From the Center for Blood Research, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, July 5, 2000, and in revised form, September 13, 2000
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ABSTRACT |
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The leukocyte integrin
The integrin Comparisons among leukocyte integrins suggest that
A key question of current integrin research is the nature of the
structural alterations in "inside-out signaling" that enables ligand binding by the extracellular domain in response to signals impinging on the cytoplasmic/transmembrane domains. Electron microscopy reveals an overall integrin structure of a globular head region connected to the cell membrane by two stalk regions (16). The head
region binds ligand and contains domains from the N-terminal portions
of both the The stalk regions provide the crucial link between the signals
impinging on the Here, we have defined regions of the integrin Cell Lines and Monoclonal Antibodies--
293T cells (human
renal epithelial transformed cells) were grown in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (FBS), nonessential amino acids (Life Technologies, Inc.),
2 mM glutamine, and 50 µg/ml gentamicin. The mouse
anti-human Human/Chicken or Human/Mouse Chimeric Transfection--
Plasmids for transfection were purified by
QIAprep Spin Kit or Maxi Kit (Qiagen, Chatsworth, CA). 293T cells were
transiently transfected with human Flow Cytometry--
Cells were washed twice with L15 medium
supplemented with 2.5% fetal bovine serum (L15/FBS). Cells
(106) were incubated with 50 µl of primary antibody (20 µg/ml purified mAb, or 1:100 dilution of ascites) on ice for 30 min.
Cells were then washed three times with L15/FBS, followed by incubation
with 50 µl of a 1:20 dilution of fluorescein
isothiocyanate-conjugated goat anti-mouse IgG (Zymed
Laboratories Inc., San Francisco, CA) for 30 min on ice. After
washing three times with L15/FBS, cells were resuspended in 200 µl of
cold phosphate-buffered saline and analyzed on a FACScan (Becton
Dickinson, San Jose, CA). Antigen expression is presented as mean
fluorescence intensity of cells.
iC3b-coated Erythrocyte Binding Assay--
As described
previously (7, 14)), sheep erythrocytes (Colorado Serum Co., Denver,
CO) were washed, resuspended to 6 × 108 cells/10 ml
in buffer 1 (Hanks' balanced salt solution, 15 mM HEPES,
pH 7.3, and 1 mM MgCl2), and sensitized with 80 µl of IgM anti-Forssman mAb M1/87 culture supernatant for 1 h at
room temperature (E-IgM). The cells were then washed and resuspended in
1.8 ml of buffer 2 (Hanks' balanced salt solution, 15 mM
HEPES, pH 7.3, 1 mM MgCl2, and 1 mM
CaCl2), supplemented with 200 µl of C5-deficient human
serum (Sigma). After incubation at 37 °C for 1 h, the resulting E-IgM-iC3b were washed twice and resuspended in 6 ml of buffer 2.
To assay the binding of Cysteine-rich Regions of the Activation of
The activating region in the C-terminal cysteine-rich region in the
mouse Chicken Residues in Cysteine-rich Repeats 2 and 3 Activate
Within the activating region defined in chicken Residues 4 and 22 in the N-terminal Cysteine-rich Region of Chicken
In region 1-29 of the Activating Mutations Expose the CBR LFA-1/2 Epitope in the
C-terminal Cysteine-rich Region of Among the Activating substitutions in the N-terminal region were localized within
the PSI domain (Fig. 6). Human/chicken
substitutions T4P and T22A each synergized with the C-terminal region
and, when present together, gave augmented synergy. PSI domains in
integrins contain six cysteines that form intradomain disulfide bonds
and one cysteine that forms a long range interdomain disulfide (30, 31). Each of the activating substitutions neighbors a cysteine residue
(Fig. 6). The substitution T4P neighbors Cys-3, which forms the long
range disulfide bond to Cys-425, which is at the beginning of the
C-terminal cysteine-rich repeats (Fig. 6). Thus, the two regions in
which activating substitutions are found, the PSI domain and
cysteine-rich repeats, are linked by a disulfide bond and must be
neighboring domains in the three-dimensional structure of the integrin
X
2 (p150,95) recognizes the
iC3b complement fragment and functions as the complement receptor type 4.
X
2 is more resistant to
activation than other
2 integrins and is inactive in
transfected cells. However, when human
X is paired with
chicken or mouse
2,
X
2 is
activated for binding to iC3b. Activating substitutions were mapped to
individual residues or groups of residues in the N-terminal
plexin/semaphorin/integrin (PSI) domain and C-terminal
cysteine-rich repeats 2 and 3. These regions are linked by a long range
disulfide bond. Substitutions in the PSI domain synergized with
substitutions in the cysteine-rich repeats. Substitutions T4P, T22A,
Q525S, and V526L gave full activation. Activation of binding to iC3b
correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich
repeat 3. The data suggest that the activating substitutions are
present in an interface that restrains the human
X/human
2 integrin in the inactive state.
The opening of this interface is linked to structural rearrangements in
other domains that activate ligand binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
X
2 (p150,95,
CD11c/CD18) is one of four integrins that are restricted in expression
to leukocytes and have different
subunits associated with a common
integrin
2 subunit (1, 2).
X
2 is also known as the complement
receptor type 4. Integrin
X
2 is
expressed on the surface of macrophages, monocytes, granulocytes, and
certain activated and B lymphocyte subpopulations (3-6). Upon
activation,
X
2 binds to its ligands,
complement component iC3b (7-9) and fibrinogen (5, 10), and mediates leukocyte adherence to endothelium and other cells, possibly by binding
to intercellular adhesion molecule 1 (ICAM-1) (4, 11-13).
X
2 has the highest barrier to activation
of ligand binding. On cells that coexpress the integrins
X
2 and Mac-1
(
M
2), stronger cellular activation is
required to activate
X
2 than
M
2 for binding to the ligand iC3b (8).
When transfected into COS or 293T cells, the integrins LFA-1
(
L
2) and
M
2
are active in binding ligands; however,
X
2 is not (14). Construction of chimeric
M and
X
subunits showed that many
reciprocal exchanges activated ligand binding, suggesting that
structural perturbations released restraints that otherwise held
X
2 in an inactive state (14).
Interestingly, although
X
2 expressed on
COS-7 cells could not bind to the ligand iC3b, an interspecies hybrid,
X
2, comprised of chicken
2
and human
X subunits was constitutively active in
binding iC3b (7). By contrast, human
M/chicken
2 and human
M/human
2
integrin heterodimers bound iC3b equally well. Studies with
mAb1 map ligand binding to
the I domain of the
X
2
X
subunit (7). These findings suggest that an intersubunit restraint on
X conformation is loosened with the chicken
2 subunit so that
X can more readily adopt the conformation that binds iC3b. It may be significant in light
of this finding and the finding that
X
2
is more difficult to activate than
L
2 or
M
2 that the association between
X and
2 is more difficult to disrupt with
denaturing conditions than the association between
L and
2 or between
M and
2
(15).
and
subunits. Seven 60-amino acid repeats in the
N-terminal half of the
subunit have been predicted to fold into a
-propeller domain (17). A subset of integrin
subunits, including
the
X subunit, contains an I domain inserted between
-sheets 2 and 3 of the
-propeller domain. The I domain has a
structure like small G proteins with a metal ion-dependent adhesion site at the top of the domain where ligand is bound (18, 19).
A conformational change at the MIDAS that regulates ligand binding is
linked structurally to a large movement of the C-terminal
-helix
that connects the bottom of the I domain to the
-propeller domain
(19-23). A domain in the
subunit has a predicted fold that is like
the I domain and a MIDAS-like site (18, 24-26). This
subunit
I-like domain associates with the side of the
subunit
-propeller
domain at
-sheets 2 and 3 (27, 28) and is thus near to the
subunit I domain, which links to
-sheets 2 and 3 at the top of the
-propeller domain.
and
subunit transmembrane and cytoplasmic domains and the conformational changes that occur in the ligand-binding head region. In the
subunit, the stalk region appears to consist of
the region C-terminal to the predicted
-propeller domain. The stalk
region is predicted to consist of domains with a two-layer
-sandwich
structure (29). Four subregions of the
M stalk have been
defined with mAb epitopes, three of which react with mAbs whether or
not the
subunit is coexpressed. In the
subunit, the stalk
region appears to consist of the cysteine-rich regions that precede and
follow the I-like domain, i.e. residues 1-103 and 342-678
in
2. These cysteine-rich regions are linked by a long
range disulfide bond defined in
3 that is predicted to
link Cys-3 and Cys-425 in
2 (30). The N-terminal
cysteine-rich region of residues ~1-50 shares sequence homology with
membrane proteins including plexins, semaphorins, and the
c-met receptor (31). This region has two predicted
-helices and has been termed the "PSI domain" for
plexins, semaphorins, and
integrins. The segment from residues 425 to 590 has a
cysteine content of 20% and is composed of four cysteine-rich repeats.
The first repeat is less similar to the others and at its N-terminal
end contains the cysteine that disulfide bonds to the PSI domain.
Several monoclonal antibodies that activate integrins or report
conformational changes have been mapped to the C-terminal region of the
subunit that includes the cysteine-rich repeats (28, 32-37) and to
the N-terminal cysteine-rich region (33). Many of these mAbs recognize
epitopes that become exposed after integrin activation. One of these,
mAb KIM127 to the
2 subunit, is not dependent on
association with the
subunit for reactivity and indeed reacts
better with the free
2 subunit than with the integrin
heterodimer (38). Thus, structural changes in the stalk region
that include exposing antibody epitopes on the integrin
subunit are
associated with integrin activation.
2 subunit
involved in regulating ligand binding by
X
2. Ligand binding is activated when the
human
X subunit is complexed with the chicken
2 subunit (7). We hypothesized that this reflects a
release of structural contacts between the human
X and
human
2 subunits that normally restrain
X
2 in a nonligand binding conformation. To map these contacts within the
2 subunit, we have
utilized chicken/human
2 chimeras. We map the key
differences and provide direct evidence that residues that restrain
ligand binding by
2 are present in both the N-terminal
cysteine-rich PSI domain and the C-terminal cysteine-rich repeats.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
X mAb CBRp150/2E1 (7) and the anti-human
2 mAbs KIM185 (39) and CBR LFA-1/2 (40) have been
previously described.
2
Constructs--
Human or chicken
2 cDNA were
inserted in vector AprM8 (41). Chimeras and substitution
mutants were generated by polymerase chain reaction overlap extension
(42). Briefly, 5' and 3' end primers were designed to include unique
restriction sites. Mutations were introduced by a pair of inner
complementary primers. After a second round of polymerase chain
reaction, the products were digested and ligated with the corresponding
predigested plasmids. All constructs were verified by DNA sequencing.
X and wild-type or
mutant
2 constructs using calcium phosphate (43, 44).
Medium was changed after 7-11 h. Cells were harvested for analysis
48 h after transfection.
X
2 to iC3b, 293T
cells transfected with recombinant
X
2
were plated on 12-well polylysine-coated plates for at least 4 h
prior to the experiment. After washing with buffer 2, the cells were
incubated together with 200 µl of E-IgM-iC3b for 30 min at 37 °C.
Unbound erythrocytes were removed by washing three times, and rosettes
(>10 erythrocytes/293T cell, >100 cells examined) were scored with microscopy.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 Subunit Regulate
Integrin
x
2 Binding to iC3b--
To
locate regions in the integrin
2 subunit that restrain
activation of
X
2, interspecies
human/chicken chimeric
2 subunits were made. Chimeras
were named according to the species origin of their segments. For
example, h103c indicates that residues 1 to 103 are human and residues
104 to the C-terminal end are chicken. Each construct was cotransfected
with the human
X subunit into 293T cells. Proper
expression was confirmed by immunostaining with antibody CBRp150/2E1 to
the
X subunit. All human/chicken
2
chimeras studied here were expressed as well as human
2
in
X
2 complexes. The percentage of
293T transfectants expressing
X
2 ranged
from 68 to 85% for chimeras and wild type in all experiments described
below. Transfectants were assayed for activation of ligand binding by
rosetting with erythrocytes sensitized with iC3b (E-IgM-iC3b). The
percentage of rosetting cells was normalized to the percentage
of
X
2+ cells for each
construct. Transfectants expressing hybrid
X
2 (human
X/chicken
2) but not transfectants expressing human
X
2 formed rosettes with E-IgM-iC3b,
confirming previous observations with COS-7 cell transfectants (7). The
chimeras mapped activation of ligand binding by chicken
2 to two regions, residues 1-71 and residues 421-610
(Fig. 1). The importance of residues
421-610 was shown by activation of iC3b rosetting by chimeras h103c
and h421c but not by chimeras h610c, h103c421h, and c71h610c. Residues 1-71 were not sufficient by themselves to activate iC3b binding as
shown with chimera c71h; however, they augmented rosetting when present
in combination with residues 421-610 (Fig. 1). Thus, with residues
421-610 of chicken origin in chimeras h103c and h421c, about 40% of
transfectants rosetted with E-IgM-iC3b. With both residues 1-71 and
421-610 of chicken origin in chimeras c678h and c71h421c, about 80%
of transfectants rosetted. This was the same level as with the
wild-type chicken
2 subunit. Thus, activation was a
synergistic effect of the N- and C-terminal cysteine-rich regions of
the chicken
2 subunit.
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Fig. 1.
Regions 1-71 and 421-610 of the
chicken 2 subunit activate
X
2
binding to iC3b. The indicated human/chicken
2
chimeras were cotransfected with the human
X subunit
into 293T cells. The percentage of transfected cells rosetting with
>10 iC3b-sensitized erythrocytes was determined with microscopy.
Rosetting was calculated as the percentage of rosetting cells divided
by the percentage of 293T cells expressing
X
2 as determined by immunofluorescence
flow cytometry. Results are averages of three experiments.
Bars indicate standard deviation. Hu, human;
Ch, chicken
x
2 by Regions in the
Mouse
2 Subunit--
We found that
X
2 heterodimers containing human
X and mouse
2 subunits were activated for
binding to iC3b almost as well as those containing human
X and chicken
2 subunits (Fig.
2A). Human/mouse
2 chimeras showed that the region containing residues 344-612 was activating (Fig. 2A). The h98m chimera was less
activating than mouse
2, suggesting that the N-terminal
cysteine-rich region contributed to activation. Furthermore, the m122h,
m163h, m254h, m302h, and m344h chimeras showed that the N-terminal
cysteine-rich region was not sufficient for activation, similar to the
results with the chicken
2 subunit.
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Fig. 2.
Regions 1-98 and 470-538 of the mouse
2 subunit activate complement receptor
type 4 function of
x
2.
A, overall mapping. B, fine mapping in the
C-terminal region. Chimeras of the human and mouse
2
subunits were cotransfected with human
X and tested for
rosetting with iC3b-sensitized erythrocytes as described in Fig. 1.
Hu, human; Mo, mouse.
2 subunit was defined with a further series of chimeras (Fig. 2B). These narrowed activation by the
C-terminal cysteine-rich region to residues 470-538 since chimera
m122h470m was activating, whereas m122h538m was not (Fig.
2B). Furthermore, residues in two different segments,
470-502 and 502-538, were activating because chimera m122h502m was
partially activating, whereas m122h470m was fully activating, and
m122h538m was not activating.
x
2--
Mapping of the C-terminal
cysteine-rich repeat region of chicken
2 was refined
with five further chicken/human chimeric
2 constructs.
Each construct contained N-terminal residues 1-71 and various lengths
of the C-terminal cysteine-rich repeats from chicken
2
(Fig. 3A). Rosetting of
chimeras c71h446c, c71h470c, and c71h498c with E-IgM-iC3b was similar
to that of wild-type chicken
2. However, chimeras
c71h527c and c71h562c did not bind to iC3b. Therefore, residues within
region 498-527 can activate binding to the ligand iC3b.
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Fig. 3.
Specific chicken residues in cysteine-rich
repeats 2 and 3 that activate binding of
x
2
to iC3b. A, mapping within the C-terminal region.
B, mapping of individual or groups of C-terminal residues
that activate
X
2 alone or in combination
with the N-terminal region. Chimeric
2 subunits were
cotransfected with human
X and tested for rosetting with
iC3b-sensitized erythrocytes as described in Fig. 1. Hu,
human; Ch, chicken.
2 of
498-527, 11 residues differ between human and chicken. Groups of one to three chicken amino acid residues in this region were introduced into the human
2 subunit and their effect on binding to
iC3b was examined (Fig. 3B). In combination with chicken
residues 1-71, four groups of amino acid substitutions were
activating: Q510T/Y511F/E513D in repeat 2 and T516N/I517M, R521F/Y522H,
and Q525S/V526L in repeat 3. Chimera c71h/Q525S/V526L was as
active as chicken
2. The four activating groups of
residues were also tested in the absence of any other chicken residues.
In this situation, only the mutation Q525S/V526L was activating, and
its activity was reduced compared with c71h/Q525S/V526L (Fig.
3B).
2 Activate
x
2 in Synergy
with the C-terminal Cysteine-rich Region--
Mapping of the
N-terminal cysteine-rich region was refined with three chicken/human
chimeras that included different portions of the N-terminal region in
combination with the synergistic C-terminal segment (Fig.
4A). All three
2 chimeras, c71h421c, c50h421c, and c29h421c, activated
binding to E-IgM-iC3b to the same extent as chicken
2.
Thus, residues within the first 29 amino acids of the
2
subunit are sufficient to synergistically activate
X
2.
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Fig. 4.
Chicken residues 4 and 22 of the
2 subunit activate iC3b binding in
synergy with residues 525 and 526. A, fine mapping of
the N-terminal region in synergy with the C-terminal region.
B, mapping of individual N-terminal residues in synergy with
residues 525 and 526. Details are as described in Fig. 1.
Hu, human; Ch, chicken.
2 subunit, 11 residues differ
between the human and chicken. Groups of these residues were
substituted with chicken sequence in combination with the mutation
Q525S/V526L in the C-terminal cysteine-rich region in each construct
(Fig. 4B). Most of the mutants rosetted E-IgM-iC3b no better
than the parent Q525S/V526L mutant. However, mutants
Q1A/T4P/Q525S/V526L and T4P/Q525S/V526L but not Q1A/Q525S/V526L were
more active than Q525S/V526L, implicating the substitution T4P in
activation. Similarly, mutants T22A/Q25K/Q525S/V526L and
T22A/Q525S/V526L but not Q25K/Q525S/V526L were more active than
Q525S/V526L, implicating T22A. Moreover, the combination of mutations
T4P and T22A was even more active, and the mutant T4P/T22A/Q525S/V526L
was as active as chicken
2 (Fig. 4B).
Therefore, four chicken residues, two each in the N-terminal and
C-terminal cysteine-rich regions of
2, are sufficient to maximally activate iC3b rosetting by
X
2.
2--
Several mAbs
that activate
2 integrins map to the C-terminal
cysteine-rich region of the
2 subunit (28, 37). The
mouse/human substitutions recognized by these mAbs map very near to the
substitutions Q525S/V526L that activate
X
2. Specifically, mAb KIM185 recognizes residues 581-604 and mAb CBR LFA-1/2 recognizes residues 534 and 536.2 Recognition by mAb CBR
LFA-1/2 correlates with the activation status of
2
integrins;
L
2 and
M
2, which are active in 293T cell
transfectants, are recognized well by CBR LFA-1/2, whereas
X
2, which is inactive in 293T cells, is
recognized poorly2 (Fig. 5).
We examined the effect of activating mutations on expression of CBR
LFA-1/2 and KIM185 epitopes (Fig. 5). The KIM185 epitope was expressed
equally well by wild-type and mutant
X
2.
By contrast, activating mutations induced exposure of the CBR LFA-1/2
epitope (Fig. 5). The Q525S/V526L mutation partially exposed the CBR
LFA-1/2 epitope, whereas the T4P/T22A/Q525S/V526L mutation maximally
exposed the epitope, i.e. to the same level as seen with
KIM185 mAb. Exposure of the CBR LFA-1/2 epitope correlated with
activation of binding to iC3b (Fig. 5). Therefore, the mutations cause
structural rearrangements in the stalk region that lead to exposure of
the CBR LFA-1/2 epitope and are linked to activation of ligand binding.
Furthermore, binding of the CBR LFA-1/2 and KIM185 mAbs demonstrates
that the mutations do not disrupt the structure of the cysteine-rich
repeats.
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Fig. 5.
Activation of iC3b binding by individual
amino acid substitutions in the PSI domain and cysteine-rich repeat 3 correlates with exposure of the CBR LFA-1/2 epitope. Mutant or
wild-type 2 subunits were cotransfected with human
X into 293T cells. The transfectants were immunostained
with mAbs KIM185 or CBR LFA-1/2 followed by immunofluorescence flow
cytometry. Epitope expression is normalized to the percentage of cells
binding mAb CBR p150/2E1 to the
X subunit. Binding to
iC3b was quantified as described in Fig. 1. Hu, human.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 integrins,
X
2 is the most resistant to activation
and to dissociation of its
and
subunits. Here, we have identified specific amino acid residues that restrain
X
2 in a conformation in which it does not
bind its ligand, iC3b. We extended previous observations with the
chicken
2 subunit (7) by showing that pairing of human
X with
2 from another species, the mouse,
also activates binding to iC3b. Interspecies
2 subunit chimeras associated with human
X subunits demonstrated
that the C-terminal cysteine-rich repeats from mouse or chicken were
sufficient for partial activation and that the N-terminal cysteine-rich
PSI domain was insufficient for activation but synergized with the C-terminal cysteine-rich repeats.
subunit.
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Fig. 6.
Sequence differences of the human
2 PSI domain and cysteine-rich repeats
with the mouse and chicken and localization of activating
mutations. Only residues that differ in the mouse or chicken
sequences are shown. Underlined residues: tested, no effect.
Black inverse residues: activating. Gray inverse
residues: activating in synergy with other residues. Underlining
or inverse font is extended to include the group of residues that was
tested. The long range disulfide bond between Cys-3 and Cys-425 is
shown. Dots above the sequence mark residue positions that
are multiples of ten. hu, human; mo, mouse;
ch, chicken.
Activating substitutions within the C-terminal region localized to
cysteine-rich repeats 2 and 3 (Fig. 6). One segment containing activating mouse substitutions localized wholly within repeat 2, whereas another included portions of repeats 2 and 3 (Fig. 6). Fine
mapping of three groups of chicken substitutions that activated
X
2 in synergy with chicken residues in
the PSI domain showed that one group mapped to repeat 2 and two groups
mapped to repeat 3 (Fig. 6). One pair of substitutions that was
sufficient for activation of
X
2,
Q525S/V526L, mapped to repeat 3. We cannot exclude the presence of
activating substitutions in repeat 1 because all chimeras in which
repeat 1 was mouse or chicken also contained repeats 2 and 3 from mouse
or chicken, which were activating by themselves. However, the segments
following the PSI domain and preceding repeat 1 were not activating.
Furthermore, repeat 4 and more C-terminal segments were not activating.
The species-specific differences in repeat 4 are greater than in
repeats 2 and 3 (Table I); therefore, an
insufficiency of species-specific differences cannot explain the lack
of activation by repeat 4.
|
What is the mechanism of integrin activation by species-specific
substitutions in the PSI domain and cysteine-rich repeats 2 and 3? Many
other observations suggest that integrins are restrained in their
resting state in a conformation that does not bind ligand and that a
wide variety of perturbations can activate ligand binding. Our results
suggest that the PSI domain and cysteine-rich repeats 2 and 3 have an
important function in restraining integrins in their resting, inactive
state. These restraints are overcome when the and
subunits are
from different species; therefore, it appears that there are direct
interactions between these
subunit domains and the
subunit that
constrain integrins in the inactive configuration. The substitutions
are unlikely to disrupt the overall conformation of these domains
because they are naturally occurring variations between species.
Furthermore, we demonstrated that mAb CBR LFA-1/2, which binds to
species-specific residues in repeat 3,2 binds well when the
activating mutations Q525S and V526L are present in repeat 3. Therefore, it appears that the activating mutations we have defined are
within or near an interface between the
2 and
X subunits. The findings suggest that in resting
integrins, there are contacts of the PSI domain and cysteine-rich
repeats 2 and 3 with the
subunit and that these contacts help
restrain ligand binding. It appears that certain species-specific
substitutions disrupt this interaction and, thereby, lower the
activation energy required for activation of ligand binding. Binding of
iC3b by
X
2 maps to the
X I
domain (7). Conformational shifts around the MIDAS in I domains
regulate ligand binding and are linked to a large movement of the
C-terminal
-helix of the I domain that connects to other integrin
subunits (19-23). Therefore, it appears that an alteration in contacts
in the stalk region between the
subunit and the PSI domain and the
cysteine-rich repeats in the
subunit are linked to conformational
rearrangements in the ligand-binding domains in the headpiece of integrins.
The loss of the restraints that keep X
2
in an inactive state appears to reflect an opening up of the
interface in the stalk region based on exposure of the epitope for the
mAb CBR LFA-1/2. This mAb can activate integrins
L
2 and
M
2
(40). It showed little reactivity with wild-type
X
2; however, introduction of activating
amino acid substitutions Q525S/V526L in cysteine-rich repeat 3 exposed
the CBR LFA-1/2 epitope, and addition of the T4P/T22A substitutions
fully exposed the epitope. Exposure correlated with iC3b binding.
The CBR LFA-1/2 mAb maps to residues 534 and 5362,
and nearby residues 525 and 526, to which activating mutations map in
repeat 3. It is unlikely that there is a significant conformational change in this repeat because its structure is constrained by four
disulfide bonds. Therefore, we envision a movement apart or change in
orientation of the
and
subunits that exposes the CBR LFA-1/2
epitope in repeat 3.
Other studies also imply a structural restraint on integrin activation
that is localized in the cysteine-rich regions of the subunit.
Activation of the integrin LFA-1 (
L
2)
expressed on COS cells was induced if the C-terminal cysteine-rich
repeat region of the
2 subunit was replaced by that of
1 (45). A point mutation that introduces a
N-glycosylation site into the beginning of cysteine-rich repeat 4 of the
3 subunit activated integrins
IIb
3 and
v
3 (46). Furthermore, disruption of the long range disulfide bond between
the PSI domain and the cysteine-rich repeats resulted in increased
ligand binding affinity of
IIb
3 (47).
Moreover, treatment with reducing agents, such as dithiothreitol,
induced the active conformation of
1 integrin (33) and
increased platelet aggregation through the
IIb
3 integrin (48). Recently, an
anti-
1 antibody with an activation-dependent
epitope has been mapped to the N-terminal cysteine-rich region,
suggesting a role of this region as a regulatory site for integrin
activation (33). In addition, several monoclonal antibodies against the
C-terminal cysteine-rich regions of
1 (32, 34),
2 (37), and
3 (49) integrins have been
described as activating mAbs with respect to their ability to promote
ligand binding. A plausible explanation is that these mAbs selectively
bind to the open conformation of the stalk region and thus stabilize
integrins in this conformation and induce linked rearrangements in the
ligand-binding domains. Indeed, activating mAbs to both the
1 and
2 cysteine-rich regions have been
found to bind better to isolated
subunits than
complexes,
implying that they favor an open conformation (26, 37, 38, 50).
In contrast to domains in the globular headpiece of integrins, the
stalk regions do not appear to directly bind ligand but instead appear
to regulate ligand binding and to relay activation signals impinging on
the cytoplasmic and transmembrane domains of the integrin and
subunits. We have identified specific amino acid residues in the PSI
domain and cysteine-rich repeats 2 and 3 of the
subunit that form
part of the interface between the
and
subunits in the stalk
region that restrains conformational movements in the ligand-binding
headpiece. It would be very interesting to learn which regions of the
subunit participate in this interface and the molecular details of
how structural alterations are communicated from one domain to another
in integrins.
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ACKNOWLEDGEMENT |
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We thank Mark Ryan for assistance with fluorescence-activated cell sorter analysis.
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FOOTNOTES |
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* This project was supported by National Institutes of Health Grant CA 31799.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Center for Blood
Research, Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3030; E-mail: springer@sprsgi.med.harvard.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M005868200
2 C. Lu, M. Ferzly, J. Takagi, and T. A. Springer, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; FBS, fetal bovine serum; c, chicken; h, human; m, mouse.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Springer, T. A. (1990) in Clinical Aspects in Immunology (Lachmann, P. J. , Peters, D. K. , Rosen, F. S. , and Walport, M. J., eds), 5th Ed. , pp. 199-219, Blackwell Scientific, Oxford |
2. | Van der Vieren, M., Trong, H. L., Wood, C. L., Moore, P. F., St. John, T., Staunton, D. E., and Gallatin, W. M. (1995) Immunity 3, 683-690[Medline] [Order article via Infotrieve] |
3. |
Miller, L. J.,
Schwarting, R.,
and Springer, T. A.
(1986)
J. Immunol.
137,
2891-2900 |
4. |
Keizer, G. D.,
Borst, J.,
Visser, W.,
Schwarting, R.,
de Vries, J. E.,
and Figdor, C. G.
(1987)
J. Immunol.
138,
3130-3136 |
5. | Postigo, A. A., Corbi, A. L., Sanchez-Madrid, F., and De Landazuri, M. O. (1991) J. Exp. Med. 174, 1313-1322[Abstract] |
6. | Hogg, N., Takacs, L., Palmer, D. G., Selvendran, Y., and Allen, C. (1986) Eur. J. Immunol. 16, 240-248[Medline] [Order article via Infotrieve] |
7. |
Bilsland, C. A. G.,
Diamond, M. S.,
and Springer, T. A.
(1994)
J. Immunol.
152,
4582-4589 |
8. | Myones, B. L., Dalzell, J. G., Hogg, N., and Ross, G. D. (1988) J. Clin. Invest. 82, 640-651[Medline] [Order article via Infotrieve] |
9. | Ross, G. D., Reed, W., Dalzell, J. G., Becker, S. E., and Hogg, N. (1992) J. Leukocyte Biol. 51, 109-117[Abstract] |
10. | Loike, J. D., Sodeik, B., Cao, L., Leucona, S., Weitz, J. I., Detmers, P. A., Wright, S. D., and Silverstein, S. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1044-1048[Abstract] |
11. | Keizer, G. D., te Velde, A. A., Schwarting, R., Figdor, C. G., and de Vries, J. E. (1987) Eur. J. Immunol. 17, 1317-1322[Medline] [Order article via Infotrieve] |
12. |
Stacker, S. A.,
and Springer, T. A.
(1991)
J. Immunol.
146,
648-655 |
13. | te Velde, A. A., Keizer, G. D., and Figdor, C. G. (1987) Immunology 61, 261-267[Medline] [Order article via Infotrieve] |
14. | Diamond, M. S., Garcia-Aguilar, J., Bickford, J. K., Corbi, A. L., and Springer, T. A. (1993) J. Cell Biol. 120, 1031-1043[Abstract] |
15. | Sanchez-Madrid, F., Nagy, J., Robbins, E., Simon, P., and Springer, T. A. (1983) J. Exp. Med. 158, 1785-1803[Abstract] |
16. |
Weisel, J. W.,
Nagaswami, C.,
Vilaire, G.,
and Bennett, J. S.
(1992)
J. Biol. Chem.
267,
16637-16643 |
17. |
Springer, T. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
65-72 |
18. | Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638[Medline] [Order article via Infotrieve] |
19. | Emsley, J., Knight, C. G., Farndale, R. W., Barnes, M. J., and Liddington, R. C. (2000) Cell 101, 47-56[Medline] [Order article via Infotrieve] |
20. | Lee, J.-O., Bankston, L. A., Arnaout, M. A., and Liddington, R. C. (1995) Structure 3, 1333-1340[Medline] [Order article via Infotrieve] |
21. |
Oxvig, C.,
Lu, C.,
and Springer, T. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2215-2220 |
22. |
Li, R.,
Rieu, P.,
Griffith, D. L.,
Scott, D.,
and Arnaout, M. A.
(1998)
J. Cell Biol.
143,
1523-1534 |
23. | Shimaoka, M., Shifman, J. M., Jing, H., Takagi, J., Mayo, S. L., and Springer, T. A. (2000) Nat. Struct. Biol. 7, 674-678[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Tozer, E. C.,
Liddington, R. C.,
Sutcliffe, M. J.,
Smeeton, A. H.,
and Loftus, J. C.
(1996)
J. Biol. Chem.
271,
21978-21984 |
25. | Tuckwell, D. S., and Humphries, M. J. (1997) FEBS Lett. 400, 297-303[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Huang, C.,
Zang, Q.,
Takagi, J.,
and Springer, T. A.
(2000)
J. Biol. Chem.
275,
21514-21524 |
27. |
Puzon-McLaughlin, W.,
Kamata, T.,
and Takada, Y.
(2000)
J. Biol. Chem.
275,
7795-7802 |
28. |
Zang, Q.,
Lu, C.,
Huang, C.,
Takagi, J.,
and Springer, T. A.
(2000)
J. Biol. Chem.
275,
22202-22212 |
29. |
Lu, C.,
Oxvig, C.,
and Springer, T. A.
(1998)
J. Biol. Chem.
273,
15138-15147 |
30. | Calvete, J. J., Henschen, A., and González-Rodríguez, J. (1991) Biochem. J. 274, 63-71[Medline] [Order article via Infotrieve] |
31. | Bork, P., Doerks, T., Springer, T. A., and Snel, B. (1999) Trends Biochem. Sci. 24, 261-263[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Faull, R. J.,
Wang, J.,
Leavesley, D. I.,
Puzon, W.,
Russ, G. R.,
Vestweber, D.,
and Takada, Y.
(1996)
J. Biol. Chem.
271,
25099-25106 |
33. |
Ni, H.,
Li, A.,
Simonsen, N.,
and Wilkins, J. A.
(1998)
J. Biol. Chem.
273,
7981-7987 |
34. | Takagi, J., Isobe, T., Takada, Y., and Saito, Y. (1997) J. Biochem. (Tokyo) 121, 914-921[Abstract] |
35. |
Bazzoni, G.,
Shih, D.-T.,
Buck, C. A.,
and Hemler, M. A.
(1995)
J. Biol. Chem.
270,
25570-25577 |
36. | Shih, D. T., Edelman, J. M., Horwitz, A. F., Grunwald, G. B., and Buck, C. A. (1993) J. Cell Biol. 122, 1361-1371[Abstract] |
37. | Stephens, P., Romer, J. T., Spitali, M., Shock, A., Ortlepp, S., Figdor, C., and Robinson, M. K. (1995) Cell Adhes. Commun. 3, 375-384[Medline] [Order article via Infotrieve] |
38. |
Huang, C.,
Lu, C.,
and Springer, T. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3156-3161 |
39. | Andrew, D., Shock, A., Ball, E., Ortlepp, S., Bell, J., and Robinson, M. (1993) Eur. J. Immunol. 23, 2217-2222[Medline] [Order article via Infotrieve] |
40. | Petruzzelli, L., Maduzia, L., and Springer, T. (1995) J. Immunol. 155, 854-866[Abstract] |
41. |
Huang, C.,
and Springer, T. A.
(1995)
J. Biol. Chem.
270,
19008-19016 |
42. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
43. | Heinzel, S. S., Krysan, P. J., Calos, M. P., and DuBridge, R. B. (1988) J. Virol. 62, 3738-3746[Medline] [Order article via Infotrieve] |
44. | DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., and Calos, M. P. (1987) Mol. Cell. Biol. 7, 379-387[Medline] [Order article via Infotrieve] |
45. | Douglass, W. A., Hyland, R. H., Buckley, C. D., Al-Shamkhani, A., Shaw, J. M., Scarth, S. L., Simmons, D. L., and Law, S. K. A. (1998) FEBS Lett. 440, 414-418[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Kashiwagi, H.,
Tomiyama, Y.,
Tadokoro, S.,
Honda, S.,
Shiraga, M.,
Mizutani, H.,
Handa, M.,
Kurata, Y.,
Matsuzawa, Y.,
and Shattil, S. J.
(1999)
Blood
93,
2559-2568 |
47. | Liu, C. Y., Sun, Q. H., Wang, R., Paddock, C. M., and Newman, P. J. (1997) Blood 90, 573 (abstr.) |
48. | Zucker, M. B., and Masiello, N. C. (1984) Thromb. Haemostasis 51, 119-124[Medline] [Order article via Infotrieve] |
49. |
Du, X.,
Gu, M.,
Weisel, J. W.,
Nagaswami, C.,
Bennett, J. S.,
Bowditch, R.,
and Ginsberg, M. H.
(1993)
J. Biol. Chem.
268,
23087-23092 |
50. |
Luque, A.,
Gomez, M.,
Puzon, W.,
Takada, Y.,
Sanchez-Madrid, F.,
and Cabanas, C.
(1996)
J. Biol. Chem.
271,
11067-11075 |