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INTRODUCTION |
Cell adhesion mediated by cell-surface receptors in the integrin
family regulates a host of cellular events including cell migration,
spreading, proliferation, apoptosis, and gene induction (1-4). By
inside-out signaling, many cellular agonists "activate" integrins,
i.e. they trigger increased ligand binding and cell adhesion
capability. In some cases, "activation" of these integrins is
accompanied by changes in their affinity for ligands and/or for ligand
mimetic monoclonal antibodies (5). Then upon ligand binding, integrins
undergo pronounced conformational changes, resulting in the appearance
of Ligand-Induced Binding
Sites (LIBS)1 on
1 (6-8),
2 (9), and
3
(10-13) integrins as detected by specific monoclonal antibodies.
Integrin-mediated ligand binding requires divalent cations, and
Ca2+, Mg2+, and especially Mn2+ can
themselves cause pronounced integrin conformational changes. Notably,
many "LIBS" epitopes regulated by ligand binding are also regulated
positively or negatively by divalent cations, thus leading to renaming
(6) as "CLIBS" (Cation and Ligand-Influenced Binding Sites). At
least six non-overlapping CLIBS epitopes have been defined, with three
sites located in
1 (6-8, 14-17) and three other sites
in the
3 subunit (18, 19). Integrin
subunit sites
regulated by ligands and divalent cations have also been described (9,
12, 20, 21). Thus, major changes occur throughout the integrin molecule
upon ligand and/or divalent cation binding. Displacement of divalent
cations may itself be a key feature of ligand binding (22). Integrin
1 CLIBS epitopes, such as those defined by mAb 15/7 (7)
and 9EG7 (23), were suggested to be "activation epitopes," but
subsequent reports showed very poor correlation with integrin
activation (6, 16).2 The mAb
9EG7 and/or 15/7 epitopes can be induced not only by ligand or
Mn2+ but also by functionally disruptive cytoplasmic tail
deletion or integrin denaturation (7, 16, 23). Thus, CLIBS epitopes may
in general be reporters of integrin unfolding (25). Because epitopes
recognized by mAb 9EG7 and 15/7 are induced upon ligand occupancy of
4
1 and
5
1,
it has been widely assumed that these epitopes would be diagnostic for
ligand occupancy of all
1 integrins. Here we show a wide
variation in CLIBS epitope induction on different
1
integrins, indicating that CLIBS epitopes are not useful for determining the extent of Mn2+ or ligand binding for
several
1 integrins. However, our CLIBS epitope results
do suggest that
1 integrins differ widely in the extent
to which
-
chain unfolding accompanies occupancy by ligand or
Mn2+. In addition, we have used the 9EG7 CLIBS epitope to
investigate anomalous regulation of
4
1 by
calcium.
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EXPERIMENTAL PROCEDURES |
Antibodies, Ligands, and Cells--
The anti-
1
monoclonal antibodies used were as follows: 9EG7, rat anti-mouse, with
cross-reactivity for human and hamster
1 (23); mAb 13, rat anti-human (26); and the mouse anti-human antibodies 15/7 (27) and
A-1A5 (28). Other antibodies (all mouse anti-human) include: A2-IIE10,
anti-
2 (29); A3-IIF5, anti-
3 (30); B5G10,
anti-
4 (31); A5-PUJ2, anti-
5 (32); A6-ELE, anti-
6 (33); and negative control antibodies P3
(34) and rat anti-E-selectin (from Dr. Pnina Brodt, McGill
University).
The GRGDSP peptide was purchased from Life Technologies, Inc.
Recombinant soluble VCAM-1 (35) was a gift from Dr. Roy Lobb (Biogen,
Cambridge, MA). Rat collagen type I was obtained from Collaborative
Biomedical Products (Bedford, MA). Laminin-1, from mouse
Engelbreth-Holm-Swarm sarcoma, was from Life Technologies, Inc., and
purified laminin-5 was from Dr. R. Burgeson. K562 cells were cultured
in RPMI 1640 with 10% fetal bovine serum and transfected by
electroporation with
2,
3, or
4 cDNA in the pFneo vector or with
6
cDNA in the pRc/CMV vector as described previously (36, 37).
Flow Cytometry--
Cells were sequentially washed with
phosphate-buffered saline and Tris-buffered saline (24 mM
Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl) and
resuspended with Tris-buffered saline containing 5% bovine serum
albumin and 0.02% sodium azide. Cells were then incubated with
agonists (MnCl2, ligands, EDTA, or EGTA) for 15-30 min at
37 °C. Aliquots of 2 × 105 cells were then
incubated for 45 min on ice with 9EG7 or other primary antibodies (2 µg/ml). Cells were washed three times with Tris-buffered saline
containing 1% bovine serum albumin and 0.02% sodium azide and
incubated for 45 min on ice with fluorescein isothiocyanate-conjugated
goat anti-mouse or anti-rat IgG (Sigma). At least 3000 cells were then
analyzed using a FACScan machine (Becton Dickinson, Oxnard, CA) to
obtain mean fluorescence intensity values. To calculate percentages of
2
1,
3
1,
4
1, and
6
1 bearing the 9EG7 epitope in K562 cells, we solved for 9EG7% of
z according to Equation 1.
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(Eq. 1)
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To obtain the percentage of
5
1
expressing 9EG7 (9EG 7% of
5), we determined 9EG7
levels relative to total
1 (measured using mAb 13) in a
cell line (K562) that only expresses
5
1.
Z represents the mean fluorescence intensity value for
2,
3,
4, or
6 in each K562 transfectant. Levels of 9EG7 were
measured after the addition of MnCl2, ligands, EDTA, or
EGTA. However, levels of each
chain and total
1 were
determined using saturating amounts of the appropriate antibodies,
added to cells untreated with any ligands, chelators, or divalent
cations. In the transfectants, expression of
Z was
typically 3-5-fold greater than
5 and was always at
least 2-fold greater.
Construction of Chimeric Molecules--
Cloning of human
2 (38),
3 (39), and
4 (40)
cDNA, as well as the generation of the chimeric constructs X2C4 and
X2C5 (36), have been previously described. Construction of chimeric X2C3 was carried out by overlap extension (41). Two segments were
generated in separate PCR reactions, each containing one set of outer
and inner primers and either
2 or
3
cDNA. For
2 cDNA we used the oligonucleotide
primers 5'-CAG-TTG-TAA-TGC-AGA-TAT-CAA-TCC-3' (corresponding to nucleotides 3099-3122 of
2 and
containing the underlined EcoRV restriction site) and
5'-GAA-GCC-GCA-CTT-CCA-TAA-AAT-TGC-AAC-3' (corresponding to a cohesive
end for nucleotides 3127-3119 of
3 and 3513-3496 of
2). For
3 cDNA we used the primers
5'-GCA-ATT-TTA-TGG-AAG-TGC-GGC-TTC-TTC-3' (corresponding to a cohesive
end for nucleotides 3499-3507 of
2 and 3113-3130 of
3) and 5'-GGT-CTA-GAA-GTC-ACA-CCA-GGT-GGG-3' (corresponding to nucleotides 3270-3252 of
3 and
introducing an XbaI site). A second PCR reaction was
performed using these two segments and the two outer primers. The
resulting
2/
3 chimeric product was
digested with EcoRV and XbaI, ligated to
2 cDNA, and finally cloned in pFneo (42). The X2C6
was prepared similarly by overlap extension PCR, starting with an
2 cDNA clone (38) and
6 cDNA from
reverse transcriptase-PCR of mRNA from HT1080 cells.
To make the X3C0 construct, the
3 subunit cytoplasmic
domain was truncated just after the GFFKR sequence by creating a
termination codon at position 3130 of
3 cDNA. The
stop codon (underlined) was introduced into the antisense primer
5'-G-AGT-GCG-GGC-TCA-TCG-CTT-GAA-GAA-GCC-GC-3' corresponding to nucleotides 3146-3120. The upstream primer
(5'-GC-AGG-CGC-CGA-CAG-CTG-GAT-CCA-GGG-3') corresponds to nucleotides
2688-2713. The resulting PCR product was inserted into full-length
3 cDNA in the pFneo vector. The construction of
4 mutants D408E, N283E, D346E, D489E, D698E, D811E, and
the combined D489E/D698E/D811E mutant (LEV) have been detailed
elsewhere (43, 44).
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RESULTS |
Manganese-dependent Induction of the 9EG7 Epitope
Differs among
1 Integrins--
In K562 cells,
Mn2+ induces the 9EG7 epitope on the
1
subunit of the
5
1 integrin (6). To
evaluate 9EG7 epitope expression on other
1 integrins,
we compared K562 cells (which express only
5
1) with K562-
2,
K562-
3, and K562-
4 transfectants. In the absence of added divalent cations, relatively low levels of the 9EG7
epitope were detected in K562-
2, K562-
3,
or K562 cells (Fig. 1A, left
panels), whereas moderate constitutive levels were seen in
K562-
4 cells. However, in the presence of 5 mM Mn2+, the 9EG7 epitope was increased greatly
on K562-
4 cells, moderately on K562 and
K562-
2 cells, but hardly at all on K562-
3
cells. A Mn2+-induced conformational change was also
detected using mAb 15/7, and again it was most obvious for
K562-
4 cells, less obvious for K562 and
K562-
2 cells, and not at all obvious for
K562-
3 cells (Fig. 1B).

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Fig. 1.
Differences among 1 integrins
in 9EG7 and 15/7 epitope expression. K562 cells transfected with
2, 3, or 4 integrin cDNA or control vector (K562) were incubated in the absence of added cations (Control) or in the presence of 5 mM MnCl2. Then cells were analyzed by flow
cytometry for surface staining with mAb 9EG7 (dotted line)
or rat anti- 1 mAb 13 (broken line)
(A) and with 15/7 (dotted line) or mouse
anti- 1 A-1A5 (broken line) (B).
Negative control staining (solid lines) was with rat
anti-E-selectin (A) or mouse P3 antibody
(B).
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For K562-
2, K562-
3, and
K562-
4 transfectants analyzed in Fig. 1, induction of
the 9EG7 and 15/7 epitopes is partly due to a small contribution of
endogenous
5
1. Therefore, to obtain a
more precise assessment of epitope induction on individual
1 integrins in transfected cells, we subtracted the
contribution of endogenous
5
1 in
subsequent experiments. A more detailed analysis revealed that
stimulation of the 9EG7 epitope by Mn2+ (up to 10 mM) was dose-dependent, and at all
concentrations tested, induction was highest for
4
1, intermediate for
5
1, low for
2
1, and essentially undetectable on
6
1 or
3
1
(Fig. 2A). A very similar
hierarchy of Mn2+-dependent 9EG7 expression was
found upon comparing Chinese hamster ovary (CHO) cells that
constitutively express
5
1 with
CHO-
2, -
3, and -
4
transfectants (not shown).

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Fig. 2.
Effects of Mn2+ on 9EG7
expression and cell adhesion. A, cells were stimulated with
manganese and analyzed for 9EG7 expression by flow cytometry. Levels of
9EG7 (% ) are presented relative to specific levels of
2 1, 3 1,
4 1, or 6 1,
after subtraction of the contribution of background
5 1 (see "Experimental Procedures"). B, manganese stimulates 3 1
adhesive function. Adhesion of K562- 3 cells to a
laminin-5-containing matrix (deposited by A431 cells as described (30))
was determined at varying Mn2+ levels. Also,
K562- 3 cells (and control K562 cells) in 1 mM Mn2+ were tested in an adhesion assay (6) on
purified laminin-5 (coated at 5 µg/ml).
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Although Mn2+ stimulated little or no 9EG7 epitope
expression on
2
1,
6
1, and
3
1,
the cation did efficiently stimulate cell adhesion mediated by these
integrins. For example, adhesion of K562-
3 cells to a
laminin-5 containing matrix (made by A431 cells) was
dose-dependently induced by Mn2+, with maximal
stimulation at
0.1 mM (Fig. 2B). Also,
Mn2+ (1 mM) strongly stimulated adhesion of
K562-
3 cells, but not control K562 cells, to purified
laminin-5 (Fig. 2B). Elsewhere, 1 mM
Mn2+ stimulated a >22-fold increase in affinity of
purified soluble
3
1 for
laminin-5.3 In other
experiments 0.1-1 mM Mn2+ stimulated to a
similar extent K562-
2 cell adhesion to collagen, K562-
4 adhesion to VCAM-1, K562-
6
adhesion to laminin-1, and untransfected K562 cell adhesion to
fibronectin (not shown). Thus, failure to induce the 9EG7 epitope (Fig.
2A) is not due to lack of integrin responsiveness to
Mn2+.
Immunoprecipitation results also showed preferential recognition of
specific Mn2+-treated 
1 combinations by
mAb 9EG7. Previously it was shown that 9EG7 immunoprecipitated a major
fraction of
4
1 from a
Mn2+-treated lysate (23). In the presence of 1 mM Mn2+, the 9EG7 mAb precipitated a moderate
amount (~10% of total
5
1 available) of
5
1 from a lysate of
125I-labeled K562 cells (not shown). In sharp contrast, the
9EG7 mAb precipitated little if any
3
1
from K562-
3 cell lysate in 1 mM
Mn2+ (not shown).
Influence of Integrin
Chain Cytoplasmic Domains on
Mn2+-induced 9EG7 Expression--
Exchange of integrin
chain cytoplasmic domains may alter integrin function and presumably
also conformation (46). Thus, we hypothesized that variable induction
of 9EG7 might be due to variations among
chain cytoplasmic domains.
However, exchange of the integrin
2 cytoplasmic domain
with the
3 or
6 tail did not cause 9EG7
expression to decrease (see K562-X2C3 and K562-X2C6 in Table
I, top). Conversely, exchange with the
4 or
5 tails (K562-X2C4 and -X2C5) did
not cause a marked increase in 9EG7 epitope expression. Similarly,
deletion of the
3 tail (K562-X3C0) did not prevent the
9EG7 epitope on
3
1 from being largely
unresponsive to Mn2+ (Table I, bottom).
Differential Induction of the 9EG7 Epitope by Soluble
Ligands--
The 9EG7 epitope is also induced by soluble ligand (6),
thus providing an opportunity for further comparisons among
1 integrins. In the presence of 5 mM
Mg2+, which by itself has no effect on 9EG7 expression, CS1
peptide (DELPQLVTLPHPNLHGPEILDVPST, not shown) or VCAM-1 (Fig.
3) induced a high level of 9EG7 on
4
1. Similarly, fibronectin induced a moderate level of 9EG7 on
5
1, and
collagen I induced a low 9EG7 level on
2
1. However, soluble laminin-1 and
laminin-5 induced little, if any, 9EG7 epitope on
6
1 and
3
1,
respectively (Fig. 3). Also, the GD6 laminin peptide (at 0.5 mg/ml)
that was described as a ligand for
3
1
(47) induced no 9EG7 expression on K562-
3 cells (not
shown). In control experiments, mock-transfected K562 cells showed no
induction of 9EG7 in response to maximal doses of soluble VCAM-1,
collagen I, laminin-1, or laminin-5 (not shown).

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Fig. 3.
Induction of the 9EG7 epitope by soluble
ligands. K562- 4, K562, K562- 2,
K562- 6, and K562- 3 cells were
preincubated with soluble VCAM-1, fibronectin, collagen I, laminin-1,
or laminin-5, respectively, in 5 mM Mg2+ for 10 min at 37 °C. The mAb 9EG7 (2 µg/ml) was then added, and cell
suspensions were incubated for an additional 45 min on ice, stained,
and analyzed by flow cytometry.
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Failure to induce 9EG7 on
6
1 or
3
1 was not due to inactivity of the
respective integrins. Under experimental conditions similar to those
used in Fig. 3 (i.e. 5 mM Mg2+, 2 µg/ml 9EG7), K562-
6 and K562-
3 cells
displayed strong cell adhesion to laminin-1 and laminin-5, respectively
(not shown). This adhesion was specifically mediated by
6
1 or
3
1
since it was not seen in control K562 cells (not shown). Furthermore, we show elsewhere that
3
1 binds to
laminin-5 in the presence of mAb 9EG7, with a Kd of
~0.088 µM.3 Thus at 0.3 µM
laminin-5 (as used in Fig. 3), the majority of
3
1 should be occupied by ligand.
As an extension of results showing 9EG7 induction by Mn2+
(Fig. 2A) or by ligand (Fig. 3), we next analyzed effects of
Mn2+ and ligand (at 0.3 µM) added together.
Stimulation of 9EG7 was already maximal (near 60%) when 0.3 µM VCAM-1 was added to K562-
4 cells in the
presence of 0.05 mM Mn2+ (Fig.
4). In comparison, a 20-fold greater
level of Mn2+ (1.0 mM) was required to support
comparable levels of 9EG7 (50-60%) on
5
1,
2
1, and
6
1 when stimulated with 0.3 µM fibronectin, collagen, or laminin-1, respectively. In
contrast,
3
1 still showed minimal 9EG7
expression even in the presence of 1.0 mM Mn2+
plus 0.3 µM laminin-5. To summarize, results of 9EG7
induction by Mn2+ (Fig. 2A), ligand (Fig. 3), or
both together (Fig. 4) show a generally consistent hierarchy among the
different
1 integrins.

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Fig. 4.
Induction of the 9EG7 epitope by ligand plus
manganese. K562 transfectants were preincubated (10 min at
37 °C) with Mn2+ and either 0.3 µM ligand
( ) or control buffer ( ). The mAb 9EG7 was then added (as in Fig.
3), and flow cytometry was carried out.
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Divalent Cation Chelating Agents Variably Affect 9EG7 Expression on
1 Integrins--
Major differences in 9EG7 expression among
1 integrins were also observed upon incubation with EDTA
or EGTA. Consistent with previous results (6), 9EG7 expression was
moderately induced on
5
1 by EDTA (Fig.
5A) or EGTA (Fig.
5B). In comparison, the 9EG7 epitope was induced to a lower
extent on
2
1 and hardly at all on
6
1 and
3
1
(Fig. 5, A and B). On
4
1, the 9EG7 epitope was diminished,
rather than induced, upon the removal of divalent cations. For all
integrins tested, similar effects were seen with EDTA and EGTA (Fig. 5,
A and B), indicating that calcium, rather than
magnesium or other divalent cations, is playing a key regulatory role.

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Fig. 5.
Effect of divalent cation chelation on 9EG7
expression. The various K562 cell transfectants were pretreated
(30 min, 37 °C) with EDTA (A) or EGTA (B) and
then analyzed for 9EG7 expression.
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Further Exploration of Unique Properties of
4
1--
Consistent with results in Figs.
2 and 5B,
5
1 in K562 cells
showed a moderate increase in the 9EG7 epitope upon Mn2+ or
EGTA stimulation (Fig. 6A).
Also,
4
1 in Molt-4 cells showed a much
stronger response to Mn2+ but showed a decrease rather than
increase in response to EGTA (Fig. 6C). These results
confirm for
4
1 in Molt-4 cells (where it
is the only integrin) the results seen for
4
1 in K562-
4 cells (Figs.
2 and 5B) where it is expressed together with
5
1. Shown in Fig. 6B is the
cumulative 9EG7 expression for both
5
1 and
4
1 in K562-
4 cells.
Notably, no net effect of EGTA is seen, presumably because positive
effects on
5
1 are balanced by negative effects on
4
1. However, subtraction of
the
5
1 contribution (Fig. 6D),
based on results seen with the K562 reference cells (Fig.
6A), reveals the specific contribution of
4
1 (Fig. 6E). Notably, the
results for
4
l in Fig. 6E
(after calculation) are very similar to the results in Fig.
6C (where no calculations are necessary). Together, these
results not only confirm the unique sensitivity of
4
1 to EGTA but help to validate the
method utilized for subtraction of background contributions due to
5
1 in the various K562 transfectants.

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Fig. 6.
Regulation of the 9EG7 epitope on K562,
K562- 4, and Molt-4 cells. 9EG7 expression is
indicated as percent of total 1 on K562 (A),
K562- 4 (B), and Molt-4 cells (C).
In the right panel, total 9EG7 expression on
K562- 4 cells (B) has been separated into
percent of 5 1 (D) and percent
of 4 1 (E). Each experiment represents the mean ± S.D, for n = 3.
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To highlight further the unique properties of
4
1, we compared the effects of
Ca2+ on ligand-induced 9EG7 expression for the various
integrins. Calcium markedly inhibited the expression of 9EG7 induced on
5
1 by the GRGDSP ligand mimetic peptide
(Fig. 7A). Similarly, calcium inhibited collagen-dependent induction of 9EG7 epitope on
2
1 in K562-
2 cells (not
shown). In contrast, Ca2+ stimulated 9EG7 expression
induced by soluble VCAM-1 (Fig. 7B). These results are
generally consistent with the divergent effects of Ca2+
inferred from Fig. 5B and further demonstrate that
4
1 conformations are regulated in a
specialized manner, distinct from other
1 integrins.

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Fig. 7.
Effects of calcium on ligand-induced 9EG7
expression. After preincubation in the presence or absence of 1.5 mM CaCl2, K562 cells were then incubated (20 min, 37 °C) with GRGDSP peptide (A), and
K562- 4 cells were incubated with soluble VCAM-1
(B).
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Effects of
4 Extracellular Domain Mutations on 9EG7
Expression--
The
4 mutations N283E, D346E, and
D408E, each within putative divalent cation sites, each have similar
negative effects on
4-dependent cell
adhesion (43, 48). However, neither the high constitutive expression of
9EG7 on
4
1 in K562 cells nor the high
level of Mn2+ induction were appreciably altered by the
D408E mutation (Fig. 8A) or
the N283E and D346E mutations (not shown).

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Fig. 8.
Effects of 4 mutations on 9EG7
expression. Constitutive and Mn2+-induced 9EG7
expressions were analyzed in K562- 4 cells for wild type
4 compared with the 4 D408E mutant
(A), or compared with the D489E, D698E, and D811E single
mutants, and the LEV triple mutant (D489E/D698E/D811E)
(B).
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Unlike other integrin
subunits, the
4 sequence
contains three extracellular "LDV" motifs (40). Within these
motifs, mutations D489E and D698E each caused severe impairment of
4-dependent cell adhesion, whereas D811E had
no negative effect (44). Upon analysis of 9EG7 expression (Fig.
8B), we found that
4 D489E and D811E
mutations caused little or no loss of the 9EG7 epitope, either
constitutively present or induced by Mn2+. In comparison,
the D698E mutation caused a marked loss in the constitutive level of
9EG7 on
4
1. Furthermore, when all three mutations were present at once (LEV mutant), the loss of constitutive 9EG7 expression was even more obvious. However, these deficits in
constitutive expression were substantially overcome upon the addition
of 1 mM Mn2+.
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DISCUSSION |
mAb 9EG7 and 15/7 Are Not Universal Reporters of Either Ligand
Occupancy or Mn2+ Activation of
1
Integrins--
Epitopes defined by anti-
1 mAb 9EG7 and
15/7 were initially suggested to correlate with integrin activation,
but several striking counter-examples emerged in subsequent studies (6, 7, 16, 23).2 More recently, such antibodies have been shown
to recognize ligand-induced binding sites on
4
1 and
5
1
(6, 7), thus causing them to be assigned to the LIBS or CLIBS category
(25). Such results have produced the expectation that CLIBS antibodies
would be universal reporters of ligand occupancy for
1
integrins. Here we show that expression of the 9EG7 epitope is not
indicative of the extent of ligand occupancy, especially for the
2
1,
6
1, and
3
1 integrins. At near-saturating ligand
binding conditions, the 9EG7 epitope was indeed induced on the majority
of
4
1 integrins and on a significant
fraction of
5
1 integrins. However in
contrast, the epitope was induced minimally on
2
1 and
6
1
and not at all on
3
1.
At the high levels of soluble ligand utilized, in the presence of 9EG7
(Fig. 3), we should be near saturation for all of the ligand-integrin
combinations tested. In this regard, we are aided by the fact that the
9EG7 antibody not only detects ligand occupancy, but itself stimulates
ligand binding (6),3 and thus resembles other
integrin-activating antibodies that increase affinity for soluble
ligands by 5-50-fold (49, 50). The 9EG7 mAb decreases the
3
1/laminin-5 binding
Kd from >600 down to ~88
nM,3 which is well below the 300 nM
dose of laminin-5 used in Fig. 3. Likewise, mAb 9EG7 markedly
stimulated
4
1 binding to soluble VCAM-1.
Although VCAM binding in the absence of 9EG7 (Mg2+
conditions) was so weak that it was not measurable, binding in the
presence of 9EG7 yielded a Kd of ~50
nM (not shown), which is comparable to VCAM-1 binding
affinities of 10-100 nM reported elsewhere (48, 51). Thus,
the 600 nM soluble VCAM-1 used in Fig. 3 is again well
above saturation. In the presence of activating antibodies,
Kd values of 9 and 18 nM have been
observed for fibronectin binding to
5
1
(49) and laminin binding to
2
1 (50). Thus
we can assume that ligand doses of 1200 nM should be well
above saturation for these integrins also. Although estimated
Kd values are not yet available for soluble
laminin-1 binding to activated
6
1, this
is a strong interaction (52) that is highly stimulated by mAb 9EG7
(53), and again the Kd values should be well below
the 600 nM dose of laminin-1 used in Fig. 3.
It is even more likely that saturation was achieved when soluble
ligands were added in the presence of 9EG7 plus Mn2+ (as in
Fig. 4). Notably, in those conditions we continued to see a similar
hierarchy of 9EG7 induction (
4
1 >
5
1,
2
1,
6
1, >
3
1).
For
3
1 binding to laminin-5, 9EG7 alone
reduces the Kd by >7-fold (down to ~88
nM), and 1 mM Mn2+ alone reduces
the Kd by >22-fold (down to ~27
nM).3 Thus we suspect that both 9EG7 and
Mn2+ together would yield a Kd of <5
nM, but nonetheless 300 nM laminin-5 added
under those conditions (Fig. 4) still showed no induction of the 9EG7
epitope.
A similar hierarchy of CLIBS epitope induction
(
4
1 >
5
1 >
2
1 >
6
1 >
3
1) was seen in response to ligand
plus Mg2+, ligand plus suboptimal Mn2+ levels,
Mn2+, or stimulation by Mn2+ alone. Wide
differences in integrin responses to Mn2+ seen for K562
cells were confirmed using
subunit-transfected CHO cells, and
results obtained by immunoprecipitation of solubilized integrins
matched those obtained by flow cytometric analysis at the cell surface.
Also, Mn2+ stimulation results obtained with the 9EG7
antibody were confirmed using mAb 15/7.
Results shown here clarify our understanding of the stimulatory effects
of Mn2+ on integrin function. From previous results
(e.g. Refs. 6 and 7), it appeared that induction of CLIBS
epitopes by Mn2+ may be an integral feature of integrin
stimulation. Now for the first time, we show definitively that
Mn2+-induced function does not necessarily correlate with
the induction of CLIBS-type epitopes. As shown elsewhere, 1 mM Mn2+ strongly stimulated the affinity of
purified recombinant soluble
3
1 integrin
for laminin-5,3 whereas we show here that even 10 mM Mn2+ failed to induce any expression of the
9EG7 epitope on cellular
3
1
(e.g. Fig. 2A), and 1.0 mM
Mn2+ did not induce 9EG7 epitope expression on solubilized
3
1 (not shown). Likewise, 0.1-1
mM Mn2+ greatly activated ligand binding by
solubilized
2
1 (54) and
6
1 (55), but 0.1-10 mM
Mn2+ had negligible effect on 9EG7 epitope expression by
cell surface
2
1 or
6
1. Thus, 9EG7 (and 15/7) epitope
expression is clearly dissociated from integrin activation by
Mn2+.
A practical implication of our results is that
1 CLIBS
epitopes are reporters of ligand occupancy and/or Mn2+
stimulation only for a few
1 integrins (e.g.
4
1 and
5
1). In this regard, it is not surprising that most reports describing expression of the 9EG7 or 15/7 epitopes have involved leukocytes expressing
4
1 (7, 27, 56, 57).
Conversely, analysis of the other
1 integrins with the
available "anti-CLIBS" reagents may lead to a substantial
underestimation of ligand binding events and underestimation of
integrin activation by Mn2+.
What Is the Meaning of 9EG7 Epitope Expression?--
If CLIBS
epitopes do not accurately report either ligand occupancy or integrin
activation, does that mean that they are irrelevant? Our analysis of
CLIBS epitopes provides important information regarding integrin
conformational unfolding that transcends considerations of only ligand
binding affinity and occupancy. The constellation of events that leads
to CLIBS epitope expression on
1 (e.g.
denaturation, absence of an
chain, ligand binding, etc.) suggests a
general correlation with
-
chain unfolding (25). The similar
hierarchy of epitope exposure ([
4
1] >
5
1 >
2
1 >
6
1 >
3
1)
induced by three different agents (Mn2+, ligand, and EGTA)
emphasizes that there may be fundamental differences in conformational
flexibility among these integrins. Consistent with
4
being most flexible, and most readily able to "uncover"
1 CLIBS epitopes, the
4 subunit is also
most easily dissociated from
1. Dissociation of
4
1 readily occurs at elevated pH
(8.5-9.0) and often occurs even at neutral pH (31, 58). In contrast, the
2
1 subunits remain substantially
associated, and
3
1 is completely
associated even after elution from mAb beads at pH 11.5 (59).
As shown schematically (Fig. 9) we
indicate that each integrin can readily bind ligand and bind
Mn2+. However, there is wide variation in the extent to
which these events are accompanied by conformational unfolding to
expose CLIBS epitopes. At one extreme,
4
1
undergoes dramatic changes in response to either ligand or
Mn2+ binding. At the other extreme, the conformation of
3
1 is minimally altered, even when both
ligand and Mn2+ are added (and bound) simultaneously. It is
not integrin activation or ligand binding that differs, but rather the
conformational changes that accompany these events.

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Fig. 9.
Schematic diagram of integrin conformational
changes. Each 1 integrin can bind ligand or be
activated by Mn2+. However, there is wide variation in the
extent to which these events are accompanied by conformational changes.
For example, 4 1 shows the most
conformational change (and the most induction of 9EG7 epitope), whereas
3 1 shows negligible conformational change
(and minimal induction of 9EG7 epitope). Also, it should be noted that
CLIBS epitopes can sometimes be constitutively expressed, even in the
absence of ligand occupancy or Mn2+ (e.g. 9EG7
on 4 1).
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The
2,
3,
4,
5, and
6 tails have diverse sequences and
differentially regulate functions such as cell migration, focal adhesion formation, and collagen gel contraction (60, 61). Also,
exchange of cytoplasmic tails may alter adhesive function, through a
change in extracellular ligand binding affinity (46). However, our tail
deletion and exchange results show that specific
tails do not
control the extracellular domain conformational changes that we have
measured. Hence, the differing degrees of conformational flexibility
seen among the
4
1,
5
1,
2
1,
6
1, and
3
1
integrins may be intrinsic to the extracellular domains.
Within the
1 molecule, 9EG7 binds to a site within
residues 495-602 (6), whereas 15/7 mapped within residues 354-425
(16). Both of these regions may lie within the membrane-proximal
integrin stalk and are presumed to be distant from ligand and divalent cation binding sites present within the integrin globular head (62,
63). According to a current paradigm, long range propagation of
conformational changes within extracellular domains may control integrin cytoplasmic domain "outside-in" signaling events (19). Thus, we hypothesize that integrins showing extensive extracellular conformational changes (e.g.
4
1 and
5
1)
might transmit a larger magnitude of conformational change across the
plasma membrane. This would potentially then lead to greater
-cytoplasmic tail exposure and enhanced formation of signaling
complexes of the type stimulated by ligand occupancy (64).
Specialized Regulation of
4
1
Conformation--
Integrin exposure to EGTA or EDTA yielded 9EG7
hierarchy (
5
1 >
2
1 >
6
1 >
3
1) comparable to that seen with ligand
or Mn2+, except that
4
1 gave
an atypical response. Among the
1 integrins tested, a
positive effect of calcium on 9EG7 epitope expression was seen only for
4
1. This positive effect was manifested
as follows: (i) an atypically high level of constitutive 9EG7 on
4
1 that was inhibitable by EGTA, and (ii)
calcium enhancement of 9EG7 epitope expression induced by soluble
VCAM-1. These results are consistent with the ability of calcium to
support
4
1-mediated adhesion to VCAM-1
(65, 66). In sharp contrast, 9EG7 epitope expression on the
2
1 and
5
1
integrins was promoted by EGTA treatment and thus was inhibited by
calcium. Thus, calcium may stabilize inactive conformations of those
integrins. This is consistent with the inhibitory effects of calcium on
the adhesive functions of
2
1,
5
1, and
6
1
(67-69). Also, 9EG7 expression on
3
1 was
not promoted by calcium, in agreement with the general failure of
calcium to support
3
1-mediated cell
adhesion (30, 70).
The D698E mutation in
4 caused a loss of constitutive
9EG7 epitope expression (Fig. 8B) together with a loss of
VCAM-1 binding (44). Treatment with EGTA also diminishes both 9EG7
expression and VCAM-1 binding. From these results, we propose that
calcium may help to stabilize an active conformation of
4
1. This specialized utilization of
Ca2+ may allow
4
1 on
leukocytes to maintain a constitutive level of activation in the
presence of high plasma calcium.
Because our D698E mutation and EGTA have similar negative effects on
constitutive 9EG7 expression and VCAM binding, we hypothesize that
Asp-698 could play a role in the specialized interaction of
4
1 with calcium. Notably, Asp-698 in
4 is part of a
DXXXXXDXSSXS sequence that partly
resembles a consensus DXXXXXDXSXS
region that is present in the I domains of six different integrin
chains, and within all eight integrin
chains (Fig.
10). Within
chains or
chain I
domains, Asp, Ser, and Ser residues (at positions 7, 9, and 11 in Fig.
10) play critical roles in divalent cation and ligand binding. Among
chains 1-9, only
2 and
9 have Asp residues comparable to Asp-698 in
4, and only
9 has a sequence in that region
(DXXXXXDXSXXS) partly resembling the
DXXXXXDXSSXS sequence in
4. Within
4, further mutations
(e.g. at positions 1, 9, 10, and 12) will be required to
verify the importance of the
4
DXXXXXDXSSXS sequence. Also it remains
to be determined whether this region directly binds to
Ca2+.

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Fig. 10.
Putative novel divalent cation binding site
within the 4 subunit. Consensus I domain and chain sequences that contact divalent cation are derived from
alignments of six different I domains and eight different integrin sequences (44, 71). The nine different sequences (24, 45, 72, 73)
have been described and aligned previously. Consensus residues are
enclosed within double lines. Conservative substitutions are
enclosed within single lines. Serines offset by one position
are shaded.
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In conclusion, we have demonstrated that monoclonal antibodies such as
9EG7 and 15/7 are not universal indicators of ligand occupancy or
Mn2+ activation but nonetheless provide important insights
into integrin function. First, we have provided evidence for major
differences among
1 integrins in the extent to which
conformational changes may accompany Mn2+,
Ca2+, and ligand binding. Second, our results support the
hypothesis that differences in extracellular conformational changes may
be highly relevant toward understanding fundamental differences in outside-in signaling. Third, only through analysis of the 9EG7 epitope
have we discovered that atypical positive effects of calcium on
4
1 functions are paralleled by the
presence of a conformation that requires ASP-698 in
4.
We thank Bob Burgeson (MGH East, Charlestown,
MA) for purified laminin-5; Roy Lobb (Biogen) for VCAM-1; Dietmar
Vestweber (Institute of Cell Biology, Münster, Germany) for mAb
9EG7; Ted Yednock (Athena Neurosciences) for mAb 15/7; Amy Skubitz
(University of Minnesota) for GD6 peptide; and Isao Tachibana for
assistance with control experiments.