(Received for publication, August 4, 1995)
From the
The monoclonal antibody 9EG7 has been previously found to
recognize an epitope induced by manganese on the integrin chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B.,
and Vestweber, D.(1993) Proc. Natl. Acad. Sci. U. S. A. 90,
9051-9055). Here we show that treatment of
integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This
epitope was also induced upon EGTA treatment to remove calcium, and the
addition of calcium inhibited 9EG7 epitope induction by manganese or by
ligand. Further emphasizing the importance of the 9EG7 epitope, the
9EG7 antibody itself stimulated adhesion mediated by multiple
integrins, and conversely, ligands for
,
,
, and
all stimulated 9EG7 expression. Together these results support a
model whereby (i) calcium inhibits
integrin function
because it prevents the appearance of a conformation favorable to
ligand binding and (ii) manganese enhances
integrin
function because it induces the same favorable conformation that is
induced by adding ligand, or removing calcium. Notably, other
-stimulating agents (magnesium and mAb TS2/16) did not
induce 9EG7 expression unless ligand was also present. Thus, although
9EG7 may reliably detect the ligand-bound conformation of
integrins, its expression does not always correlate with integrin
``activation.'' Finally, mouse/chicken
chimeric molecules were used to map the 9EG7 epitope to
residues 495-602 within the cysteine-rich
region, and antibody cross-blocking studies showed that the 9EG7
epitope is distinct from all previously defined human
epitopes.
Adhesive events mediated by transmembrane
heterodimers in the integrin family are dynamically
regulated(1, 2) . Dramatic changes in
integrin-mediated adhesive functions can be observed upon cell
differentiation(3, 4) , during hemostatic (5) and immunological (6) responses, and upon
triggering with various cellular agonists(2, 7) .
Studies with the platelet integrin
have indicated that both integrin activation (8, 9, 10) and ligand binding (11, 12) are associated with conformational changes,
and that an ``activated'' integrin sometimes resembles a
``ligand-bound'' integrin(13) .
The study of variable integrin activation states has been facilitated by the use of monoclonal antibodies that selectively recognize distinct integrin conformations. Notably, the expression of several integrin epitopes is regulated by cell triggering with various agonists, divalent cation gain or loss, ligand binding, or combinations of these events(9, 11, 12, 14, 15, 16, 17, 18, 19) . Also, several antibodies can stimulate integrin adhesive functions, presumably by stabilizing an active conformation(4, 12, 15, 20, 21, 22, 23, 24, 25, 26) . Interestingly, some antibodies not only recognize epitopes induced by ligand, but also stimulate integrin function themselves, whereas other antibodies apparently have only one or the other of these properties.
The mechanism for ligand binding and activation of integrins has not been well studied, partly because few
antibodies that specifically recognize activated or ligand-bound forms
have been available. One possible ``activation-specific''
antibody (called 15/7) has been found to selectively recognize
on activated T cell subsets in
vivo(27) . Also, two recently described antibodies (9EG7
and SG/7) define
neoepitopes induced by divalent
cations. The former recognizes an epitope up-regulated in response to
Mn
treatment(28) , whereas the latter defines
a
epitope induced by either Mn
or
Ca
, but not Mg
(29) .
However, the association of these epitopes with integrin functions has
not been extensively studied. Also, there are two antibodies that
recognize ligand-induced
epitopes(30, 31) . In other studies of
integrins, the inhibitory effects of
Ca
(32, 33, 34, 35, 36) ,
and the stimulatory effects of manganese (34, 35, 37, 38, 39) have
often been noted, but few mechanistic insights have emerged.
Because
little is known regarding integrin conformational
changes, we have utilized the 9EG7 mAb
to study events
accompanying
integrin activation and ligand binding.
We define ``activation'' as an increase in the potential of an integrin to bind ligand, and/or to mediate the more complex
function of cell adhesion. We wish to clearly distinguish
``activation'' as a distinct phase that precedes
``ligand binding.''
Here we have found that the 9EG7
antibody defines a conformation of fundamental
importance because (i) the epitope is negatively regulated by calcium,
(ii) it is induced by manganese, (iii) it is induced by the binding of
all
integrin ligands tested, and (iv) the 9EG7
antibody itself stimulates all
adhesive functions
tested. Finally, we mapped the 9EG7 epitope to a site within
, distinct from that recognized by all other known
anti-human
antibodies, and not previously shown to be
regulated by divalent cations and ligand binding.
Figure 1:
mAb 9EG7
stimulation of K562 cell adhesion to fibronectin. K562 cells were
preincubated for 15 min at 37 °C with mAb 9EG7 in the presence of
Mn at the indicated concentrations and then tested
for adhesion to fibronectin (coated at 5
µg/ml).
Figure 2:
Comparative effects of
Mn, Mg
, TS2/16, and PMA on K562
cell adhesion and 9EG7 epitope expression. K562 cells were washed twice
in PBS and then were stimulated with Mn
(panel
A), Mg
(panel B), mAb TS2/16 (panel
C), or PMA (panel D). The experiments represented in panels C and D were carried out in the presence of 1
mM MgCl
and 1 mM CaCl
. Cells
were stained with 9EG7 or mAb 13 and analyzed by flow cytometry to
determine the percent of 9EG7 expression relative to total
stained by mAb 13 (left y axes). Bound TS2/16 did not
interfere with detection of 9EG7 because they bind to non-overlapping
epitopes (see Table 2and ``Discussion''), and we used
a fluorescein isothiocyanate-conjugated second antibody specific for
rat (9EG7) but not mouse (TS2/16) primary antibodies. Cell adhesion was
measured (in the absence of 9EG7) as indicated under ``Materials
and Methods'' (right y axes).
Figure 3:
Effects of Mn and
soluble ligand on 9EG7 surface expression. K562 cells were analyzed by
flow cytometry for surface expression of the 9EG7 epitope in the
presence of: no pretreatment (control), 5 mM MnCl
,
1 mM GRGESP control peptide, or 1 mM GRGDSP. Cell
staining with the mAb 9EG7 (solid lines) is flanked by
negative control staining (sparse dotted line) and mAb 13
staining of total
integrin (tight dotted
line).
Similar to Mg, the stimulatory antibody
mAb TS2/16 (Fig. 2C) and the phorbol ester PMA (Fig. 2D) induced little or no 9EG7 expression, but did
promote cell adhesion to fibronectin in a dose-dependent manner.
Because integrin-mediated adhesion requires divalent cations, 1 mM MgCl
and 1 mM CaCl
were added
during the adhesion assays and were also present during the analysis of
9EG7 epitope expression (Fig. 2, C and D). If
MgCl
and CaCl
were omitted, TS2/16 and PMA
still failed to induce 9EG7 epitope expression (data not shown).
Figure 4:
Effect of fibronectin and RGD peptide on
9EG7 expression. K562 cells were incubated (in the presence of 1 mM MgCl and 1 mM CaCl
) with either
soluble human fibronectin for 30 min at 37 °C (panel A) or
GRGDSP and GRGESP peptides for 5 min at room temperature (panel
B). The percent of 9EG7 relative to total
expression was determined by flow
cytometry.
Figure 5:
Effect of EDTA and EGTA on 9EG7 epitope
expression. K562 cells were washed in PBS and then resuspended in TBS
(+5% BSA and 0.02% NaN) containing either EDTA or
EGTA. After 30 min at 37 °C cells were analyzed by flow cytometry
to determine the percent of 9EG7 relative to total
expression. In parallel experiments cells were incubated with 2.5
mM EDTA for 30 min at the indicated temperatures (inset).
Also,
the effect of EGTA on 9EG7 epitope expression was fully reversible.
Incubation of K562 cells with 2.5 mM EGTA (for 30 min at 37
°C) caused elevated 9EG7 expression (from 5.2% up to 32.6%).
Continued incubation for another 30 min in 2.5 mM EGTA, or
after addition of buffer that slightly diluted the EGTA (to 2.3
mM), did not markedly alter 9EG7 expression. In contrast,
subsequent addition of Ca (to 12.5 mM, for
30 min at 37 °C) lowered 9EG7 expression back to a basal level
(4.1% relative to total
, Fig. 6A).
Remarkably, simply removing EGTA by washing cells and resuspending them
in PBS for an additional 30 min also resulted in a loss of much of the
9EG7 epitope (down to 9.5%). Because this occurred even when the PBS
was pretreated with Chelex 100 resin, we suspect that the calcium
responsible for this effect is derived from the cell, rather than
buffer contamination. This result is consistent with very tight binding
of Ca
to
integrins
(
) on K562 cells, as also suggested
from Fig. 5.
Figure 6:
9EG7 epitope expression induced by
Mn and soluble ligand is reversed by
Ca
. K562 cells (in a 100-µl volume) were
preincubated for 30 min at 37 °C with either 2.5 mM EGTA (A), 5 mM Mn
(B), or 25 µM GRGDSP peptide (C).
Then, additional 10-µl volumes of control buffer, CaCl
(to 12.5 mM), or EGTA (to 2.5 mM) were added,
and the cells were incubated for another 30 min before being assayed
for 9EG7 expression by flow cytometry. In one experiment (part
A), cells were washed and resuspended for 30 min in PBS that had
been treated with Bio-Rad Chelex 100 resin (buffer +
wash).
Notably, 9EG7 expression was also elevated to a
roughly similar extent upon preincubation with either 5 mM
MnCl or 25 µM GRGDSP peptide (Fig. 6, B and C). Again, this expression was nearly
completely reversed upon the addition of 12.5 mM Ca
. When 2.5 mM EGTA was added
subsequent to treatment with either 5 mM MnCl
or
25 µM GRGDSP peptide, no further increase in the 9EG7
epitope was observed. These results suggest that Ca
had already been depleted due to incubation with 5 mM MnCl
or 25 µM GRGDSP peptide.
Detailed
Ca titrations confirmed that the addition of
Ca
(at >0.1 mM) could reverse both the
stimulatory effects of 5 mM Mn
(Fig. 7A) and 25 µM GRGDSP ligand (Fig. 7B). At lower doses of Mn
and
GRGDSP, the percent inhibition by Ca
was even more
pronounced (data not shown). In contrast, Mg
had only
a very minor inhibitory effect on Mn
stimulation, and
in fact, Mg
exerted a slight to moderate stimulatory
effect above that seen with 25 µM GRGDSP alone.
Figure 7:
Effects of calcium and magnesium on
manganese-induced and ligand-induced 9EG7 expression. K562 cells were
washed twice with PBS then stimulated for 30 min at 37 °C with
either 5 mM MnCl (A) or 25 µM GRGDSP peptide (B) and then incubated for 25 min at 37
°C with CaCl
or MgCl
at the indicated
concentrations. In the absence of added CaCl
or
MgCl
, expression of the 9EG7 epitope was determined by flow
cytometry to be 39.7 and 37.6 mean fluorescence intensity units (in A and B, respectively).
Because 9EG7 did not overlap strongly with any previously defined
epitope, precise localization required a chimeric mapping approach. For this, previously described mouse/chicken
chimeras (50) were utilized, since 9EG7 recognizes mouse
(28) but fails to recognize chicken
(data not shown). As shown in a reprecipitation
experiment (Fig. 8), the mouse/chicken MC3 chimera was isolated
from NIH-3T3 cells by the CSAT mAb (lane 1), and then
reprecipitated using the 9EG7 mAb (lane 6), but not by a
negative control antibody (lane 5). In contrast, 9EG7 did not
recognize the MC5 chimera (lane 8) after it was first isolated
using W1B10 (lane 3). In control precipitations (lanes 2 and 4), no chimeric
was present in the
lanes. Also, control experiments using polyclonal
anti-
sera confirmed that
integrins
were indeed available for reprecipitation (lanes 7 and 9). As indicated in the schematic diagram (Fig. 8C), these results are consistent with 9EG7
binding to
between residues 495 and 602.
Figure 8:
Epitope mapping for mAb 9EG7. A,
chimeras were precipitated from NIH-3T3 cells using
anti-chicken
antibodies CSAT (lane 1) and
W1B10 (lane 3), respectively. As a control, mouse
was precipitated from the same lysates (lanes 2 and 4). B, reprecipitation of material precipitated by
CSAT (lanes 5-7) or by W1B10 (lanes 8 and 9) was carried out using either negative control mAb P3 (lane 5), mAb 9EG7 (lanes 6 and 8) or an
antiserum to the cytoplasmic domain of
(lanes 7 and 9). Note that the diminished intensity of the
bands seen by the anti-
tail serum
is likely due to competition by a pool of immature
subunit that
is not surface labeled. C, a schematic diagram of the MC3 and
MC5
chimeras is shown.
Here we have characterized a novel integrin
epitope that is highly relevant to ligand binding and adhesive
functions. The 9EG7 epitope was induced by all
integrin ligands tested (GRGDSP peptide, fibronectin, soluble
collagen, kalinin, VCAM-1, and CS1 peptide) and the antibody itself
could stimulate the adhesive function of all
integrins tested, most likely by stabilizing a conformation
favorable to ligand binding. In contrast to the previous
report(28) , we saw no evidence for blocking of
integrin-mediated adhesion.
Notably, binding of ligand to integrin also triggered the appearance of the 9EG7 epitope. The
addition of excess Ca
could reverse this effect, but
under these conditions, levels in the mM range were required.
We assume that ligand binding is inhibited by this excess
Ca
because (i) this is far in excess of the amount
needed to prevent 9EG7 binding in the absence of ligand, and (ii)
binding of 9EG7 itself is not appreciably increased in affinity (data
not shown). In this regard, there is recent biophysical evidence for a
mechanism whereby bound ligand can displace
integrin
cations (52) from a site (aa 118-131) that is very well
conserved in
(aa 129-142), and also required
for ligand binding to
integrins(53) .
The
inhibitory effect of Ca on several
integrin functions(32, 33, 34, 35, 36, 54) and on
some
integrin functions (55) has been well
established. Now we have the fundamental new insight that
Ca
but not Mg
may act largely by
obstructing the appearance of a conformation (defined by 9EG7) that is
favorable for ligand binding. In this regard, it is likely that the
enhanced cell migration associated with a lower
Ca
/Mg
ratio in wound fluid (56) probably involves a more favorable
ligand-binding conformation such as described here.
It has been well
established that Mn is a strong stimulator of
integrin
function(34, 35, 37, 38, 39) .
Now we gain a new insight into this activity since Mn
not only supports ligand binding, but also, by itself, stabilizes
an epitope favorable to ligand binding, and causes a diminished
inhibitory effect of Ca
. In contrast, Mg
is able to support ligand binding, but does not appear to have
these other activities. It has been suggested elsewhere (55) that Mn
could reach 1-12
µM in many tissues, and up to 50 µM upon bone
resorption. These levels approach the point at which Mn
begins to stimulate
-dependent
cell adhesion and 9EG7 expression (i.e. see Fig. 2A).
Rather than defining an activation epitope, the
9EG7 epitope can be better characterized as a ``ligand-induced
binding site'' or ``LIBS,'' such as have been described
for integrins(11, 57) . However,
even the term LIBS is only partially correct, since in the absence of
ligand, both the addition of Mn
and removal of
Ca
also induce 9EG7 expression (see below).
Although the phorbol ester PMA stimulated adhesive activity, it did
not directly stimulate 9EG7 expression, nor did it synergize with
soluble fibronectin to give increased 9EG7 expression. We are not sure
why PMA was previously found to cause increased 9EG7
expression(28) , except that possibly peripheral blood T cells
could differ from K562 cells in this regard. Nonetheless, our results
are consistent with previous findings that PMA could stimulate adhesion
without increasing integrin ligand binding
affinity(59, 60) . Rather than altering affinity, PMA
could alter receptor clustering as previously reported(61) ,
thus leading to enhanced avidity for ligand. Consonant with this,
reduced clustering of
integrin did
not change 9EG7 expression, but caused markedly diminished cell
adhesion.
Likewise,
chain cytoplasmic tail deletion
did not change Mn
-inducible 9EG7 expression, despite
causing diminished adhesive function(62) .
All of these
results reinforce the idea that there are at least three ways to
increase the functional potential of integrins.
First, agents such as Mn
increase ligand binding
potential as well as 9EG7 expression; second, agents such as
Mg
and TS2/16 increase ligand binding potential
without directly inducing 9EG7 expression; and third, agents such as
PMA can stimulate cell adhesion without either stimulating ligand
binding or inducing 9EG7 epitope expression.
In preliminary data, we
have found that increasing doses of Mn or GRGDSP
peptide uniformly increased the number of 9EG7 binding sites (relative
to total
), without appreciably altering the apparent K
(
10 nM) for 9EG7 binding to
(data not shown). This result is consistent with each
individual receptor existing in either a ``+'' or
``-'' state with regard to the 9EG7 epitope. Notably,
we have never observed 9EG7 expression to reach more than 40-50%
relative to the total
expression, when measured
either by flow cytometry or by immunoprecipitation from solution (data
not shown). In this regard, several other antibodies that report
ligand-induced or activated integrin conformations also fail to bind to
more than 50% of the integrins(9, 17, 55) .
As yet, no adequate explanation for this widely observed phenomenon has
been offered. Possibly, it may be an intrinsic property of integrins to
exist in multiple conformations, with it being very difficult to shift
equilibrium completely toward the conformation resembling the
ligand-bound state.
Notably, several anti- LIBS
antibodies have been mapped to
regions
602-690(19, 67) , or 422-490(19) ,
both of which are mostly non-overlapping with the 495-602 region
in
. Thus, between the
and
subunits, there now have been described a total of
five different LIBS sites. Notably, these same five sites on the
or
subunits are also sensitive to
divalent cations as indicated for
aa
1-6(19) ,
aa
422-490(12, 19) ,
aa
602-690(67, 68) ,
aa
207-218(29, 42, 63) , and
aa 495-602 (described here). Underscoring the fact that
these cation- and ligand-regulated sites are apparently inseparable, we
refer to them here as cation- and ligand-influenced binding sites, or
CLIBS. These sites, labeled as
-CL1-
-CL3 and
-CL1 and
-CL2, are
shown schematically in Fig. 9.
Figure 9:
Schematic diagram of the human subunit. Residue numbers that define mapping sites are listed
along the top of the bar. The three shaded areas represent (left to right) the putative cation binding site
(130-138), the cysteine-rich region (442-629), and the
transmembrane domain (709-731). Above the bar, CLIBS sites
defined here and elsewhere (see ``Discussion'') are marked as
-CL1,
-CL2,
-CL3,
-CL1, and
-CL2 to indicate the three distinct CLIBS
sites in
and two CLIBS sites in
,
respectively.
Together these results
emphasize that conformational changes due to ligand and divalent cation
binding events are not limited to a small area within the primary
structure. Rather, our results help to demonstrate that profound
changes occur throughout the integrin molecule upon ligand binding
and/or cation manipulation. This point is additionally reinforced by
several anti-integrin chain epitopes that also are both induced
by ligand, and regulated by divalent cations (e.g. Refs. 11,
18, 69, 70).
In conclusion, the 9EG7 antibody defines a novel
epitope near the cysteine-rich region of the integrin, now known for the first time to be conformationally
involved in both ligand and cation regulation. As such, 9EG7 has been
exceptionally useful. For example, it has allowed us to demonstrate
that a fundamental role for Ca
may be to
counterbalance those agents (including ligand) and Mn
that stimulate 9EG7 expression. In addition, it has allowed us to
elucidate three categories of function-activating stimuli. Finally, the
appearance of the 9EG7 epitope not only provides a useful tool for
studying the regulation of
integrin function, but
also it implies that ligand binding may expose this epitope, which
could potentially play a role in interactions with other integrins or
other proteins, and ultimately in outside-in signaling.