(Received for publication, January 6, 1997, and in revised form, February 24, 1997)
From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom, the § Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, and the ¶ Max-Planck-Institut für Biochemie, 82152 Martinsreid, Germany
The integrin 1
1 is a cell surface receptor
for collagens and laminin. The
1 subunit contains an A-domain, and
the A-domains of other integrins are known to mediate ligand binding.
To determine the role of the
1 A-domain in ligand binding and the
extent to which it reproduced the ligand binding activity and
specificity of the parent molecule, we produced recombinant
1
A-domain and tested its ability to bind collagens and laminin. In solid
phase assays, the A-domain from
1 was found to bind to collagen I, collagen IV, and laminin in a largely cation-dependent
manner. The
2 A-domain, from the
2
1 integrin, also bound to
these ligands, but the binding hierarchy differed from that seen for
1. This is the first demonstration of laminin binding by A-domains.
Specificity of A-domain-ligand binding was further investigated using
the triple-helical proteolytic fragment of collagen IV, CB3, and its subfragments, F1 and F4.
1 A-domain bound to all three fragments, while the
2 A-domain bound CB3 less well and exhibited little binding to F1 and no binding to F4. These differences mirror previous reports of distinct integrin binding sites in collagen IV and for the
first time identify a limited proteolytic fragment of a ligand that
contains integrin A-domain binding activity. To gain insight into the
contribution that the A-domain makes to ligand binding within the whole
integrin heterodimer, we measured binding constants for
A-domain-collagen interactions using surface plasmon resonance
biosensor technology. The values obtained were similar to those
reported for intact integrin binding, suggesting that the A-domain is
the major collagen binding site in the
1
1 and
2
1
integrins.
Integrins are a family of heterodimeric cell surface
receptors, responsible for cell-cell and cell-extracellular matrix interactions. The specificity and regulation of these interactions is
critical to many biological processes, including embryonic cell
migration, wound healing, and the immune response (1). The integrin
family contains at least 16
subunits, seven of which contain an
~200 amino acid inserted domain in their N-terminal region (I or
A-domain) (2, 3). This domain is homologous to the von Willebrand
factor A-domain, a module also found in a number of other membrane,
plasma, and matrix proteins (2, 4).
Mapping studies that have localized the epitopes of anti-functional
monoclonal antibodies to the A-domains of the 1,
2,
L,
M,
and
X integrins initially suggested a role for A-domains in ligand
binding (5-10). More recently, it has also been demonstrated that
isolated recombinant A-domains from
2,
L, and
M are capable of
binding ligands (11-15).
The integrin 1
1 is a receptor for collagen I, collagen IV, and
laminin (16). These interactions, like all integrin-ligand binding
events, are cation-dependent and require Mg2+
or Mn2+; Ca2+, however, does not support
binding (17). A similar pattern of ligand binding is found for the
closely related integrin
2
1, which also interacts with collagen
I, collagen IV, and laminin (16).
1
1 and
2
1 have, however,
been shown to differ in their relative affinities for ligands, with
1
1 binding collagen IV and laminin with higher affinity (18).
Integrins
1
1 and
2
1 also bind to a proteolytic fragment of
collagen IV, CB3 (19), but are believed to have distinct binding sites
within this fragment (18).
To investigate the role of the 1 A-domain in ligand binding, we have
generated recombinant A-domain in a bacterial expression system and
tested its binding to a range of integrin ligands. The specificity of
A-domain ligand binding has also been examined by comparing recombinant
1,
2, and
M integrin A-domains. We find that integrin
1
A-domain binds to collagen I, collagen IV, and laminin in a saturable,
concentration-dependent manner and that the binding can be
inhibited by an anti-functional anti-
1 monoclonal antibody
(mAb).1
2 A-domain also binds these
ligands, while
M A-domain does not. The
M A-domain does, however,
bind to fibrinogen, while the
1 and
2 A-domains do not. This
binding is largely cation-dependent, and real time binding
studies using surface plasmon resonance (SPR) analysis suggests that
cation, A-domain, and ligand may form a ternary complex. By using
proteolytic fragments of collagen IV, the ligand specificity of the
closely related
1 and
2 A-domains has been compared. The results
indicate that recombinant A-domains retain the specificity reported for
intact receptors.
Oligonucleotides were synthesized on an Applied
Biosystems 391 DNA synthesizer. Antibodies were obtained from the
following sources: monoclonal mouse anti-human integrin 1, 5E8D9
(Upstate Biotechnology Inc., New York); monoclonal mouse anti-human
integrin
2, Gi9 (The Binding Site, Birmingham, United Kingdom.) and
16B4 (previously reported as 1C11) raised as described by Mould
et al. (20); monoclonal mouse anti-human integrin
M, 44 (Autogen Bioclear, Devizes, UK); anti-glutathione
S-transferase (GST) rabbit polyclonal antibody (Autogen
Bioclear). Rat tail tendon type I collagen, human placental type IV
collagen, and human fibrinogen were obtained from Sigma, Poole, Dorset,
UK. Rat laminin was obtained from Life Technologies Ltd., Paisley, UK.
The collagen IV fragments CB3, F1, and F4 were produced as described
previously (18, 19).
The production
of recombinant 2 A-domain has already been described (12). The
1
and
M A-domains were produced in a similar manner. DNA coding for
the A-domains from integrins
1 and
M was produced by reverse
transcriptase-polymerase chain reaction (RT-PCR). Total RNA prepared
from the A375 human melanoma cell line (for
1) or human buffy coat
lymphocytes (for
M) was a gift of L. J. Green (University of
Manchester, UK). First strand cDNA was generated using a 3
primer
spanning the predicted end of the integrin A-domains and incorporating
a SalI site (
1, 5
-TTTGTCGACTCAGGCTTCCAGG GCAAATATTCTTTCTCC-3
;
M,
5
-TTTGTCGACTCAACCCTCGATCGCAAAGATCTTCTCCCG-3
). Polymerase chain
reaction (PCR) amplification of this cDNA was then carried out
using the thermostable proof-reading DNA polymerase Pfu (Stratagene
Ltd., Cambridge, UK) with the above 3
primer and a 5
primer designed
to produce DNA coding for 17 amino acids preceding the predicted start
of the integrin A-domain (21) and including a BamHI
site (
1, 5
-TTTGGATCCGTCAGCCCCACATTTCAAGTCGTGAATTCC-3
;
M,
5
-TTTGGATCCAACCTACGGCAGCAGCCCCAG-3
). 50 cycles, each consisting of 1 min at 94 °C, 1 min at 55 °C, and 2.5 min at 72 °C, were carried out. PCR products, of the correct Mr,
were then excised from a 1% agarose gel. After digestion with
BamHI and SalI, the products were ligated into
pUC119 and used to transform Escherichia coli strain
DH5
F
. A-domain sequences from transformants were sequenced by the
dideoxy chain termination method of Sanger (22) and compared with the
published sequences (
1 and
M, see Refs. 23 and 24, respectively).
The sequenced DNA was then subcloned into the expression vector
pGEX-4T3 (25) (Pharmacia, Milton Keynes, UK). Transformants were
screened, and protein was produced as described for
2 (12) except
that dithiothreitol was not required for GST removal from
1 and
M
A-domains. For experiments using fusion protein, a single glutathione
affinity step was performed and thrombin digestion was not carried out.
The recombinant A-domains produced were 214 (
1), 224 (
2), and 211 (
M) amino acids long. N-terminal amino acid sequencing of
thrombin-cleaved
1 and
M A-domains confirmed the predicted
starting sequence.
A-domain-GST fusion proteins, collagen I, and fibrinogen were biotinylated as follows. Protein was diluted to 1 mg/ml in 150 mM NaCl, 10 mM Tris-HCl, pH 7.5 (TBS), and dry sulfo-N-hydroxysuccinimido-biotin (Pierce, Chester, UK) was added to give a ratio of 1:1 (w/w) protein:biotin. The mixture was incubated for 1 h at room temperature and then dialyzed against TBS (or 0.1 M acetic acid for collagen I) to remove unincorporated biotin.
Ligand Binding AssaysBinding of soluble ligand to
immobilized A-domain was measured using assays adapted from Tuckwell
et al. (12). A-domain fusion proteins (10 µg/ml in PBS
without divalent cations, PBS) were coated to 96-well microtitre
plates (Immulon 4, Dynatech, Billingshurst, UK) overnight at 4 °C.
Wells were then blocked with 50 mg/ml BSA in TBS for 1 h at room
temperature and, washed twice with TBS, and biotinylated ligand in TBS,
1 mg/ml BSA, plus cation, was added for 3 h at 37 °C. Plates
were washed three times with TBS, 1 mM MnCl2,
and ExtrAvidin peroxidase (Sigma; 10 µg/ml in TBS, 1 mM
MnCl2) was added for 15 min. After washing three times with
TBS, 1 mM MnCl2, bound ligand was visualized
with 0.1 M sodium acetate, 0.05 M
NaH2PO4, 2 mM
2
2
-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 0.03%
H2O2. Wells were read at 405 nm on a plate reader.
Assays measuring binding of soluble A-domains to immobilized ligand were carried out essentially as above, but plates were coated with ligand (usually at 30 µg/ml) instead of fusion protein and binding of biotinylated A-domain fusion protein was measured. For antibody detection of unbiotinylated A-domain fusion protein binding to ligand, the assay was performed as above up to the streptavidin step where 5 µg/ml anti-GST antibody in TBS, 1 mg/ml BSA, 1 mM MnCl2 was added for 1 h instead. Wells were washed three times with TBS, 1 mM MnCl2, and 1:500 peroxidase-linked goat anti-rabbit antibody (Sigma) in TBS, 1 mg/ml BSA, 1 mM MnCl2 was added for 45 min. Wells were washed three times with TBS, 1 mM MnCl2, and binding was detected with ABTS as above.
Surface Plasmon ResonanceThe kinetic parameters (apparent
association and dissociation rate constants ka and
kd, respectively) and the apparent equilibrium
constant (KD) for A-domain binding to collagen I and
IV were measured using SPR on a BIAcoreTM (Biacore, St.
Albans, UK). This biosensor device was used in accordance with the
manufacturer instructions. Briefly, collagen I or IV was covalently
coupled via primary amine groups to the dextran matrix of a CM5 sensor
chip. A-domain solution was flowed over the chip at 5 µl/min, and
binding was measured as a function of time. TBS, 1 mM
MnCl2 was used as running buffer throughout, and injections
of TBS, 10 mM EDTA were used to remove bound A-domain, regenerating the surface for further binding experiments. The curve
fitting software, BIAevaluation, was used to fit these results to the
simple first order interaction model A + B AB. This produced values
for ka and kd, allowing the
apparent equilibrium constant KD to be derived from
kd/ka. To measure binding in the
presence of divalent cations other than Mn2+, a 10-min
injection of TBS, 10 mM EDTA was passed over the chip (to
remove any residual Mn2+) immediately prior to injection of
A-domain in the specified cation.
The
reverse transcriptase-polymerase chain reaction was used to generate
integrin 1 A-domain cDNA from A375 human melanoma cell line RNA
and integrin
M A-domain cDNA from human buffy coat lymphocyte
RNA. After cloning into pUC119, sequencing revealed that the
M
A-domain cDNA matched the published sequence (24). Sequencing of
1 A-domain cDNA revealed three differences from the published
sequence (23): 1) an inserted Thr at position 502; 2) a deleted Ala at
position 511, which put the sequence back in frame after the earlier
insertion, and 3) a Cys to Thr mutation at position 674. These
differences result in two changes in the predicted amino acid sequence
of the protein, a lysine to glutamate at position 170 and a threonine
to isoleucine at 228, numbered from the start of the mature
polypeptide. Repeating the RT-PCR using two different A375 RNA samples
produced the same sequence, and RT-PCR using human smooth muscle RNA
also gave the same sequence. As all PCR reactions used a proof-reading
DNA polymerase and produced the same sequence from different RNA
samples, we believe that it represents an accurate human
1 integrin
sequence.
Integrin A-domain cDNAs were cloned into the pGEX-2T3
expression vector and used to transform E. coli. After
induction, transformants expressed GST-A-domain fusion proteins of
~50 kDa as reported for the 2 A-domain (12). Fusion proteins were
purified on glutathione-agarose columns and were either used directly
or cleaved with thrombin and passed through a second
glutathione-agarose column to produce purified 25 kDa A-domain.
N-terminal sequencing of purified A-domains showed that cleavage had
occurred at the expected site. SDS-PAGE showed the recombinant protein
was at least 90% pure (data not shown).
All A-domains reacted specifically with previously characterized mAbs:
the 2 A-domain bound a number of anti-
2 integrin mAbs, including
Gi9, an anti-functional mAb. The
1 A-domain was recognized by the
anti-functional anti-
1 integrin mAb 5E8D9 in ELISA, and
M
A-domain bound the anti-functional anti-
M mAb 44 (data not
shown).
The integrins 1
1 and
2
1 are reported to be
collagen receptors, while integrin
M
2 binds non-collagenous
ligands. To investigate the role of A-domains in binding collagen, the
binding of biotinylated collagen I to A-domain fusion proteins was
measured. Fig. 1 shows that the A-domain fusion proteins
from
1 and
2 integrins support dose-dependent,
saturable binding of collagen I, while the
M A-domain exhibits very
little binding. Collagen I shows a higher maximal binding to the
1
A-domain than to the
2 A-domain. The observed differences in binding
were not due to different coating efficiencies of the GST-fusion
proteins as the coating concentration chosen (10 µg/ml) gave very
similar, almost maximal, coating of all three fusion proteins to the
plate as measured by anti-GST antibody in ELISA (data not shown). The
differences between
1 and
2 A-domains may instead reflect
differences in the amount of correctly folded A-domain in the different
samples rather than a difference in the number of binding sites per
A-domain. Binding of collagen I was dependent on the conformation of
the triple helix, as heat denaturation of the collagen I at 50 °C
for 30 min inhibited its binding to
1 and
2 A-domains (data not
shown).
Fibrinogen Binds the
To confirm the specificity of
the A-domains for their ligands, we investigated binding of the known
M
2 ligand fibrinogen to all three A-domains. Biotinylated
fibrinogen bound to the
M A-domain in the presence of 1 mM Mn2+ but did not bind to
1 or
2
A-domains, above the GST control (Fig. 2). Interestingly
the binding to GST was higher than to BSA. Fibrinogen binding to
M
A-domain was reduced to GST levels by 5 mM EDTA indicating
that the binding was cation-dependent. The binding of
biotinylated fibrinogen to
M A-domain was also reduced to the levels
of the GST control by the anti-functional anti-
M mAb 44 (data
not shown).
Cation-dependence of Collagen I Binding to A-domains
Having
shown that the M A-domain interaction with fibrinogen required
divalent cations, we investigated the effect of different divalent
cations on biotinylated collagen I binding to
1 and
2 A-domains.
Fig. 3A shows that collagen binding to
1
A-domain was completely inhibited by EDTA and, therefore, requires
divalent cations. However, the nature of the divalent cation had little effect as Ca2+, Mg2+, and Mn2+ all
supported similar levels of binding. Of the three cations, Mn2+ supported the highest levels of binding. This cation
profile does not match that reported for the whole integrin, where
Mg2+ was required for ligand binding and Ca2+
did not support binding (17). Collagen binding to
2 A-domain was
inhibited by EDTA (Fig. 3B); however, in this case
Ca2+ supported only very little binding, and
Mg2+ and Mn2+ produced identical levels of
collagen binding. This is in agreement with previously published data
(12). Thus, while the A-domains from integrins
1 and
2 both bind
collagen I, they differ in their cation specificities.
The binding of soluble A-domain to immobilized collagen was also
investigated. This was carried out using biotinylated A-domain fusion
proteins or by antibody detection of the GST moiety on the A-domain
fusion protein. Both methods demonstrated
concentration-dependent saturable binding of 1 A-domain
to collagen I; however, EDTA only partially inhibited this interaction
(Fig. 4A). Inhibition was similar in both
biotinylation and antibody detection assays, with slightly more
inhibition seen for the antibody detection assays (data not shown). In
both assays, inhibition was maximal at lower A-domain protein
concentrations. Similar results were obtained for
2 A-domain binding
to collagen I (data not shown).
The discrepancy between cation-dependence of collagen binding to immobilized A-domain and A-domain binding to immobilized collagen is difficult to explain. The interaction appeared to be specific as, under these conditions, binding was still dependent on the triple-helical conformation of collagen and the native A-domain structure, as heat denaturation of either component strongly inhibited binding (Fig. 4B, and data not shown).
To address the apparent discrepancies in solid phase assay results, the
cation dependence of the A-domain-collagen interaction was further
investigated using SPR measurements on a BIAcore. Collagen I was
covalently immobilized onto the sensor chip, and binding of A-domain
was measured. These measurements were performed for both 1 and
2
A-domain fusion proteins at a range of concentrations (3-100 µg/ml).
Fig. 5 shows a typical sensorgram of
1 A-domain binding to collagen I in the presence of 1 mM
Mn2+. Association and dissociation phases are marked and
removal of bound A-domain with 10 mM EDTA is shown. EDTA
injection removed 86 ± 2% of the bound
1 A-domain (mean ± S.E.; n = 33) and 82 ± 8% of the bound
2
A-domain (mean ± S.E.; n = 10), indicating that
the presence of divalent cation is required for maintenance of the
bound complex, not simply for A-domain-collagen binding. GST failed to
bind collagen in these assays. Binding of fusion proteins in the
presence of different cations was also investigated, and, as already
seen for biotinylated collagen binding to A-domains, 1 mM
Mn2+, Mg2+, and Ca2+ all supported
1 A-domain binding while only Mn2+ and Mg2+
supported
2 A-domain binding. No binding occurred without addition of divalent cations (data not shown). This supports the ligand-binding assay results that demonstrated a cation-dependent
interaction.
In addition to
collagen I, 1
1 is also reported to bind collagen IV and laminin
(16). The binding of collagen IV could not be studied using immobilized
A-domains because collagen IV is a poor biotinylation
substrate.2 Antibody detection of
1
A-domain fusion protein binding to immobilized collagen I, collagen IV,
and laminin showed that the
1 A-domain bound all these ligands in a
concentration-dependent, saturable manner (Fig.
6A). The
1 A-domain exhibited very similar
levels of binding to collagen I and collagen IV, with laminin bound to a lesser extent. In addition, CB3, an integrin-binding proteolytic fragment of collagen IV, supported a similar level of binding to intact
collagen IV although binding was less at lower A-domain concentrations.
To test the specificity of the A-domain binding, we investigated cation
dependence and antibody inhibition at low A-domain concentrations. Fig.
6B shows that 0.2 µg/ml of biotinylated
1 A-domain
bound collagen I, collagen IV, laminin, and CB3 and that binding was
inhibited by EDTA and the anti-
1 integrin mAb 5E8D9.
Collagen IV and Laminin Are Ligands for the
The
integrin 2
1 is also a receptor for laminin and collagen IV, so we
investigated the binding of
2 A-domain to these ligands and compared
it with
1 A-domain. Fig. 7 shows that
2 A-domain bound to collagen IV and laminin; however, in this case, collagen I was
a better ligand than collagen IV and laminin. Furthermore, CB3
supported much less binding than collagen IV, unlike the case with
1. Thus, while
1 and
2 A-domains bound the same range of
ligands, comparison of Figs. 6A and 7 indicate that the
relative binding to these ligands differed between the two A-domains.
GST showed only background levels of binding to laminin, collagen I,
and collagen IV (data not shown).
Proteolytic fragments of collagen IV were used to
further investigate the differences between 1 and
2 A-domains and
the integrin binding sites in collagen IV. As described above, both
1 and
2 A-domains bound to CB3, which is consistent with results obtained for whole integrins in solid phase binding assays and cell
attachment assays (12, 18, 19). Further proteolysis of this fragment
produces four smaller fragments, F1-F4, and investigation of integrin
binding sites in these fragments demonstrated that both
1
1 and
2
1 were able to bind to F1 but only
1
1 bound to F4 (18). F4
lacks the N- and C-terminal regions present in CB3 and F1, which are
thought to contain the
2
1 recognition site. Antibody detection of
fusion protein binding demonstrated that
1 A-domain bound both F1
and F4 in a concentration-dependent saturable manner, while
2 A-domain bound only poorly, if at all, to F1 and not at all to F4.
This binding was inhibited by 5 mM EDTA (data not
shown).
Binding of 1 and
2 A-domain fusion proteins to
collagen I and IV was investigated on a BIAcore. BIAevaluation, the
kinetic analysis curve-fitting software supplied with the BIAcore, was used to determine the ka and kd
values. Prior to fitting the association and dissociation phases of the
curve, the binding of A-domain to a blank sensor chip was measured and subtracted from the binding curve. Binding to the uncoated surface was
negligible. Binding of a range of fusion protein concentrations (3-100
µg/ml) to collagen I and IV-coated chips was measured. Covalent
coupling of collagen I to the chip was much more efficient than for
collagen IV, and consequently, signals obtained for binding to collagen
I were higher than those to collagen IV. Binding of
2 A-domain to
collagen IV could be detected using the BIAcore; however, signals were
too low to allow accurate curve fitting. Values obtained are shown in
Table I. The BIAevaluation software provides a number of
statistical parameters to judge the fitting of the binding model to the
experimental data. These indicated that a simple association model
provided a good approximation to the experimental data; however,
residual plots, comparing the fitted data with experimental results,
indicated that a more complex interaction may occur. That the kinetic
constants produced provide a good measure of the molecular interactions
involved was shown by comparison of a simulated binding curve, produced
using the calculated constants, with the experimental results (Fig.
8). This demonstrated that both curves were very
similar. The binding curves obtained for A-domain binding to
immobilized collagens in solid phase assays can also be fitted to
produce apparent KD values for the interaction (26,
27). These values are shown in Table I and exhibit generally good
agreement with those obtained using the BIAcore.
|
We have produced a recombinant A-domain from the 1 integrin and
compared its ligand binding characteristics with recombinant A-domains
from the
2 and
M integrins. Our key findings are that (i) the
1 integrin A-domain is a largely cation-dependent ligand binding domain, (ii) the
1 and
2 A-domains show similar but distinct ligand binding specificities, and (iii) measurement of binding
affinities for A-domain-collagen interactions produces values
comparable with those reported for integrin-collagen interactions, suggesting that A-domains are the major collagen binding sites in
integrins.
Recombinant integrin A-domains and solid phase ligand-binding assays
have been used to investigate A-domain-ligand interactions. The results
showed that recombinant A-domains from 1 and
2 integrins bind
collagen I, collagen IV, laminin, and the collagen IV fragment CB3, but
not the
M
2 ligand fibrinogen. This is the first direct demonstration that the
1 A-domain is a ligand-binding module, and
the first observation of laminin binding by A-domains. Interestingly, while the
1 and
2 A-domains were found qualitatively to bind the
same ligands, their binding profiles were quantitatively different, and
their binding sites within collagen IV appeared to be separate. The
specificity of these interactions was confirmed by demonstrating the
requirement for native structure of both ligand and A-domain and
through antibody inhibition studies.
Published reports of the ligand specificity of 1
1 and
2
1
match these findings with isolated A-domains. Thus, cell binding, antibody blocking, affinity chromatography, and solid phase assays have
variously been employed to demonstrate
1
1 binding to collagen types I, III, IV, and VI and to laminin (5, 17, 18, 28-31). In
addition, the CB3 fragment of collagen IV and fragments F1 and F4 of
CB3 have been shown to contain the
1
1 binding site (18, 19). The
2
1 integrin binds a similar range of ligands (16); however, the
affinity of interaction with collagens is different from that of
integrin
1
1, and
1
1 appears to bind laminin better than
2
1 (18). The binding site for
2
1 in collagen IV has also
been localized to the CB3 fragment (19); however, the
1
1 and
2
1 binding sites are apparently separate (18). The A-domains thus
mimic almost all of the ligand binding activity of the intact
1
1
and
2
1 integrins and appear to be key ligand-recognition modules
within the intact heterodimer.
To characterize the A-domain-collagen interactions further and address
the relative contribution of the A-domain to integrin-ligand binding
affinity, the kinetics of binding were measured using SPR. These
results showed that the 1 A-domain has a higher affinity for
collagens than does the
2 A-domain. Comparison of apparent binding
affinities produced from analysis of solid phase binding data indicated
a similar pattern of binding; however, the differences between
1 and
2 binding were less notable. The apparent binding constants for
1
1 binding to collagen IV have been measured using both BIAcore
and solid phase binding and inhibition
assays.3 These techniques produced
KD values in the range of 1-5 nM, which
is in good agreement with the figures obtained for
1 A-domain
binding to collagen IV (6-34 nM; Table I). Kern et al. (18) have obtained similar values for
1
1 binding to CB3; however, this varied from 1 to 30 nM depending on the
divalent cations present.
2
1 binding to CB3 showed similar
affinity but was more sensitive to cations (1-110 nM)
(18). We measured the affinity of
2 A-domain for collagen IV as 115 nM. Taken together, these findings indicate that the
binding affinity of
1 A-domain and
1
1 binding to collagen IV
are similar, suggesting that the A-domain is the major ligand binding
site in the integrin. The variation in affinity reported for the intact
receptor is dependent on divalent cation and suggests that the binding
site can be regulated by different cations. As discussed below, this
may suggest allosteric regulation of the A-domain by cation-binding
regions lying outside the A-domain. As the recombinant A-domain is free
of this regulation, it may account for the variation between
KD values measured in the presence of
Mn2+ for the A-domains and those reported for whole
integrins. With this in mind, it should be noted that while the simple
association model closely approximates the BIAcore data, some
non-random variation from the model suggests that a more complex
interaction may occur; this may be due to conformational changes in the
A-domain.
Integrins are known to require divalent cations for ligand binding, and
a number of regions have been proposed to act as cation-binding sites.
The crystal structures of the M and
L A-domains have now been
solved and show a single divalent cation-binding site at one end of the
domain (4, 32). While it is accepted that intact integrins require
cations for ligand binding, cation dependence, independence, and
partial dependence have all been reported for isolated A-domain binding
to ligands (11, 12, 33, 34). Results reported here show largely
cation-dependent binding; however, some variation in
cation-dependence was observed. Binding of soluble A-domain to
immobilized collagen is only partially cation-dependent, while binding of biotinylated collagen to immobilized A-domain is
completely cation-dependent. BIAcore analysis of
A-domain-collagen binding showed an absolute requirement for divalent
cations, and binding that had already taken place could be reversed
with EDTA, suggesting that there is a requirement for cation to remain
bound during the integrin-ligand interaction. This suggests that
divalent cations are normally required for A-domain ligand binding;
however, some cation-independent binding may be seen due to non-native conformations of the recombinant A-domains.
The exact role of divalent cations in ligand binding remains unclear as
it is difficult to determine whether the cation binding produces a
ligand binding conformation in the A-domain or is itself required as a
bridge between ligand and A-domains. Data from mutagenesis and peptide
binding studies (14, 11) suggest that cation is not absolutely required
for A-domain-ligand binding but that it normally regulates
integrin-ligand binding. Qu and Leahy (35) have recently shown that the
crystal structures of the recombinant L A-domain in the presence and
absence of divalent cation are very similar, suggesting that the
cation-dependence of ligand binding is not due to stabilization of a
ligand binding conformation. The presence or absence of divalent cation
does, however, have a profound effect on the charge distribution on the
cation binding face of the A-domain. As epitope mapping and mutagenesis
studies suggest that the ligand binding and cation binding sites are
located on the same face of the molecule, surface charge might account for the cation requirement of ligand binding. Our finding that cation
was required to maintain ligand binding is consistent with both this
and a cation bridge model. In some recombinant A-domains, non-native
structures may mean that even in the absence of divalent cation the
conformation and charge distribution are such that ligand binding can
occur.
In the intact integrin, the situation is further complicated by the
presence of other cation-binding sites in the and
subunits.
Cation regulation of the whole integrin does not always match that seen
in the isolated A-domain. For example, it is reported that
Ca2+ will not support collagen binding by
2
1, or
1
1; indeed, Ca2+ may actually inhibit
Mg2+-induced collagen binding (18, 17). We have shown that,
while Ca2+ will support only low levels of collagen binding
to
2 A-domain, it will allow binding to
1 A-domain. It is
possible that, while the A-domain in the intact
1
1 will bind
Ca2+, potentially permitting ligand binding,
Ca2+ binding at other sites on the integrin normally
precludes binding.
In conclusion, we have demonstrated that the A-domain from 1
integrin can bind ligands and that laminin is a ligand for A-domains. We demonstrate that distinctions between
1
1 and
2
1 binding to collagen IV are also observed with isolated A-domains and that the
affinity of A-domain binding to collagen is similar to that reported
for whole integrin binding. Finally, we show that A-domain-ligand binding is largely divalent cation-dependent and suggest
that cation, ligand, and A-domain form a ternary complex.
We are grateful to Dr. Linda Green for the gifts of RNA and to Dr. Sylvie Cot from Biacore for critical review of BIAcore results.