From the
Cell adhesion mediated by leukocyte integrin CR3 (CD11b/CD18,
The leukocyte integrin, complement receptor type 3 (CR3,
The ability of CR3
to bind its ligands is low in resting cells, but it is transiently
enabled by stimulation (Wright and Meyer, 1986; Lo et al.,
1989a, 1989b). For example, stimulation of PMN with phorbol ester
causes a dramatic rise in adhesivity after 20 min and a return to base
line by 40 min (Wright and Meyer, 1986). Similar activation-dependent
binding has also been found in several other integrins (Dustin and
Springer, 1989; O'Toole et al., 1990; Shaw et
al., 1990), and various models have been proposed to explain this
phenomenon (Hynes, 1992; Smyth et al., 1993; Diamond and
Springer, 1994). Since changes in adhesivity may occur without a change
in receptor number, the ``affinity modulation model'' has
received a significant amount of study. In this model, the avidity of
individual receptors is thought to shift reversibly from low to high.
Shifting of the affinity to the high level allows adhesion, and
shifting back to low affinity allows detachment. Reliable estimates of
CR3 affinity, however, have not been made, and studies with other
integrins have suggested that the affinity may be too low to measure
readily (Horwitz et al., 1985; Lollo et al., 1993).
In the present study, we describe a novel, soluble, monomeric ligand
for CR3 and an enzymatic microassay to study the affinity of individual
receptors. Our data make the surprising finding that the affinity of
CR3 for C3bi is very high. Moreover, inactive receptor exhibited a
kinetic block in binding, not a change in binding energetics. The
implications of these findings for the affinity modulation model are
discussed.
Calf intestinal alkaline phosphatase (AP), n-succinimidyl 3-(2-pyridithio)propionate (SPDP), and bovine
serum albumin were purchased from Pierce. Iodoacetamide, n-octyl
IB4 (anti-CD18, Wright et al.(1983)), OKM-1 (anti-CD11b, Wright et
al.(1983)), 44a (anti-CD11b, Dana et al.(1986)), TS1/22
(anti-CD11a, Davignon et al. (1981)), W6/32 (anti-class 1
histocompatability Ag, Barnstable et al.(1978)), and 3C10
(anti-CD14, Van Voorhis et al. (1983)) were as described
previously, and are all available from American Type Culture Collection
(Rockville, MD). KIM-127 (anti-CD18, Robinson et al.(1992))
was a gift from Dr. M. Robinson (Celltech Ltd., United Kingdom).
Anti-C3bi was from Quidel (San Diego, CA). Alkaline
phosphatase-conjugated goat anti-mouse IgG was from Bio-Rad.
C3bi was conjugated to calf intestinal AP through a disulfide
bridge. A sulfhydryl was first introduced into AP with the bifunctional
cross-linker, SPDP. 2-Pyridyldisulfide-AP was prepared by incubating 5
mg of AP in 1 ml of PBS with 25 µl of 20 mM SPDP diluted
in Me
C3bi with a
single free sulfhydryl was purified from normal human plasma by
modifying the method reported by Ross et al. (1987). Briefly,
50 ml of normal human plasma was treated with a final concentration of
20 mM iodoacetamide for 1 h at 37 °C to block all free
sulfhydryl groups. After extensive dialysis, the plasma was incubated
with 2 g of activated thiol-Sepharose for 2 h at 37 °C. The
Sepharose activates the complement cascade, and because proteolytic
activation of C3 leads to the liberation of a free sulfhydryl (Pangurn,
1986), the C3 is captured in disulfide linkage with the Sepharose.
After thorough washing, C3bi was eluted with 10 mML-cysteine in 20 mM Tris, pH 7.4. C3bi was
further purified with a Mono Q column eluted with a gradient (0-1 M) of NaCl in 20 mM Tris, 5 mM EDTA, 0.02%
NaN
CR3 was purified from PMN lysates by one of the following two
methods.
To measure the binding of C3bi-AP to purified CR3, 72-well
terasaki plates (Robbins Scientific, Mountain View, CA) were coated
with 10 µl/well (
The method for quantitating the amount of CR3 bound to coated
terasaki plates was as described previously (Van Strijp et
al., 1993). Briefly, CR3-coated plates were incubated with
Protein concentration was determined by Bio-Rad protein assay
(Bio-Rad Laboratories, Richmond, CA). Bovine serum albumin was used as
a standard. Data are presented as the mean of three wells ± S.D. of a representative experiment (n = 4).
C3bi-AP bound to surfaces coated with CR3 but not to uncoated
surfaces (Fig. 2A, first and lastcolumns). The CR3-dependent binding of C3bi-AP was
completely inhibited by mAb IB4 (anti-CD18) and 44a or OKM1 (both
anti-CD11b), while mAb W6/32 (anti-HLA class-1 antigen) and TS1/22
(anti-CD11a) were ineffective. The binding of C3bi-AP conjugates to CR3
was also dose-dependently blocked by unlabeled C3bi (Fig. 2B). C3bi-AP binds at least as avidly as unlabeled
C3bi, and attachment of AP thus appears not to decrease the affinity of
C3bi for CR3. Additional studies showed that unconjugated AP failed to
bind to CR3, and the amount of C3bi-AP binding varied with the dose of
CR3 used to coat the surfaces (data not shown). These studies
established that C3bi-AP bound specifically to CR3.
To determine the binding properties of leukocyte integrin
CR3, we developed a novel, soluble, monomeric probe, C3bi-AP, and a
binding assay using purified immobilized CR3. Our procedures obviate
problems such as pinocytic uptake and degradation of ligand that are
inherent in cell-based assays, especially with interactions that
require warm temperatures. They further obviate problems inherent in
measurements of multimeric ligands and yield the first equilibrium
measurements on CR3 suitable for rigorous quantitative analysis. Using
these reagents, we determined that the stoichiometry of the binding of
C3bi to CR3 is 1:1, that binding shows an absolute requirement for
divalent cations, and that binding is of high affinity.
Previous
work has shown that CR3 can be isolated in a form that is capable
(Diamond etal., 1990) or incapable (Van Strijp et al., 1993) of binding multimeric ligand, and we confirmed
these findings here using our monomeric probe. We further showed that
addition of mAb KIM-127 to purified CR3 caused a shift from an inactive
to an active state. This result indicates that KIM-127 acts directly on
CR3 to enable its ligand binding properties. Importantly,
KIM-127-induced binding exhibited an affinity for C3bi that is
identical with the affinity for active receptor preparation, suggesting
that the site exposed by this mAb is similar to that exposed during
activation of receptors by physiological stimuli.
KIM-127 could
enable binding either by contributing sufficient energy to change
receptor affinity or by lowering the activation energy required for
binding. Studies reported here suggest that KIM-127 does not modulate
affinity. If KIM-127 drove a conformational change that raised the
affinity of the C3bi binding site, then the binding energy for C3bi
conferred by binding of mAb would be balanced by a decrease in the
binding energy for KIM-127. Such reciprocal changes in binding energy
are observed in allosteric proteins (e.g. hemoglobin) for
ligands (e.g. 2,3-diphosphoglycerate and O
The most
surprising finding of the study is that the binding affinity of CR3 for
C3bi is very high (K
The above studies necessitate a revision of current thinking
regarding control of integrin-mediated cell adhesion. The widely held
affinity modulation model calls for increases in integrin affinity to
enable cell adhesion and for a reversal of this process to cause
detachment. Data in this paper suggest that neither of the two
propositions of this model are tenable. Studies with our model system
show that the activation of receptors does not result from changes in
affinity of CR3 but from a change in the activation energy for shifting
receptor from inactive to active state. A second revision concerns the
mechanism by which adhesion is reversed. Since our data indicate that
release of bound ligand requires significant energy input, cell
detachment must involve a process mechanistically distinct from
receptor activation and cannot result from a reversal of receptor
activation. One potential mechanism for reversing adhesion may involve
removal of divalents, since divalent cations are required for binding
of C3bi-AP to CR3 (Fig. 2C) and removal of divalent
cations reverses binding.
The final
observation derived from Fig. 9is that receptors might be
incapable of binding ligand and mediating adhesion for two distinct
reasons. They may be in the inactive state or in a ligand-occupied
state. Experiments are currently ongoing to determine the state of
receptors on living PMN.
We thank D. S. Miller for assistance in preparation of
inactive CR3. We also thank Dr. M. K. Robinson (Celltech Ltd., UK) for
the generous gift of mAb KIM-127, Dr. P. A. Detmers for critical
reading of the manuscript, and our colleagues for valuable discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) may be rapidly modulated without
changes in receptor number, and transient changes in adhesivity are
thought to be driven by reversible alteration of the affinity of CR3
for ligand. Here we measure the binding affinity of CR3 using purified
active and inactive receptor and the ligand, C3bi, coupled to alkaline
phosphatase. Immobilized, active CR3 bound saturably and with high
affinity (12.5 ± 4.7 nM). In contrast, inactive CR3
exhibited no measurable binding. High affinity binding could be
restored by the addition of the activating anti-CR3 monoclonal antibody
KIM-127 to inactive CR3. Since the affinity of KIM-127 for active and
inactive receptor was identical, it cannot contribute the energy to
convert a low affinity receptor into a high affinity receptor. Rather,
KIM-127 appears to facilitate binding of C3bi by lowering the
activation energy for the shift from an inactive to an active state.
These results suggest that CR3-mediated binding and detachment of cells
is not driven by a reversible change in affinity but by two
mechanistically distinct processes, an energetically neutral activation
step for binding and an energy-dependent step that reverses binding of
ligand.
(
)Mac-1, CD11b/CD18,
) is expressed by polymorphonuclear
leukocytes (PMN), monocytes, and natural killer cells and mediates the
binding of cells to surfaces coated with a wide variety of ligands,
including C3bi (Wright et al., 1983), fibrinogen (Wright et al., 1988), intercellular adhesion molecule-1 (Diamond et al., 1990), and unknown ligands on endothelial cells (Lo et al., 1989a; Diamond et al., 1990). CR3 thereby
plays a critical role in the adhesion of PMN to the vasculature, to
opsonized particles, and to extracellular matrix.
Reagents and Monoclonal Antibodies
-D-glucopyranoside, Sepharose CL-4B-200,
activated thiol-Sepharose, and cyanogen bromide-activated Sepharose
were from Sigma. Dulbecco's phosphate-buffered saline (PBS) was
from Biowhittaker (Walkersville, MD). Attophos was from JBL Scientific,
Inc. (San Luis Obispo, CA). All protease inhibitors were from
Calbiochem-Novabiochem Co. (La Jolla, CA).
Constructing a Soluble, Monomeric Probe: C3bi-Alkaline
Phosphatase
SO. After a 30-min incubation at 37 °C, excess
cross-linker was removed with a NAP-25 (Pharmacia LKB Biotechnology,
Uppsala, Sweden) desalting column equilibrated with PBS, 1 mM EDTA. The extent of derivitization was measured using an molar
extinction coefficient of 8.08
10
M
cm
at 343 nm for
released 2-pyridyldisulfide (Carlsson et al., 1978) and was
found to be 1.24 mol of 2-pyridyldisulfide/mol of AP.
, pH 7.4. Purified C3bi was then mixed with equimolar
2-pyridyldisulfide-AP and rotated at room temperature for 24 h.
Approximately 80% of C3bi became bound to AP. Conjugated and free
proteins were separated with a Mono Q column eluted with a gradient
(0-1 M) of NaCl in 20 mM Tris, 0.02%
NaN
, pH 7.4, and results of conjugation were analyzed by
electrophoresis on a 8-25% native gels (see Fig. 1). The
presence of a single band of conjugate suggests stoichiometric coupling
of C3bi and AP, and this inferrence is confirmed by the observation
that the mobility of the conjugate is slower than either of the two
reactants, and its R
is consistent with a
1:1 complex. The purity of the conjugate was also verified by
immunoprecipitation. Incubation of conjugate with immobilized mAb
against C3bi removed over 95% of AP activity, while with irrelevant
mAb, AP in the supernatant remained unchanged (data not shown).
Figure 1:
Conjugation of C3bi to AP. C3bi
purified from normal human plasma was conjugated to calf intestinal
alkaline phosphatase as described under ``Materials and
Methods.'' Proteins were analyzed on native gels (8-25%)
stained with Coomassie Brilliant Blue. LaneA,
molecular mass markers (Thy, thyroglobulin, 669 kDa; Fer, ferritin, 440 kDa; Cat, catalase, 232 kDa; LD, lactase dehydrogenase, 140 kDa; BSA, bovine serum
albumin, 67 kDa); laneB, purified AP; laneC, purified C3bi; laneD, purified
C3bi-AP conjugate.
Purification of CR3
Antibody Affinity Chromatography
Pellets of PMN,
prepared as described previously (Van Strijp et al., 1993),
were lysed at 2 10
cells/ml with lysis buffer (100
mM Tris, pH 8.0, 150 mM NaCl, 2 mM MgCl
, 1% Triton X-100, 0.02% NaN
, protease
inhibitors (1 µg/ml each of antipain, benzamidine, chymostatin,
leupeptin, and pepstatin, 0.24 units/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM pefablock)) for 1
h at 4 °C with stirring. The lysate was then centrifuged at 35,000
g for 30 min. The supernatant was passed over an IB4
(anti-CD18) immunoaffinity column that was prepared by coupling IB4 to
cyanogen bromide-activated Sepharose. The column was prewashed with
equilibration buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM MgCl
, 0.1% Triton X-100). The
column was loaded at 1 ml/min and sequentially washed with
equilibration buffer and buffer composed of 50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 2 mM MgCl
, 1% n-octyl
-D-glucopyranoside. CR3 was eluted with
buffer containing 50 mM triethylamine, pH 11.0, 150 mM NaCl, 2 mM MgCl
, 1% n-octyl
-D-glucopyranoside into tubes with neutralizing buffer
(15% by volume 1 M Tris-HCl, pH 7.4). Aliquots were made and
stored at -80 °C. The purity of CR3 was analyzed on
SDS-polyacrylamide gels (4-15%) stained with silver. Strong bands
corresponding to the
and
chain of CR3 were observed at 165
and 95 kDa, and the identity of
chain (165 kDa band) was
confirmed with Western blot using polyclonal anti-CR3 (data not shown).
Although PMN contain low amounts of CD11a/CD18 and CD11c/CD18, our
preparation showed no CD11c and only a weak CD11a band (<5% of the
density of the CD11b band). This preparation will bind C3bi and is
referred to as ``active'' CR3.
Ligand Affinity Chromatography
CR3 was purified on
immobilized C3bi as described previously (Van Strijp et al.,
1993). Briefly, C3bi-coated Sepharose beads were prepared by incubating
normal human plasma with Sepharose CL-4B-200 for 1 h at 37 °C. The
beads were incubated with PMN lysate for 20 min, and, after thorough
washing, CR3 bound to C3bi-coated beads was eluted using 10 mM EDTA. Receptor isolated in this way fails to bind C3bi and is
referred to as ``inactive'' CR3.
Binding of C3bi-AP to Immobilized CR3
10 µg/ml) of purified CR3 diluted in PBS,
0.02% NaN
for 2 h at 4 °C. Wells were aspirated and
then blocked with 10% dry milk diluted in PBS for 1 h at 4 °C.
After four washes with PBS, a total of 10 µl of C3bi-AP diluted in
PBS with 0.1% of milk was added to each well. Plates were incubated at
37 °C for 20 min, unless indicated otherwise. Following four washes
with saline, 5 µl/well of a fluorogenic substrate for AP, Attophos,
was added. The fluorescence signal generated at 21 °C was detected
at time intervals with a Cytofluor
2300 fluorescence plate
reader (Millipore Corp., Bedford, MA). The amount of C3bi-AP bound to
CR3-coated plates was expressed as the change in arbitrary fluorescence
units/time interval. The actual value of fmol of C3bi-AP bound per well
was calculated from a standard curve relating enzyme activity to µg
of conjugate. Each sample was assayed in triplicate.
Quantitation of CR3 on Plates
I-labeled mAb anti-CR3 (
I-44a, iodinated
with Na
I by the Iodogen method (Fraker and Speck, 1978))
at 37 °C for 60 min. After thorough washing, plates were dried. The
amount of
I-44a bound was determined by counting
individual wells in a
-counter. Results were expressed in cpm, and
the actual value of fmol of
I-44a bound per well was
calculated from the specific activity of the
I-44a.
Protein Concentration Assay and Data Analysis
C3bi-AP Binds CR3 Specifically
C3 binds
covalently to surfaces through an ester link (Law and Levine, 1977; Law et al., 1979). In this process, an intrachain thiol ester in
C3 undergoes a transesterification reaction yielding an ester link to a
hydroxyl (or to water) and a free thiol (Abbas et al., 1991).
We have used this free thiol to conjugate alkaline phosphatase to C3bi.
Since the free thiol residue of C3bi is only four amino acids away from
the normal site of attachment to surfaces (Khan and Erickson, 1982), it
represents a sterically favorable site to attach a bulky group.
Analysis of the conjugate indicates that it is a soluble monomeric
molecule with a 1:1 ratio between C3bi and AP (Fig. 1). We
measured binding of C3bi-AP to purified CR3 by incubating C3bi-AP in
plates coated with purified CR3. The CR3 used for this study was
purified using mAb affinity chromatography, and this purification
produces a population of receptors that avidly bind C3bi (see below).
Figure 2:
C3bi-AP specifically binds CR3. CR3-coated
plates were incubated with C3bi-AP (15 nM) for 20 min at 37
°C in the presence of the indicated mAbs (10 µg/ml) (A) or in the presence of increasing concentrations of
unconjugated C3bi (B, plates coated with () or without
(
) CR3). These incubations were done with buffer containing 1
mM Ca
and 1 mM MgCl
.
The requirement for divalent cations was measured in parallel
incubation with buffer containing 1 mM each of the indicated
divalent cations (C). After thorough washing, the binding of
C3bi-AP to plates was quantitated by enzyme assay as described under
``Materials and Methods.'' Parallel experiment showed the
binding rate to be linear at this time
point.
Binding of C3bi-AP to CR3 Is Divalent
Cation-dependent
To determine the cation dependence of binding
of C3bi-AP to CR3, binding studies were conducted in the presence of 1
mM each of Ca, Mg
, and
Mn
at 37 °C for 20 min (Fig. 2C).
Omission of divalent cations completely inhibited the binding of
C3bi-AP to purified CR3. In the presence of 1 mM Ca
or Mg
, binding was
partially restored, but the most efficient binding was observed in the
presence of Mn
or the combination of all three
divalents. These results are consistent with our previous studies
showing that binding of C3bi-coated particles to CR3 on cells (Wright
and Silverstein, 1982) and plastic (Van Strijp et al., 1993)
is divalent cation-dependent and are also consistant with the known
divalent cation binding properties of CR3 (Michishita et al.,
1993).
Binding of C3bi-AP to CR3 Is Temperature-dependent and
Requires a High Activation Energy
We have previously shown that
binding of C3bi-coated erythrocytes to CR3-bearing cells (Wright and
Jong, 1986) or CR3-bearing plastic surfaces (Van Strijp et
al., 1993) could not be observed at low temperature. To determine
if this temperature dependence was a property of CR3 or the C3bi-coated
erythrocytes, binding of C3bi-AP to CR3 coated plates was studied at
various temperatures with 1 mM each of Ca and Mg
. The rate of binding of C3bi-AP to CR3
was greatest at 37 °C, but was nearly absent when the temperature
was lowered to 17 °C (Fig. 3A) and could not be
observed at 0 °C (data not shown). The strong temperature
dependence thus suggests a high activation energy for the reaction
between C3bi and CR3. Arrhenius analysis of the data in Fig. 3A yielded an activation energy (E
) of 15 ± 2 kcal/mol (Fig. 3B).
Figure 3:
Effects of incubation temperature on the
binding of C3bi-AP to CR3. A, plates coated with () or
without (
) CR3 were incubated with C3bi-AP (15 nM) for
20 min at the indicated temperatures. Parallel experiments showed that
the binding rate was linear at this time point. After thorough washing,
the binding of C3bi-AP to plates was quantitated by enzyme assay at 21
°C as described under ``Materials and Methods.'' B, data in A were plotted by the method of
Arrhenius.
Binding of C3bi-AP to CR3 Is Time-dependent and High
Affinity
The specificity, divalent cation, and temperature
dependence for C3bi-AP binding to CR3-coated plates indicated that the
soluble monomeric probe, C3bi-AP, had binding properties identical to
those of immobilized C3bi (Wright and Silverstein, 1982; Van Strijp etal., 1993). We may thus use soluble C3bi-AP to
estimate the binding constant of CR3 for C3bi. To ensure the accuracy
of our estimation, the kinetics of C3bi binding to CR3 were first
studied by incubating 12 nM C3bi-AP with CR3-coated plates for
increasing times. Binding was time-dependent with an association rate (k) of
6
10
M
min
(Fig. 4). Binding reached steady state at 60 min.
Figure 4:
Kinetics of C3bi-AP binding to CR3. Plates
coated with () or without (
) CR3 were incubated with
C3bi-AP (12 nM) at 37 °C for increasing times. After
thorough washing, the binding of C3bi-AP to plates was quantitated by
enzyme assay. k
was calculated using this kinetic
data as described by Lauffenburger and Linderman
(1993).
To
measure the affinity of CR3 for C3bi, increasing concentrations of
C3bi-AP were incubated with plates coated with or without CR3 for 60
min at 37 °C. Binding of C3bi-AP to CR3 was dose-dependent and
saturable (Fig. 5A). For affinity estimation, values
from plates without CR3 coating were treated as nonspecific binding and
were subtracted to yield specific binding. Nearly identical nonspecific
values were obtained when excess unlabeled C3bi or mAb anti-CR3 was
used for competing the specific binding of C3bi-AP to CR3-coated plates
(data not shown). Scatchard analysis (Fig. 5B) revealed a K of 12.5 ± 4.7 nM.
Figure 5:
Dose-dependent binding of C3bi-AP to CR3. A, plates coated with () or without (
) CR3 were
incubated with increasing concentrations of C3bi-AP for 60 min at 37
°C. After thorough washing, the binding of C3bi-AP to plates was
quantitated by enzyme assay. B, the data in A were
plotted by the method of Scatchard after subtraction of
background.
The binding of C3bi-AP to CR3 appears to occur in a 1:1 ratio. When
50 nM C3bi-AP (a saturating concentration) was incubated with
CR3-coated plates for 60 min, the amount of C3bi-AP bound to CR3 was
estimated to be 7 fmol/well, while the number of CR3 molecules coated
on the plate was 10 fmol/well as determined by I-labeled
anti-CR3 (
I-44a) (data not shown).
C3bi-AP Does Not Bind Inactive CR3
Cells may
express CR3 that is active or inactive in mediating cell adhesion
(Wright and Meyer, 1986; Diamond and Springer, 1993; Elemer and
Edgington, 1994). The CR3 used in the above studies was isolated by
method using mAb affinity chromatography that yielded constitutively
active receptors, and this activity was demonstrated here by the avid
binding of C3bi-AP (Fig. 5A). We have previously
described a method for purification of inactive CR3 using affinity
chromatography on C3bi-Sepharose (Van Strijp et al., 1993).
This procedure yields receptor that neither binds C3bi-coated particles
(Hermanowski-Vosatka et al., 1992; Van Strijp et al.,
1993) nor CBRM1/5,(
)a monoclonal antibody that
detects an activation epitope of CR3 (Diamond and Springer, 1993).
Studies using this inactive receptor preparation showed that 12 nM C3bi-AP failed to bind above background (Fig. 6, firstcolumn). Similar results were obtained in experiments
using higher concentrations of C3bi-AP (100 nM) or incubations
as long as 18 h (data not shown). These findings indicate that inactive
CR3 either fails to bind C3bi or binds with a very low affinity.
Figure 6:
Binding of C3bi-AP to inactive CR3. Plates
coated with inactive CR3 (purified by ligand affinity chromatography)
were incubated with C3bi-AP (15 nM) at 37 °C for 30 min in
the presence of the indicated mAbs (10 µg/ml except IB4 that was 50
µg/ml). After thorough washing, the binding of C3bi-AP to plates
was quantitated by enzyme assay.
To
verify that our inactive CR3 is functional and capable of being
activated, we employed the monoclonal antibody KIM-127. This antibody
against CD18 strongly increases CR3-mediated PMN adhesion to
protein-coated surfaces without influencing the expression of
integrin (Robinson et al., 1992), a result
we have confirmed (data not shown). To determine if the effects of
KIM-127 are caused by direct action of the antibody on the receptor,
plates coated with inactive CR3 were incubated with C3bi-AP in the
presence or absence of KIM-127. The addition of KIM-127 led to strong
binding of C3bi-AP. The binding enabled by KIM-127 was totally blocked
by mAb IB4 (anti-CD18) (Fig. 6). This finding indicates that our
inactive CR3 retained the capacity to bind C3bi, and that KIM-127 acted
directly on CR3 to change its binding properties. The ability of
KIM-127 to enhance binding of C3bi-AP to inactive CR3 was
dose-dependent, with a maximal effect at
1 nM (data not
shown). The curve for enhanced binding of C3bi-AP to CR3 closely
matched the curve for binding of KIM-127 to CR3 (see Fig. 7below).
Figure 7:
Effect of mAb KIM-127 on binding of
C3bi-AP to CR3. A, binding of C3bi-AP to CR3. Plates coated
with (,
) or without (
) CR3 were incubated with
increasing concentrations of C3bi-AP for 20 min at 37 °C in the
presence (
,
) or absence (
) of KIM-127 (10 µg/ml).
After a thorough washing, the binding of C3bi-AP to plates was
quantitated by enzyme assay. B, the data in A were
plotted by the method of Scatchard after subtraction of
background.
KIM-127 Increases Binding of C3bi to CR3 without Changing
Affinity
Results from the above experiments indicate that the
capacity of inactive CR3 to bind monomeric ligand may be dramatically
altered by changes, presumably conformational in nature, induced by
binding of KIM-127. Equilibrium measurement of the binding in the
presence of 10 µg/ml of KIM-127 yielded K = 15.9 ± 3.4 nM. This value is not
significantly different from the K
of
active CR3 in the absence of KIM-127 (12.5 ± 4.7 nM).
This result suggests that KIM-127 does not influence the energetics of
the interaction of CR3 and C3bi. To further ask if KIM-127 alters the
affinity of active receptors, increasing concentrations of C3bi-AP were
incubated with plates coated with an active CR3 preparation in the
presence or absence of KIM-127, and binding was measured (Fig. 7). The addition of KIM-127 to CR3-coated plates increased
the number of binding sites for C3bi-AP to CR3, but the shape of the
binding curve indicated a single population of CR3 with similar
affinity in all cases. Further studies showed identical temperature
dependence of binding in the presence and absence of KIM-127 (data not
shown). These observations suggest first that the active CR3
preparation contains two populations, a population of active receptors
that bind C3bi, and an inactive population that only binds after
treatment with KIM-127. In four experiments, the increase in C3bi
binding sites caused by KIM-127 varied from 30 to 100%, suggesting that
15-50% of the receptors in this preparation were inactive but
susceptible to activation by KIM-127. More importantly, the results
suggest that KIM-127 does not modulate the binding affinity of CR3 for
C3bi. It is either high (12
16 nM) or unmeasurable, with
no middle value.
KIM-127 Does Not Contribute to the Energetics of Binding
of C3bi-AP to CR3
Inactive CR3 has unmeasurable affinity for
C3bi, while the active receptor has an affinity of 12 nM, a
change consonant with a G
of approximately
-11 kcal/mol. To test the hypothesis that KIM-127 contributes
this energy, we measured the binding affinity of KIM-127 for active and
inactive CR3. The affinities, roughly estimated from concentrations for
half-maximal binding, were practically identical (
1 nM)
for active and inactive CR3 (Fig. 8). If KIM-127 contributed the
11 kcal/mol needed for binding, its affinity would change from
1
nM for the active receptor to
80 mM for the
inactive receptor. This large change would have been easily observed in
our studies. Additional studies showed that ligation of CR3 with C3bi
also had no effect on the binding of KIM-127 (not shown). These results
suggests that KIM-127 affects binding of C3bi to CR3 without
contributing to the energetics of binding.
Figure 8:
Binding of mAb KIM-127 to active and
inactive CR3. Plates coated with active (, purified by mAb
affinity chromatography), inactive CR3 (
, purified by ligand
affinity chromatography), or without CR3 (
) were incubated with
increasing concentrations of mAb KIM-127 for 30 min at 37 °C. After
four washes with PBS, alkaline phosphatase-conjugated goat anti-mouse
IgG (1:2000 dilutions, Bio-Rad) was added, and plates were incubated
for another 30 min at 37 °C. After a thorough washing, the binding
of mAb KIM-127 to plates was quantitated by enzyme
assay.
(Kilmartin, 1976)). To determine if KIM-127 makes such an
energetic contribution, we measured its affinity for both active and
inactive receptors. No difference in affinity was observed (Fig. 8). It is thus clear that KIM-127 mediates alterations in
binding activity without significant energy input, and we may draw
active CR3 and inactive CR3 at equivalent positions on an energy
diagram (Fig. 9).
Figure 9:
Energetics of leukocyte integrin CR3.
Inactive CR3 and active CR3 are isoenergetic, but equilibrium is not
established because of a high activation energy for conversion of these
two species. Inactive CR3, once formed, may thus persist for long
periods, even in the presence of C3bi. KIM-127 has the same binding
affinity for both active and inactive CR3, but it facilitates the
interconversion of these two species. Thus the equilibrium distribution
of inactive and active CR3 is not changed by KIM-127, but the
nonequilibrium distribution can be changed by KIM-127 acting to
catalyze conversion of inactive CR3 to active CR3. Binding of C3bi to
active CR3 is energetically very favorable and will draw the reaction
to the right. We speculate that inactive and active CR3 at the cell
surface are also isoenergetic, that they are also separated by a high
activation energy barrier, and that receptor activation is occasioned
by facilitation of the interconversion of these
forms.
An important consequence of the above
finding is that cells may alter the binding properties of CR3 without a
need for transmitting a large amount of energy from the cytoplasmic
side of the membrane to the exoplasmic surface. Indeed, it is difficult
to imagine a propagated conformational change carried by the two
membrane-spanning peptides of CR3 delivering an energetically large
signal to the exoplasmic domain. While the shift from inactive to
active receptor is energetically neutral, the transition appears to
have a very high activation energy since it does not occur to a
significant degree during a prolonged incubation (18 h, 37 °C) in
the presence of ligand. Under these conditions, binding of ligand would
be expected to ``trap'' any receptor that may become active
during the incubation. We have thus drawn a high activation energy
profile for the transition from inactive to active CR3 and have
depicted KIM127 as lowering this barrier (Fig. 9). The action of
KIM-127 thus resembles that of a catalyst and could be mediated by
exposure of a physically hidden binding site for C3bi.
10
M). We further showed that the
activation energy for binding is also high (E
15 kcal/mol), and we may thus draw the energy diagram
in Fig. 9. Several conclusions derive from this rendering of
binding energetics. The first is that, while switching from inactive to
active receptors is energetically neutral, removal of ligand bound to
receptor is energetically expensive. A consequence of this is that
spontaneous disassociation of ligand from receptor will be slow. From
the on rate (Fig. 4) and the affinity (Fig. 5), we
calculate that more than 20 min is required for spontaneous
disassociation of one-half of the ligand, and preliminary studies of
the disassociation of C3bi-AP in the presence of excess unlabeled C3bi
are consistent with this estimate (not shown). The slow spontaneous
disassociation of ligand from CR3 stands in contrast with the
observation that during chemotaxis of PMN, CR3 may bind substrate-bound
ligand and release the ligand in less than 1 min (Nahas et
al., 1971). We have also observed that C5a-stimulated PMN may bind
to endothelial cells via CR3 and may fully detach within 7 min (Lo et al., 1989b), that C5a-stimulated PMN may bind C3bi-coated
erythrocytes and release the erythrocytes within 5 min,
(
)and that PMN bind C3bi-coated epithelial monolayers
via CR3 but promptly detach upon stimulation (Cramer et al.,
1986). These observations indicate that detachment of PMN from a
ligand-coated surface is not likely to be caused by spontaneous release
of ligand from CR3 and is likely to require an energy-dependent step.
(
)An alternative
mechanism to reverse binding could involve proteolytic degradation of
the receptor or ligand. The nature of the process by which ligand is
removed from receptor is currently under study.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.