From the Department of Vascular Biology, American Red Cross Holland Laboratory, Rockville, Maryland 20855
Received for publication, September 29, 2000, and in revised form, February 16, 2001
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ABSTRACT |
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The central region (residues 125-385) of
the integrin Central to the ligand binding function of
Recently, we have deployed homolog-scanning mutagenesis (28)
to identify several segments critical to Fg and C3bi binding within the
Materials--
Human kidney 293 cells and the expression vector,
pCIS2M, were gifts from Dr. F. J. Castellino (University of
Notre Dame, Notre Dame, IN). The cDNAs of CD11b and CD18 were
obtained from Dr. B. Karan-Tamir (Amgen, Thousand Oaks, CA). The
recombinant Site-directed Mutagenesis and Development of Stable Cell
Lines--
The detailed procedures used for homolog-scanning
mutagenesis and to establish stable cell lines expressing wild-type and mutant Surface Labeling and Immunoprecipitation--
Cells expressing
wild-type and mutant C3bi Binding and Adhesion to Fg--
The ligand binding activity
of the FACS Analysis--
A total of 106 cells expressing
wild-type or mutant Homolog-scanning Mutagenesis of the
Surface Expression and Heterodimer Formation--
A large number
of natural mutations occur within the Epitope Mapping of Function-blocking mAbs--
To help locate the
functional sites within the Role of the
In addition to the segment Pro192-Glu197, five
other segments were recognized by the nine The Influence of Ca2+ on the Conformation of the
To explore the possibility that the Ca2+ binding site
reported by these two mAbs is located within the MIDAS motif
(D134XSXS) of the
Several studies have reported that ligand binding by the
In this work, we have probed the function of the hydrophilic
surface of the putative A number of studies have demonstrated the importance of the central
region (residues 125-385 in It was proposed recently that the central region within the 2 subunit is postulated to adopt an
I-domain-like fold (the
2I-domain) and to play a
critical role in ligand binding and heterodimer formation. To
understand structure-function relationships of this region of
2, a homolog-scanning mutagenesis approach, which
entails substitution of nonconserved hydrophilic sequences within the
2I-domain with their homologous counterparts of the
1I-domain, has been deployed. This approach is based on
the premise that
1 and
2 are highly
homologous, yet recognize different ligands. Altogether, 16 segments
were switched to cover the predicted outer surface of the
2I-domain. When these mutant
2 subunits
were transfected together with wild-type
M in human 293 cells, all 16
2 mutants were expressed on the cell
surface as heterodimers, suggesting that these 16 sequences within the
2I-domain are not critically involved in heterodimer
formation between the
M and
2 subunits.
Using these mutant
M
2 receptors, we have
mapped the epitopes of nine
2I-domain specific mAbs, and
found that they all recognized at least two noncontiguous segments
within this domain. The requisite spatial proximity among these
non-linear sequences to form the mAb epitopes supports a model of an
I-domain-like fold for this region. In addition, none of the mutations
that abolish the epitopes of the nine function-blocking mAbs, including segment Pro192-Glu197, destroyed ligand
binding of the
M
2 receptor, suggesting
that these function-blocking mAbs inhibit
M
2 function allosterically. Given the
recent reports implicating the segment equivalent to Pro192-Glu197 in ligand binding by
3 integrins, these data suggest that ligand binding by
the
2 integrins occurs via a different mechanism than
3. Finally, both the conformation of the
2I-domain and C3bi binding activity of
M
2 were dependent on a high affinity
Ca2+ binding site (Kd = 105 µM), which is most likely located within this region of
2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M
2 is a member of the
2 integrin subfamily, which includes
L
2 (LFA-1, CD11a/CD18),
X
2 (p150,95, CD11c/CD18), and
D
2. Like all integrins, the
2 subfamily members are expressed on cell surfaces as
heterodimers, but their expression is restricted primarily to
leukocytes.
M
2 plays a multifunctional
role on leukocytes. As examples, this integrin is important in
leukocyte adhesion and transmigration through endothelium, in
activation of neutrophils and monocytes, in phagocytosis of foreign
material, and in apoptosis (1, 2). A wide variety of protein and
non-protein ligands have been identified that interact with
M
2, with representative examples
including fibrinogen (Fg)1
(3), ICAM-1 (4), C3bi (5), zymosan (6), and neutrophil inhibitory
factor (7). C3bi and Fg are two particularly important ligands of
M
2; C3bi is critical to phagocytosis of
opsonized foreign particles, and Fg, which interacts with
M
2 via its
-module (8), is involved in
leukocyte adhesion and migration.
M
2 is its I(A) domain. The
MI-domain is an inserted segment of ~200
amino acids and is highly homologous to several I-domains found in
integrin
subunits (9). The three-dimensional structures of several I-domains (
M,
L,
X,
2, etc.) have been solved (10-13). These I-domains are
composed of six or seven
-helices and six
-sheets arranged in a
Rossman-type fold. A cation binding site, termed the MIDAS motif, is
located within the I-domain. In the MIDAS motif, cation coordination is
provided by a DXSXS sequence and by other two
distant (in terms of primary sequence) oxygenated residues (10). In
addition to the
subunits with their I-domains, the
subunits
also contribute to ligand binding to integrins. Studies of the
subunits have been focused primarily on their central regions (residues
~125-385 in a typical
subunit of >700 amino acids). This region
is predicted to contain a MIDAS motif, and candidate residues for
cation coordination have been identified by mutagenesis (14-17).
Protein sequence analysis suggests that this region may also fold into
an I-domain-like structure (10, 18). However, due to the low homology
between the I-domains of the
and
subunits, it is uncertain
whether this putative I-domain region does, indeed, fold into an
I-domain, or merely contains a MIDAS motif. What is clear is that this
region does play a critical role in mediating ligand binding to
integrins. In
3, it was reported that bound RGD peptides
can be cross-linked to this region (19, 20). Substituting this segment
within the
1I- or
5I-domain with its
homologous counterpart from
3 imparts
3
ligand specificity to the
1 or
5 integrin
(21, 22). A natural mutation of Arg214 to Gln in
3 abolishes ligand binding of
IIb
3, and a synthetic peptide containing
the sequence of
3 (211) blocks Fg binding to
purified
IIb
3 (23). Similar observations
implicate the
1I-domain in the ligand binding functions
of the
1 integrins. For example, it was shown that both
activating and inhibiting mAbs recognize a small stretch of
1 (residues 124-160 and 207-218) (24, 25). Recently,
the D134XSXS sequence of the proposed
MIDAS motif within
2 was implicated in the binding of
Fg, C3bi, and ICAM-1 to
M
2 (26, 27).
These data indicate that this putative I-domain is important to ligand binding functions of the
2 integrins as well.
MI-domain (8, 29). This approach entails switching sequences within the
MI-domain to their homologous
sequences within the
LI-domain. This approach is
feasible because the
MI- and
LI-domains
are highly homologous, but
M
2 and
L
2 recognize different ligands. In the
study reported here, we have applied this same strategy to the putative
2I-domain region. Our data are consistent with folding
of the region into an I-domain-like structure. However, our results
suggest that ligand recognition by the region of the
2
subunit is achieved in a distinct fashion from that involved in ligand
recognition by the
3 integrins. In addition, we show
that the epitopes of several blocking mAbs map to this region but their
inhibitory activity is likely to be achieved via an allosteric
mechanism. Finally, we show that the conformation and ligand binding
functions of the
2I-domain are enhanced selectively by
Ca2+, suggesting a unique cation-specific effect on the
2I-domain. Taken together, these results provide insight
into the structure-function relationships of
M
2, which may also extend to other
integrins in general.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-module of Fg was provided by Dr. Medved (American Red
Cross, Rockville, MD). The mAbs used in this study were obtained from the following sources. mAb 6.5E was provided by Dr. D. P. Andrew (Amgen Inc., Boulder, CO); mAb MHM23 was from Dako (Carpinteria, CA);
IB4 and TS1/18 were from the ATCC (Rockville, MD); mAb 44 was from
Sigma; CLB-LFA-1/1,54 (CLB54) was from RDI (Flanders, NJ); YFC118.3 and
R3.3 were from Chemicon (Temecula, CA); H20A was from VMRD Inc.
(Pullman, WA); 6.7 was from PharMingen (San Diego, CA); 685A5, MEM-48,
and 7E4 were from Biodesign (Kennebunk, ME).
M
2 receptors in human kidney 293 cells have been published (30). Similar methods were used to express
the
M
2 heterodimer and the single
2 subunit on the surface of the Chinese hamster ovary
cells. To obtain cell lines with similar expressions, each mutant cell
line was subcloned by cell sorting using an
M-specific mAb (2LPM19c). Up to 20 colonies were selected and analyzed for integrin expression by FACS analysis. Cells with receptor expression levels similar to wild-type
M
2 were
chosen, and five different subclones were used for the subsequent
studies reported in this work. To exclude the possibility of subcloning
artifacts, all studies were repeated using the original pool of each
mutant receptor.
M
2 were washed once
with DPBS, biotinylated with EZ-link Sulfo-NHS-LC-Biotin
(sulfosuccinimidyl 6-biotinamidohexanoate, Pierce), and lysed with a
solution containing 20 mM Tris-Cl, 150 mM NaCl,
pH 7.4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 mM benzamidine, 25 µg/ml soybean trypsin
inhibitor, and 20 µg/ml leupeptin. The cell lysates were subjected to
immunoprecipitation with an
M-specific mAb 44a and a
2-specific mAb 6.7. The immunoprecipitates were analyzed
on 7% acrylamide gels, and the surface-expressed
M
2 was visualized by Western blotting
using a horseradish peroxidase-avidin conjugate.
2 mutants was assessed using two classic
M
2 ligands, C3bi and Fg, according to our
published methods (27). For adhesion of
M
2-expressing cells to Fg, the recombinant
-module (10 µg/ml) was deposited at the center of each
well in a 24-well non-tissue culture polystyrene plate. After blocking
with 400 µl of 0.05% polyvinylpyrrolidone in DPBS, a total of 2 × 106 cells in Hank's balanced salt solution containing 1 mM Ca2+ and 1 mM Mg2+
was added to each well and incubated at 37 °C for 20 min. The unbound cells were removed by three washes with DPBS, and the adherent
cells were quantified by cell-associated acid phosphatase as described
previously (27).
M
2 in Hank's
balanced salt solution containing 1 mM Mg2+ and
1 mM Ca2+ was incubated with 1 µg of mAb for
30 min at 4 °C. A subtype-matched mouse IgG served as a control.
After washing with PBS, cells were mixed with FITC-conjugated
goat-anti-mouse IgG(H+L) F(ab')2 fragment (1:20 dilution)
(Zymed Laboratories Inc.), and kept at 4 °C for another 30 min. Cells were then washed with PBS and resuspended in 500 µl of DPBS. The FACS analyses were performed using FACScan (Becton-Dickinson), counting 10,000 events. Mean fluorescence intensities were quantified using the FACScan program, and these values
were used to compare
M
2 expression levels
or the reactivity of the different
M
2
mutants with specific mAbs.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2I-domain--
As shown in Fig.
1, the purported I-domain within integrin
2 shares considerable sequence homology with the
corresponding region of the
1 subunit. The major
sequence differences are confined to regions that are predicted to be
hydrophilic and surface-oriented based on hydropathy plots and
molecular modeling, and, thereby, are the segments that are likely to
contribute to the unique functions of the
2 integrins.
For example, the
2 subunit partners with an entirely
separate set of
subunits from
1, and the
2 integrins recognize a set of ligands very distinct
from the
1 integrins (there is no known peptide sequence
recognized by both
1 and
2 integrins).
Based on the sequence homology between the
1I- and
2I-domains, we sought to systematically probe the
function of the hydrophilic and unique segments of this region
(residues 125-385) using homolog-scanning mutagenesis. Accordingly, we
replaced 16 non-conserved segments of three to nine residues within the
2I-domain with the corresponding segments from the
1 subunit (Fig. 1). These 16 segments covered the entire
hydrophilic region of the
2I-domain predicted from
hydropathy plots and molecular modeling. The primers used for
mutagenesis are listed in Table I. The
DNA sequence of the entire I-domain was confirmed for each mutant
before and after transfer back into the pCIS2M expression vector
containing the cDNA of
2.
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Fig. 1.
Sequence alignment between the putative
1I- and
2I-domains. The amino acid
residues are from 141 to 395 for the
1I-domain and from
125 to 380 for the
2I-domain (the numbering is based on
the entire protein sequence including the signal peptide). The
conserved residues are underlined, and the mutated segments
are shown in brackets.
Primers used in the homolog-scanning mutagenesis of the putative
2I-domain
2I-domain, which
abolish surface expression and/or heterodimer formation (31-37).
Nevertheless, when the
2 mutants were co-transfected with wild-type
M in human kidney 293 cells, all 16 mutants were expressed on the cell surface as heterodimers and the
subunits had appropriate molecular weights. As shown in Fig.
2, immunoprecipitation of surface-labeled
cells with 44a, a mAb specific for the
M subunit, yielded two bands of ~165 kDa (
M) and 95 kDa
(
2) on SDS-PAGE. The patterns were similar to those
obtained for wild-type
M
2 (27). In
addition, FACS analyses were conducted on these 16 mutants using a
panel of
2-specific mAbs (Table
II). All 16
2 mutants were
recognized by three different mAbs to the
2 subunit MEM48, 7E4, and 6.7, as well as by the
M-specific mAb
44. To exclude selection artifacts, we established at least five
independent stable cell lines for each mutant
2 integrin
that expressed similar levels of receptors on their cell surfaces, as
judged by FACS analysis using mAb 44. Heterodimer formation, as well as
other results described below, was similar for all five clones.
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Fig. 2.
Surface expression and heterodimer formation
of the 16 2I-domain mutants.
The 16
2 homolog-scanning mutants were co-transfected
with wild-type
M into human 293 cells, and stable cell
lines were established. A total of 5 × 106 cells
expressing the wild-type (wt) or mutant
M
2 were surface-labeled by biotin and
then lysed. The biotinylated
M
2 was
immunoprecipitated with 5 µg of an
M-specific mAb
(44a) and analyzed on 7% SDS-PAGE. Lane 1,
M
2(Arg144-LysK148);
lane 2,
M
2(Leu154-Glu159);
lane 3,
M
2(Glu162-Glu164);
lane 4,
M
2(Asn181-Asp185);
lane 5,
M
2(Pro192-Glu197);
lane 6,
M
2(Gln199-Ala203);
lane 7,
M
2(Asn213-Glu220);
lane 8,
M
2(Pro247-Glu249);
lane 9,
M
2(Ala262-Asp265);
lane 10,
M
2(Asp290-Glu298);
lane 11,
M
2(Gly305-His309);
lane 12,
M
2(Ser324-Thr329);
lane 13,
M
2(Thr334-Ile336);
lane 14,
M
2(Glu344-Asp348);
lane 15,
M
2(His354-Asn358);
lane 16,
m
2,
His371-Lys379.
Reactivity of function-blocking monoclonal antibodies with the
2I-domain mutants
M
2-expressing cells. A "+" indicates that the
mean fluorescence intensity of the mAb is at least 10 times that of the
IgG control. A "
" indicates that the mean fluorescence intensity
of the mAb is no more than that of the IgG control.
2I-domain, we sought to map
the epitopes of several
2-specific function-blocking mAbs: MHM23, IB4, 6.5E, TS1/18, CLB54, YFC118.3, R3.3, H20A, 685A5, and
7E4. The ability of these mAbs to block
2 integrin
functions, such as
M
2-mediated adhesion
and C3bi binding and
L
2-mediated binding
to ICAM-1, has been well documented (38-42). Representative FACS
analyses using mAb IB4 with five of the
M
2 mutants are shown in Fig.
3A and a summary of the FACS
analyses for all 16 mutants and 12
2-specific mAbs is
shown in Table II. Among these 12 mAbs, 3 (6.7, MEM-48, and 7E4)
reacted well with all 16 mutants, but not the mock-transfected 293 cells. The other nine mAbs recognized the
2I-domain, and
their epitopes consisted of at least two noncontiguous sequences. For
example, mAb IB4 reacted well with wild-type
M
2, and mutants
M
2(Leu154-Glu159),
M
2(Asn213-Glu220)
and
M
2(His354-Asn358),
but its binding to the two mutants
M
2(Arg144-Lys148)
and
M
2(Pro192-Glu197)
was ablated (Fig. 3A), suggesting that these two segments
(Arg144-Lys148 and
Pro192-Glu197) contribute to the epitope of
IB4. As shown in Table II, in addition to IB4, mAbs MHM23, H20A, R3.3,
and perhaps 6.5E also depended on segments
Arg144-Lys148 and
Pro192-Glu197 for their interactions with
M
2. mAb 685A5 required segments Arg144-Lys148,
Pro192-Glu197, and
Asn213-Glu220; mAb TS1/18 required segments
Leu154-Glu159 and
Glu344-Asp348; mAb CLB54 required segments
Leu154-Glu159 and
His354-Asn358; and finally mAb YFC118.3
required segments Arg144-Lys148,
Leu154-Glu159, and
His354-Asn358. These epitopes can be roughly
divided into two different groups (see Fig. 8). The first contains
segments Leu154-Glu159,
Glu344-Asp348, and
His354-Asn358, and is important for
M
2 interaction with mAbs TS1/18, CLB54, and YFC118.3, and the second contains segments
Arg144-Lys148,
Pro192-Glu197, and
Asn213-Glu220, and is important for
M
2 binding of mAbs MHM23, H20A, IB4, R3.3, and 685A5. To further support our epitope mapping results and
this grouping of the mAbs, we performed two additional experiments. First, competition was performed between mAbs MHM23, IB4, and R3.3 from
group 2, TS1/18 from group 1, and 7E4, which recognizes an epitope that
is likely located outside of the
2I-domain. In these
experiments,
M
2-expressing cells were
incubated first with the competitor mAb, IB4, R3.3, TS1/18, or 7E4, and
then the reporter mAb MHM23 was added. Binding of MHM23 was measured by FACS analysis, and the results are shown in Fig. 3B. As
predicated, mAbs IB4 and R3.3, which belong to the same group as MHM23
(group 2), blocked more than 95% of the binding of mAb MHM23 to
M
2. In contrast, mAb TS1/18 (group 1) and
mAb 7E4 had little effect on MHM23 binding. The specificity of these
assays was confirmed by the ability of unlabeled MHM23 but not a
control IgG to block the binding of the fluorescence-labeled MHM23 to
the cells. Second, the ability of mAb IB4 to block adhesion of
M
2-expressing cells to a representative
ligand, the
-module of fibrinogen, was assessed using wild-type and
two different
M
2 mutants. As shown in
Fig. 3C, cells expressing these three different
M
2 receptors all adhered well to the
-module in the presence of a control IgG. Addition of mAb IB4
completely inhibited adhesion of cells expressing the wild-type and one
of the mutant receptors
M
2(Leu154-Glu159).
However, mAb IB4 had no effect on adhesion by the second mutant
M
2(Arg144-Lys148).
These results are consistent with the FACS data presented in Fig.
3A, which show that the epitope of mAb IB4 was destroyed in
mutant
M
2(Arg144-Lys148)
but not in mutant
M
2(Leu154-Glu159).
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Fig. 3.
Epitope mapping of the
2I-domain function-blocking mAbs.
A, representative FACS analysis using mAb IB4.
M
2-expressing 293 cells (106)
were incubated with 1 µg of mAb at 4 °C for 30 min. After three
washes with PBS, the cells were stained with FITC-conjugated goat
anti-mouse IgG and analyzed using FACScan. An isotype-matched IgG was
used as a control. B, competition among different
function-blocking mAbs for binding to
M
2.
The
M
2-expressing cells were first
incubated with an IgG control, or with mAbs IB4, R3.3, MHM23, TS1/18,
and 7E4 individually for 30 min at 4 °C, and then incubated with a
MHM23-FITC conjugate for additional 30 min. After washing with PBS,
bound MHM23 was measured by FACS analysis. C, inhibition of
cell adhesion to the
-module of Fg by mAb IB4. A total of 2 × 106
M
2-expressing cells was
incubated with either a control IgG (open bar) or
mAb IB4 (filled bar) for 30 min at 4 °C, and
then added to 24-well non-tissue culture polystyrene plates, which were
pre-coated with recombinant
-module (10 µg/ml) and subsequently
blocked with 0.05% polyvinylpyrrolidone in DPBS. After incubation at
37 °C for 20 min, the unbound cells were removed by three washes
with DPBS and the adherent cells were quantified by cell-associated
acid phosphatase. For each cell line, the number of adherent cells in
the presence of the control IgG was taken as 100%. Asterisk
(*) indicates a value of less than 1%. Data shown are the means ± S.D. of three independent experiments.
2I-domain in Ligand Binding--
A
short disulfide loop of 7-8 amino acids has been implicated in the
ligand binding functions of the
3 integrins (21, 22, 43). This disulfide loop is conserved in the
2 subunit,
corresponding to Pro192-Glu197 within the
putative
2I-domain. Given the high degree of homology between the
2 and
3 subunits, we tested
the hypothesis that segment Pro192-Glu197 is
also important to the ligand binding function of
M
2. The
-module of Fg and C3bi were
used as model
M
2 ligands, and we assessed
their interactions with the mutant
M
2(Pro192-Glu197),
in which this segment was replaced with its homologous counterpart of
the
1 subunit. We expected that this mutant would be
defective in Fg and C3bi binding, should
Pro192-Glu197 constitute a part of the ligand
binding site within
M
2. As shown in Fig.
4 (A and B), this
mutant bound C3bi and interacted with the
-module similarly to
wild-type
M
2, suggesting that this
sequence is not directly involved in ligand binding by
M
2. The specificity of the C3bi binding
assay was confirmed using mock-transfected 293 cells and by inhibition
experiments with EDTA. In addition, the specificity was further
verified by blocking experiments using the
M-specific
mAb 44a; addition of 44a blocked more than 90% C3bi binding to both
the wild-type and the mutant receptors. Similarly, the specificity of
the adhesion to the
-module was confirmed by blocking experiments
with EDTA (data not shown) and mAb 44a. Thus, the contribution of the
2 subunit to ligand binding is different from that of
3, suggesting that ligand binding to the
2 integrins has different requirements.
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Fig. 4.
Ligand binding to
2I-domain mutants containing the
epitopes of function-blocking mAbs. A, C3bi binding.
Biotinylated EC3bi (2 × 107) were added to 2 × 105 cells expressing
M
2,
which had been pre-seeded onto polylysine-coated 24-well plates. After
60 min at 37 °C, the amount of bound EC3bi was determined using
avidin-alkaline phosphatase and p-nitrophenylphosphate,
measuring the absorbance at 405 nm. The value for wild-type
M
2 was taken as 100%. Specificity was
demonstrated by addition of 1 mM EDTA (shown with
asterisks) and further verified by blocking experiments with
an
M-specific mAb 44a; addition of mAb 44a blocked more
than 90% C3bi binding to wild-type and three representative mutants:
M
2(Arg144-Lys148),
M
2(Pro192-Glu197),
and
M
2(Leu154-Glu159).
Data are the means ± S.D. of three to six independent
experiments. B, Fg adhesion. Adhesion of
M
2-expressing cells to the
-module of
Fg was performed as described in Fig. 3C except that the
number of adherent cells expressing wild-type
M
2 was taken as 100%. Specificity was
verified using
M-specific function-blocking mAb 44a
(filled bar). Data are the means ± S.D. of
three to six independent experiments.
2-blocking
mAbs (MHM23, IB4, 6.5E, TS1/18, CLB54, YFC118.3, R3.3, H20A, and
685A5). To determine if these segments contribute directly to the
ligand binding function of
M
2, we
repeated the above ligand binding experiments with the five
2 mutants:
M
2(Arg144-Lys148),
M
2(Leu154-Glu159),
M
2(Asn213-Glu220),
M
2(Glu344-Asp348),
and
M
2(His354-Asn358).
As shown in Fig. 4 (A and B), the five additional
mutants also displayed significant ligand binding activities toward
both C3bi and the Fg
-module. For C3bi binding, all five mutants
interacted well with C3bi, similar to wild-type
M
2 (Fig. 4A); for adhesion to
the Fg
-module, two mutants
(
M
2(Leu154-Glu159)
and
M
2(His354-Asn358))
behaved similarly to the wild-type receptor, whereas one mutant (
M
2(Arg144-Lys148))
adhered less (~50% of wild-type), and the other two mutants (
M
2(Asn213-Glu220)
and
M
2(Glu344-Asp348))
adhered more strongly (~300% of wild-type) to the
-module. Thus,
these results suggested that the epitopes of the above
2I-domain specific mAbs do not contribute significantly
to ligand binding per se, and, therefore their blocking
functions occur most likely via an allosteric mechanism.
2I-domain--
Ligand binding to integrins depends upon
divalent cations, and specific cations can influence ligand binding
specificity. For example, the
MI-domain adopts different
conformations in the presence of Ca2+ versus
Mn2+ (44, 45), and conformational changes are induced in
the
1I-domain by Mg2+ and Ca2+
(46, 47). In the course of our studies, we observed that binding of
mAbs YFC118.3 and TS1/18 to
M
2 was
supported by Ca2+ but not by Mg2+ and that
addition of EGTA/Mg2+ or EDTA reduced the binding of these
mAbs by 4-fold (for YFC118.3) or 5-fold (for TS1/18) (Fig.
5A). As these two mAbs
recognize different and non-contiguous regions within the
2I-domain (see Table II; TS1/18 recognizes
Leu154-Glu159 and
Glu344-Asp348, whereas YFC118.3 recognizes
Arg144-Lys148,
Leu154-Glu159, and
His354-Asn358), these results suggested that
the overall conformation of the
2I-domain is
differentially influenced by cations and that the conformation induced
by Ca2+ is required for optimal reactivity with these mAbs.
To further characterize these observations, we tested the effect of
Ca2+ concentrations on the binding of these two mAbs. A
constant concentration of each mAb of 20 nM was selected
for these analyses, which is below the concentrations of each required
for 50% of its maximal binding to
M
2.
The Ca2+ titration curve for mAb YFC118.3 is shown in Fig.
5B. Binding of the mAb increased with increasing
Ca2+ and saturated above 500 µM added
Ca2+. These data could be fitted to a single binding site
model. The estimated Kd of this Ca2+
binding site was 105 ± 9 µM. A similar
Kd for Ca2+ binding site was obtained
with TS1/18. This similarity suggests that the same Ca2+
binding site is involved in the binding of these two mAbs to
M
2.
View larger version (25K):
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Fig. 5.
Effects of Ca2+ on the binding of
mAbs TS1/18 and YFC118.3. A, binding of mAbs TS1/18 and
YFC118.3 to M
2 depends on
Ca2+.
M
2-expressing 293 cells
(106) in the presence of 1 mM Ca2+
(thick line) or 2 mM Mg2+
plus 1 mM EGTA (thin line) was
determined by FACS analysis. An isotype-matched IgG was used as a
control for each mAb (dotted line). B,
mAb YFC118.3 binding to
M
2 as a function
of different concentrations of Ca2+. Binding of mAbYFC118.3
(1 µg) to wild-type
M
2-expressing 293 cells (106) in the presence of different concentrations of
Ca2+ was determined by FACS analysis. The mean fluorescence
intensity was determined using the FACScan program. The titration data
can be fitted into a single binding site model with an apparent
Kd value of 105 ± 9 µM by
non-linear regression analysis, using an equation provided by
SigmaPlot, F = F0 + Fmax*[Ca]/(Kd+[Ca]),
where F is the total bound mAb measured by FACS,
F0 is the mAb bound in the absence of
Ca2+, and [Ca] is the Ca2+ concentration
added. C, mAbYFC118.3 binding to
M
2(S136A)-expressing cells. Binding of
mAbYFC118.3 to
M
2(S136A) in the presence
of different concentrations of Ca2+ was determined as
described for wild-type
M
2.
2I-domain, we tested YFC118.3 binding to a mutant
2, in which Ser136, a putative cation
coordination site, was replaced by Ala. As shown in Fig. 5C,
Ca2+ bound to this mutant
M
2
with a significantly (p < 0.03) reduced affinity
(Kd = 151 ± 10 µM), compared
with wild-type
M
2, indicating that the
cation binding site reported by YFC118.3 is likely located within the
2I-domain. To exclude the possibility that
Ca2+ binding to the
M subunit may
allosterically affect YFC118.3 and TS1/18 binding to
2,
we expressed the
2 subunit alone on the surface of the
Chinese hamster ovary cells. The presence of
2 and
absence of
M on the cell surface was confirmed by FACS analyses using an
M-specific mAb 44 and
2-specific mAbs 6.7 (Fig.
6A), 7E4, and MEM-48 (data not
shown). That the
2 subunit is expressed alone on the
cell surface is further supported by surface labeling and
immunoprecipitation experiments; for the
M
2-expressing cells, both mAbs 44a
(against
M) and 6.7 (against
2) yielded
two bands of ~95 and 165 kDa on SDS-PAGE, whereas for the
2-expressing cells, mAb 44a did not produce any
detectable band and mAb 6.7 yielded only a single band of 95 kDa
(
2) (Fig. 6B). These data demonstrate that
the
2 subunit is present alone on the cell surface and
not complexed with
M or any other integrin
subunits.
To see whether the single
2 subunit still contains a
high affinity Ca2+ binding site, we repeated the above
Ca2+ titration experiments with mAbs YFC118.3 and TS1/18.
Fig. 6C shows that, similar to the
M
2 heterodimer, mAb YFC118.3 bound to
single
2 in a cation-dependent manner, and
the Ca2+ titration curve can be fitted to a single binding
site model. The estimated Kd of this
Ca2+ binding site is 83 ± 2 µM, which
is very close to the Kd of 105 µM for
the heterodimeric receptor. A similar Kd for
Ca2+ binding to
2 was obtained with TS1/18.
Taking these data together, we conclude that the proper conformation
for mAb binding to the
2I-domain depends upon a
Ca2+ binding site within
2, possibly
composed of Ser136 within the proposed MIDAS motif of the
2I-domain.
View larger version (30K):
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Fig. 6.
Ca2+ binding to the
surface-expressed single 2
subunit. A, FACS analysis of surface-expressed single
2. The
2-expressing cells
(106) were incubated with 1 µg of an
M-specific mAb 44 (gray line), a
2-specific mAb 6.7 (black line),
or a control IgG (dashed line) at 4 °C for 30 min. After three washes with PBS, the cells were stained with
FITC-conjugated goat anti-mouse IgG and analyzed using FACScan.
B, surface labeling and immunoprecipitation. A total of
5 × 106 cells expressing the
M
2 heterodimer (lanes
1 and 2) or the
2 subunit alone
(lanes 3 and 4) were surface-labeled
by biotin and then lysed. The biotinylated
M
2 or single
2 was
immunoprecipitated with 5 µg of an
M-specific mAb 44a
(lanes 1 and 3) or a
2-specific mAb 6.7 (lanes 2 and
4), and analyzed on 7% SDS-PAGE. C, mAbYFC118.3
binding to the
2-expressing cells. Binding of
mAbYFC118.3 to the single
2 subunit in the presence of
different concentrations of Ca2+ was determined as
described for wild-type
M
2.
MI-,
LI-,
1I-,
2I-,
1I-, and
3I-domains
is supported by Mg2+ but not Ca2+ (47-50). In
fact, Ca2+ can inhibit ligand binding to several of these
integrins. As our data suggest that the
2I-domain
contains a unique high affinity Ca2+ binding site, we next
tested the effects of Mg2+ and Ca2+ on C3bi
binding by
M
2. As shown in Fig.
7, C3bi binding is supported by 1 mM Ca2+. This interaction can be further
increased by addition of Mg2+. However,
M
2 only exhibited minimal ligand binding
in the presence of Mg2+ alone (EGTA was added to exclude
possible contributions by Ca2+). As expected, addition of 1 mM EDTA completely abolished C3bi binding to
M
2, confirming the cation dependence of
the C3bi/
M
2 interaction. Thus, in the
case of
M
2, Ca2+ is not
inhibitory but is required for ligand binding.
View larger version (22K):
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Fig. 7.
Ca2+ dependence of C3bi binding
to
M
2.
C3bi binding to wild-type
M
2 was measured
in the presence of 1 mM Ca2+
(hatched bar), 2 mM Mg2+
plus 1 mM EGTA (gray bar), 1 mM Ca2+ plus 1 mM Mg2+
(solid bar), or 1 mM EDTA
(open bar) as described in Fig. 4. The value for
C3bi binding in the presence of both Ca2+ and
Mg2+ was taken as 100%. Specificity was verified using
mock-transfected 293 cells in the presence of 1 mM
Ca2+ and 1 mM Mg2+. Data shown are
the means ± S.D. of three independent experiments.
Asterisk (*) represents a value of less than 1%.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2I-domain (residues 125-385),
using a homolog-scanning mutagenesis approach. Our major findings are as follows. 1) The majority of the hydrophilic surface of the
2I-domain is not critically involved in heterodimer
formation between the
M and
2 subunits.
2) Although the epitopes of several function-blocking mAbs map to the
putative
2I-domain, these epitopes are not involved
directly in ligand binding to
M
2. 3) The
positioning of these epitopes is consistent with an I-domain-like fold
for this region of the
2 subunit, as proposed by several
investigators (10, 15-18, 42). 4) Of particular note, segment
Pro192-Glu197, which has been implicated in
direct ligand contact by the
3 integrins (21, 22, 43),
is not critical to ligand binding by
M
2,
suggesting a fundamental difference between the ligand binding
mechanism by
2 versus
3. 5)
The optimal conformation of the
2I-domain for C3bi
binding depends on a functional Ca2+ binding site within
the
2 subunit.
2) of the integrin
subunits in
/
association. This region of
1
(residues 121-329) forms a heterodimer with
5
(160) (47), and the same region of
3 (residues
111-318) complexes with
IIb (1) (51). In addition, many naturally occurring point mutations within this region,
Asp128, Leu149, Gly169,
Pro178, Lys196, Gly273,
Gly284, and Asn351 (31-37), preclude
cell-surface expression of the
2 integrins. Furthermore,
swapping residues V275GSDNH between human and avian
3 was found to change the specificity of
IIb/
3 association (51). Taken together,
these data strongly implicate this central region of the
subunits
in either heterodimer formation or in controlling the pairing
specificity between the
and
subunits. None of the known
subunits complex with both
1 and
2, and
thus we expected heterodimer formation would be perturbed in some of
our homolog-scanning mutants, particularly the one involving segment
Asp290-Glu298, which is homologous to
V275GSDNH of
3 (51). Nevertheless, when all
16 nonconserved segments within the
2I-domain, including
segment Asp290-Glu298, were replaced with
their counterpart sequences within the
1I-domain, surface expression and heterodimer formation were not affected, as
assessed by surface labeling and immunoprecipitation experiments. Therefore, we conclude that the majority of the hydrophilic residues of
the
2I-domain do not make a significant contribution to
the heterodimer formation and specificity pairing of the
M and
2 subunits. As most of the
hydrophobic residues are identical between
1I- and
2I-domains, it is possible that the remaining few
non-identical hydrophobic residues within the
2I-domain,
most of which have been mutated and found not critical in this study
(see Fig. 1), are responsible for the specific paring between
M and
2. Alternatively, as the C-terminal
cysteine-rich region is not involved in heterodimer formation (52, 53),
the N-terminal plexin-homologous region is a likely candidate for
determining the specificity of the heterodimer formation. It should be
noted that most of the naturally occurring point mutations that prevent
cell surface expression occur at conserved sites in the
subunit,
and these could affect the overall fold of the
I-domains, leading to
intracellular degradation (the exception to this is Lys196,
which is not conserved, but when we substituted a Glu at this position,
surface expression also was not affected).
subunits (residues 125-385 for
2) folds into an
I-domain-like structure, similar to that present in several integrin
subunits (10, 18). However, homology between the I-domains of the
and
subunits is very low, particularly in the C-terminal
portions, and conflicting views exist in the literature as to whether
this region assumes an I-domain fold or merely contains a metal binding MIDAS motif (DXSXS), such as that found in
I-domains (14-17). Using the 16 homolog-scanning mutants of the
2I-domain, we have mapped the epitopes of nine mAbs
(MHM23, IB4, 6.5E, TS1/18, CLB54, YFC118.3, R3.3, H20A, and 685A5). All
of these mAbs reactive with the putative
2I-domain
recognized epitopes that are composed of at least two non-contiguous
sequences. For discussion purposes, these epitopes can be divided into
two groups (Fig. 8): the first group
contains segments Leu154-Glu159,
Glu344-Asp348, and
His354-Asn358 as part of their epitopes. This
group includes TS1/18 (Leu154-Glu159 and
Glu344-Asp348), CLB54
(Leu154-Glu159and
His354-Asn358), and YFC118.3
(Arg144-Lys148,
Leu154-Glu159, and
His354-Asn358). The second group includes
segments Arg144-Lys148,
Pro192-Glu197, and
Asn213-Glu220 as part of their epitopes. This
second group includes MHM23, H20A, IB4, and R3.3 (
Arg144-Lys148 and
Pro192-Glu197), and 685A5
(Arg144-Lys148,
Pro192-Glu197, and
Asn213-Glu220). The spatial relationship of
these segments is consistent with the I-domain fold such that protein
folding will bring the distal segments
Leu154-Glu159,
Glu344-Asp348, and
His354-Asn358 (group 1), or
Arg144-Lys148,
Pro192-Glu197, and
Asn213-Glu220 (group 2) together into spatial
proximity to form the overlapping mAb epitopes. Our mapping results are
consistent with a very recent study, in which the epitopes of some mAbs
from group 1 were mapped and used to construct a three-dimensional
model for the
2I-domain (42). Although our results
support this model, there is one major difference. The identification
of mAbs (MHM23, H20A, IB4, R3.3, and 685A5) belonging to group 2 allows
us to define more accurately the position of the disulfide loop,
C191PNKEKEC198, within the three-dimensional
framework. The positioning of this loop is particularly important,
given the recent report implicating the segments equivalent to
Pro192-Glu197 within this loop in ligand
binding to
3 and
5 (22, 43). As shown in
Table II, segments Arg144-Lys148,
Pro192-Glu197 (within the disulfide loop), and
Asn213-Glu220 form the epitope for mAb 685A5
and must, therefore, be located in the vicinity of each other. Thus,
the disulfide loop region Pro192-Glu197 should
be positioned in the lower portion of the
2I-domain, close to segment Arg144-Lys148 (helix 1) and
segment Asn213-Glu220 (helix 2) (Fig. 8). Such
an arrangement is different from the proposed location of this
disulfide loop on the upper portion of the I-domain by Huang et
al. (42).
View larger version (59K):
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Fig. 8.
Epitopes of function-blocking mAbs in
the 2I-domain. The
structure of the
2I-domain is modeled according
to the crystal coordinates of the
MI-domain (10) and a
recently published model of the
2I-domain by Huang
et al. (42). The model is further modified based on the
epitope mapping data in Table II using the Biosym software. The
backbone of the
2I-domain is shown with helix 1 in
green, helix 2 in silver, helix 6 in
cyan,
-sheet 6 in purple, the disulfide loop
in yellow, and the bound Ca2+ in
blue. The epitopes identified in this study can be divided
in two groups. Group 1 (yellow circle) includes
mAbs TS1/18 (Leu154-Glu159 and
Glu344-Asp348), CLB54
(Leu154-Glu159and
His354-Asn358), and YFC118.3
(Arg144-Lys148,
Leu154-Glu159, and
His354-Asn358); and group 2 (cyan
circle) includes mAbs MHM23, H20A, IB4, and R3.3
(Arg144-Lys148 and
Pro192-Glu197), and 685A5
(Arg144-Lys148,
Pro192-Glu197, and
Asn213-Glu220).
Recent studies from several laboratories have implicated segment
179-183 of 3, which is homologous to
Pro192-Glu197 of
2, in ligand
binding (21, 22, 43). However, the
M
2(Pro192-Glu197)
mutant interacted well with C3bi and the
-module of Fg, similar to
the wild-type receptor, suggesting that this segment is not involved
directly in ligand contact within the
2 integrins. Thus, there appears to be a fundamental difference between the ligand binding
requirements of
3 and that of
2. Of note,
the integrins
IIb
3 and
V
3 that utilize this sequence in ligand
binding lack I-domains within their
subunits. Therefore, integrins
with or without I-domains in their
subunits may employ different
mechanisms for ligand binding. Support for this hypothesis also can be
derived from recent findings showing that the W2 and W3 repeats within the
-propeller of the
subunits are located in close proximity to
the sequence corresponding to Pro192-Glu197 of
2 within their
subunits, and together contribute to
formation of the ligand binding site (43). Since the I-domains within the
subunits are predicted to insert between the W2 and W3 repeats (54), this geometry would be altered and not be available for ligand
binding to the
2 integrins.
In this study, we mapped the epitopes of nine 2-blocking
mAbs to specific regions within the
2I-domain. The
epitopes were restricted to six segments
(Arg144-Lys148,
Leu154-Glu159,
Pro192-Glu197,
Glu344-Asp348,
Asn213-Glu220, and
His354-Asn358). To determine whether these
segments also are involved in ligand binding, we examine their binding
of C3bi and Fg, two classic ligands of
M
2. All six
2 mutants
interacted well with C3bi and the
-module of Fg, in a manner
similarly to wild-type
M
2, except mutant
M
2(Arg144-Lys148),
which exhibited 50% adhesive activity of the wild-type receptor. These
data suggest that none of these segments is critically involved in
ligand binding of
M
2. Therefore, it is
very likely that these
2I-domain specific mAbs, like the
1-specific function-blocking mAb described by Mold
et al. (55), inhibit receptor functions allosterically. We
cannot exclude the possibility that some of these mAbs sterically
hinder ligand binding. However, several activating mAbs of the
1 integrins map to the homologous region within the
1I-domain (25), suggesting that this region is
conformationally flexible, consistent with an allosteric mechanism. In
further support of this model, we found that mutant
M
2(Asn213-Glu220),
which interacted more avidly with both C3bi and the
-module (Fig. 4,
A and B), exhibited an active conformation,
judged by its reactivity toward an activation-dependent mAb
24.2 This mAb has been used
in a number of studies to probe the activated state of several
2 integrin receptors (49, 56, 57). Investigation of the
underlying mechanism of activation is currently under way.
It has been well established that integrin-ligand interactions are
cation-dependent, but the nature and location of these cation-binding sites are currently unclear. Recently, a novel cation
binding site, termed the MIDAS motif, was identified in the crystal
structures within the I-domains of several subunits and was found
to be central to ligand binding functions of these I-domains (10,
58-61). Evidence for the existence of MIDAS motifs in the I-domains of
1,
2, and
3 has also been
developed (14, 16, 17, 47, 51, 62, 64). Although the I-domains of the
and
subunits are predicted to have similar MIDAS folds, their
cation binding properties appear to differ significantly. Using several
different approaches including x-ray crystallography, circular
dichroism, and fluorescence, it appears that cation binding to the
I-domains of the
subunits and the
1,
3, and
5 subunits can lead to changes in
conformation and ligand binding activity (14, 44, 45, 47, 50, 63).
Compared with the
1 and
3 subunits, the
role of cation binding in controlling the conformation of the
2 subunit is not well understood. In this study, we
report that the binding of two mAbs (TS1/18 and YFC118.3) recognizing non-contiguous regions within the
2I-domain depend on
Ca2+ for optimal recognition of
M
2 (Fig. 5). A single Ca2+
binding site with a Kd value of ~105
µM was estimated for both mAbs. This
Kd value is very similar to that determined for
Ca2+ binding to the
LI-domain (50 µM) (45) and those obtained for Mg2+ binding
to the I-domains of
1,
2,
1, and
5 (80-100 µM) (14, 47, 50), suggesting that the cation binding site that controls the
conformation of the
2I-domain is most likely located
within the
2I-domain itself. To test this hypothesis, we
evaluated Ca2+ binding activity of
M
2(S136A), in which the predicted
coordinating residue within the MIDAS motif was changed. Using mAb
YFC118.3 and FACS analysis, we found that the Ca2+ binding
affinity obtained for this mutant
2 was significantly lower than that of wild-type
2 (151 ± 10 µM for the mutant versus 105 ± 9 µM for wild-type
2, p < 0.03) (Fig. 5C). Our results are in agreement with the
studies of Lin et al. (14), showing that mutations of the
residues within the MIDAS motif of the
5I-domain changed
the apparent affinity of Mg2+ for
v
5 from 80-180 µM to
125-300 µM. To exclude the possibility that
Ca2+ could affect YFC118.3 and TS1/18 binding to the
2I-domain allosterically by binding to
M
(via the Ca2+ binding site within either the
MI-domain or the
-propeller), we expressed single
2 on the cell surface. We found that the
2 subunit alone, in the absence of
M or
any other
subunits, still possessed a high affinity
Ca2+ binding site, which is required for optimal binding of
mAbs YFC118 and TS1/18 to the
2I-domain (Fig. 6). The
calculated Kd is 83 µM, which is very
close to that of the
M
2 heterodimer (105 µM). These data strongly suggest that the
Ca2+ binding site that promotes YFC118.3 and TS1/18 binding
to the
2I-domain is located within the
2
subunit, possibly composed of Ser136 of the MIDAS motif.
However, since mutation of Ser136 did not completely
abolish Ca2+ binding, residues outside the MIDAS motif may
also be involved in Ca2+ coordination.
Given the specificity of the 2I-domain for
Ca2+, we next tested whether this Ca2+ binding
site plays a role in ligand binding by
M
2, and found that C3bi binding to
M
2 was supported more effectively by
Ca2+ than Mg2+ (Fig. 7). In light of the report
that Ca2+ does not support C3bi binding to the recombinant
MI-domain (48), the Ca2+ binding site that
supports C3bi binding of
M
2 is likely
located within the
subunit, most probably in the
2I-domain. A similar cation-binding site was reported in
the
1 subunit that modulates both ligand binding and mAb
12G10 recognition by integrin
5
1 (46,
65). This mAb (12G10) recognizes an epitope
(Val211-Met287) within the
1I-domain similar to that of TS1/18 and YFC118.3, and
its binding depends on a single high affinity cation binding site with
a Kd of 70 µM for Ca2+
(46). Mold et al. (46) proposed that divalent cations
induced conformational changes within the
1I-domain,
leading to an unmasking of the ligand binding site within
5
1. Given the similarity between the
Ca2+ binding sites within the
1I- and
2I-domains, it is very possible that the same mechanism
is involved in the modulation of
M
2 function by Ca2+.
In summary, using homolog-scanning mutagenesis, we have systematically
probed the hydrophilic surface of the 2I-domain. Our data suggest that the majority of the hydrophilic regions of the
2I-domain are not critically involved in the specific
association of
2 with
M. Additionally, we
have mapped the epitopes of nine
2-specific mAbs into
two separate groups within the
2I-domain and showed that
the spatial arrangement of the residues that constitute these mAb
epitopes is consistent with an I-domain-like fold in this region. Most
importantly, our data strongly demonstrate that the ligand binding site
within
2 is distinct when compared with that of
3. This fact leads us to hypothesize that integrins
containing I-domains in their
subunits may utilize different
regions of the
I-domains for ligand recognition than the integrins
lacking I-domains in their
subunits. In addition, our C3bi binding
and Fg adhesion data showed that the epitopes of the nine
2I-domain specific function-blocking mAbs are not
critically involved in ligand binding, implying that they block
M
2 functions by allosteric mechanisms.
Finally, we have demonstrated that both the conformation of the
2I-domain and C3bi binding to
M
2 depend on a functional Ca2+ binding site, which is located within the
2 subunit and probably in the
2I-domain.
As C3bi binding to the
MI-domain is supported by
Mg2+, but not Ca2+ (48), our data suggest a
role for the Ca2+ binding site within the
2I-domain in C3bi-
M
2
interactions. Given the high degree of homology between all integrin
subunits, these conclusions should extend to other integrins as well.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Edward Plow for critical reading and editing of this manuscript and Dr. Tom Haas for help with protein modeling. We are also grateful for the help of Teresa Hawley with cell sorting and to Drs. Andrew and Medved for providing valuable reagents.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the American Heart Association and National Institutes of Health Grant HL61589.
To whom correspondence should be addressed: Dept. of Vascular
Biology, American Red Cross Holland Laboratory, 15601 Crabbs Branch
Way, Rockville, MD 20855. Tel.: 301-738-0657; Fax: 301-738-0465; E-mail: zhangl@usa.redcross.org.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M008903200
2 Y. Xiong and L. Zhang, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Fg, fibrinogen; DPBS, Dulbecco's phosphate-buffered saline; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
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