(Received for publication, May 29, 1996, and in revised form, January 10, 1997)
From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
The 2
1
integrin binds collagen in a Mg2+-dependent
manner that is inhibited by Ca2+. Like the intact integrin,
purified recombinant proteins containing the
2 integrin
I domain, either alone or with variable numbers of
2
integrin EF hand metal binding sites, bound collagen in a
Mg2+-dependent manner, and Ca2+ did
not support binding. However, unlike the intact integrin, Ca2+ did not inhibit the
Mg2+-dependent binding of any of the fusion
proteins to collagen. Binding to collagen was saturable and blocked by
the
2
1 function blocking antibody 6F1.
Deletional analysis demonstrated that residues present within the
amino-terminal 35 amino acids contribute to the 6F1 epitope and are
required for Mg2+-dependent collagen binding.
The results indicate that the I domain contains a Mg2+
binding site that is essential for collagen binding and that the I
domain alone is sufficient for collagen binding. Binding is markedly
enhanced in a divalent cation-dependent manner by the
addition of the first EF hand motif. Mutation of the EF hand to an
inactive form completely abrogated the effect. The sites necessary for
Ca2+ inhibition are not present within the I domain or the
adjacent region containing the three EF hand sites.
The integrins are heterodimeric cell adhesion molecules that
mediate cell-cell adhesion and adhesion between cells and the extracellular matrix. They are widely expressed and function throughout development and adulthood in a variety of normal and pathologic processes (for review, see Ref. 1). The
2
1 integrin is expressed on several
different cell types, including endothelial and epithelial cells,
fibroblasts, lymphocytes, and platelets (2). The ligand specificity of
2
1 varies with cell type. While it serves
as a collagen receptor on platelets and fibroblasts, it can serve as
both a collagen and as a laminin receptor on endothelial and epithelial
cells (3, 4).
Cell adhesion to collagen mediated by the
2
1 integrin is dependent upon the
presence of divalent cations (5). Mg2+, for example,
supported the adhesion. Ca2+ could not substitute for
Mg2+ and inhibited the
Mg2+-dependent adhesion. The adhesion of
liposomes containing purified
2
1 integrin
to collagen was also found to depend on the presence of
Mg2+ and to be inhibited by Ca2+ (6). The
inhibition of Mg2+-dependent adhesion to
collagen of liposomes containing the
2
1 integrin occurred via a simple linear noncompetitive mechanism suggesting that Mg2+ and Ca2+ exert their
effects by binding to distinct sites on the
2
1 integrin. Further evidence that
Mg2+ and Ca2+ bind to distinct sites was
obtained when limited proteolytic digestion of
2
1 gave different cleavage patterns
depending on which divalent cation was present (7).
Several potential divalent cation binding sites present in the
2 integrin subunit may mediate the distinct effects of
Ca2+ and Mg2+. Within the extracellular domain
of
2 are three EF hand motifs. These structures were
originally described as Ca2+ binding sites in regulatory
proteins (8, 9) but have since been shown to be capable of binding
other divalent cations. The
2 subunit is a member of a
subset of integrin
subunits that contain an approximately 200 amino
acid domain located near the amino terminus often referred to as the I
(or inserted) domain. Many I domains, including the
M
integrin subunit I domain, contain an additional recently described
cation binding site, the metal ion-dependent adhesion site
(MIDAS)1 motif (10). The
2 I
domain also appears to contain a MIDAS motif since all five of the
amino acids that contribute to divalent cation coordination in the
M MIDAS motif are conserved in the
2
integrin I domain.
I domains share homology with the collagen-binding A domains of von
Willebrand factor and cartilage matrix proteins suggesting that
integrin I domains may be important determinants in ligand binding. The
1 subunit has been shown to be involved with the binding
of
1
1 to its ligands, collagen, and
laminin (11). Likewise, the
M I domain is required for
the interaction of the
M
2 integrin with
its ligands, ICAM-1, iC3b, and fibrinogen (10, 12). The
L I domain also appears to be important in the binding of the integrin
L
2 with its ligands,
ICAM-1, and ICAM-3 (13, 14). Similarly, several lines of evidence
implicate the involvement of the
2 I domain in ligand
binding activity of the
2
1 integrin. First, a polyclonal antiserum directed against a bacterially expressed
2 I domain fusion protein was shown to block the
attachment of endothelial cells to gelatin, type I collagen, and
laminin (15). Second, a series of human/bovine
2
integrin chimeras was generated and used to map the epitopes recognized
by anti-human
2 integrin monoclonal antibodies that were
capable of inhibiting the ligand binding activity of the human
2
1 integrin. All of these antibodies mapped to regions within the
2 I domain, revealing the
significance of the I domain with regard to collagen recognition (16).
Finally, several mutagenesis studies have demonstrated the importance
of amino acids within the
2 integrin I domain for ligand
binding (16, 17). Recently, recombinant
2 integrin
I domain expressed in bacteria as a glutathione
S-transferase (GST) fusion protein has been shown to bind
specifically to collagen. However, two reports (17, 18) have presented
conflicting data as to whether the I domain, like the intact integrin,
binds collagen in a divalent cation-dependent manner.
To elucidate the roles of the various divalent cation binding sites
present within the 2 subunit with regard to the metal ion-dependent function of the intact
2
1 integrin, we have expressed a series
of
2 integrin I domain-containing proteins with either none, one, two, or all three of the EF hand sites. Our results indicate
an essential role for the I domain and a heretofore unrecognized role
for the first EF hand motif in divalent cation-dependent collagen binding activity.
Complementary DNAs encoding the human
2 integrin subunit I domain and the I domain with one,
two, or all three of the EF hand divalent cation binding sites were
generated by PCR using full-length human
2 integrin
cDNA as template. The proteins encoded by this series constructs
will be referred to as I, I + 1, I + 12, and I + 123. All four of the
proteins in this series begin at Ser-124 and terminate at Met-349,
Gly-516, Lys-570, and Ser-620 of the published
2
sequence (19). In addition, cDNAs encoding a shorter I domain
protein lacking the 35 amino-terminal amino acids and the analogous I + 1 protein were also prepared. These shorter proteins, referred to as
I and
I + 1, begin at Trp-159 and terminate at Met-349 and
Gly-516, respectively. Thus these two proteins lack the
DXSXS portion of the MIDAS motif. The PCR primers
were designed such that all of the amplification products would contain
a BglII restriction site at their 5
ends and a stop codon
followed by an XhoI restriction site at their 3
ends. The
PCR products were digested with BglII and XhoI,
purified in agarose gels, and cloned into BamHI and
XhoI-digested GST fusion protein expression vector pGEX-5X-1
(Pharmacia Biotech Inc.). The sequences of all cDNAs used in this
study were determined using the dideoxy chain termination method (20)
and compared with the published
2 integrin sequence
(19).
The sequences of the oligonucleotides used for PCR were as follows: I
domain forward primer, 5-GAAGATCTCTCCTGATTTTCAGCTCTCAGCCAGC-3
; I
domain reverse primer, 5
-CCGCTCGAGTCACATTTCCATCTGAAAGTTGTCTCC-3
; I + 1 reverse primer, 5
-CCGCTCGAGTCAGCCTTCAAGAAATTGGTGCTGACC-3
; I + 12 reverse primer, 5
-CCGCTCGAGTCACTTTGTGCGGATAGTGCCCTGATG-3
; I + 123 reverse primer, 5
-CCGCTCGAGTCATGACCAGAGTTGAACCACTTGTCC-3
;
I
forward primer, 5
-GAAGATCTGGGATGCAGTAAAGAATTTTTTGG.
To prepare an I + 1 protein with a mutated EF hand, pGEX-5X-1/I + 1 was
digested with PstI and XhoI, and the 357-base
pair PstI-XhoI fragment was purified in an
agarose gel. The fragment was cloned into pBlueScript KS, previously
digested with the same enzymes. The Kunkel (21) method of site directed
mutagenesis was used to create a double mutant, D272KDAKA. This
protein will be referred to as I + 1*. The oligonucleotide used for the
mutagenesis reaction was antisense; its sequence was
5
-CACGTCTGTAATGGTGGCTTTAGCCACATCAACTGAAC-3
. The mutation was verified
by sequencing, and the 357-base pair PstI-XhoI
fragment containing the double mutation was cloned back into the
pGEX-5X-1/I + 1 background. Fig. 1 shows a schematic diagram of the
constructs used in this study.
Trial inductions were performed to determine whether the selected
clones could direct expression of appropriately sized GST fusion
proteins. E. coli DH5 containing each of the plasmid
constructs was grown at 37 °C in 60 ml of 2 × YT media
supplemented with 0.2% glucose and 100 µg/ml ampicillin. Uninduced
samples were removed from each culture when the A550
reached 0.3-0.4. Isopropylthiogalactoside was then added to a final
concentration of 1 mM, and the cultures were returned to
the incubator for 3 h to allow for accumulation of the expressed
proteins. Cell lysates from the uninduced and induced samples were
analyzed by SDS-PAGE (22) followed by Coomassie Blue staining. All of
the constructs directed the expression of recombinant proteins of the
expected size. The site of accumulation and degree of solubility was
determined for a representative I domain-containing protein using a
published cellular fractionation protocol (23). Bacteria harboring the
I + 123 construct were grown and induced as described above. At the end
of the induction period, the sample was fractionated into media,
periplasmic, cytoplasmic soluble, membrane, and insoluble fractions.
Each fraction was analyzed by SDS-PAGE, followed by Coomassie Blue
staining. The bulk of the recombinant protein (95-100%) accumulated
in the insoluble fraction. The other I domain-containing proteins
accumulated in the insoluble fraction as well.
For the purification of the fusion proteins, the inductions were
performed as above except that the culture volume was increased to 500 ml. At the end of the induction period, the cells were recovered by
centrifugation at 2600 × g for 10 min. The cells were
washed twice with 10 ml of ice-cold phosphate-buffered saline (10 mM Na2HPO4, 1.8 mM
KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4) and stored at 70 °C until needed.
Insoluble fractions were prepared and washed using the Triton X-100
procedure described by Marston (24). The recombinant proteins were
solubilized in 8 M urea on ice for 1 h. After removal
of urea-insoluble material by centrifugation at 12,100 × g for 20 min, the urea was diluted to 1.33 M
with 25 mM Tris-HCl, pH 8.0, 10 mM EDTA. The
proteins were purified by affinity chromatography on
glutathione-Sepharose (Pharmacia) according to a published method (25).
Following purification, the proteins were dialyzed extensively against
TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4).
Protein yields were determined using the BCA protein assay reagent
(Pierce). Fig. 2 shows a Coomassie Blue stained SDS-PAGE gel containing
approximately 5 µg/lane of each of the recombinant proteins used in
this study. All recombinant proteins were subjected to gel filtration
analysis on a 10 × 300 mm Superose 12 column (Pharmacia)
equilibrated with TBS containing 2 mM MgCl2
(Fig. 3).
Collagen and Laminin Binding Assays
The wells of a 96-well microtiter plate (Immulon 2, Dynatech Laboratories, Inc.) were coated overnight at 4 °C with 0.1 ml of 30 µg/ml collagen I from calf skin (Sigma) in 0.09% acetic acid or with 30 µg/ml laminin I (Collaborative Biomedical Products) in TBS. The wells were washed twice with 0.15 ml TBS and then blocked for 1 h at room temperature with 0.15 ml of 100 µg/ml bovine serum albumin (ICN Biomedicals, Inc.) in TBS. Recombinant proteins were diluted to 400 nM in various wash buffers (TBS containing 0.05% Tween-20, 10 µg/ml bovine serum albumin, and either 1 mM EDTA, 2 mM CaCl2, 2 mM CaCl2 plus 2 mM MgCl2, or 2 mM MgCl2). The wells were washed once with 0.15 ml of the appropriate wash buffer, and then 0.1 ml of each recombinant protein was added and allowed to interact for 1.5 h at room temperature. Wells were then washed three times with 0.15 ml of the appropriate wash buffer, and then 0.1 ml of a 1:500 dilution of anti-GST antiserum (Pharmacia) in the appropriate wash buffer was added for 1 h at room temperature. Following this incubation, the wells were again washed three times, and then 0.1 ml of a 1:4500 dilution of pig-anti-goat secondary antibody-horseradish peroxidase conjugate (Boehringer Mannheim) in the appropriate wash buffer was added for 1 h at room temperature. The wells were again washed three times, and 0.1 ml of tetramethylbenzidine dihydrochloride (Sigma) prepared according to the manufacturer directions was added per well. After 1 h of substrate conversion, reactions were stopped with 0.025 ml of 4 N H2SO4, and the plates were read at 450 nm.
ELISAI domain-containing proteins were diluted to 10 µg/ml in TBS containing 2 mM MgCl2 and used
to coat the wells of a 96-well microtiter plate (Immulon 2, Dynatech).
Coating was carried out overnight at 4 °C with 0.1 ml of
solution/well. The wells were washed twice with 0.15 ml of TBS
containing 2 mM MgCl2 and then blocked for
1 h at room temperature with 0.15 ml of TBS containing 100 µg/ml
bovine serum albumin and 2 mM MgCl2. Primary
antibodies used include anti-GST antiserum and anti-human
2 monoclonal antibodies 6F1 and 12F1. The anti-GST
antiserum was diluted 1:2500; the monoclonal antibodies were diluted to
1 µg/ml in wash buffer (TBS containing 0.05% Tween-20, 10 µg/ml
bovine serum albumin, and 2 mM MgCl2). Following blocking, the wells were washed once with 0.15 ml of wash
buffer, and then 0.1 ml of primary antibody was added and allowed to
interact for 1 h at room temperature. The wells were washed three
times with 0.15 ml of wash buffer, and then 0.1 ml of secondary
antibody-horseradish peroxidase conjugate (pig-anti-goat for anti-GST
or goat-anti-mouse for 6F1 and 12F1) diluted 1:4500 in wash buffer was
added per well. Substrate was added, and the plates read as described
above.
The wells of a 96-well microtiter
plate were coated with collagen and blocked with bovine serum albumin
as described above. I + 1 (100 nM) was preincubated with
anti-human 2 antibodies 6F1 or 12F1 (300 nM)
for 1 h at room temperature in wash buffer (TBS containing 0.05%
Tween-20, 10 µg/ml bovine serum albumin, and 2 mM
MgCl2). The wells were washed once with 0.15 ml of wash buffer, and then 0.1 ml of I + 1/antibody mixture was added and allowed
to interact for 1.5 h at room temperature. Detection of collagen
bound I + 1 was carried out as described above. Substrate was added,
and the plates were read at 450 nm.
As an approach to assess the contributions of distinct classes of
divalent cation binding sites present within the 2
integrin subunit to the collagen binding activity of the
2
1 integrin, the domains were expressed
as recombinant GST fusion proteins containing the
2
integrin I domain alone or in combination with one, two, or all three
of the EF hand-like motifs (I, I + 1, I + 12, I + 123). In addition,
the I domain was modified by deleting 35 amino acids from its amino
terminus in two constructs (
I and
I + 1). The EF hand motif of I + 1 was modified by incorporating two D
A point mutations to render
the EF hand motif incapable of metal binding (I + 1*). The recombinant
proteins examined in this investigation are presented schematically in
Fig. 1.
After purification by affinity chromatography on glutathione-Sepharose,
the proteins were subjected to analysis by SDS-PAGE (Fig.
2). The recombinant proteins were further analyzed by
gel filtration chromatography. This analysis confirmed the purity of
the proteins in agreement with the SDS-PAGE analysis and revealed the
lack of protein aggregation with less than 2.6% of the protein running
with an apparent size larger than that predicted for the monomeric
species. Quantitative analysis of I domain and I + 1 proteins revealed
that 95 and 96%, respectively, of the protein applied to the column
eluted in the monomer peaks. Representative profiles for the I, I + 1, I + 1 and I + 1* proteins are shown in Fig. 3.
Similar chromatographic profiles were obtained for each of the other
proteins used in this study (data not shown).
The proteins were then tested for collagen binding activity. To assess
the divalent cation specificity, if any, collagen binding assays were
conducted in the presence of 2 mM EDTA, 2 mM
Ca2+ or 2 mM Mg2+. The ability of
Ca2+ to inhibit any Mg2+-dependent
collagen binding was assessed by carrying out the binding assay in the
presence of 2 mM of both Ca2+ and
Mg2+. The results are shown in Fig. 4. GST
alone did not bind specifically to collagen under any of the divalent
cation conditions. As previously observed with the intact
2
1 integrin, all four of the proteins containing an intact I domain bound collagen in a
Mg2+-dependent manner. As also observed with
the intact integrin, Ca2+ did not support the collagen
binding activity of any of the I domain-containing proteins. However,
unlike the intact
2
1 integrin, Mg2+-dependent collagen binding activity of
the I domain-containing proteins was not inhibited by Ca2+.
Addition of the first EF hand motif onto the I domain appeared to
markedly enhance Mg2+-dependent collagen
binding activity as revealed by an increased extent of binding. This
was most apparent in the I + 1 and I + 12 proteins.
The necessity of an intact I domain for the collagen binding activity
of the constructs was examined with two fusion proteins containing
truncated I domains in which the amino-terminal 35 residues were
deleted (I and
I + 1). The deleted region contained the
DXSXS sequence, a region thought to be critical
for the structural integrity of the MIDAS motif (10). As expected and
as shown in Fig. 5, both the I and I + 1 constructs
bound collagen in a Mg2+-dependent manner.
However, neither of the truncated constructs (
I and
I + 1) bound
collagen. Thus an intact MIDAS motif is required for
Mg2+-dependent collagen binding activity.
The contribution of the putative metal binding sequences present within the first EF hand motif to the enhanced collagen binding activity of the I + 1 protein relative to the I domain was examined by mutating two aspartate residues essential for metal binding activity of the motif (Asp-272 and Asp-274) to alanines to create the I + 1* protein. Unlike the wild-type EF hand motif, the mutated EF hand conferred no enhanced collagen binding activity upon the I domain (Fig. 5). The collagen binding activity of I + 1* was comparable with that of I domain alone. Thus, sequences within the EF hand motif that confer metal binding properties upon the motif, are essential for the enhancement of collagen binding activity.
The enhanced collagen binding activity of the I + 1 construct relative
to that of the I domain alone was examined in greater detail and over a
range of concentrations (Fig. 6). Both the I and I + 1 proteins bound to collagen in a concentration-dependent and
saturable manner. Whereas half-maximal binding of the I domain protein
to collagen occurred at 820 nM, the half-maximal binding of
the I + 1 construct was observed at 87 nM.
Mg2+ concentrations required for half-maximal collagen
binding were determined for I domain, I + 1, I + 12, and I + 123 proteins and found to be 0.54, 0.31, 0.61, and 1.17 mM,
respectively (Fig. 7). Mn2+ was also shown
to support collagen binding of each of these constructs (Fig.
8). Approximately 0.5 mM Mn2+
was required for half-maximal collagen binding.
Anti-human 2 integrin monoclonal antibodies 6F1 and 12F1
were tested for their ability to bind the I,
I, I + 1, and
I + 1 fusion proteins. The results are shown in Fig.
9A. All of the proteins were recognized by
the anti-GST antiserum to similar extents, indicating that comparable
quantities of the proteins were coated onto the microtiter wells.
Monoclonal antibodies 6F1 and 12F1 both bound equivalently to the I and
I + 1 proteins that contained intact I domains. Neither bound to the
I and
I + 1 proteins containing truncated I domains. Both of the
antibodies also effectively recognized the I + 1*, I + 12, and I + 123 proteins (data not shown). The same patterns of reactivity were
observed in the presence of either 2 mM Mg2+ or
2 mM EDTA (data not shown). Thus, the two distinct complex, conformation-dependent epitopes recognized by the 6F1 and
12F1 antibodies (26, 27) are retained to comparable extents in the I
and I + 1 constructs, suggesting that the conformation of the I domain
in these two proteins is intact. Antibody 6F1 which inhibits the
binding of the intact
2
1 integrin to
collagen (26), also effectively blocked the binding of the I + 1 protein to collagen. 12F1, an antibody that does not inhibit the
binding of the intact
2
1 integrin to
collagen (28), similarly failed to inhibit the binding of I + 1 protein to collagen although, as shown in Fig. 9A, the 12F1
antibody effectively bound to the I + 1 protein. The effects of the 6F1
and 12F1 antibodies on collagen binding activity of the I + 1 protein
are shown in Fig. 9B.
Since the I and I + 1 proteins exhibited rather different collagen
binding activities, we next examined the binding of the two proteins to
laminin, a second ligand for the 2
1
integrin. When examined in either Mg2+ or
Mn2+-containing buffers, the I and I + 1 proteins bound
laminin to comparable extents (Fig. 10). Unlike binding
to collagen, however, binding to laminin was markedly enhanced in the
presence of Mn2+. Both I and I + 1 proteins were similarly
affected.
The adhesion of cells to collagen via the
2
1 integrin requires the presence of
Mg2+ (5). Ca2+ is incapable of supporting
2
1 integrin-mediated adhesion to collagen
and inhibits the Mg2+-dependent adhesion.
Liposomes containing purified
2
1 integrin demonstrated identical metal ion dependence (6). Recently, the I
domains of several integrins have been shown to be important determinants of ligand binding (10-14). Evidence of the involvement of
the
2 I domain in collagen recognition includes:
(a) an anti-
2 antiserum blocks endothelial
cell attachment to collagen (15); (b) several
2
1 integrin function blocking antibodies
map to the I domain (16); and (c) purified recombinant
2 I domain binds specifically to collagen (17, 18). The
crystal structure of the I domain of the related
M
integrin subunit has been solved and found to contain a single novel
Mg2+ binding site that involves residues that are widely
separated in the primary sequence (10). In addition to the
Mg2+ binding MIDAS motif within the I domain, the
2 integrin subunit contains three EF hand-like metal
binding sites in close proximity to the I domain. In the present study,
we have prepared a series of
2 I domain-containing GST
fusion proteins with various numbers of EF hand sites for the purpose
of establishing the contributions of the divalent cation binding sites
present within the I and EF hand domains of the
2
integrin subunit to the metal and ligand binding properties of the
integrin.
Like the intact integrin, each of the I domain-containing proteins
bound collagen in a Mg2+-dependent manner. As
also observed with the intact integrin, Ca2+ could not
substitute for Mg2+. Unlike the intact integrin, however,
Ca2+ did not inhibit the
Mg2+-dependent binding to collagen of any of
the I domain-containing proteins. These data suggest that the sites
responsible for Ca2+ inhibition of
Mg2+-dependent collagen binding activity are
either not present within the I domain or the region containing the
three EF hand structures or that their inhibitory effects are not
manifested outside of the context of the intact integrin. The finding
that isolated I domain binds collagen in a
Mg2+-dependent manner is in agreement with the
recent work of Tuckwell, et al. (18) but contrasts with
results from Kamata and Takada (17), who found that independently
expressed I domain bound collagen in a divalent cation-independent
manner. The reason for the discrepancy is not apparent but may
represent an adverse consequence of the iodination of the recombinant I
domain protein. We have previously observed deleterious effects on
2
1 integrin structure and ligand binding
activity as a result of iodination (7). The results of our binding
studies using the
I and
I + 1 proteins strongly support the
conclusion that the MIDAS site present within the I domain is critical
for the collagen binding activity of the
2 I domain.
A marked enhancement of collagen binding activity was consistently observed upon the addition of the first EF hand motif to the I domain. To determine if the increased binding was contingent upon the divalent cation binding activity of the EF hand, an I + 1 protein with a mutated EF hand was prepared. To ensure complete inactivation the EF hand site in I + 1, aspartates at positions 3 and 5 were mutated to alanines. Mutation of the EF hand site to an inactive form completely abrogated the increase in collagen binding observed for I + 1, reducing its collagen binding activity to that of the I domain. This finding indicates that the enhancement was likely a direct result of Mg2+ binding to the EF hand rather than to divalent cation-independent interactions of other regions of the added sequence.
Mg2+ concentrations required for half-maximal binding to
collagen of I domain, I + 1, I + 12, and I + 123 proteins ranged from 0.3 to 1.2 mM. These values are similar to those observed
for half-maximal 2
1 integrin-mediated
adhesion of platelets and of other cells to collagen (5, 29).
Mn2+ has been shown to support the adhesion of cells to
collagen at significantly lower concentrations than Mg2+
(10-30 µM) (29, 30). However, the I domain fusion
proteins required approximately 0.5 mM Mn2+ for
half-maximal collagen binding. This concentration is similar to that of
Mg2+ rather than the ten-fold lower concentration observed
with the intact integrin. These data suggest that for the I
domain-containing proteins, Mn2+ is substituting for
Mg2+.
Recently, the I domain from the M integrin subunit has
been crystallized in the presence of a limiting concentration of
Mn2+ (31), and the structure obtained from this analysis
was compared with that obtained when the protein was crystallized in
the presence of Mg2+ (10). A change in conformation as well
as a change in the way the metal ion was coordinated was revealed when
the protein was crystallized in the presence of Mn2+. The
differing divalent cation-dependent structures may
potentially reflect affinity modulation of the integrin. Our data
indicate that the conformational changes observed in the I domain are
themselves insufficient to confer significantly altered collagen
binding activity on the I domain protein(s). In contrast,
Mn2+ did markedly enhance binding of the I and I + 1 proteins to laminin. In this regard, it is noteworthy that only the
most activated form of the
2
1 integrin is
thought to exhibit laminin binding activity (32).
The antibody binding experiments are significant in their own right.
Both the 6F1 and 12F1 monoclonal antibodies bound to the proteins
containing an intact 2 integrin I domain (I, I + 1, etc.) in the absence of the
1 integrin subunit
indicating that the
1 subunit is not necessary for
formation of the epitope recognized by these two antibodies. Deletion
of the amino-terminal 35 amino acids of the I domain (residues 124-158
of the published
2 integrin sequence (19)) to form the
I and
I + 1 proteins resulted in loss of reactivity with both the
6F1 and 12F1 antibodies. Since the deletion destroyed the integrity of
the MIDAS motif, it was conceivable that critical divalent
cation-dependent structures essential for reactivity with
the antibodies may have also been destroyed. This seemed unlikely,
however, since the same patterns of antibody reactivity were observed
in both EDTA and Mg2+-containing buffers. These data,
therefore, suggest that in addition to the region of residues 173-259
identified by Kamata, et al. (16) in their study of
human/bovine chimeric
2 integrin, an additional
determinant present within residues 124-158 makes an important
contribution to the apparently complex epitopes recognized by the 6F1
and 12F1 antibodies.
In summary, the results of this investigation indicate, in agreement
with other recent studies, that the 2 integrin subunit I
domain is sufficient for collagen binding activity. The data obtained
in this study indicate that, while there appears to be a
Mg2+ binding site within the I domain that is critical for
collagen binding, the sites responsible for Ca2+ inhibition
are not present within the I domain or the region containing the three
EF hand structures or that additional portions of the intact integrin
are also required in conjunction with Ca2+ binding to
observe the inhibitory effect. These results are consistent with our
earlier observation that Mg2+ and Ca2+ exert
their effects by binding to distinct sites on
2
1 (6, 7).
Finally, our studies reveal several differences between the binding of the I and I + 1 proteins to collagen and laminin. Unlike binding to collagen, which was equivalent in the presence of Mg2+ or Mn2+, binding of both the I and I + 1 proteins to laminin was greatly enhanced in the presence of Mn2+. Furthermore, whereas binding to collagen was considerably enhanced by the addition of the first EF hand motif to the I domain, binding to laminin was essentially unaltered. The structural basis underlying these apparent mechanistic differences warrants further exploration.
We are grateful to Drs. Barry S. Coller and Virgil L. Woods, Jr., for generous gifts of the monoclonal antibodies 6F1 and 12F1, respectively. We thank Nancy L. Mathis for technical assistance.