtained with
purified mutant C-D loop VCAM-Fc fusion proteins has extended our
understanding of
4 integrin binding requirements. Mutation of
Arg36 to alanine had no effect, while replacement with a
glutamate markedly affected
4
1 interactions. This indicates that
the physical characteristics of the arginine side chain are not
absolutely required for integrin-VCAM-1 engagement, however, a negative
charge in this position (glutamate) appears to adversely affect the
recognition of adjacent residues. Analysis of the x-ray crystal
structure of VCAM-1 in this region reveals that Arg36 is in
close proximity to the acidic side chain of Asp40 (see Fig.
8) and therefore might feasibly influence the pKa of
the crucial Asp40 carboxyl group and consequently integrin
active site engagement. Alternatively, alterations at position 36 might
affect the conformation of neighboring side chains within the C-D loop.
Mutagenesis data on amino acid Gln38 suggest an important
role in
4
1-VCAM-1 binding, since changing the glutamine to a
charged residue (glutamate or lysine) severely affected
4
1
binding. However, removal of the glutamine side chain (Q38G) was found
to convert VCAM-1 into a super-adhesive
4
1 ligand and replacement
of glutamine with an aliphatic residue (leucine) resulted in a further
enhancement of integrin binding activity. When a phenylalanine was
substituted into this position a slight decrease in
4
1 binding
capacity was observed. At least two explanations can be drawn from
these data: first, part of the integrin active site might comprise a
restricted pocket with hydrophobic characteristics and thus have a
preference for interacting with hydrophobic residues. Alternatively,
residues in position 38 might not be directly recognized by the
4
1 active site but might confer subtle structural alterations on
the remainder of the C-D loop, thus modifying integrin binding. It is
notable that the MAdCAM-1 C-D loop possesses a glycine in a homologous
position to Gln38, providing an explanation for the
super-adhesive phenotype of VCAM-Fc chimera B.
Residues at VCAM-1 positions 39, 40, and 43 are critical for 4
1
binding. Several mutations of Ile39 were tested: replacing
isoleucine with a charged residue or an alanine residue severely
affected
4
1 binding ability, however, replacement with valine
only resulted in a slight decrease in activity. These data are again
consistent with a hydrophobic pocket at or near the
integrin-ligand-binding site. Charged residues or side chains too small
to fill this pocket result in compromised integrin interactions, while
longer, branched side chains such as valine appear to be sufficient to
permit essentially unperturbed
4
1 binding. Previous reports in
which Asp40 was replaced with alanine, asparagine, or
lysine resulted in abolition of VCAM-1 adhesive activity (16, 18, 19),
however, mutation of Asp40 to glutamate was reported to
have no detectable affect on
4
1 binding (19). Here, we observed a
marked effect on
4
1-VCAM-1 binding when Asp40 was
mutated to glutamate. In addition VCAM-1 mutants R36A and P42G have
been reported previously to perturb
4
1 binding (19, 22), an
explanation for these discrepancies is unclear. Previous mutagenesis
studies coupled with sequence alignment data have identified the
4
binding motif within VCAM-1 as IDSP; however, we report here that
Leu43 is crucial for integrin-VCAM-1 binding and therefore
propose that the adhesion motif should be extended to include this
residue: i.e. IDSPL.
Analysis of the effects of residues in the C-D loop region on
4
7-VCAM-1 binding revealed essentially the same results as
4
1 binding, however, there were several key differences: (i) the
Q38G mutation slightly reduced
4
7 interactions, while Q38L only
slightly enhanced
4
7 binding, (ii) mutant I39V had markedly reduced
4
7 binding activity, and (iii) D40E abolished
4
7-VCAM-1 interactions. These data suggest subtle differences
between the ligand-binding sites of
4
1 and
4
7 and imply
that
4
1 may possess a slightly deeper pocket to accommodate the
crucial acidic group at position 40.
Mutation of analogous C-D loop residues within MAdCAM-1 confirmed that
both IgCAM C-D loops have essentially identical integrin binding
characteristics. Again point mutations provided an explanation for
results obtained with VCAM-1/MAdCAM-1 chimeras: amino acid Gly39 (analogous to VCAM-1 Gln38) when mutated
to glutamine almost abolished 4
7 binding. Interestingly the
native VCAM-(Gln38)-Fc and MAdCAM-1-Fc chimera A possessed
4
7 binding activity, suggesting that the increased loop size of
this construct comprised a more ideal adhesion structure than
MAdCAM-G39Q-Fc. The importance of this region of MAdCAM-1 has recently
been confirmed by mutagenesis and peptide inhibition studies: MAdCAM-1
residues Leu40, Asp41, Thr42, and
Leu44 were found to be required for full
4
7 binding
activity (45).
These data demonstrate for the first time the effects of performing
full IgCAM C-D loop swaps. However, partial swaps have been
investigated previously; the central portion of the ICAM-1 Ig domain 1 C-D loop has been used to replace the analogous region of VCAM-1 and
was found to have no negative effect on 4
1 binding (18). Our data
with the same mutant in our VCAM-Fc construct (Q38IDS/GIET)
demonstrated identical integrin binding activity (data not shown).
Analysis of the effects of point mutations described above now provides
an explanation for these observations: the overall phenotype of the
Q38IDS/GIET mutation is a composite effect of Q38G (super-adhesive) and
D40E (marked negative effect), thus the overall effect produces a
native phenotype. We further investigated the ability of the ICAM-1
(and ICAM-3) C-D loop sequences to substitute for that of VCAM-1 by
performing whole loop swaps (8 VCAM-1 C-D loop amino acids replaced
with 8 ICAM-1 (or ICAM-3) C-D loop amino acids, see Fig. 1). The
resulting constructs failed to bind two anti-VCAM-1 mAbs (4B2 and 1E10,
see Table I) and failed to bind
4
1 (data not shown). These data
suggest that the ICAM-1 and ICAM-3 C-D loops, although similar to that
of VCAM-1 in terms of sequence and function, may adopt markedly
different conformations.
Taken together, these data provide an insight into the structural
requirements of 4 integrins for the C-D loops of VCAM-1 and MAdCAM-1
and also identify mutant forms of VCAM-1 capable of conferring a
super-adhesive phenotype. In an effort to identify those sites outside
the C-D loop that differentially affect
4
1/
4
7 binding, we
performed an extensive survey of the contribution of VCAM-1 Ig domain 1 and 2 amino acids. We targeted residues that were conserved between
species, surface-exposed, and confined to the C-D loop face of the
molecule according to the VCAM-1 x-ray crystal structure (29, Fig. 8).
When purified mutant VCAM-Fc proteins were tested for their ability to
bind
4
1 and
4
7 in cell-based assays, striking differences
between
4
1 and
4
7 binding were observed. Although both
integrins had an acute requirement for residues within the C-D loop,
their respective adhesion footprints over VCAM-1 Ig domains 1 and 2 differed in two major respects. First, key Ig domain 1 residues
required for
4
1-VCAM-1 interactions outside the C-D loop included
Leu70, Thr72, Glu81, and
Glu87 but only Glu87 appeared to be involved in
4
7-VCAM-1 binding. Second, mutation of amino acids at three
positions in Ig domain 2 (Asp143, Ser148, and
Glu150) had a marked, but not severe effect, on
4
1
binding whereas these residues have a much more pronounced affect on
4
7 binding (Fig. 11). Furthermore, mutation of two residues,
Lys152 and Glu155, had a marked effect on
4
7 binding reinforcing the importance of this region for
4
7-VCAM-1 binding. In addition, several residues were found to
augment integrin binding with effects analogous to the Q38G/Q38L
mutants. In all instances augmentation sites (Thr74,
Leu80, Lys147 (both
4
1 and
4
7), and
Thr151 (
4
7 only)) lie spatially close to residues
that affect integrin interactions (Fig. 11). The identification of
Thr151 as an augmentation site in the case of
4
7-VCAM-1 binding further implies that residues in this region of
VCAM-1 Ig domain 2 have a greater role for
4
7 interactions
compared with
4
1. Although the mechanism of integrin-binding
augmentation is unclear, it is likely that changes at these positions
confer slight structural changes which lead to enhanced integrin
recognition of nearby contact sites. The main evidence to support this
hypothesis comes from the finding that replacement of the native
residue with amino acids of very different physical characteristics are
capable of producing the same result, e.g. L80A, L80K (Fig.
9).
Overall these data suggest an important shift in the "binding
footprint" between 4
1 and
4
7.
4
1 primarily binds to
VCAM-1 Ig domain 1 via a group of residues comprised of the C-D
adhesion loop and three further adjacent Ig domain 1 sites with a
contribution from several Ig domain 2 residues, whereas
4
7, apart
from residues within the Ig domain 1 C-D loop has a reduced requirement
for sites within Ig domain 1 but a pronounced requirement for residues that locate to the C
-E loop/E
-strand of Ig domain 2. Conflicting data regarding the role of Leu70 for
4
7 binding have
been reported: VCAM-L70N was found to abolish both
4
1 and
4
7 interactions (22). Since both Leu70 mutants tested
here, L70A and L70K perturbed
4
1 but not
4
7 binding, an
explanation for this discrepancy is not clear, however, enzyme-linked
immunosorbent assay data suggest that the L70N mutant may perturb
structure since two anti-functional mAbs bind with reduced affinity
relative to a native VCAM-1 construct (56 and 42%; Ref. 22).
One of the most important implications of our findings is that the C-E
loop of Ig domain 2 contributes to the regulation of integrin-IgCAM
specificity. This hypothesis is strikingly reinforced by sequence
alignment studies between VCAM-1 and MAdCAM-1 (29). The C
-E loop of
MAdCAM-1 Ig domain 2 is highly acidic and contains a sequence of three
consecutive glutamate residues (murine MAdCAM-1, Ref. 24) or five
consecutive glutamate residues (human MAdCAM-1, Ref. 51). Since
4
7 binding is highly sensitive to the removal, by site-directed
mutagenesis, of acidic residues from the C
-E loop of VCAM-1 Ig domain
2, this suggests a key role for this region in governing
MAdCAM-1-
4
7 interactions. We therefore examined the effect of
truncating the MAdCAM-1 Ig domain 2 C
-E loop by removing residues
143-150 (including the acidic sequence of amino acids predicted to be
inserted residues in comparison to VCAM-1 Ig domain 2 C
-E loop, Ref.
29). In both
4
1- and
4
7-dependent assays, cell
binding activity was abolished, confirming the role of this
region in integrin interactions (Fig. 12).
Comparison of integrin-IgCAM interactions with other ligands reveals
striking parallels. Several integrins interact with the extracellular
matrix protein fibronectin including 5
1,
V
1,
IIb
3,
and
V
3 and all four integrins require the tripeptide motif RGD
displayed by the 10th type III repeat of fibronectin (FnIII(10))
(6-10). Site-directed mutagenesis, domain deletion, and peptide-based
approaches have subsequently identified a second binding site (sequence
PHSRN, termed the synergy site) located in the 9th type III repeat
(46-52). The x-ray crystal structure of this region of fibronectin
(FnIII(7-10)) has recently been solved and reveals a tandem array of
fibronectin type III repeats; each composed of an anti-parallel array
of
-strands analogous to Ig domains (53). The RGD motif lies in the
F-G loop of FnIII(10) and the synergy sequence is found in the C-E loop
and part of E
-strand. Comparison of the arrangement of active sites
in fibronectin with that of VCAM-1 highlights three similarities: (i)
both sets of adhesion motifs are on surface-exposed loops, (ii) both
molecules display active sites on the same face of adjacent domains,
and (iii) the functional groups of the key amino acids in each motif are approximately 30-40 Å apart. Thus, two different integrin ligands
appear to share a broadly similar "binding topology" composed of a
dominant adhesion motif (IDSPL, VCAM-1, and RGD, fibronectin) coupled
to second "synergy site" that may regulate integrin specificity (Ig
domain 2 C
-E loop-E strand, VCAM-1 and III(9) C-E loop and part of E
-strand, fibronectin).
Historically, peptide-based approaches have dominated the identification of integrin ligand active sites, and this has led to the perception that integrins principally recognize discrete peptide motifs such as RGD and LDV. However, data, largely provided by site-directed mutagenesis studies, is now accumulating to suggest that integrin recognition sites on ligands are more complex and are comprised of a dominant acidic peptide flanked by additional contact residues. In the future it will be of interest to assess the relative contribution of functionally important residues in providing the free-energy of interaction between integrin and ligand, an approach successfully used to identify residues responsible for human growth hormone-human growth hormone receptor interactions (54). In turn these findings, together with the use of reporter mAbs (such as those that recognize integrin neo-epitopes arising as a result of ligand engagement), might provide clues as to the connection between ligand engagement by integrin and the conformational responses that occur prior to signal transduction across the plasma membrane.
We are grateful for the assistance of Linda Berry, Katherine Clark, Ian Hesketh, and Drs. John Clements and Tim Dudgeon for helpful advice and discussions. We also thank Drs. Paul Mould and Danny Tuckwell for advice and critical reading of the manuscript.