(Received for publication, May 11, 1995; and in revised form, September 7, 1995)
From the
``Activation'' of integrins is involved in the
dramatic transition of leukocytes and platelets from suspension to
adhesion. The integrin is not known
to take part in this sort of transition, even though it shares its
subunit with
, the activable
integrin on platelets. In the context of a constitutively adhered cell,
changes in activation state may be more subtle in their effects, but
nonetheless important in regulating cell behavior. We hypothesized that
can undergo conformational changes
analogous to those associated with
activation. Accordingly, we examined
on the surface of M21 cells (a human melanoma cell line) and
found that, like
, it can undergo
conformational changes upon binding of a ligand analog and can be
activated for ligand binding and migration by a monoclonal antibody
directed against
. Modulation of the binding of this
activating antibody, AP5, ligand binding, and antibody-mediated
activation all are associated with a discrete cation-binding site
shared in both
and
. Based on a measured K
, this site has an apparent K
for calcium of approximately 20
µM. At physiological levels of calcium, about 40% of the
total
on a cell's surface is
in a conformation detected by AP5. The data suggest a model for both
and
function in which the molecule can
exist in either of (at least) two conformational states, one stabilized
either by AP5 or ligand binding, refractory to calcium binding, and
enhanced for ligand recognition, the other stabilized by calcium
binding and refractory to AP5 and ligand binding. Functional analysis
suggests that AP5 activates
by
preventing occupation of this calcium site, and that the activated form
of
differs functionally from the
basal form. The active form is more conducive to migration and the
basal to tight adhesion.
Integrins comprise a large family of heterodimeric cell-surface receptors involved in cell-matrix and cell-cell interactions. These receptors mediate adhesion and modulate the cell's responses to various adhesive ligands (for review, see (1) ). The integrin's role in modulating information flow recently has become a field of growing interest. Several potentially significant biochemical changes are known now to be regulated by adhesion events involving integrins, including changes in intracellular pH, changes and oscillations in intracellular free calcium, and phosphorylation on tyrosine of a number of proteins (1, 2, 3, 4, 5, 6) (for review, see (7) ).
Integrins bind divalent cations, which
are required for integrin function(8) . In addition to this
basic requirement of divalent cations, specific divalent cations have
been suggested to regulate integrins. For example, manganese
(Mn) appears to enhance the adhesive properties of
many integrins (9, 10, 11) although the
mechanism of this enhancement is not clear. A model has been proposed
by Smith and co-workers (9, 12) that a cation must be
displaced from the integrin
as
ligand binds. In this model, a calcium or other divalent cation is
bound to the integrin and is necessary for proper function, but must be
displaced as ligand binds.
Several integrins are conformationally
complex molecules. That is, they can exist in different conformational
states, or activation states, that affect ligand recognition. Examples
of this included and some
and
integrins on
leukocytes(13, 14, 15, 16) . In the
case of
, binding of ligand
analogs, such as RGD-containing peptides, induces a conformational
change that can be detected by a change in the Stokes radius of the
receptor(17) . This change occurs concomitantly with the
presentation of ``LIBS,'' or ligand-induced binding sites,
which are epitopes recognized by monoclonal antibodies known as
anti-LIBS antibodies(18, 19) .
``Activation'' of
results in a conformational change that can be detected by
changes in fluorescence resonance energy transfer (14) and
occurs concomitantly with increased efficiency of ligand binding and
changes in ligand specificity (reviewed in (16) ). It is
important to recognize that the so-called ``resting'' or
``inactive'' state of
is
a functional integrin(3, 20, 21) . Therefore,
we refer to it as the ``basal'' state. The binding of some
anti-LIBS antibodies results in changes in
consistent with activation; that
is, the antibodies stimulate binding of ligand ( (19) and
references therein).
We recently showed for the case of
that the conformational state can
affect not only ligand recognition, but ``outside-in''
signals as well(20) . We have hypothesized that integrins in
general exist on the cell surface as subpopulations in different
conformation, or activation, states with specific ligand-binding and
signaling functions. Implicit in this hypothesis is the assumption that
integrins on constitutively adhered cells can undergo changes in
conformation, or activation state, similar to those seen for
. Although the role of integrin
activation in the case of integrins that mediate the conversion from
suspension to adhesion in blood cells is obvious, the effect of
integrin activation in the context of an adherent cell might be less
dramatic.
To explore this possibility, we have taken advantage of a
conformation-sensitive antibody we have described previously. This
antibody, AP5, binds a linear epitope corresponding to the first 6
amino acids of the subunit(22) . It is an
anti-LIBS for
, that is its epitope
is presented following binding of soluble ligand analogs. It also is an
activating antibody; it stimulates the binding of soluble fibrinogen
either to platelets or to a cell line expressing
(20, 22) . Since
the presentation of LIBS following ligand binding is a measure of a
conformational change, we tested whether this conformational change
occurred in
on the surface of the
human melanoma cell line M21 and found that it did. AP5 also activates
, as measured by increased the
efficiency of cell adhesion to various ligands. Moreover, pretreatment
with AP5 resulted in an increase in M21 cell migration. We found that a
discrete calcium-binding site residing both in
and
was involved in regulating the conformational changes associated
with LIBS presentation and activation. Calcium inhibits the
presentation of the AP5 epitope on
with a K
of about 20
µM. Functional analysis suggests a model wherein AP5
activates
by preventing occupation
of an inhibitory calcium-binding site. Further, the binding
characteristics of the active and basal receptors were investigated in
intact cells. The active receptor promoted efficient, but weak,
interactions with matrix and favored migration. The basal receptor, in
contrast, promoted stronger adhesion and was less conducive to
migration. This result indicates that activation does not represent a
simple ``on/off'' switch of the integrin, but rather a change
in function and a resulting change in cell behavior.
Results were inspected to ensure that a homogeneous, monophasic distribution of fluorescence was obtained, and mean fluorescence intensity (MFI) was extracted from the histogram by the Lysys-II program.
where S and S
are the
slopes of the line without and with inhibitor, respectively, and
[I] is the concentration of inhibitor present. This was
necessary because the units of binding, MFI, could not be related
directly to concentration of antibody-antigen complex with our
instrumentation. However, the ratio of the slopes is dimensionless and
could be used to calculate K
. ``Without
inhibitor'' is 1 µM Ca
, since this
concentration resulted in maximal binding.
Figure 9: A comparison of adhesion under increasing stringency. For this experiment, adhesion to Opn, plus or minus AP5 pretreatment, was carried out as for Fig. 2except in complete DPBS. Following a 30-min incubation, each of four plates was subject to the indicated G force for 5 min. Values are average of 6 wells ± 1 S.D. &cjs2110;, -AP5; &cjs2108;, +AP5.
Figure 2: Adhesion of M21 cells to Vn and Opn was quantitated either with or without pretreatment with AP5 (100 µg/ml). Values are averages of 6 points ± 1 S.D.
Figure 1:
A, cells were exposed to FITC-labeled
AP5 at 10 µg/ml in DPBS on ice in the presence or absence of the
cyclic RGD analog, G4120 (40 µM) as indicated. The control
antibody was FITC-labeled anti- (CP8). Histograms represent fluorescence of 10,000 individual
cells. B, MFI was calculated from similar histograms at the
indicated concentrations of AP5, in the presence or absence of G4120,
as indicated.
Since AP5 is an activating antibody for
, we next wanted to determine if it
could activate
as well.
has been reported to support
adhesion to many extracellular matrix proteins, including vitronectin
(Vn), fibrinogen, fibronectin, laminin, denatured collagen I, and
osteopontin (Opn)(9, 11) . However, binding studies
with
often were performed under
different conditions. For example,
will only support adhesion to fibrinogen in the presence of the
divalent cation manganese (Mn
)(11) .
Similarly,
binding to Opn also is
stimulated by Mn
and inhibited by
Ca
(9) .
We therefore thought it likely
that binding to these substrates
might be regulated by activation and the alternate cations may induce a
kind of activation, as has been suggested ( (9) and references
therein). Accordingly, we examined the ability of AP5 to stimulate
adhesion of M21 cells to Vn or Opn in the absence of Mn
and in low Ca
(40 µM).
Mg
was included at 400 µM in these
experiments (see ``Materials and Methods''). The assay for
adhesion we employed was that of Calof and Lander(24) , which
includes a low speed centrifugation of the inverted plate to remove
unbound or loosely adhered cells. The results, expressed as the percent
of cells that remain adhered following 5 min at 100
g,
are shown in Fig. 2. AP5 increased the adhesion of cells to both
substrata under these conditions. We have performed more conventional
plate assays with similar results on fibrinogen as well (data not
shown). Adhesion to fibrinogen, Opn, and Vn in these latter assays was
inhibited completely by inclusion of 40 µM G4120 to block
. LM609 (150 µg/ml), a blocking,
anti-
antibody, inhibited 100% of the
adhesion to Opn and about 85% of adhesion to Vn. This residual adhesion
probably was due to
on the surface
of M21(11, 25) . For this reason, Opn adhesion was
used in most subsequent assays.
Figure 3:
Cells
were exposed to the indicated concentrations of FITC-labeled AP5 in the
presence or absence of G4120 (40 µM) either in DPBS or
DPBS lacking calcium. Bound AP5 was quantitated by flow cytometry and
is expressed as MFI for 10,000 cells. The data for the DPBS case are
exactly the same as those presented in Fig. 1B. ,
-Ca, -G4120;
, +Ca, -G4120;
,
-Ca, +G4120;
, +Ca,
+G4120.
Calcium is not exerting its effect on AP5 directly.
In equilibrium dialysis experiments, no binding of Ca to
AP5 was detected at concentrations that inhibit binding of AP5 to
(data not shown). Also, since the
effect of G4120 clearly is on the receptor, we would expect low calcium
and G4120 to have additive effects on the overall binding of the
antibody if the effect of the calcium were on the antibody.
Furthermore, results from detailed analysis of the inhibition (below)
are inconsistent with calcium exerting its effect on AP5.
Fig. 4shows the effect of calcium concentration over a range
from 100 nM to 2 mM on the binding of AP5. Starting
with a calcium-minus ``DPBS,'' buffers of the indicated
calcium concentration were made. The binding of AP5 is maximal at
approximately 1 µM Ca and is inhibited
significantly at 10 µM.
Figure 4: FITC-labeled AP5 (130 µg/ml) was exposed to M21 cells in a buffer based on DPBS, but made with the indicated concentrations of calcium. Washes were carried out in the same buffer as the incubations. MFI of 10,000 cells is presented. Maximal binding of AP5 occurs at approximately 1 µM calcium.
We performed a similar test of the effect of calcium on AP5 binding over a range of AP5 concentrations from 5-140 µg/ml, which is shown in Fig. 5A. These data show that even at high levels of calcium (900 µM) at which the inhibitory effect of calcium has reached a plateau, AP5 still binds in a dose-dependent manner. The shape of the curves suggests that there are two different effects of calcium, one in the range of 1-100 µM and a second effect at the higher concentrations.
Figure 5:
A, AP5 binding at various concentrations
of AP5 (4.4-140 µg/ml) and at various calcium concentrations
from 4-900 µM (in a DPBS-based buffer) was
quantitated by flow cytometry, and expressed as MFI for 5,000 cells versus calcium concentration (on a log scale). AP5
concentrations (mg/ml): , 140;
, 70;
, 35;
,
17.5;
, 8.75;
, 4.4;
, control. B, the same
data were plotted as 1/MFI versus 1/[AP5] for the
indicated calcium concentrations. As suggested by the shape of the
curves in A, these plots reveal two discrete effects of
calcium, a competitive inhibition between 4 and 100 µM,
and an essentially irreversible inhibition of AP5 binding to
approximately 60% of the total receptor at concentrations of calcium
above 100 µM. Calcium concentrations (mM):
, 0.1;
, 0.033;
, 0.011;
, 0.004;
,
0.3;
, 0.9.
We examined the inhibition of AP5 binding by
calcium by expressing the data as a double-reciprocal plot of
1/[AP5] by 1/MFI for the indicated concentrations of calcium
between 1 and 100 µM. This can be done because the off
reaction of the antibody is not significant over the period of the
experiment and thus the binding could be considered to be all forward
(not shown, but note that a linear double reciprocal plot could not be
obtained if a significant off reaction existed). The results shown in Fig. 5B show that AP5 and calcium binding are mutually
exclusive in this range. That is, calcium inhibits AP5 binding and vice
versa. This generally is termed ``competitive'' inhibition.
Such an effect does not necessarily imply that the two molecules
compete for the same site, but does require that the two sites be
linked structurally. From the ratio of the slopes of the lines, we
calculated the K of calcium. Data from several
independent experiments, including that shown in Fig. 5B, resulted in a calculated K
of 22 ± 8 µM.
At higher concentrations
(300 and 900 µM), the character of the inhibition changes;
this is the second, high concentration effect mentioned above. The
predominant effect at high concentrations of calcium is that the number
of sites available for AP5 binding (indicated by the y-intercept) is decreased to approximately 40% of the total.
Note that two lines corresponding to 300 and 900 µM calcium are essentially identical, indicating that the inhibitory
effect of Ca on AP5 binding had reached a plateau,
and that this resistant 40% remains accessible to AP5 even at high
calcium concentrations.
In true competitive inhibition, the off-rate of AP5 should be insensitive to calcium. The point at which the lines intersect falls slightly to the left of the y axis. This result, consistent in several experiments, could be explained if calcium had a small effect on the off rate of AP5 such that bound AP5 was being lost during the high calcium wash steps. Again, this off-rate would have to be extremely low, or the linear double-reciprocal plots would not be obtained. The left-shift was eliminated if washes were carried out in low calcium buffer (data not shown), indicating that the effect was occurring largely during the wash steps.
Our data indicate that calcium and AP5 do not sterically compete for the same physical site on the receptor, yet inhibit each other's binding. This is likely to occur via conformational changes in the receptor. Nonetheless, our data demonstrate that the calcium- and AP5-binding sites are linked structurally.
Figure 6:
AP5 binding to M21 cells was quantitated
at the indicated concentrations of Mg (supplied as
sulfate), in DPBS containing either 1 or 100 µM calcium.
Magnesium had no effect either on the binding of AP5 or on the
inhibition of AP5 binding caused by
calcium.
In
contrast, manganese does reverse the inhibitory effect of calcium. Fig. 7shows that, at low calcium, manganese does not increase
binding of AP5 to M21 cells (in fact, it has a small, but reproducible,
inhibitory effect). However, the inhibitory effect of calcium is
abrogated partially by 2 mM Mn. These data
are consistent with manganese inhibiting the binding of calcium to its
site, though the mechanism cannot be determined. Mn
may bind to a separate site and inhibit calcium binding
noncompetitively, or it may compete directly with calcium.
Figure 7:
An
experiment similar to that presented in Fig. 4(AP5 binding as a
function of calcium concentration) was performed either in the presence
or absence of 2 mM MnCl. Inclusion of manganese
has no effect on AP5 binding at low concentrations, but partially
overcomes the inhibition of AP5 binding detected at higher calcium
concentrations.
Figure 8:
Migration of M21 cells on Opn, measured in
a Transwell assay. Quantitation was achieved by counting fixed and
stained cells on the bottom surface of the membrane following a 20-h
incubation. Numbers are the average number of cells counted in
15,400 fields plus or minus on standard deviation. &cjs2110;, no AP5;
&cjs2108;, +AP5
An advantage of the Calof and Lander (24) adhesion assay is that the ``wash'' step is centrifugation of the plate in the inverted position, which can be varied in a precise manner. This allows us to measure both the efficiency and strength of adhesion of an intact cell. We compared the ability of the basal and active receptors to bind to Opn under increasing G force. The results, shown in Fig. 9demonstrate the somewhat surprising result that the active receptor is less able to resist force and therefore mediates weaker binding to Opn. As G force is increased, the added adhesion of AP5-treated cells is lost. Thus, adhesion via the active receptor is efficient but weak.
In this work, we report that the integrin
, like
, undergoes conformational changes
related to LIBS presentation and activation. AP5, an antibody that
activates
, also can activate
as measured by increased
-mediated adhesion of cells to
various ligands and by increased migration. These changes in
conformation associated with LIBS presentation and activation can be
regulated by a discrete cation-binding site, the K
of which is expected to be approximately 20-25 µM for calcium, based on our K
data. Functional
analysis suggests that AP5 activates
by preventing the inhibitory effects of calcium. Finally, our
data indicate that activation of integrins can be more than a simple
on/off switch. Activation of
results
in changes in the character of adhesion and in increased migration.
We have reported previously that RGD analogs increase both the
apparent affinity and total number of sites bound by
AP5(20, 22) . Our data here suggest that low calcium
recapitulates this effect. At low levels of calcium, below 100
µM, AP5 and calcium appear to compete for binding of
. However, at calcium concentrations
between 300 µM and 900 µM, levels still below
physiological calcium, the inhibition no longer appears competitive;
increasing levels of AP5 no longer can overcome the inhibition. This
could be due to two separate cation-binding sites, both affecting AP5
binding. Rather than propose that AP5 is sensitive to calcium binding
at two sites, we think the simpler explanation for this is that at
millimolar calcium concentrations, the 20 µM calcium-binding site is occupied effectively 100% of the time,
thereby not allowing AP5 access to 60-70% of the receptor at all.
At concentrations of calcium above 300 µM, only about
30-40% of the total is
accessible to AP5 at all, and this 30-40% remains accessible even
up to 10 mM calcium (not shown, but note that there is
essentially no change in the binding of AP5 between 300 and 900
µM calcium). Thus, AP5 reveals two distinct populations of
on the cell surface under
physiological calcium conditions. The AP5-positive population of
receptor must be maintained in that conformation by some mechanism,
perhaps regulated by the cell. The significance of the AP5-accessible
population is not yet known, although it should be noted here that it
is not strictly speaking an ``activated'' population.
The
inhibition of AP5 binding by calcium at low concentrations shows the
hallmarks of competitive inhibition. However, we have shown that AP5
binds a linear epitope comprising the first 6 amino acids of
GPNICT(22) . This sequence is extremely
unlikely to bind a divalent cation directly, since it lacks any acidic
residues and is near a basic residue, Arg-8. Furthermore, the data in Fig. 5, when examined in detail, suggest that AP5 and calcium do
not compete directly for the same site. Residue Cys-5 is predicted to
take part in a long-range disulfide bond (27) which suggests
that this epitope is part of a complex tertiary structure. A likely
explanation for the mutually exclusive binding of calcium and AP5 is
that each binds only one of two conformations of the receptor and, when
bound, stabilizes that conformation. In stabilizing its preferred
conformation of the receptor, each molecule inhibits the binding of the
other.
Since this calcium site is present in both
and
, it must be a structure common to
both heterodimers. It seems likely that this site resides at least in
part within the
molecule itself, although the
and
chains may be directly
involved.
A specific cation-binding domain has been proposed for the
subunit(28, 29) . This site was
originally proposed to be an ``E-F hand'' type of
cation-binding domain, but mutational analysis was not consistent with
this hypothesis(28, 29) . This same region of
shares considerable secondary structure similarity
with another divalent cation-binding domain, the ``A'' domain
of the integrin
subunit MAC-1. The crystal structure of this
domain recently has been solved(30) . Coordination of the metal
in this domain is incomplete, with the last coordination site provided
by the adjacent molecule in the crystal lattice. This is a
symmetry-related artifact, and the authors suggest that in the normal
case this last coordination site is provided by ligand. The authors
name this motif a MIDAS domain, for metal ion-dependent adhesion site.
A second possible E-F hand metal coordination site has been proposed
to comprise residues Ser-211-Gln-227 in
(28) . Interestingly, this sequence resides
within the overall structure containing the putative
``MIDAS'' domain in
(30) in a
proposed hydrophilic loop between
-helix 4 and
-strand D in
the secondary structure prediction. Cierniewski et al.(28) reported that a short peptide corresponding to this
sequence did not bind terbium, a trivalent surrogate for calcium.
However, the failure of a short peptide to bind a trivalent cation such
as terbium does not exclude the possibility that the same sequence in
the context of the whole protein coordinates divalent cations.
As an
assay, AP5 binding has the advantage of permitting assessment of
calcium binding within the intact heterodimer. Therefore, analysis of
mutants in the subunit, or even in the
and
subunits, should be useful in defining
the location of the inhibitory calcium-binding site and in testing
which model (the MIDAS or the E-F hand) best fits the cation-binding
site in
.
Smith and co-workers (9) have reported that manganese stimulates binding of ligand
to and that calcium is a mixed-type
inhibitor of the manganese-mediated stimulation. They also report (26) that calcium directly inhibits
binding to osteopontin. Our data
show that manganese antagonizes the ability of calcium to inhibit AP5
binding. It seems likely that our observation and those of Smith and
co-workers (9) are related and that manganese and calcium
antagonize each other's binding to
. Whether this is by direct
competition for the same site or not, we currently cannot say.
Available data on integrin-ligand
interactions on cells indicates that the effect of activation is on the
kinetics of the interaction, not the thermodynamics. For example, Cai
and Wright (31) showed that antibody binding could not
contribute the energy necessary to account for the increase in ligand
binding observed for activated if the increase was
due to an increase in affinity (that is, a change in the thermodynamics
or a
G). To explain some of the functional
distinctions between the active and basal forms of
, we offer the following formalism:
where I and L are integrin and ligand, [IL] is an
intermediate bound form, and IL* is a functionally irreversible bound
form. k is a zero order rate constant with an
unknown T
. The reaction that would be given by a k
is negligible and may require input
chemical energy. Note that the procession to irreversible binding may
go through more than one intermediate state, with associated rate
constants.
The less efficient but strong adhesion via the basal
receptor could be explained if k
k
and k
is relatively
low and rate-limiting for formation of LI*. The initial interaction is
slow, and effectively all receptors that reach [IL] proceed
to IL* or irreversible binding. The efficient but weak adhesion via the
active receptor could be explained if the active conformation resulted
in increased k
and k
,
such that k
is significant compared to k
. Alternatively, k
could be
increased and k
significantly decreased with the
same net result. In either case, k
becomes
rate-limiting for formation of the IL*. Thus, with no change in
affinity and therefore no requirement that the activating antibody
contributes energy, the observed properties of the active receptor
could be achieved.
A commonly held view is that
integrins that mediate adhesion of constitutively adhered cells must be
activated, since they function. We find, however, that both the active
and basal forms of are functional,
but are functionally distinct. In the case of M21 cells, activation
appears to stimulate adhesion that is more efficient, but weaker, than
adhesion via the basal integrin and to facilitate migration. Therefore,
activation is not a simple on/off switch.
Implicit in our model is
the prediction that a given integrin exists on the surface of the cell
in different populations, which generally have been known as
``activation states.'' In fact, subsets of integrins can be
distinguished antigenically, for example, by AP5, as well as
biochemically and functionally. For example,
is phosphorylated in response to
various stimuli(34) , and ADP-ribosylation has been reported on
another integrin,
(35) . In
these cases, the biochemical modification has been detected on a subset
only, between 5 and 40% of that integrin. Substoichiometric
modification of an integrin makes sense if the modified integrin has a
specific function not requiring all the receptors; for example,
initiating a specific signaling cascade. In fact, our recent work on
suggests that integrins in
different activation states are associated with different signaling
pathways (20) .
The conclusion we draw from all of these data is that integrins are structurally complex, existing in discrete forms, or activation states, that differ functionally. We hypothesize that these states contribute to cell regulation both by altering ligand recognition and by initiating specific signals that lead to changes in cell behavior.