(Received for publication, June 19, 1995; and in revised form, August 4, 1995)
From the
Integrin-ligand interactions are known to be dependent on
divalent cations, although the precise role of cations in ligand
binding is still unclear. Using the interaction between 5
1
and fibronectin as a model system, we have performed a comprehensive
analysis of the effects of Mn
, Mg
,
and Ca
on ligand binding. Each cation had distinct
effects on the ligand-binding capacity of
5
1: Mn
promoted high levels of ligand binding, Mg
promoted low levels of binding, and Ca
failed
to support binding. Studies of the effects of different combinations of
cations on ligand binding indicated that the cation-binding sites
within
5
1 are not all identical, or of broad specificity, but
instead each site shows a distinct preference for one or more cations.
Ca
strongly inhibited Mn
-supported
ligand binding, but this inhibition was noncompetitive, suggesting that
Ca
recognizes different cation-binding sites to
Mn
. In contrast, Ca
acted as a
direct competitive inhibitor of Mg
-supported ligand
binding, implying that Ca
can displace
Mg
from the integrin. However, low concentrations of
Ca
greatly increased the apparent affinity of
Mg
for its binding site, suggesting the existence of
a distinct high affinity Ca
-binding site. Taken
together, our results imply that the ligand-binding capacity of
5
1 can be regulated in a complex manner through separate
classes of binding sites for Mn
,
Mg
, and Ca
.
Cell adhesion is of fundamental importance to many normal
biological processes, including wound healing, embryonic cell
migration, and the function of the immune system. Conversely, aberrant
cell adhesion contributes to the pathogenesis of a large number of
common human disorders such as rheumatoid arthritis, atherosclerosis,
and tumor cell metastasis in cancer. Many cell-cell and cell-matrix
interactions are mediated by members of the integrin superfamily of
cell-surface receptors. Integrins are heterodimers that have
been classified into eight different groups according to the identity
of their
subunit. The
1 family is the principal group of
cell-matrix receptors (Hynes, 1992, 1994; Ruoslahti et al.,
1994).
Integrin-ligand interactions are dependent on divalent
cations, but the precise role of divalent cations in ligand binding has
not yet been elucidated. The N-terminal portion of the integrin
subunits comprises seven homologous, tandemly-repeated domains of
50 amino acid residues. Domains 4-7 (or in some subunits,
5-7) contain sequence motifs similar to the
Ca
-binding EF-hands in proteins such as calmodulin.
However, the integrin divalent cation-binding motif differs from
classical EF-hand sequences in that it lacks an essential oxygenated
residue at the -z coordination position. Hence, it has
been proposed that integrin ligands, such as RGD, may supply a crucial
aspartate residue to complete the coordination geometry of the divalent
cation (Corbi et al., 1987; Humphries, 1990). This hypothesis
therefore suggests that divalent cations may act as bridge between
ligand and receptor.
Chemical cross-linking experiments have
provided direct evidence for the role of integrin EF-hand-like sites in
ligand binding. Specifically, the binding site of a sequence from the
fibrinogen chain has been mapped to the fifth repeat of
IIb
(D'Souza et al., 1990), and a peptide corresponding to
the EF-hand-like sequence in this repeat bound to fibrinogen in a
divalent-cation-dependent manner (D'Souza et al., 1991).
Similarly, cross-linking of an RGD peptide to
V
3 localized
the ligand-binding site between the second and sixth domains of the
V subunit (Smith and Cheresh, 1990).
The importance of cation
coordination in ligand binding by integrins has been demonstrated
directly by the covalent coupling of Co(III) to v
3 (Smith and
Cheresh, 1991). In addition, a recombinant fragment of
IIb that
spans the EF-hand-like domains has been shown to contain multiple
Ca
binding sites (Gulino et al., 1992), and
a modeling study of hybrid integrin-calmodulin EF-hands predicts the
integrin loop to support divalent cation chelation (Tuckwell et
al., 1992).
A highly conserved region is found toward the N
terminus of integrin subunits suggesting that this sequence may
be functionally important. Cross-linking studies have shown that this
region in the
3 subunit is proximal to the ligand-binding site
(D'Souza et al., 1988; Smith and Cheresh, 1988). In
addition, mutation of oxygenated residues in this region (Loftus et
al., 1990; Bajt and Loftus, 1994) results in a receptor that is
deficient in binding both cation and ligand. The sequence containing
these residues shows some homology to a cation-binding sequence found
in integrin I domains (Michishita et al., 1993; Bajt and
Loftus, 1994; Lee et al., 1995). In an important recent
advance, a synthetic peptide comprising this region of
3 (residues
118-131) was shown to bind both the Ca
analogue
Tb
and RGD peptides (D'Souza et al. 1994). Ligand binding caused displacement of cation from this
peptide, and a similar displacement of cations from
IIb
3 by
ligands was also observed. Based on these results, a mechanism of
integrin-ligand binding has been proposed, termed the ``cation
displacement hypothesis'' (D'Souza et al., 1994).
In this mechanism, cation, ligand, and receptor initially form a
ternary complex in which ligand is bridged to the integrin through the
cation, and cation is subsequently displaced from the ligand-binding
site.
Cation binding by integrins has also been shown to be
associated with conformational changes. For example, expression of the
epitope recognized by monoclonal antibody (mAb) ()24 on
L
2 is dependent on Mg
(or
Mn
), and mAb 24 epitope expression correlates with
the ability to bind ligand (Dransfield and Hogg, 1989; Dransfield et al., 1992a, 1992b). Hence, an alternative hypothesis for
the role of divalent ions in integrin function is that cation binding
is required to cause a conformational change in the integrin that
renders it competent to bind ligand.
A number of important questions
concerning the cation-binding sites on integrins are currently
unresolved. First, do the cation-binding sites bind only one type of
divalent cation, or do they all have a broad specificity? Second, is
there only a single cation-binding site involved in ligand recognition
or can occupancy of more than one site support ligand binding? Third,
why does Mn confer a much higher ligand-binding
affinity on many integrins than Ca
or Mg
(Gailit and Ruoslahti, 1988; Altieri, 1991; Elices et
al., 1991; Dransfield et al., 1992b; Kern et
al., 1993; Sanchez-Aparicio et al., 1993)?
The
extracellular matrix glycoprotein fibronectin has served as a prototype
substrate for the study of integrin-ligand interactions, and several
regions of the molecule have been shown to be responsible for its
adhesive activity. One domain that is recognized by a wide variety of
cell types lies close to the center of the fibronectin subunit and
contains the tripeptide RGD as a key active site (Pierschbacher and
Ruoslahti, 1984; Yamada and Kennedy, 1984). The integrin 5
1
is the major receptor for this central cell-binding domain (CCBD) and
is expressed on many cell types. Here we have studied the role of
Mn
, Mg
, and Ca
in
modulating
5
1-fibronectin interactions. We show that either
Mn
or Mg
, but not
Ca
, can support ligand binding. However,
Ca
strongly modulates ligand binding supported by
Mn
or Mg
and acts as a direct
competitive inhibitor of Mg
-supported binding but not
of Mn
-supported binding. Our results suggest the
existence of distinct classes of cation-binding sites for each divalent
ion.
Pooled fractions were
then mixed with 2 ml of mAb 16-Sepharose (5 mg IgG/ml Sepharose) for 2
h on ice. The suspension was then packed into a 0.8-cm diameter column
and washed with 12 ml of buffer C. Bound material was eluted with
buffer D, and 0.5-ml fractions were collected and neutralized with 0.1
ml of 1 M Tris-HCl, pH 8.2. Aliquots of the fractions (25
µl) were analyzed by SDS-polyacrylamide gel electrophoresis using a
6% nonreducing resolving gel. The only bands detected by Coomassie Blue
staining were those corresponding to expected positions of the 5
and
1 subunits.
5 and
1 were the only integrin subunits
detected in the eluted fractions by ELISA.
Figure 1:
Effect of Mn,
Mg
, and Ca
on the binding of CCBD
fibronectin fragment to
5
1 integrin in a solid phase assay (A) and on the attachment of K562 cells to CCBD fragment (B). Binding of biotinylated CCBD fragment in A or
cell attachment in B was measured for a range of
concentrations of each individual cation.
, Mn
;
, Mg
;
,
Ca
.
Figure 2:
Effect of Ca on
Mn
-supported binding of CCBD fragment to
5
1. The ability of Ca
to interfere with
ligand binding supported by 100 µM Mn
was measured for a range of Ca
concentrations.
In the experiment shown above, the level of ligand binding in 8
mM Ca
alone was 0.032 ±
0.010.
Figure 3:
A,
effect of 8 mM Ca on the binding of CCBD
fragment to
5
1 at varying Mn
concentrations.
, Mn
alone;
,
Mn
with 8 mM Ca
. B, double-reciprocal plot of the data shown in A. By
linear regression analysis, the two lines intersect approximately on
the x axis, indicative of noncompetitive inhibition. r
values are 0.997 (Mn
alone)
and 0.997 (Mn
with 8 mM
Ca
).
Figure 4:
Effect of Ca on the
binding of CCBD fragment to
5
1 supported by a low
concentration of Mg
. The ability of Ca
to modulate ligand binding was examined for a range of
Ca
concentrations.
, 50 µM Mg
with Ca
;
,
Ca
alone.
Figure 5:
A, effect of 0.25 mM Ca on the binding of CCBD fragment to
5
1 in the presence of varying concentrations of
Mg
.
, Mg
alone;
,
Mg
with 0.25 mM Ca
. B, double-reciprocal plot of the data shown in A. By
linear regression analysis, the two lines intersect above and to the
right of the origin, indicative of a mixed-type inhibition. r
values are 0.997 (Mg
alone)
and 0.955 (Mg
with 0.25 mM
Ca
).
Figure 6:
A, comparison of the effects of 0.2 mM and 8 mM Ca on the binding of CCBD
fragment to
5
1 in the presence of varying concentrations of
Mg
.
, Mg
with 0.2 mM Ca
;
, Mg
with 8
mM Ca
. B, double-reciprocal plot of
the data shown in A. By linear regression analysis, the two
lines intersect approximately on the y axis, indicative of a
competitive inhibition. r
values are 0.973
(Mg
with 0.2 mM Ca
) and
0.999 (Mg
with 8 mM Ca
).
Similar results were
obtained in the two assay systems (Fig. 1, A and B). These data suggest that cell-surface 5
1 and
5
1 in solid phase assays behave in a similar manner with
respect to the divalent cation dependence of receptor-ligand
interactions. Mn
, Mg
, and
Ca
had markedly different effects on
5
1-fibronectin interactions. Both Mn
and
Mg
promoted ligand binding, although Mn
supported higher levels of binding than Mg
. In
contrast, Ca
supported little or no binding. In the
cell attachment assay, low levels of attachment were observed in the
absence of cations, and this level was decreased with increasing
concentrations of Ca
. Comparison of the concentration
of Mn
and Mg
to give half-maximal
ligand binding in the solid phase assay (Table 1) suggested that
the affinity of Mn
for its binding site(s) on
5
1 was
40-fold higher than that of Mg
.
Scatchard-type analysis of the binding curves (not shown) indicated
that there was only a single site (or a single class of sites) for
Mn
and Mg
on
5
1 for which
cation occupancy supports ligand binding. Such sites have been termed
``ligand-competent'' sites (Smith et al., 1994).
Since the solid phase assay was found to be highly reproducible, and also avoided possible artifacts from cation effects on cellular components other than integrins, we chose to use this assay for a detailed study of the effects of combinations of cations on ligand binding.
To further analyze the effects of
Ca on Mn
-supported ligand-binding,
we examined the effects of varying the concentration of Mn
at constant Ca
. Fig. 3A shows
the inhibition of ligand binding by 8 mM Ca
.
Ca
greatly reduced the maximal level of ligand
binding but did not significantly alter the concentration of
Mn
required for half-maximal ligand binding. A
double-reciprocal plot of these data (Fig. 3B)
indicated that the inhibition observed at high Ca
concentrations is noncompetitive in nature. A detailed analysis
of the effects of lower Ca
concentrations on
Mn
-supported ligand binding (not shown) suggested
that Ca
binding at multiple sites on the integrin was
responsible for its inhibitory effects; however, Ca
binding to any of these sites did not decrease the apparent
affinity of Mn
for its ligand-competent site. An
important inference from these studies is therefore that,
Ca
does not compete with Mn
for
binding to the Mn
ligand-competent site on
5
1, and therefore appears to bind to different sites.
To analyze the inhibition of
Mg-supported ligand-binding by high Ca
concentrations (the second phase in Fig. 4) we compared
the effect of 0.2 mM Ca
(a concentration
that caused maximal stimulation of binding supported by low
Mg
concentrations) with that of 8 mM (Fig. 6A). High concentrations of Ca
increased the concentration of Mg
required for
half-maximal ligand binding but did not affect the maximal amount of
ligand bound at high Mg
concentrations. The
reciprocal plot (Fig. 6B) shows that the inhibition of
ligand binding at high Ca
concentrations is
competitive in nature. By nonlinear regression analysis, the K
value was calculated as
2 mM.
Further analysis of the inhibition of Mg
-supported
ligand binding at different Ca
concentrations (not
shown) indicated that this inhibition is directly competitive, i.e. Ca
is able to compete with Mg
for binding to the Mg
ligand-competent site.
However, when this site is occupied by Ca
, the
integrin fails to bind ligand.
Taken together, these data suggest: (a) that there is a Ca-binding site of high
affinity, the occupancy of which converts the Mg
ligand-competent site into a high-affinity binding site for
Mg
and (b) that Ca
is,
however, also able to competitively inhibit the binding of
Mg
to its ligand-competent site. However, since
Ca
binds this latter site with only low affinity,
high concentrations of Ca
are required to oppose the
high affinity binding of Mg
. Importantly, the
observation that Ca
can act as a direct competitive
inhibitor of Mg
-supported binding but not of
Mn
-supported binding also suggests that the
ligand-competent sites for these two ions may be distinct.
In this report, we have performed a comprehensive analysis of
the effects of Mn, Mg
, and
Ca
ions on the ligand-binding capacity of the
integrin
5
1. Our data show the following. (a) Only
Mn
and Mg
support ligand binding.
Although Ca
does not support ligand binding, it
strongly modulates ligand binding supported by Mn
or
Mg
. (b) Ca
is a
noncompetitive inhibitor of Mn
-supported ligand
binding, suggesting that it does not compete with Mn
for binding to the Mn
ligand-competent site. (c) Ca
can either enhance or inhibit
Mg
-supported binding, depending on the concentrations
of each ion. The results suggest that Ca
can compete
directly with Mg
for binding to the Mg
ligand-competent site, but Ca
binding to a
separate high affinity site also greatly increases the affinity of
Mg
for its ligand-competent site. Taken together,
these findings indicate that
5
1 possesses several distinct
cation-binding sites, each of which has a different specificity for
Mn
, Mg
, and Ca
.
Our studies of the effects of Mn,
Mg
, and Ca
on fibronectin binding
to
5
1 showed that this interaction was strongly promoted by
Mn
, and to a lesser extent by Mg
.
This pattern has been observed for many other integrins including
1
1 (Luque et al., 1994),
2
1 (Staatz et
al., 1989; Kern et al., 1993),
3
1 (Weitzman et al., 1993),
6
1 (Sonnenberg et al.,
1988),
V
1 (Kirchhofer et al., 1991), and
L
2 (Dransfield et al., 1992b). Ca
does, however, support ligand binding by a small number of
integrins, including those of the
3 family (Kirchhofer et
al., 1991; Smith et al., 1994). In a previous study
(Gailit and Ruoslahti, 1988), Mn
and Mg
were found to support ligand binding by purified
5
1 in
liposomes, with similar values for the cation concentrations required
for half-maximal ligand binding as reported here. However,
Ca
was also found to support ligand binding in the
above study. Based on our findings that Ca
can
synergize with low concentrations of Mg
, this result
may have been due to contamination of the Ca
samples
with low concentrations of Mg
. Similarly, an
explanation for the low levels of K562 cell attachment we observed in
the absence of exogenous cations or in the presence of low
Ca
concentrations is probably that small amounts of
Mg
are released from the cells during the time course
of the experiment, high concentrations of Ca
were
observed to inhibit this effect. A recent study of myeloid cell
adhesion to fibronectin confirms our finding that Ca
alone does not support
5
1-ligand interactions and that
Ca
also inhibits Mn
- and
Mg
-supported adhesion (Davis and Camarillo, 1993).
We found that the affinity of 5
1 for Mn
was
40-fold greater than that for Mg
. It is a
common feature of integrin-ligand interactions that typically
>10-fold lower concentrations of Mn
are required
to support ligand binding than Mg
(Altieri, 1991;
Dransfield et al., 1992b; Kern et al., 1993;
Michishita et al., 1993; Luque et al., 1994; Smith et al., 1994), indicating that many other integrins also
contain one or more high affinity Mn
-binding sites.
An important implication from our observation that Ca
could competitively inhibit Mg
-supported ligand
binding but not Mn
-supported binding is that there
may be separate ligand-competent sites on
5
1 for
Mn
and Mg
. This may shed light on
why Mn
-supported ligand binding is of much higher
affinity than that supported by Mg
. We have also
found that Mn
causes a larger increase than
Mg
in the expression of an activation epitope on
5
1 recognized by the mAb 12G10 (Mould et al., 1995), (
)suggesting that Mn
is better than
Mg
at stabilizing a conformational change in the
integrin required for ligand recognition.
The existence of a
separate high affinity Ca-binding site, distinct from
the Mg
ligand-competent site, was suggested by the
observation that low concentrations of Ca
greatly
increased the apparent affinity of Mg
for its
ligand-competent site. In summary, our studies suggest that at least
three distinct cation-binding sites on
5
1 are involved in the
regulation of integrin activity; a tentative model of these sites is
shown in Fig. 7. Site 1 binds Mn
with high
affinity; Ca
does not appear to compete with
Mn
for binding to this site. Site 2 binds
Mg
with low affinity. Both sites 1 and 2 are
ligand-competent sites (Smith et al., 1994). Since, at high
concentrations, Ca
acts as a direct competitive
inhibitor of Mg
binding, Ca
can
also bind to site 2, although with low affinity. Site 3 is a
Ca
-binding site of high affinity with characteristics
of the ``effector'' site proposed by Smith et
al.,(1994). Ca
binding to this site dramatically
increases the affinity of Mg
for site 2.
Ca
binding at sites 2 and 3 (or possibly at
additional sites) may be responsible for its ability to
noncompetitively inhibit ligand binding supported by
Mn
. Our results clearly indicate that the
cation-binding sites on
5
1 are not all equivalent, neither
are they of broad specificity, but instead each site shows a distinct
selectivity for one or more cations. Binding of one cation to its
site(s) can also affect the affinity of a second cation for its
site(s); a similar cooperativity in cation binding has been observed
for proteins such as calmodulin that contain multiple EF-hands
(Strynadka and James, 1989). How the proposed cation-binding sites in Fig. 7correspond to the putative divalent cation-binding sites
in the
5 and
1 subunits and the molecular basis of their
specificity will be the subject of future investigations. Such studies
have the prospect of maping key sites involved in modulating integrin
function.
Figure 7:
Model of the type and specificity of
cation-binding sites in 5
1. Occupancy of site 1 by
Mn
, or occupancy of site 2 by Mg
,
renders the integrin competent to bind ligand. Although Ca
can compete with Mg
for binding to site 2,
Ca
occupancy of this site does not permit ligand
binding. Site 3 is a Ca
-binding site of high
affinity; occupancy of this site by Ca
increases the
affinity of Mg
for site 2 (K
40 µM). The term ``site'' in
the above context could refer either to individual cation-binding sites
or to classes of site; our data do not allow us to distinguish between
these possibilities.
The model shown in Fig. 7is similar in several
respects to that proposed to explain the effects of Ca and Mn
on ligand binding to
V
3 (Smith et al., 1994). In this study, Ca
was found
to be a mixed-type inhibitor of Mn
-supported
fibrinogen binding to
V
3: low concentrations of
Ca
slightly increased the affinity of Mn
binding to
V
3 but decreased the amount of ligand bound
at saturating Mn
concentrations. Since high
concentrations of Ca
completely inhibited ligand
binding, it was suggested that Ca
could also
competitively inhibit the binding of Mn
to its site.
Based on these results, a two-site model for
V
3 was proposed
in which Ca
binds to an effector site and
Mn
(or Ca
) to a ligand-competent
site (Smith et al., 1994). However, in a recent study of
osteopontin binding to
V
3 (Hu et al., 1995), it was
found that Ca
only decreased the association-rate of
Mn
-supported ligand binding but had no effect on the
dissociation-rate, suggesting that Mn
and
Ca
must bind to different sites on the integrin, with
one cation influencing the on-rate and the other the off-rate of ligand
binding. Hence this latter report endorses the concept that there are
cation-binding sites on integrins that selectively bind only one type
of divalent ion.
Our model of the cation-binding sites in
5
1 may be broadly applicable to the other integrins that show
similar divalent-cation requirements for ligand binding. For example,
it has been shown for the
L
2-ICAM-1 interaction that high
concentrations of Ca
can compete with
Mg
, but not with Mn
, for binding to
the integrin (Jackson et al., 1994). Low concentrations of
Ca
can, however, synergize with low concentrations of
Mg
to increase ligand binding (Marlin and Springer,
1987). Hence
L
2 may have a similar arrangement of
cation-binding sites to
5
1.
One intriguing implication
from our results is that there may be one cation-binding site on the
integrin that selectively binds Mn. Whether or not
Mn
(or other transition metals) have a role in the
regulation of integrin activity in vivo has been a matter of
some speculation. It has been estimated that the concentration of
Mn
in tissues is in the range 1-14 µM (Smith et al., 1994). Significant binding of fibronectin
to
5
1 was induced by these concentrations of Mn
in vitro, hence Mn
could potentially
act as a physiological effector of
5
1. Since the majority of
Mn
in the body is sequestered in bone, Mn
might also be an important regulator of integrins during bone
resorption. Alternatively, the effects of Mn
observed in vitro could be fortuitous and not relevant in
vivo. If it is possible to identify the
Mn
-binding site in
5
1 (e.g. through site-directed mutagenesis), then several approaches could
be adopted to resolve this question. This will be the subject of future
work.
In conclusion, we have shown that Mn,
Mg
, and Ca
appear to recognize
different sites on
5
1 and that each ion has distinct effects
on the capacity of
5
1 to bind ligand. Since these cations
have the ability to differentially regulate
5
1 function, it
will be important in the future to examine the in vivo consequences of fluctuations in divalent cation concentrations on
5
1-mediated cell adhesion and migration, for example, during
wound healing (Banai et al., 1990; Sank et al.,
1989).