| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
2 Subunit Reveals Regions that Become Exposed Upon Receptor Activation1
Center for Blood Research, Department of Pathology, Harvard Medical School, Boston, MA 02115
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subunits appear to convey signals impinging on the cytoplasmic domains
to the ligand-binding headpiece of integrins. We have examined the
functional properties of mAbs to the stalk region and mapped their
epitopes, providing a structure-function map. Among a panel of 14 mAbs
to the 2 subunit, one, KIM127, preferentially bound to
L2 that was activated by mutations in the
cytoplasmic domains, and by Mn2+. KIM127 also bound
preferentially to the free 2 subunit compared with
resting L2. Activating 2
mutations also greatly enhanced binding of KIM127 to integrins
M2 and X2.
Thus, the KIM127 epitope is shielded by the subunit, and becomes
reexposed upon receptor activation. Three other mAbs, CBR LFA-1/2,
MEM48, and KIM185, activated L2 and bound
equally well to resting and activated L2,
differentially recognized resting M2 and
X2, and bound fully to activated
M2 and X2.
The KIM127 epitope localizes within cysteine-rich repeat 2, to residues
504, 506, and 508. By contrast, the two activating mAbs CBR LFA-1/2 and
MEM48 bind to overlapping epitopes involving residues 534, 536, 541,
543, and 546 in cysteine-rich repeat 3, and the activating mAb KIM185
maps near the end of cysteine-rich repeat 4. The nonactivating mAbs,
6.7 and CBR LFA-1/7, map more N-terminal, to subregions 344–432 and
432–487, respectively. We thus define five different 2
stalk subregions, mAb binding to which correlates with effect on
activation, and define regions in an interface that becomes exposed
upon integrin activation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
L2, CD11a/CD18) is
one of four integrins that are restricted in expression to leukocytes
and have different subunits associated with a common integrin
2 subunit in heterodimers
(1, 2, 3). LFA-1 is expressed on all leukocytes, and is
important in essentially all cell-cell interactions by immune cells,
including Ag-specific interactions and binding of leukocytes in the
circulation to the endothelium. The counterreceptors for LFA-1 are
ICAMs that are members of the Ig superfamily. Other leukocyte integrins
include M2 and
X2, which are
primarily expressed on myeloid lineage cells and bind ligands including
ICAM-1, the complement component iC3b, and fibrinogen. In common with
many other integrins, the leukocyte or 2
integrins must be activated before they bind ligands, through a process
termed inside-out signaling. For example, Ag recognition by receptors
linked to tyrosine kinases, or recognition of chemoattractants by G
protein-coupled receptors, activates intracellular signaling pathways
that in turn activate adhesiveness through LFA-1 on a timescale of less
than 1 s. In this way, LFA-1 acts as an adhesion servomotor under
the control of other cell surface receptors, enabling dynamic
modulation of cellular adhesion and migration.
A key question of current integrin research is how inside-out signals
are transduced from the cytoplasm to the ligand-binding domains,
despite the presence of long and subunit stalk regions that
intervene between the transmembrane domains and the ligand-binding
headpiece. Electron microscopy of integrins reveals an overall
structure with a globular headpiece connected to the plasma membrane by
two long stalks each 
16 nm long (4). The headpiece
binds ligand and contains domains from the N-terminal portions of both
the and subunits. The N-terminal region of the integrin
subunits contains seven repeats of 60 aa each, and has been
predicted to fold into a seven-bladed -propeller domain
(5) containing Ca2+-binding
-hairpin loop motifs (6). About one-half of the
integrin subunits, including the L,
M, and X subunits,
contain a domain of 200 aa that is inserted between -sheets 2 and
3 of the -propeller domain, and is termed the inserted (I) domain.
The I domain has a structure similar to small G proteins, with a metal
ion-dependent adhesion site
(MIDAS)4 at the top of
the domain in which ligand is bound (7, 8). A
conformational change at the MIDAS that regulates ligand binding is
linked structurally to a large movement of the C-terminal -helix,
which connects the bottom of the I domain to the -propeller domain
(8, 9, 10, 11, 12). Integrin subunits contain an evolutionarily
well-conserved domain of 250 residues in their N-terminal portion
that has been predicted to have an I domain-like fold, and a MIDAS-like
site (7, 13, 14, 15, 16). This subunit I-like domain
associates with the side of the subunit -propeller domain at
-sheets 2 and 3 (17, 18), and is thus near to the
subunit I domain that links to -sheets 2 and 3 at the top of the
-propeller domain.
The stalk regions provide the crucial link between signals impinging on
the and subunit transmembrane and cytoplasmic domains and the
conformational changes that occur in the ligand-binding headpiece. In
the subunit, the stalk region appears to consist of the region
C-terminal to the predicted -propeller domain, and contains 500
residues. Four subregions of the M stalk
region have been defined with mAb epitopes, three of which react with
mAb whether or not the subunit is coexpressed. The stalk region
is predicted to consist of domains with a two-layer -sandwich
structure (19). In the subunit, the stalk region
appears to consist of regions that precede and follow the I-like
domain; i.e., residues 1–103 and 342–678 in
2. These regions include segments that are
cysteine rich, and are linked by a long-range disulfide bond defined in
3 that is predicted to link
Cys3 and Cys425 in
2 (20). The N-terminal
cysteine-rich region of about residues 1–50 shares sequence homology
with membrane proteins including plexins, semaphorins, and the
c-met receptor (21). This region has two
predicted -helices, and has been termed the PSI domain for plexins,
semaphorins, and integrins. The region from residues 425 to 590 has a
high cysteine content (20%), and is composed of four 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.
The subunit stalk region appears to be important for regulating
ligand binding in the headpiece. Several Abs that activate integrins or
bind to activated forms of integrins have been mapped to these regions.
The mAb LIBS2, which increases the affinity of the platelet integrin
IIb3 for fibrinogen,
maps to an 89-aa region next to the transmembrane region
(22). The mAb TASC promotes cell adhesion to laminin, and
binds to the region from residues 493 to 602 in the chicken
1 subunit (23). Another
1 integrin-activating mAb, QE.2E5, binds to
the 1 cysteine-rich repeats (24).
mAb 9EG7 recognizes a 1 subunit epitope
between residues 495 and 602 that becomes exposed after activation of
1 integrins, and activates ligand binding by
multiple 1 integrins (25).
Similarly, mAb AG89, which maps to residues 426–587 in the
1 subunit, recognizes an epitope that becomes
exposed after activation of 1 integrins
(26). Four activating mAbs to the
2 subunit, KIM127, KIM185, CBR LFA-1/2, and
MEM48 (27, 28, 29, 30), bind to the 2
cysteine-rich region (18, 31). Thus, structural changes in
the stalk region that include exposing Ab epitopes on the integrin
subunit are associated with integrin activation. However, the location
of these subunit epitopes has not been narrowed to specific
cysteine-rich repeats, e.g., residues 406–570 to which KIM127 maps
include repeats 1, 2, 3, and 4 (31). In many cases, the
epitopes have been mapped not to the cysteine-rich repeats themselves,
but to regions that include the cysteine-rich repeats and adjacent
N-terminal or C-terminal segments.
In this study, we define specific amino acid residues and domains in
the integrin 2 stalk region that are exposed
during activation, and to which activating Abs bind. We demonstrate
that mAb KIM127 preferentially reacts with activated
2 integrins and the free
2 subunit compared with resting
2 integrins. We also demonstrate differences
between resting L2,
M2, and
X2 in the exposure of
the CBR LFA-1/2, KIM185, and MEM48 epitopes on the
2 stalk. We map the epitopes of these four
activating mAbs, and the epitopes of two mAbs that are not activating.
KIM127 recognizes residues 504, 506, and 508 in cysteine-rich repeat 2,
two activating mAbs recognize three to five residues in cysteine-rich
repeat 3, and another activating mAb binds near the end of
cysteine-rich repeat 4. By contrast, two nonactivating mAbs bind to
subregions between the I-like domain and the region to which activating
mAbs bind. We thus provide a structure-function map for the
2 stalk region, and identify residues in
interfaces that become exposed upon integrin activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The murine mAbs TS1/18, CBR LFA-1/7, and CBR LFA-1/2 to human
2 (CD18) were described previously (30, 32). The mAbs KIM127 (27) and KIM185
(28) were kindly provided by M. Robinson (Celltech,
Berkshire, U.K.). MEM48 (33) was generously provided by V.
Horejsi (Institute of Molecular Genetics, Prague,
Czechoslovakia). YFC51 and YFC118 (34) were kindly donated
by G. Hale (University of Oxford, Oxford, U.K.). The mAbs 6.7
(35), GRF1 (36), CLB54 (37), and
CLBLFA-1/1 (38) were obtained from the Fifth International
Leukocyte Workshop. The murine mAbs TS1/22 and TS2/4 to human
L, and the myeloma IgG1 X63 were described
previously (32).
Cell lines
Human embryonic kidney 293T cells were cultured in DMEM
supplemented with 10% FBS, 2 mM glutamine, and 50 µg/ml gentamicin.
JY cells (human B lymphoblastoid cell line) were cultured in RPMI
1640/10% FBS, 50 µg/ml gentamicin. Jurkat-2.7 (J2.7)
transfectants and K562 transfectants that express wild-type or mutant
LFA-1 were described previously (39), and cultured in RPMI
1640/10% FBS supplemented with 4 µg/ml puromycin.
Construction of 2 mutants
The wild-type human and mouse 2 subunit
cDNAs were contained in the expression vector AprM8, a derivative of
CDM8 (40). Chimeric human and mouse
2 subunits were generated by overlap extension
PCR (41, 42). For making chimeras with mouse sequence at
the N-terminal portion and human sequence at the C-terminal portion,
outer left and outer right primers for overlap PCR were used that were
designated mEcoRV and hNOTI, respectively. mEcoRV
was 5' to the EcoRV site at nucleotide 310 in the mouse
2 cDNA, and hNOT I was 3' to the stop codon of
the human 2 cDNA, and contained a stop codon,
a NheI recognition sequence, and a NotI site. The
inner primers designed for each individual chimera contained
overlapping sequences. The first PCRs used the mouse and human
2 cDNA to amplify the mouse and human
sequences, respectively. The second PCR product was digested with
EcoRV and NotI, and the 2.1-kb
EcoRV-NotI fragment was swapped into the
same sites in the wild-type mouse 2 cDNA. The
unique NheI site was used for mutant identification. A
similar approach was used to make chimeras with the N-terminal portion
of human sequence and C-terminal mouse sequence. Briefly, the outer
left PCR primer, hBsrG, was 5' to the BsrG I site at nucleotide 947 of
the human 2 cDNA, and the outer right primer,
mNot I, was complementary to the mouse 2 cDNA,
and contained a stop codon followed by a NheI site and a
NotI site. The second PCR product was cut with BsrG I and
NotI, and the 1.4-kb BsrG I and NotI fragment was
swapped into the wild-type human 2 cDNA at the
same sites. Overlap extension PCR was also used to make human to mouse
amino acid substitution mutants. The outer left and right PCR primers
were hBsrG and hNOT I, respectively. The inner PCR primers were
designed to contain the desired mutations. The second PCR product was
cut with BsrG I and NotI, and swapped into the wild-type
human 2 cDNA at the same sites. All mutations
were confirmed by DNA sequencing.
Transient transfection of 293T cells
The 293T cells were transfected using the calcium
phosphate precipitate method (43, 44). Briefly, 7.5 µg
wild-type or mutant 2 cDNA and 7.5 µg
L cDNA were used to cotransfect one 6-cm plate
of 70–80% confluent cells. Two days after transfection, cells were
detached from the plate with HBSS containing 5 mM EDTA and washed twice
for flow cytometric analysis.
Flow cytometry
Cells were harvested and washed twice with L15 medium (Sigma, St. Louis, MO) containing 2.5% FBS (L15/FBS). Cells (105) were incubated with primary Ab in 100 µl L15/FBS on ice for 30 min, except for KIM127. Incubation with mAb KIM127 was conducted at 37°C for 30 min (27). The mAbs TS1/18, CBR LFA-1/7, CBR LFA-1/2, KIM127, KIM185, MEM48, YFC51, YFC118, TS1/22, and TS2/4 were used as purified IgG at 15 µg/ml, and the International Leukocyte Workshops mAbs 6.7, GRF, CLBLFA-1/1, and CLB54 were used at 1/100 dilution. The control IgG X63 was used as 1/5 dilution of hybridoma supernatant. Cells were then washed twice with L15/FBS and incubated with FITC-conjugated goat anti-mouse IgG (heavy and light chain; Zymed Laboratories, San Francisco, CA) for 30 min on ice. After washing, cells were resuspended in cold PBS and analyzed on a FACScan (BD Biosciences, San Jose, CA).
Cell adhesion
Cell adhesion to purified ICAM-1 was described previously (39). Briefly, cells were labeled with 2',7'-bis-(carboxyethyl)-5 (and -6)-carboxyfluorescein, acetoxymethyl ester, and resuspended to 106/ml in L15/FBS. Cell suspensions (50 µl) were mixed in ICAM-1-coated wells with an equal volume of L15/FBS containing activating or control mAb. After incubation at 37°C for 30 min, unbound cells were washed off on a Microplate Autowasher (Biotek Instruments, Winooski, VT). The fluorescence content of total input cells and the bound cells in each well was quantitated on a Fluorescent Concentration Analyzer (Idexx, Westbrook, ME). The bound cells were expressed as a percentage of total input cells.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 subunit
The mAbs were tested for differential reactivity with mutationally
activated and wild-type LFA-1. L subunit
mutations with a complete truncation of the cytoplasmic domain,
L1090*, and an internal deletion of the
conserved GFFKR motif in the cytoplasmic domain,
L
GFFKR, activated binding to ICAM-1 of
L2 heterodimers
expressed in several cell types, including K562 cells and J2.7 cells
(39). These mutants also express the activation-dependent
m24 epitope (45). Of a panel of 14 mAbs to the
2 subunit, one mAb, KIM127, stained
transfectants expressing wild-type
L2 much more weakly
than the other mAbs (Fig. 1
). By
contrast, KIM127 bound almost as well to the constitutively active
L2 mutants as the
other mAbs (Fig. 1). KIM127 mAb bound 7.5- and 5.3-fold better to
L1090* 2 and
LGFFKR 2
complexes, respectively, than to wild-type
L2 (Table I). By contrast, the other 13 mAbs to
2 bound equally well to wild-type
L2 and the
constitutively active mutants (Table I and Fig. 1). These included five
other mAbs to the C-terminal region of 2, mAbs
6.7, MEM48, CBR LFA-1/7, CBR LFA-1/2, and KIM185.
|
|
L2 can also be
activated by truncation of the 2 cytoplasmic
domain, i.e., by mutation 2702*
(46) (our unpublished data). Although KIM127 bound
less well than other mAbs to 293T transfectants expressing wild-type
L2, it bound to
L2 containing the
2702* mutation as well as other mAbs (Table II). In all cells examined that
constitutively expressed LFA-1, including JY lymphocytes (see below),
KIM127 mAb bound markedly less well than other Abs to
L2. Furthermore, in
all transfectants examined, including J2.7 cells and K562 cells (see
below), KIM127 bound less well than other Abs to wild-type
L2, and bound
markedly better to L2
containing activating mutations in the L or
2 cytoplasmic domains. These results
demonstrate that the KIM127 epitope is more exposed in mutationally
activated L2 than in
the resting, wild-type
L2 molecule.
|
2 subunit can be expressed in the absence
of integrin subunits in transfected cells, although with less
efficiency than as an complex (47). Therefore, we
examined binding of KIM127 mAb to the 2
subunit expressed alone on the surface of 293T transfectants. KIM127
bound to 2 expressed in isolation from
L as well as the other mAbs to the
2 C-terminal region, 6.7, MEM48, CBR LFA-1/7,
CBR LFA-1/2, and KIM185 (Table II
). Thus, in resting, wild-type
L2, the KIM127
epitope is shielded by the L subunit. Induction of KIM127 epitope exposure by Mn2+ and PMA
Mg2+ in the absence of
Ca2+, and Mn2+ have
previously been reported to activate adhesiveness of LFA-1 (45, 48). Mn2+ greatly increased KIM127 binding
to wild-type LFA-1 expressed on the surface of K562 transfectants,
whereas binding of other 2 mAbs was not
affected (Fig. 2). KIM127 binding was
increased 8.3-fold in the presence of Mn2+.
Mg2+ in the absence of Ca2+
also increased KIM127 binding, but to a lesser degree than
Mn2+ (data not shown). Similar results were
obtained with JY cells (data not shown). Therefore, activation of LFA-1
ligand binding by Mn2+, and
Mg2+ in the absence of
Ca2+, correlates with increased expression of the
KIM127 epitope.
|
2-fold, whereas binding of other mAbs was unchanged (Fig. 3). Although this increase was
consistently less than that seen with Mn2+, it
was significant and reproducible.
|
The mAbs KIM127, KIM185, CBR LFA-1/2, and MEM48 have all
previously been reported to increase
L2-mediated cell
adhesion and cell aggregation (27, 28, 29, 30), but have not been
compared with one another. We compared these mAbs for their ability to
activate LFA-1-dependent binding to ICAM-1. K562 transfectants
expressing wild-type
L2 did not bind to
ICAM-1 in the presence of the nonbinding control IgG X63 or mAbs 6.7 or
CBR LFA-1/7 to the C-terminal region of 2
(Fig. 4). By contrast, KIM127, CBR
LFA-1/2, MEM48, and KIM185 greatly increased binding of cells to
ICAM-1. Furthermore, the four mAbs activated
L2 binding to ICAM-1
to a similar level (Fig. 4). The four mAbs also activated
L2 binding to ICAM-1
to a similar level in J2.7 transfectants (data not shown).
|
L2,
M2, and X2,
whereas three other activating 2 mAbs bind
differentially to these integrins
We compared the reactivity of KIM127 and the three other
2-activating mAbs for L2,
M2, and
X2 (Table III). KIM127 reacted weakly with resting
L2,
M2, and
X2, compared with mAb
CBR LFA-1/7. By contrast, the activating 2702*
truncation induced KIM127 reactivity to the same level as mAb CBR
LFA-1/7 for all three complexes (Table III). The mAbs CBR
LFA-1/2, MEM48, and KIM185 reacted with the three resting
complexes differentially. As described above, all three mAbs bound to
L2 as well as CBR
LFA-1/7. However, CBR LFA-1/2 showed markedly reduced binding to
M2 and
X2 (Table III). MEM48
showed weak binding to
X2 and moderate
binding to M2,
whereas KIM185 bound to
M2 weakly. By
contrast, binding of CBR LFA-1/2, MEM48, and KIM185 to
L2702*,
M2702*, and
X2702* was comparable
with mAb CBR LFA-1/7. The results demonstrate that the KIM127 epitope
is shielded in all three 2 integrins in the
resting state, and that epitopes of CBR LFA-1/2, MEM48, and KIM185 are
differentially exposed in resting
L2,
M2, and
X2. Differences in
shielding may reflect differences in the structures of the
L, M, and
X subunits.
|
2 subunit
To understand the molecular basis of
L2 activation, we
mapped the epitopes of KIM127, MEM48, CBR LFA-1/2, and KIM185. For
comparison, we also mapped two nonactivating mAbs, 6.7 and CBR LFA-1/7.
These mAbs had previously been shown to bind within a segment in the
2 subunit lying between the I-like domain and
the transmembrane domain (15, 16, 31). A series of human
and mouse chimeric 2 subunits were
constructed. In these 2 chimeras, a segment of
the human sequence in the region from residues 344–621 was
progressively replaced by the corresponding mouse sequence (Fig. 5). Reciprocal exchanges were also made
in which mouse sequence was progressively replaced with human sequence.
The chimeric 2 subunits were coexpressed with
the human L subunit in 293T cells, and mAb
reactivity with the
L2 complex was
determined by immunofluorescence flow cytometry. All
2 chimeras were expressed on the cell surface
at levels comparable with that of human 2. The
overall results are summarized in Table IV, and raw immunofluorescence flow
cytometry data are shown for the mutants that were key for mapping
KIM127 (Fig. 6). Although KIM127 bound
less well to L2 in
293T transfectants than other mAbs, such as the positive control mAb
TS1/18 to 2, the reactivity of KIM127 was far
above background, allowing mutations that abolished its binding to be
easily identified (Fig. 6). The epitopes of the six mAbs localized to
five different subregions, residues 344–432 (mAb 6.7), residues
432–487 (mAb CBR LFA-1/7), residues 487–538 (mAbs KIM127 and CBR
LFA-1/2), residues 487–581 (mAb MEM48), and residues 581–621 (mAb
KIM185) (Table IV).
|
|
|
2 sequences (Fig. 7). First, this region was divided into
10 subregions that contained either one amino acid substitution, or two
or three nearby amino acid substitutions. These 10 mutants were tested
for effect on Ab binding. Subsequently, single amino acid substitutions
were made within any region containing two or three substitutions that
was found to affect mAb binding.
|
. mAb KIM127 binding localized to
residues 504, 506, and 508. The mutation G504N/L506E/Y508F completely
abolished binding, while substitutions in the other subregions had no
effect. The three individual amino acid substitutions G504N, L506E, and
Y508F each partially reduced binding of mAb KIM127, with G504N
(Gly
Asn) having the largest effect.
|
The mAb CBR LFA-1/2 mapped to a set of residues overlapping with the MEM48 epitope. Binding of mAb CBR LFA-1/2 was abolished by mutation L534S/F536N and reduced to 40% of wild type by mutation R541S/H543K/F546Y. The single substitution F536N reduced CBR LFA-1/2 binding to 40% of wild type, whereas the single substitution L534S had no effect. Although the L534S substitution had no effect on its own, it synergized with F536N, because the L534S/F536N mutation had a more severe effect on CBR LFA-1/2 binding than the F536N mutation. Although the mutation R541S/H543K/F546Y reduced CBR LFA-1/2 binding, individual substitutions at residues 541, 543, and 546 had no effect. Therefore, the CBR LFA-1/2 epitope includes residues Phe536 and to a lesser extent Leu534, and some combination of residues Arg541, His543, and Phe546.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
subunits appears to have an
important function in inside-out signaling. The stalk region conveys
signals that impinge on integrin cytoplasmic domains to the
ligand-binding integrin headpiece. For the first time, we have mapped
Abs to specific amino acid residues and specific cysteine-rich repeats
in this region of integrin subunits. Previously, certain Abs to
integrin subunits have been reported to either activate ligand
binding by integrins, or to bind in a manner that is dependent on
integrin activation or binding to ligand. We have directly compared
four such Abs to the integrin 2 subunit.
The original report on mAb KIM127 showed that it bound to the integrin
2 subunit and promoted LFA-1- and
Mac-1-mediated cell-adhesive events (27). We found that
KIM127 bound weakly to wild-type
L2 on the surface of
JY cells, and a number of
L2-transfected cell
lines, including J2.7 cells, 293T, and K562 cells. The level of
KIM127 binding to wild-type
L2 was only 10–15%
of that found with other 2 mAbs. However,
KIM127 binding to the same cells transfected with constitutively active
L2 bearing
L1090*, LGFFKR, or
2702* mutations was greatly increased, to the
same level as seen for typical mAb to the 2
and L subunits. These
L2 mutants
constitutively bind to ICAM-1, and express the activation epitope
defined by the m24 mAb (39 and our unpublished
data). Although increased exposure of the KIM127 epitope
correlates with the expression of the m24 epitope, KIM127 differs from
m24 in its ability to activate 2 integrins.
Furthermore, mAb m24 differs from the mAbs investigated in this study,
because it maps to the I-like domain of 2
(58). Binding of mAb KIM127 to wild-type
L2 was greatly
increased in the presence of Mn2+, and to a
lesser degree in the combined presence of Mg2+
and absence of Ca2+. PMA consistently increased
KIM127 expression by a significant, but markedly lesser amount than
Mn2+. The greater effect of
Mn2+ than PMA is consistent with induction of the
m24 epitope by Mn2+, but not PMA
(51); however, results with the KIM127 epitope differ in
that PMA induces a small, but consistent increase in expression. We
compared KIM127 with three other mAbs that were previously reported to
activate cell adhesion mediated by 2 integrins
(27, 28, 29, 30). We found that mAbs KIM185, CBR LFA-1/2, and
MEM48 bound equally well to wild-type and mutationally activated
L2, and activated
ligand binding by wild-type
L2 to a similar
extent. By contrast, KIM127 bound to a much lower extent to wild-type
L2, yet activated
ligand binding by wild-type
L2 to the same
extent. It is likely that KIM127 activates adhesion by binding to and
stabilizing a complex between
L2 and an ICAM. It is
well known that many mAbs that appear specific for activated integrins
bind to ligand-induced binding sites or LIBS (52).
Interesting differences were noted among the four activating mAbs
in their ability to bind to
L2,
M2, and
X2. For all three
wild-type integrins, there was little expression of the KIM127 epitope,
and the activating truncation of the 2
cytoplasmic domain induced full expression. However, wild-type
L2,
M2, and
X2 differed in
expression of the MEM48, CBR LFA-1/2, and KIM185 epitopes. These three
epitopes were all well expressed on wild-type
L2, but not on
M2 and
X2. The KIM185
epitope was not well expressed on resting
M2, but was on
resting X2. CBR
LFA-1/2 and MEM48 were more similar to one another in reactivity than
to KIM127 or KIM185, correlating with binding to overlapping epitopes.
Both CBR LFA-1/2 and MEM48 mAbs bound to resting
L2, bound little to
resting X2, and
showed intermediate reactivity with resting
M2, with MEM48
reacting better than CBR LFA-1/2. The activating truncation of the
2 cytoplasmic domain induced full expression
of all four of these epitopes on
L2,
M2, and
X2. The differences
between L2,
M2, and
X2 in constitutive
expression of the epitopes are interesting. Differences are consistent
with our finding that the KIM127 epitope is shielded by the
L subunit in 2,
because the subunits differ in sequence and thus would be expected
to differ in shielding. Differences in shielding could result both from
subtle differences in conformation of the stalk regions of the
subunits, and differences between the subunits in the way their
stalk regions interact with the three different cysteine-rich repeats,
to which these mAbs differentially map, repeats 2, 3, and 4.
Our studies provide a structure-function map for the C-terminal
portion of the extracellular domain of the 2
subunit. Previously, little has been known about this segment, which
appears to correspond to a stalk structure seen in electron micrographs
of integrins. This region from residues 344 to 678 is often referred to
as cysteine rich; however, the cysteine-rich repeats themselves
correspond only to residues 425–591, and the regions from 344 to 425
and 591 to 678 have a lower cysteine content (Fig. 7). The segment from
residues 344–678 contains 20 predicted -strands and no predicted
-helices, suggesting that it contains -sandwich domains
(16).
We have divided the 2 C-terminal region into
five different mAb-defined segments, and show that the segment to which
a mAb binds correlates with its ability to activate integrin
adhesiveness (Fig. 7). The epitope of mAb 6.7 maps to residues
350–432, which correspond largely to the regions between the I-like
domain and cysteine-rich repeat 1. mAb CBR LFA-1/7 maps largely to
cysteine-rich repeat 1, and to a portion of cysteine-rich repeat 2
(residues 432–487). Neither of these Abs activates integrin
adhesiveness. By contrast, all four mAbs that map to three more
C-terminal segments that include cysteine-rich repeats 2, 3, and 4
activate 2 integrin adhesiveness. The binding
sites for three of these mouse anti-human mAbs were narrowed down
with individual humanmouse amino acid substitutions. KIM127
recognizes three residues, Gly504,
Leu506, and Tyr508. These
residues are located wholly within cysteine-rich repeat 2 (Fig. 7).
mAbs MEM48 and CBR LFA-1/2 recognize overlapping epitopes located
wholly within cysteine-rich repeat 3. mAb MEM48 recognizes residues
Leu534, Phe536, and
His543. mAb CBR LFA-1/2 recognizes residue
Phe536, to a lesser extent residue
Leu534, and a combination of two or more of the
residues Arg541, His543,
and Phe546. mAb KIM185 was mapped to residues
581–621. Together with a previous report localizing the C-terminal
boundary of the KIM185 epitope to residue 604 (16), this
shows that it recognizes a segment of 23 aa corresponding to the end of
cysteine-rich repeat 4 and the beginning of the next region (Fig. 7).
These results suggest that in particular cysteine-rich repeats 2 and 3,
and possibly cysteine-rich repeat 4, are important in
2 integrin activation, whereas the more
N-terminal regions of residues 344–432 and 432–487 have less or
little function in 2 integrin activation.
For several reasons, we favor the interpretation that in resting
integrins, there is an interaction of the subunit with
cysteine-rich repeats 2 and 3 (and possibly 4) of the
2 subunit, and that upon activation this
interface opens and becomes more exposed. Four disulfide bonds are
present in each of these cysteine-rich repeats (20), and
this suggests that these domains are rigid and unlikely to undergo any
significant conformational change independently of interactions with
the subunit. We found that all of the epitopes are present on the
isolated 2 subunit, whereas exposure of the
KIM127 epitope is greatly reduced in the
L2,
M2, and
X2 complexes.
Furthermore, exposure of the CBR LFA-1/2, MEM48, and KIM185 epitopes is
greatly reduced in either or both of
M2 and
X2. Other studies
suggest that interactions in integrin subunit stalk regions
restrain integrins in an inactive state, and that there is a loosening
in this region upon activation. Species-specific substitutions in the
2 subunit activate ligand binding by
X2, and appear to
define an interaction interface between the X
and 2 subunits that restrains
X2 in an inactive
conformation (53). These substitutions map to the PSI
domain and cysteine-rich repeats 2 and 3, and the most activating
substitutions were Gln525Ser and
Val526Leu, in cysteine-rich repeat 3. These
substitutions exposed the CBR LFA-1/2 epitope (53), which
we also map within cysteine-rich repeat 3, to residues 534–546. In
other studies, activation of
L2 expressed on COS
cells was induced if the C-terminal cysteine-rich-repeat region of the
2 subunit was replaced by that of
1 (54). A point mutation that
introduces a N-glycosylation site into the beginning of
cysteine-rich repeat 4 of the 3 subunit
activates integrins
IIb3 and
V3
(55).
Several lines of evidence suggest that the epitopes in the
2 cysteine-rich repeats are shielded by the
subunit and not some other associating molecule. First, the
L, M, and
X subunits differed in shielding of the CBR
LFA-1/2, MEM48, and KIM185 epitopes. Second, in cell transfection
experiments, these epitopes and the KIM127 epitope only became shielded
when an subunit was cotransfected with the
2 subunit. Furthermore, no other associating
molecules were seen in immunoprecipitation experiments, in which KIM127
mAb was found to immunoprecipitate the unassociated
2 subunit precursor well, but the
L2 complex poorly
(15).
Association between the and subunit stalk regions and the
and subunit cytoplasmic/transmembrane domains may be linked.
Previous work has suggested interactions between the and
subunit cytoplasmic/transmembrane domains that include complementary
negatively and positively charged residues, and restrain integrins in
an inactive state (56, 57). Consistent with this,
mutations that activate 2 integrins, such as
truncation of the L or
2 cytoplasmic domains and removal of the GFFKR
sequence, would disrupt and subunit cytoplasmic/transmembrane
interactions. We now show that these mutations also expose epitopes in
cysteine-rich repeats 2 and 3, and near the end of cysteine-rich repeat
4. Our data suggest that these are important regulatory sites in the
2 subunit for
L2 activation. We
propose that in the resting integrin state (closed conformation), the
C-terminal stalk-like regions of the and 2
subunits are kept close together by the transmembrane and cytoplasmic
domains, and epitopes in cysteine-rich repeats 2, 3, and 4 are shielded
by the subunit. We further propose that in the active state (open
conformation), the release of the interactions between the and
subunit cytoplasmic/transmembrane domains results in a movement apart
of the stalk-like regions of the and subunits, thus exposing
epitopes in cysteine-rich repeats 2, 3, and 4. The Abs to these
epitopes may activate by acting like a wedge to keep the and
subunit stalk-like regions apart. The KIM127 epitope appears to be the
most resistant to exposure, because it is shielded in all three resting
2 complexes that we examined.
Interestingly, binding of the KIM185 mAb to a site that we map near the
end of cysteine-rich repeat 4 can expose the KIM127 epitope, which we
map to cysteine-rich repeat 2 (28). The opening of the
stalk region may in turn alter the relative orientations of the and
subunits in the ligand-binding integrin headpiece. 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 domains (8, 9, 10, 11, 12). It
appears that an alteration in contacts in the stalk region between the
cysteine-rich repeats in the subunit and the subunit is linked
to conformational rearrangements in the ligand-binding domains in the
headpiece of integrins. Our data provide new insights into the
molecular mechanisms for integrin activation.
|
Footnotes |
|---|
2 Current address: Millennium Pharmaceuticals, 75 Sidney Street, Cambridge, MA 02139.
3 Address correspondence and reprint requests to Dr. Timothy A. Springer, Department of Pathology, Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Room 251, Boston, MA 02115.
4 Abbreviations used in this paper: MIDAS, metal ion-dependent adhesion site; PSI, plexins, semaphorins, and integrins.
Received for publication November 11, 2000. Accepted for publication February 5, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2-integrins and their cellular ligands. Eur. J. Biochem. 245:215.[Medline]
-subunits into a -propeller domain. Proc. Natl. Acad. Sci. USA 94:65.-hairpin loop better resembles integrin sequence motifs than the EF-hand. Cell 102:275.[Medline]
subunit of integrin CR3 (CD11b/CD18). Cell 80:631.[Medline]
21. Cell 101:47.[Medline]
IIb3 is dependent on a MIDAS-like domain in the 3 subunit. J. Biol. Chem. 271:21978. subunit: evidence for the presence of a von Willebrand factor A domain. FEBS Lett. 400:297.[Medline]
2 subunit requires association with the subunit. Proc. Natl. Acad. Sci. USA 94:3156.2 subunit: a model for the I-like domain. J. Biol. Chem. 275:21514.IIb3. J. Biol. Chem. 275:7795. subunit contacts the subunit -propeller domain near -sheet 3. J. Biol. Chem. 275:22202.-propeller domain and C-terminal region of the integrin M subunit. J. Biol. Chem. 273:15138.-subunits of the integrin family. Biochem. J. 274:63.
IIb3. J. Biol. Chem. 268:23087.1 subunit by epitope mapping. J. Cell Biol. 122:1361.1 integrin monoclonal antibody binds to the cysteine-rich repeats in the 1 chain. J. Biol. Chem. 271:25099.1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium. J. Biol. Chem. 270:25570.1 integrin revealed by a monoclonal antibody recognizing conformation-dependent epitope. J. Biochem. 121:914.-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events. J. Immunol. 148:1080.[Abstract]
lymphocytes are required for antibody-dependent cellular cytotoxicity (killer cell activity). Eur. J. Immunol. 14:518.[Medline]
subunit cytoplasmic domain regulates the assembly and adhesiveness of integrin lymphocyte function-associated antigen-1 (LFA-1). J. Immunol. 159:268.[Abstract]
subunits. EMBO J. 8:3759.[Medline]
1, 2, and 7 integrins differentially regulate LFA-1 function in K562 cells. Mol. Biol. Cell 8:719.[Abstract]
2 subunit that restrain activation of the integrin X2. J. Biol. Chem. 276:6922.2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, L2, CD11a/CD18) heterodimer formation and ligand binding. FEBS Lett. 440:414.[Medline]
3 subunit activates integrins IIb3 and V3. Blood 93:2559.L2 I domain with disulfide bonds reveals functional relationships among integrin domains. Proc. Natl. Acad. Sci. USA 98:2393.This article has been cited by other articles:
|
|
 |
|
  C. T. Lefort, Y.-M. Hyun, J. B. Schultz, F.-Y. Law, R. E. Waugh, P. A. Knauf, and M. Kim Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1 J. Immunol., November 15, 2009; 183(10): 6460 - 6468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Schack, R. Stapulionis, B. Christensen, E. Kofod-Olsen, U. B. Skov Sorensen, T. Vorup-Jensen, E. S. Sorensen, and P. Hollsberg Osteopontin Enhances Phagocytosis through a Novel Osteopontin Receptor, the {alpha}X{beta}2 Integrin J. Immunol., June 1, 2009; 182(11): 6943 - 6950. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Vararattanavech, X. Lin, J. Torres, and S.-M. Tan Disruption of the Integrin {alpha}L{beta}2 Transmembrane Domain Interface by {beta}2 Thr-686 Mutation Activates {alpha}L{beta}2 and Promotes Micro-clustering of the {alpha}L Subunits J. Biol. Chem., January 30, 2009; 284(5): 3239 - 3249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Askari, P. A. Buckley, A. P. Mould, and M. J. Humphries Linking integrin conformation to function J. Cell Sci., January 15, 2009; 122(2): 165 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Valdramidou, M. J. Humphries, and A. P. Mould Distinct Roles of {beta}1 Metal Ion-dependent Adhesion Site (MIDAS), Adjacent to MIDAS (ADMIDAS), and Ligand-associated Metal-binding Site (LIMBS) Cation-binding Sites in Ligand Recognition by Integrin {alpha}2{beta}1 J. Biol. Chem., November 21, 2008; 283(47): 32704 - 32714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-L. Tang, A. Vararattanavech, and S.-M. Tan Urokinase-type Plasminogen Activator Receptor Induces Conformational Changes in the Integrin {alpha}M{beta}2 Headpiece and Reorientation of Its Transmembrane Domains J. Biol. Chem., September 12, 2008; 283(37): 25392 - 25403. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mor-Cohen, N. Rosenberg, M. Landau, J. Lahav, and U. Seligsohn Specific Cysteines in {beta}3 Are Involved in Disulfide Bond Exchange-dependent and -independent Activation of {alpha}IIb{beta}3 J. Biol. Chem., July 11, 2008; 283(28): 19235 - 19244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Carreno, D. Li, M. Sen, I. Nira, T. Yamakawa, Q. Ma, and G. B. Legge A Mechanism for Antibody-mediated Outside-in Activation of LFA-1 J. Biol. Chem., April 18, 2008; 283(16): 10642 - 10648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Li, Y. Li, W. Wu, W. R. Gordon, D. W. Chang, M. Lu, S. Scoggin, T. Fu, L. Vien, G. Histen, et al. Modulation of Notch Signaling by Antibodies Specific for the Extracellular Negative Regulatory Region of NOTCH3 J. Biol. Chem., March 21, 2008; 283(12): 8046 - 8054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Stapulionis, C. L. Pinto Oliveira, M. C. Gjelstrup, J. S. Pedersen, M. E. Hokland, S. V. Hoffmann, K. Poulsen, C. Jacobsen, and T. Vorup-Jensen Structural Insight into the Function of Myelin Basic Protein as a Ligand for Integrin {alpha}M{beta}2 J. Immunol., March 15, 2008; 180(6): 3946 - 3956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Nakayama, F. Yoshizaki, A. Prinetti, S. Sonnino, L. Mauri, K. Takamori, H. Ogawa, and K. Iwabuchi Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms J. Leukoc. Biol., March 1, 2008; 83(3): 728 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Morin, P. W. Oakes, Y.-M. Hyun, D. Lee, Y. E. Chin, M. R. King, T. A. Springer, M. Shimaoka, J. X. Tang, J. S. Reichner, et al. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration J. Exp. Med., January 21, 2008; 205(1): 195 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Vorup-Jensen, L. Chi, L. C. Gjelstrup, U. B. Jensen, C. A. Jewett, C. Xie, M. Shimaoka, R. J. Linhardt, and T. A. Springer Binding between the Integrin {alpha}Xbeta2 (CD11c/CD18) and Heparin J. Biol. Chem., October 19, 2007; 282(42): 30869 - 30877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shi, S. Y. Foo, S.-M. Tan, E. P. Mitchell, S. K. A. Law, and J. Lescar A Structural Hypothesis for the Transition between Bent and Extended Conformations of the Leukocyte beta2 Integrins J. Biol. Chem., October 12, 2007; 282(41): 30198 - 30206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Dileepan, S. C. Kachlany, N. V. Balashova, J. Patel, and S. K. Maheswaran Human CD18 Is the Functional Receptor for Aggregatibacter actinomycetemcomitans Leukotoxin Infect. Immun., October 1, 2007; 75(10): 4851 - 4856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cheng, S.-Y. Foo, M.-L. Shi, R.-H. Tang, L.-S. Kong, S. K. A. Law, and S.-M. Tan Mutation of a Conserved Asparagine in the I-like Domain Promotes Constitutively Active Integrins {alpha}Lbeta2 and {alpha}IIbbeta3 J. Biol. Chem., June 22, 2007; 282(25): 18225 - 18232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Gupta, A. Gylling, J. L. Alonso, T. Sugimori, P. Ianakiev, J.-P. Xiong, and M. Amin Arnaout The {beta}-tail domain ({beta}TD) regulates physiologic ligand binding to integrin CD11b/CD18 Blood, April 15, 2007; 109(8): 3513 - 3520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Y. Park, M. Amin Arnaout, and V. Gupta A Simple, No-Wash Cell Adhesion-Based High-Throughput Assay for the Discovery of Small-Molecule Regulators of the Integrin CD11b/CD18 J Biomol Screen, April 1, 2007; 12(3): 406 - 417. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Shimaoka, M. Kim, E. H. Cohen, W. Yang, N. Astrof, D. Peer, A. Salas, A. Ferrand, and T. A. Springer AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes PNAS, September 19, 2006; 103(38): 13991 - 13996. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, W. Yang, M. Kim, C. V. Carman, and T. A. Springer Regulation of outside-in signaling and affinity by the beta2 I domain of integrin {alpha}Lbeta2 PNAS, August 29, 2006; 103(35): 13062 - 13067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Evans, A. McDowall, P. C. Taylor, N. Hogg, D. O. Haskard, and R. C. Landis Shedding of lymphocyte function-associated antigen-1 (LFA-1) in a human inflammatory response Blood, May 1, 2006; 107(9): 3593 - 3599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Salas, M. Shimaoka, U. Phan, M. Kim, and T. A. Springer Transition from Rolling to Firm Adhesion Can Be Mimicked by Extension of Integrin {alpha}Lbeta2 in an Intermediate Affinity State J. Biol. Chem., April 21, 2006; 281(16): 10876 - 10882. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Heit, P. Colarusso, and P. Kubes Fundamentally different roles for LFA-1, Mac-1 and {alpha}4-integrin in neutrophil chemotaxis J. Cell Sci., November 15, 2005; 118(22): 5205 - 5220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Shi, K. Sundramurthy, B. Liu, S.-M. Tan, S. K. A. Law, and J. Lescar The Crystal Structure of the Plexin-Semaphorin-Integrin Domain/Hybrid Domain/I-EGF1 Segment from the Human Integrin {beta}2 Subunit at 1.8-A Resolution J. Biol. Chem., August 26, 2005; 280(34): 30586 - 30593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.-H. Tang, E. Tng, S. K. A. Law, and S.-M. Tan Epitope Mapping of Monoclonal Antibody to Integrin {alpha}L {beta}2 Hybrid Domain Suggests Different Requirements of Affinity States for Intercellular Adhesion Molecules (ICAM)-1 and ICAM-3 Binding J. Biol. Chem., August 12, 2005; 280(32): 29208 - 29216. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Sarantos, S. Raychaudhuri, A. F. H. Lum, D. E. Staunton, and S. I. Simon Leukocyte Function-associated Antigen 1-mediated Adhesion Stability Is Dynamically Regulated through Affinity and Valency during Bond Formation with Intercellular Adhesion Molecule-1 J. Biol. Chem., August 5, 2005; 280(31): 28290 - 28298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Ehirchiou, Y.-M. Xiong, Y. Li, S. Brew, and L. Zhang Dual Function for a Unique Site within the {beta}2I Domain of Integrin {alpha}M{beta}2 J. Biol. Chem., March 4, 2005; 280(9): 8324 - 8331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Vorup-Jensen, C. V. Carman, M. Shimaoka, P. Schuck, J. Svitel, and T. A. Springer Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin {alpha}X{beta}2 PNAS, February 1, 2005; 102(5): 1614 - 1619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Tng, S.-M. Tan, S. Ranganathan, M. Cheng, and S. K. A. Law The Integrin {alpha}L{beta}2 Hybrid Domain Serves as a Link for the Propagation of Activation Signal from Its Stalk Regions to the I-like Domain J. Biol. Chem., December 24, 2004; 279(52): 54334 - 54339. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kim, C. V. Carman, W. Yang, A. Salas, and T. A. Springer The primacy of affinity over clustering in regulation of adhesiveness of the integrin {alpha}L{beta}2 J. Cell Biol., December 20, 2004; 167(6): 1241 - 1253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Xie, M. Shimaoka, T. Xiao, P. Schwab, L. B. Klickstein, and T. A. Springer The integrin {alpha}-subunit leg extends at a Ca2+-dependent epitope in the thigh/genu interface upon activation PNAS, October 26, 2004; 101(43): 15422 - 15427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lu, M. Shimaoka, A. Salas, and T. A. Springer The Binding Sites for Competitive Antagonistic, Allosteric Antagonistic, and Agonistic Antibodies to the I Domain of Integrin LFA-1 J. Immunol., September 15, 2004; 173(6): 3972 - 3978. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Calvete Structures of Integrin Domains and Concerted Conformational Changes in the Bidirectional Signaling Mechanism of {alpha}IIb{beta}3 Experimental Biology and Medicine, September 1, 2004; 229(8): 732 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bouaouina, E. Blouin, L. Halbwachs-Mecarelli, P. Lesavre, and P. Rieu TNF-Induced {beta}2 Integrin Activation Involves Src Kinases and a Redox-Regulated Activation of p38 MAPK J. Immunol., July 15, 2004; 173(2): 1313 - 1320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bjorklund, P. Heikkila, and E. Koivunen Peptide Inhibition of Catalytic and Noncatalytic Activities of Matrix Metalloproteinase-9 Blocks Tumor Cell Migration and Invasion J. Biol. Chem., July 9, 2004; 279(28): 29589 - 29597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-H. Luo, K. Strokovich, T. Walz, T. A. Springer, and J. Takagi Allosteric {beta}1 Integrin Antibodies That Stabilize the Low Affinity State by Preventing the Swing-out of the Hybrid Domain J. Biol. Chem., June 25, 2004; 279(26): 27466 - 27471. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Yang, M. Shimaoka, A. Salas, J. Takagi, and T. A. Springer Intersubunit signal transmission in integrins by a receptor-like interaction with a pull spring PNAS, March 2, 2004; 101(9): 2906 - 2911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Yang, M. Shimaoka, J. Chen, and T. A. Springer Activation of integrin {beta}-subunit I-like domains by one-turn C-terminal {alpha}-helix deletions PNAS, February 24, 2004; 101(8): 2333 - 2338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fukuda and G. W. Schmid-Schonbein Regulation of CD18 expression on neutrophils in response to fluid shear stress PNAS, November 11, 2003; 100(23): 13152 - 13157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kim, C. V. Carman, and T. A. Springer Bidirectional Transmembrane Signaling by Cytoplasmic Domain Separation in Integrins Science, September 19, 2003; 301(5640): 1720 - 1725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. Xiong, J. Chen, and L. Zhang Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions J. Immunol., July 15, 2003; 171(2): 1042 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Marwali, J. Rey-Ladino, L. Dreolini, D. Shaw, and F. Takei Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity Blood, July 1, 2003; 102(1): 215 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Takagi, N. Beglova, P. Yalamanchili, S. C. Blacklow, and T. A. Springer Definition of EGF-like, closely interacting modules that bear activation epitopes in integrin beta subunits PNAS, September 25, 2001; 98(20): 11175 - 11180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-M. Tan, M. K. Robinson, K. Drbal, Y. van Kooyk, J. M. Shaw, and S. K. A. Law The N-terminal Region and the Mid-region Complex of the Integrin beta 2 Subunit J. Biol. Chem., September 21, 2001; 276(39): 36370 - 36376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fagerholm, N. Morrice, C. G. Gahmberg, and P. Cohen Phosphorylation of the Cytoplasmic Domain of the Integrin CD18 Chain by Protein Kinase C Isoforms in Leukocytes J. Biol. Chem., January 11, 2002; 277(3): 1728 - 1738. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
