From the
Leukocyte function-associated antigen-1 (LFA-1) is a cell
surface adhesion receptor for intercellular adhesion molecule-1, -2,
and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of
the LFA-1
Leukocyte function antigen-1 (LFA-1),
LFA-1 binds to three distinct ligands: ICAM-1,
-2, and -3
(1, 4, 5, 6) . LFA-1/ICAM-1
interactions are the best understood and data from a number of groups
indicates that the I-domain of CD11a plays a critical role in this
interaction. For example, we and others have shown that a number of
monoclonal antibodies (mAbs) that block LFA-1 binding to ICAM-1 map to
the CD11a I-domain
(7, 8) . In addition, CD11a
I-domain-IgG chimeras bind specifically to and can inhibit T-cell
interactions with ICAM-1
(8, 9) . To date, the amino
acids within the intact or isolated I-domain, which are critical to
ICAM-1 binding, have not been defined.
In previous studies, using
site-directed mutagenesis, we localized the epitopes recognized by
eight mAbs to murine and human CD11a (MHM-24, 50G1, CLB LFA 1/2, 32E
6.2, 25.3, TS 1/22, M17, I 21/7) that blocked the binding of LFA-1 to
its ligand ICAM-1. Residues critical for the binding of all eight mAbs
were localized to an 80-amino acid region of the I-domain. Within this
region three discrete epitopes were localized and specific amino acids
residues essential for the binding of all eight mAbs were
identified
(7) . These epitopes may define residues within the
I-domain that contact ICAM-1, since Lollo et al.(10) suggest that at least one of the antibodies, M17, blocks
CD11a function by competitive inhibition of ligand binding.
Other
investigators
(11) have identified a novel cation binding site
in the I-domain of the closely related integrin Mac-1 (CD11b/CD18) that
is essential for the binding of divalent cations as well as iC3b. Mac-1
also binds ICAM-1 and this interaction also appears to be mediated by
the I-domain of CD11b
(12) . It is not known whether CD11b
possesses a distinct binding site for each ligand or whether the
different ligands recognize the same or overlapping sites. In the
present study, we analyzed the ligand binding properties of two sets of
CD11a variants. One set, in which part or all of the human I-domain was
replaced with murine sequences, suggested that the human ICAM-1 binding
site of CD11a is not contained within the I-domain epitopes recognized
by mAbs that block LFA-1 function. A second set was generated in which
amino acids conserved throughout CD11a, -b, and -c I-domains were
mutagenized. With these constructs, two aspartic acids (Asp-137 and
Asp-239) were identified as important for binding of LFA-1 to ICAM-1. A
third residue, Pro-192, appears to play a role in maintaining the
I-domain conformation.
The generation of the H/M chimeras has been described
previously
(7) . The H11a.D137A (D137A), H11a.D137K (D137K),
H11a.S139A (S139A), H11a.P192A (P192A), H11a.D239A (D239A), H11a.D239K
(D239K) constructs contain the indicated point mutations in the human
CD11a I-domain and were generated by oligonucleotide-directed
mutagenesis using pRKLFA
Transfected cells were detached with 5
mM EDTA/PBS and resuspended at a concentration of 200,000
cells/100 µl of adhesion buffer (140 mM NaCl, 20
mM HEPES, pH 7.5, 0.2% glucose) containing the specified
concentration of divalent cations and antibodies. Antibodies were bound
in buffer containing 1 mM each of MgCl
We next examined
amino acids Pro-192 and Asp-239 in CD11a, which are conserved in CD11
I-domains and have been shown to be important for Mac-1 binding to
Mn
Mutating Pro-192 to
Ala also completely destroyed the ability of LFA-1 to bind to ICAM-1
(Fig. 3, A-C). No obvious changes in the gross
morphology of this mutant were observed since the antibodies to CD18
and the antibodies to CD11a that map outside the I-domain bound at
levels comparable to wild-type CD11a/CD18 (). Mutation of
residue 192 did appear to alter the conformation of the I-domain as
mAbs 32E6, 50G1, and TS 1/22 showed a 28-70% reduction in
binding. Immunoprecipitations of the P192A variant also showed dramatic
reduction in binding of all the I-domain antibodies, which was not the
case for any of the other mutants (data not shown). These data suggest
that the loss of ICAM-1 binding by this mutant is due to conformational
changes within the I-domain. Mutating the analogous Pro in CD11b
resulted in less drastic changes in Mac-1 binding to iC3b, but did
reduce the binding of isolated CD11b I-domain to Mn
Our results demonstrate the
importance of Pro-192 and the aspartic acids, Asp-137 and Asp-239, for
LFA-1 binding to ICAM-1. Pro-192 appears to play a structural role, as
evidenced by the significant changes in I-domain mAb binding to the
P192A mutant. While we cannot rule out a structural function for
Asp-137 and Asp-239, substitution of alanine at either of these
positions resulted in moderate or no changes in mAb binding. This
suggests that loss of ICAM-1 binding in the D137A and D239A mutants is
not a consequence of gross conformational changes in the I-domain.
Recently, the significance of these aspartic acids in other I-domain
containing integrins,
Taken together,
these data suggest that by isolating all or part of these I-domains
away from their full-length receptors (Mac-1,
We gratefully acknowledge Dr. Yvette van Kyook for
providing us with the mAb L16; Dr. James Hildreth for providing us with
the mAbs MHM-23, MHM-24, and H52; and Dr. Bradley McIntyre for
providing us with the mAbs 3D6, 32E6, and 50G1. We also thank the
Genentech oligonucleotide synthesis group and the Genentech flow
cytometry laboratory.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit (CD11a), we recently identified the epitopes
recognized by eight monoclonal antibodies against CD11a that inhibit
LFA-1 binding to ICAM-1. In this report, we determined that replacement
of the entire human I-domain with the entire murine I-domain in CD11a
completely abrogated LFA-1 binding to human ICAM-1 without affecting
the gross conformation or heterodimer formation of LFA-1, as assayed by
antibody binding. In order to assess which residues of the I-domain are
responsible for binding to ICAM-1, we tested the ability of a panel of
human/murine I-domain chimeras to bind to human ICAM-1. When complexed
with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to
wild-type CD11a/CD18, indicating that the residues in these chimeras
which differ in human and murine I-domains may not play a critical role
in LFA-1 binding to ICAM-1. A series of point mutations of residues
that are conserved between murine and human CD11a I-domains, as well as
between CD11b and CD11c, were also generated. Substitution of alanine
for proline at position 192 in the human CD11a I-domain abrogated
adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this
was due to conformational changes within the I-domain. Mutation of the
aspartic acids at positions 137 and 239 to either alanine or lysine
completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine
mutants was not significantly altered. This suggests that these
aspartic acids are required for binding of human LFA-1 to human ICAM-1.
(
)
like Mac-1 and p150/95, is a member of the
integrin family. It is expressed exclusively on leukocytes and
mediates a range of adhesive interactions including priming of T cells
by antigen presenting cells, migration of leukocytes across the
endothelium, and the binding of effector cells (e.g. cytotoxic
T cells) to their targets
(1, 2, 3) . Like all
integrins, LFA-1 is a noncovalent heterodimer
consisting of an unique
chain (
, CD11a) of
approximately 180 kDa and a common
chain (
,
CD18) of approximately 95 kDa. The CD11a subunit, as well as CD11b,
CD11c, and the
integrin-associated
chains,
and
, contains an approximately
200-amino acid domain located amino-terminal to the divalent cation
binding domain which is know as the ``inserted/interactive''
domain (I-domain)
(1) . This conserved motif is thought to
participate in ligand binding of all I-domain containing
integrins
(3) .
Generation of the Mutants
The expression vector
used to produce membrane-associated CD11a (pRKLFAm) closely
resembles that described previously for the expression of
membrane-associated Mac-1 (13). To generate the pRKCD11a.mu3 (mu3)
construct, the entire murine CD11a I-domain was synthesized de novo by oligonucleotide-directed mutagenesis using uracil containing
human CD11a I-domain cDNA as a template
(14) . Briefly, the human
CD11a I-domain was generated by polymerase chain reaction from a
plasmid containing the entire extracellular domain of CD11a and
subcloned into a vector containing the M13 origin of replication
(pLFAhuID). The fidelity of the polymerase chain reaction was confirmed
by sequencing using Sequenase version 2.0 (U. S. Biochemical Corp.). A
series of I-domain oligonucleotides were generated to incorporate the
murine-specific I-domain amino acids into the human CD11a I-domain. The
oligonucleotides were pooled and the murine I-domain (pLFA.IDmu3) was
generated by oligonucleotide-directed mutagenesis in two steps.
Additionally, the first PflM 1 site 3` to the I-domain was abolished.
The clones generated at each step were sequenced to confirm that the
oligonucleotides had been incorporated correctly. The murine I-domain
from pLFA.IDmu3 was isolated by digestion with Nar 1 and PflM 1 and the
fragment was inserted into the human CD11a plasmid
(pRKLFA
) at the comparable Nar 1 and PflM 1 sites.
Correct insertion of the murine I-domain was confirmed by sequencing.
as the template
(14) .
Transfections
Human 293 cells were transfected by
a standard calcium phosphate method as described
previously
(15) . Briefly, 1 day prior to transfection, 293 cells
were seeded at 30-40% confluence in 10-cm plates and incubated
overnight. For each condition, 10 µg of pRKCD18, 1 µg of
pAdvRNA, and 10 µg of the plasmid containing the specified CD11a
constructs were precipitated in CaCl and HEPES buffer and
added to the plates. Mock transfected cells were transfected with the
pRKCD18 and pAdvRNA constructs. CD18 transfected in the absence of a
CD11a construct was not expressed at the cell surface (data not shown).
After 3 days transfectants were tested for adhesion to full-length
recombinant human ICAM-1 (rICAM-1) immobilized on plastic or for
expression of the CD11a/CD18 complexes on the cell surface.
Transfection efficiencies ranged from 15 to 90%.
Adhesion Assays
Full-length human ICAM-1 was
purified from a stably transfected 293 cell line expressing rICAM-1.
Detergent extracted rICAM-1 was purified on a lentil-lectin column
followed by gel filtration chromatography on a Superdex 200 column
(Pharmacia Biotech Inc.). Details of the purification will be presented
elsewhere. The purity and concentration of the isolated protein were
determined by SDS-polyacrylamide gel electrophoresis and enzyme-linked
immunosorbent assay (T Cell Diagnostics), respectively. 350 ng of
rICAM-1 in PBS was added to each well of Nunc Maxisorb 96-well plates
and incubated overnight at 4 °C. Plates were washed and blocked
with a 2% bovine serum albumin/PBS solution for 2 h at 37 °C. The
plates were washed with PBS and 100 µl of cells were added per well
in triplicate determinations for each condition. Each experiment was
carried out at least twice.
and
CaCl
. (FACS analysis determined that the I-domain mutants
bound the anti-CD18 activating antibody, MEM-48 (16), at levels
comparable to wild-type CD11a/CD18 under these conditions (data not
shown)). The cells were added to the plates and incubated at 37 °C
for 1.5 h. Nonadherent cells were removed by three washes with PBS.
Cell attachment was measured using the p-nitrophenyl
n-acetyl-
-D-glucosaminide method of Landegren
(17). Mean OD
of triplicate determinations is presented.
For comparison 30,000 cells gave an average OD of 1.
Antibody Staining and FACS Analysis
Three days
post-transfection cells were detached and resuspended in 100 µl of
Dulbecco's modified Eagle's medium, 10% fetal bovine serum
with antibodies against CD11a or CD18. Purified mAbs to CD11a were used
at 1 µg/ml (MHM-24
(18) , M17 (ATCC, Rockville, MD), 25.3
(Amac, Inc., Westbrook, ME); L16
(19) , 32E6
(7) ) or at
1/100 dilution of ascites (TS 1/22 (ATCC, Rockville, MD); 3D6,
50G1
(7) ). MAbs to CD18 were used at 5 µg/ml (MEM-48
(R& Systems, Abingdon, United Kingdom), H52
(20) ) or 1
µg/ml (MHM-23
(18) ). Cells were incubated with antibodies on
ice for 1 h, washed, and fluorescein isothiocyanate-conjugated
secondary antibodies (goat anti-mouse (Cappell) and goat anti-rat
(Caltag)) were added at 1/500 and 1/100 dilutions, respectively. Cells
were washed, fixed in 2% paraformaldehyde, and analyzed by FACS
analysis using the FACScan Research software (Becton Dickinson).
I-domain Epitopes Recognized by Antibodies That Block
LFA-1 Function Are Distinct from Critical ICAM-1 Binding Site(s) in
CD11a
Previously, a panel of human/murine (H/M) chimeras of the
CD11a I-domain were used to localize the epitopes recognized by mAbs
that block the function of human and murine CD11a. The antibodies
recognized three discrete epitopes (IdeA, IdeB, and IdeC).
Fig. 1
shows the location of the epitopes and the chimeric
proteins used to localize them. The chimeric genes H/M 48, H/M 49, H/M
50, H/M 51, H/M 52, H/M 53, and H/M 54 all incorporated mutations in
which murine sequences were substituted into human CD11a. Of the seven
constructs tested, three (H/M 52, H/M 53, H/M 54) were effective in
blocking antibody binding. Given that the mAbs studied all inhibit
LFA-1 mediated adhesion to ICAM-1, we set out to test whether the amino
acids that disrupted antibody binding were also important in LFA-1
binding to ICAM-1.
Figure 1:
Amino acid sequences of the murine and
human CD11a I-domains and the human/murine chimeras and point mutants.
A, schematic representation of CD11a with the respective
locations of the I-domain and the metal binding (EF-hand) domains.
B, the residues of the human and murine I-domains, as well as
the chimeras and point mutations, are illustrated. In the chimeras and
point mutants a dash represents the human residue. Where the
sequences differ from the human sequence the residue is shown. The mu3
construct depicts the amino acid differences between the human and
murine I-domains. MAbs and their epitopes (IdeA, -B, -C) are
indicated.
Previous studies have demonstrated that murine
LFA-1 does not bind to human ICAM-1 and that this species specificity
is encoded in the CD11a subunit
(21) . Since the I-domains of
LFA-1, Mac-1, , and
integrins are
known to be necessary for binding of their respective ligands (ICAM-1,
iC3b, fibrinogen, collagen, and
laminin)
(12, 22, 23, 24) , we
hypothesized that this domain was responsible for the species
specificity of murine LFA-1. A cDNA fragment encoding the entire
I-domain of murine CD11a (amino acids 125-308) was synthesized
de novo using human CD11a I-domain cDNA as a template and
M13-based mutagenesis
(14) . It was inserted into the cDNA for
the full-length human CD11a at the corresponding position (amino acids
126-308), to generate the mu3 variant. The mu3 cDNA was
co-transfected with the cDNA for human CD18 into 293 cells and
transfectants were analyzed for cell surface expression, antibody
binding, and adhesion to human ICAM-1. shows the average
values from the FACS analysis of mu3/CD18 expression. The chimeric
mu3/CD18 construct was expressed at the cell surface at approximately
the same levels as wild-type human CD11a/CD18. All mAbs to human LFA-1
that mapped to the I-domain (32E6, TS 1/22, MHM-24, 25.3, and 50G1)
failed to bind mu3. Other mAbs known to bind outside of the I-domain
(L16, 3D6), bound approximately the same percentage of cells and with
approximately the same mean fluorescence intensity (data not shown) as
human CD11a/C18 expressing cells. M17, a rat mAb to murine CD11a, bound
mu3/CD18 transfectants about 50 times better than human CD11a/CD18
transfectants. MAbs to human CD18 bound the mu3/CD18 transfectants at
levels comparable to human CD11a/CD18. In addition MHM-23, which
recognizes CD18 when paired with an
chain (20),
(
)
also bound the mu3 transfectants. This demonstrates that
mu3 formed heterodimers with CD18. When transfectants were tested for
their ability to bind to human ICAM-1-coated plates, adhesion was
dependent on the presence of CD11a/CD18 at the cell surface, as mock
transfected 293 cells showed background levels of binding
(Fig. 2). Adhesion to ICAM-1 required the presence of divalent
cations. Under conditions which promoted robust binding of wild-type
CD11a/CD18, the mu3/CD18 transfectants failed to adhere to ICAM-1
(Fig. 2A). This data suggests that replacement of the
human CD11a I-domain with murine CD11a sequences yields a heterodimeric
CD11a/CD18 complex that is properly folded and exported to the cell
surface, but is unable to bind to human ICAM-1.
Figure 2:
Attachment of the human/murine I-domain
chimeras to human ICAM-1. A, cells transfected with CD18
alone, mock (), or together with wild-type CD11a (
) or
mu3 (
) were tested for their ability to adhere to human ICAM-1
in the absence and presence of 500 µM MnCl
, 5
mM MgCl
, 10 µg/ml irrelevant IgG1 or 10
µg/ml of the activating antibody, MEM-48, in the presence of 1
mM MgCl
and 1 mM CaCl
.
B, 293 cells transfected with the indicated constructs were
tested for their ability to bind to ICAM-1 in the absence (
)
and presence (
) of 200 µM MnCl
. Staining
with MHM-23 determined that the CD11a, mu3, and H/M 48-H/M 54
transfectants expressed comparable levels of LFA-1 heterodimers on the
cell surface (data not shown).
One mechanism by
which antibodies to LFA-1, such as M17, may block function is by
competitive inhibition of ICAM-1 binding to LFA-1
(10) . Since
mAbs 25.3 and TS 1/22 bind to homologous residues as M17
(7) , it
is likely that they inhibit LFA-1/ICAM-1 interactions by binding to the
same site on CD11a as ICAM-1. Hence, it might be predicted that the
chimera H/M 53, which contains the M17 epitope, would fail to bind to
human ICAM-1 when complexed with CD18. Therefore, we tested whether the
H/M chimeras disrupt the binding of ICAM-1. The H/M constructs were
transiently co-transfected with the human CD18 expression plasmid into
293 cells and assayed for cell surface expression and ICAM-1 binding.
FACS analysis of the binding of a panel of antibodies to these chimeras
was consistent with the epitope mapping studies
(7) (, Fig. 1). Mutants which contain murine
sequences at positions corresponding to I-domain antibody epitopes did
not bind the relevant antibodies, but bound other I-domain antibodies
at levels comparable to wild-type CD11a/CD18. All chimeras bound MHM-23
with values comparable to wild-type, indicating the formation of
heterodimers with CD18. Antibodies to CD18 and CD11a that map outside
the I-domain bound at levels comparable to wild-type. When the
transfectants were tested for adhesion to human ICAM-1 in the presence
of 200 µM MnCl, all of the H/M chimeras bound
to human ICAM-1 at levels comparable (within 2.5-fold) to wild-type
CD11a/CD18 (Fig. 2B). These results indicate that the
residues within these mAb epitopes are not strictly required for human
ICAM-1 binding. It cannot, however, be ruled out that these epitopes
may correspond to only part of the ICAM-1 binding domain which is
composed of multiple residues at discontinuous locations within the
I-domain. If this were the case, mutating residue(s) in a single region
would not necessarily destroy ICAM-1 binding. Preliminary evidence
supporting this hypothesis suggests that substitution of murine
residues for human residues is required at multiple locations within
the human CD11a I-domain in order to abolish ICAM-1 binding (data not
shown).
Mutating Asp-137, Pro-192, and Asp-239 in the I-domain of
CD11a Abolishes Binding of LFA-1 to ICAM-1
As described
previously, the epitopes mapped by the H/M mutants were specific for
CD11a and were not conserved in the closely related integrin, Mac-1,
that also binds ICAM-1
(7) . Assuming that ICAM-1 binds similar
sequences in CD11a and CD11b, we hypothesized that conserved sequences
located in the region of these epitopes might be important for ligand
binding. The sequence between IdeA and IdeB is highly conserved between
these two molecules and has been found to be important for ligand
binding of Mac-1 by Michishita et al.(11) . These
investigators showed that mutating D140GS of CD11b to AGA abolished
Mac-1 adhesion to iC3b. Binding of isolated I-domain to
Mn
was also abolished by this mutation.
To further localize residues in this region of CD11a involved in the
LFA-1/ICAM-1 interaction, we generated variants with the following
point mutations in the CD11a I-domain: D137A, D137K, and S139A. These
were assayed for their ability to promote binding of LFA-1 to ICAM-1 in
the presence of varying concentrations of MnCl
(Fig. 3A), MgCl
(Fig. 3B), and MEM-48 (Fig. 3C), an
antibody to CD18, which activates LFA-1
(16) . (We consistently
found higher levels of binding of wild-type LFA-1 transfectants to
ICAM-1 in the presence of MnCl
than MgCl
.) We
found that the substitution of Ala or Lys for Asp at position 137
abrogated the ability of LFA-1 to bind to ICAM-1 under any of the
conditions tested. However, substitution of Ala for Ser at position 139
had no significant effect on LFA-1 binding to ICAM-1 (Fig. 3,
A-C).
Figure 3:
Attachment of I-domain point mutants to
human ICAM-1. 293 cells were transfected with the following CD11a/CD18
constructs and tested for ability to adhere to ICAM-1 in increasing
concentrations of MnCl (A) or MgCl
(B): mock (
), wild-type CD11a (
), D137A
(
), D137K (
), S139A (
), P192A (
), D239A
(
), D239K (▾). C, the indicated transfectants were
assayed for ability to adhere to ICAM-1 in the absence (
) or
presence of 1 mM CaCl
, 1 mM MgCl
(
), 10 µg/ml MEM-48 (
), or 500 µM
MnCl
(
). Staining with MHM-23 determined that the
transfectants expressed comparable levels of LFA-1 heterodimers on the
cell surface (data not shown).
FACS analysis demonstrated that these
mutants formed heterodimers with CD18 at the cell surface, as the mAb
MHM-23 bound at levels comparable to wild-type CD11a/CD18
(). Subtle changes in the conformation of the I-domain of
these mutants were suggested by the decrease in binding of mAb TS 1/22
to the D137A variant, and in the moderate reduction in mAbs 32E6, 50G1,
and TS 1/22 binding to D137K. No decrease in antibody binding was
observed in the S139A variant. The gross conformation of these variants
appeared to be intact since the antibodies to CD18 and most of the
antibodies to CD11a bound these variants at levels comparable to
wild-type CD11a/CD18 (). These results suggest that amino
acids common to Mac-1 and LFA-1 are critical for the binding of these
integrins to distinct ligands (iC3b and ICAM-1).
and iC3b
(11) . An Ala was substituted for
Pro at position 192 and an Ala or Lys was substituted for the Asp at
position 239. Mutating Asp-239 in the CD11a I-domain to either Ala or
Lys completely abolished LFA-1 binding to ICAM-1 under the conditions
tested (Fig. 3, A-C). Binding of MHM-23 confirmed
that these mutants were expressed at the cell surface as heterodimers
with CD18. It appears that these substitutions had little or no effect
on the gross conformation of LFA-1 as judged by antibody binding
(). No reduction in binding of any of the antibodies was
observed for the D239A variant and substituting Lys at this position
reduced the binding of only TS 1/22. This is noteworthy given the
considerable distance along the primary sequence between Asp-239 and
the TS 1/22 epitope (IdeA) (Fig. 1)
(7) . It will be of
interest to determine whether Asp-239, as well as Asp-137 whose TS 1/22
binding is also perturbed, are located proximal to IdeA in the
three-dimensional structure of this receptor.
and shifted the optimal cation concentration required for
adhesion of Mac-1 expressing cells to iC3b
(11) . In CD11a, this
mutant was unable to bind to ICAM-1 even at high concentrations of
divalent cations (500 µM MnCl
or 2 mM
MgCl
) (Fig. 3, A and B). This
suggests that either the Pro-192 mutation in the CD11a I-domain exerts
a more profound effect on the I-domain structure or that the
LFA-1/ICAM-1 interaction is more sensitive to structural constraints
than the Mac-1/iC3b interaction.
and
, has been determined. Mutation of
Asp-253 in the
I-domain abrogated
binding to collagen IV and
significantly reduced
binding to
laminin
(23) . Mutation of Asp-151 and Asp-254 in the
I-domain abolished
binding to
collagen I
(22) . Interestingly, the data for both Mac-1 and
suggests that these residues are not
ligand contact points, but rather cation binding sites. In Mac-1, these
aspartic acids appear to bind cations and mutation of these residues
destroyed iC3b binding of the full-length receptor as well as the
isolated I-domain
(11, 25) . However, a 14-amino acid
peptide derived from the CD11b I-domain that includes Asp-242 bound
iC3b in a cation independent manner. Substitution of Ala for Asp at
position 242 of this peptide did not affect binding to
iC3b
(25) . For
, Kamata et
al.(22) demonstrated that mutation of the analogous
aspartic acids in full-length
abrogated collagen
binding. However the isolated I-domain bound to collagen I in a
cation-independent manner (26). Moreover, in the isolated I-domain,
mutation of these residues did not completely abrogate collagen
binding, suggesting that these aspartic acids are not critical contact
points in
adhesion to collagen.
), it is possible to promote
receptor-ligand interactions in the absence of cations. Our preliminary
data suggests that the isolated CD11a I-domain also binds ICAM-1 in a
cation-independent manner (data not shown). Hence, it appears that the
ligand binding sites in these full-length integrins are masked in the
absence of cations, and cation binding is required to expose these
sites. Conceivably the binding of cations to the above-defined aspartic
acids regulates ligand accessibility of these integrins, which bind
ligands as diverse as collagen, laminin, iC3b, and ICAM-1. The role of
these residues in direct binding of divalent cations and ICAM(s) to the
CD11a I-domain is currently being investigated. A full understanding of
these interactions is likely to require a combination of
three-dimensional structural and mutational analysis of both ICAM-1 and
LFA-1. Determination of the domains of LFA-1 that confer ligand
specificity, as well as understanding the role of amino acids that are
involved in cation binding or activation, will be important to the
development of highly specific anti-inflammatory therapeutics.
Table: 0p4in
NA,
not applicable.(119)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.