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Harvard Medical School, Center for Blood Research, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits, L (CD11a), M
(CD11b), X (CD11c), and D, to form
lymphocyte function-associated Ag
(LFA-1)3, Mac-1, p150,95, and
Dß2, respectively (1, 2). LFA-1 is a
well-characterized member of this family, and Abs directed against
LFA-1 have been used to demonstrate its role in T cell- and NK
cell-mediated killing, T and B cell interactions, and
leukocyte-endothelial cell adhesion (3). LFA-1 interacts with at least
three members of the Ig-like supergene family of proteins, ICAM-1,
ICAM-2, and ICAM-3 (4, 5, 6, 7). Ligand binding is divalent cation dependent
and requires energy but is not affected by disruption of the actin
cytoskeleton by cytochalasin B (4, 8, 9). Structurally, LFA-1 is
comprised of an subunit that contains, in its external region, a
putative ß-propeller domain within which an I domain is inserted that
is involved in ligand recognition (10, 11, 12). The subunit has a short
cytoplasmic tail of 58 amino acids that contains the membrane-proximal
GFFKR amino acid motif that is conserved among all subunits (13, 14). The ß2 subunit contains highly conserved and
cysteine-rich domains in its extracellular region and a short
cytoplasmic tail (15).
Cellular adhesion through integrin molecules involves a complex set of
events that include activation, ligand binding, and reorganization of
the cytoskeleton (16, 17). To mediate cell-cell adhesion, LFA-1
undergoes activation through an array of known signal transduction
pathways that include cross-linking of the T cell receptor/CD3, CD2,
cell surface Ig, or MHC Class II molecules, stimulation of protein
kinase C activity by phorbol esters, and activation of chemokine
receptors (17, 18, 19, 20, 21, 22). Ligand binding can also be induced by Abs against
integrin family members (23, 24, 25, 26). A panel of Abs has emerged that is
directed against both the L and ß subunits and that is
able to either tag the activation state of LFA-1 or enhance binding to
ligand (8, 11, 27, 28, 29, 30). Specifically, we have described an Ab to the
ß2 subunit that enhances binding of LFA-1-bearing cells
to ICAM-1 and ICAM-3, independently of Fc interactions or whether
dimeric or monomeric Fab preparations are used (30).
Divalent cations are critical for ligand binding and can directly
modulate binding of LFA-1 to ligand (4, 8, 30). There are metal binding
domains in both the and ß subunits of integrin family members,
and divalent cations, in particular Mg2+, are required for
ligand binding (4, 10, 13, 31, 32, 33). In contrast to Mg++,
whose presence is required but which only enhances binding in response
to activation of intracellular signaling or mAb, Mn2+ alone
is able to stimulate binding of LFA-1-bearing cells to ligand (8, 30).
Although a broad range of tools can be used to activate LFA-1, an exact understanding of the molecular steps that mediate activation has remained elusive. Protein kinase C can serve as a common second messenger for several of these stimuli; however, the use of inhibitors suggests that more than one signaling pathway may play a role in reaching the common goal of activation of LFA-1 through inside-out signaling (18). Both protein kinase and protein phosphatase activities have been implicated in the activation of integrin family members (34, 35). Activation is transient and is independent of a change in cell surface expression of LFA-1 (18). The increase in avidity of LFA-1-bearing cells for ICAM-1 may in part be due to enhanced affinity of LFA-1 for ligand (36). Detailed mutagenesis of the ß subunit has shown that activation of lCAM-1 binding through signal transduction pathways requires the presence of amino acids between residues 756 and 762 and the phenylalanine at position 766 within the ß2 cytoplasmic domain (37). Although protein kinase and phosphatase activities have been implicated in the activation of LFA-1, phosphorylation of the molecule on either serine or tyrosine residues does not appear to play a role in avidity regulation (37, 38, 39). To examine minimally required events involved in the mechanism of activation of integrin activity, we have employed the Ab CBR LFA-1/2, known to induce binding of LFA-1 to ICAM-1 and to lower the Mg2+ concentration required to form the LFA-1/ICAM-1 complex (30). We use this Ab to compare the intracellular pathways that are essential for activation of LFA-1 with those events needed for cell aggregation and to determine the structural features of LFA-1 that are required in this process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Ab CBR LFA-1/2 was prepared, isolated, subcloned, and
purified as described (30). Abs to the LFA-1 L
subunit, TS 1/22 (40) and TS 2/4 (40), to the ß2 subunit,
TS1/18 (40), to ICAM-1, RR1/1 (41) and R6.5 (41), and to Mac-1,
CBRM1/29, have previously been described (42).
Cell lines
The B lymphoblastoid cell line JY was grown in RPMI 1640 medium supplemented with 5% FCS. The EBV-transformed cell line derived from patient 2 (P2) with leukocyte adhesion deficiency (LAD) (43) was maintained in RPMI 1640 medium containing 20% FCS. Patient 2 transfected cell lines were maintained in RPMI 1640 medium, 20% FCS, and hygromycin (200 µg/ml).
Cell binding to immobilized ICAM-1
ICAM-1 was purified and adsorbed to plastic (Linbro Titertek 96-well plates, ICN/Flow, Aurora, OH) as described (18). Cells (2 ml, 2 x 107/ml in L15 medium with 2.5% FCS) were labeled with 3 µg of 2', 7'-bis-(2-carboxyethyl)-5-(and -6) carboxy-fluorescein acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, Oregon) for 20 min at 37°C in the presence of 4 µg/ml of the anti-ICAM-1 mAb R6.5 to block cell aggregation. Cells were washed three times in buffer A (L15 medium, 2.5% FCS) and resuspended at 2 x 106 cells/ml in buffer A. Cells (50 µl, 2 x 106/ml) were applied to wells containing buffer A (50 µl) alone, or buffer A with control Ab (5 µg/ml), CBR LFA-1/2 (5 µg/ml), or PMA (50 ng/ml). Nonspecific binding was assessed by comparing binding in the presence or absence of the blocking mAb to LFA-1, TS1/22 (5 µg/ml). The total fluorescent content of the cells was quantitated on a Fluorescent Concentration Analyzer (IDEXX, Westbrook, Maine), after which the plate was centrifuged at 200 x g for 2 min at 4°C. After incubation at 4°C for 20 min, the plate was transferred to 37°C for the indicated time. Unbound cells were removed by aspiration in buffer A six times with a 20-gauge needle. Bound cells were quantitated in the Fluorescent Concentration Analyzer (IDEXX) and expressed as a percentage of total input cells per sample well.
A tip plate assay was performed as described (42). Briefly, 25 µl of ICAM-1 (200 µg/ml) or BSA (200 µg/ml) in PBS were spotted on a 5-mm circle in a 60-mm polystyrene petri dish (Falcon 1007, Becton Dickinson, Lincoln Park, NJ) for 1.5 h. The plates were washed four times in buffer A and allowed to incubate in buffer A for 30 min at room temperature. Cells (1 ml, 2 x 106/ml) were then allowed to adhere in the presence of PMA or CBR LFA-1/2 mAb as described above for 10 min at 37°C. Unbound cells were removed by transfer pipet and gentle replacement with buffer A. Bound cells were quantitated by counting four to five high-powered fields/protein circle after 10 to 12 washes. Nonspecific binding was determined for each transfected cell line in the presence of the blocking Ab TS1/22 to LFA-1 and was less than five cells per high-powered field for each cell line.
Cell binding to ICAM-1 in the presence of protein kinase inhibitors, phosphatase inhibitors, or the C3 exoenzyme
The optimal concentration of inhibitors was determined by titration of ICAM-1 binding at increasing concentration of inhibitors; cell viability was >90% as assessed by trypan blue exclusion at each concentration. DMSO, used as a solvent for each inhibitor, was included in controls and was at a final concentration less than or equal to 1% v/v. Specifically, cells were incubated with BCECF and the anti-ICAM-1 Ab R6.5, as described above, and then incubated with or without staurosporine (5 µg/ml), genistein (50 µg/ml), and okadaic acid (0.75 µM), in buffer A for 30 min at room temperature. They were transferred to wells containing the respective inhibitors, and binding to ICAM-1 was assessed as above. Cells were incubated in growth medium with C3 exoenzyme for 16 h at 5, 10, 20, and 30 µg/ml. They were washed into L15 2.5% FCS, incubated with BCECF and the blocking Ab R6.5, and ICAM-1 binding was assessed as above.
Depletion of intracellular energy pools was as described (4). Cells were incubated with 2-deoxyglucose (50 mM) and sodium azide (10 mM) for 15 min at room temperature. Binding to ICAM-1 in response to phorbol esters, CBR LFA-1/2 mAb, or control Ab was determined as described above.
Expression of ß2 subunit cytoplasmic truncation mutants in an LAD cell line
The ß2 subunit cytoplasmic domain mutant cDNAs in an episomal vector with an EBV origin of replication were prepared as described (43). EBV-transformed B cells derived from LAD patient 2, P2, deficient in expression of the ß subunit (43), were transfected with 20 µg of plasmid DNA by electroporation of 2 x 107 cells in 0.8 ml PBS at 960 µF and 200 V. The cells were placed in RPMI 1640 medium containing 20% FCS for 48 h and then transferred to selection in hygromycin (200 µg/ml). After 2 weeks in culture, cells expressing LFA-1 were enriched by adherence to the anti-CD18 Ab CBR LFA-1/7. Briefly, CBR LFA-1/7 (40 µg/ml) was bound to polystyrene plates (NUNC 10, Nunc, Naperville, IL) for 30 min at room temperature. The unbound Ab was removed by washing in RPMI 1640 medium with 5% FCS, and nonspecific sites were blocked with RPMI 1640/5% FCS for 30 min at room temperature. The transfected cell lines were added to plates in 12 ml. After 5 min, nonadherent cells were removed by washing in RPMI 1640/5% FCS ten times. Adherent cells were removed with trypsin/EDTA and placed in RPMI 1640 medium containing 20% FCS and 200 µg/ml hygromycin. Continued antibiotic selection was necessary for maintenance of expression of LFA-1.
Flow cytometry was performed as described (44).
Aggregation assays were performed as described (45). Cells cultured to
a density of 5 x 105 cells/ml were collected and
resuspended in buffer A (2 x 106 cells/ml) containing
staurosporine, genistein, or okadaic acid for 30 min. Cells were also
treated with C3 exoenzyme or DMSO for 16 h. Cells were then added
to the 96-well plate (Falcon 3072, Becton Dickinson) with or without 4
µg/ml of RR1/1 mAb. After 10 min, cells were then stimulated with
control Ab (10 µg/ml), CBR LFA-1/2 (10 mg/ml), or PMA (50 ng/ml).
After 15 min, aggregation was scored as described in Table I
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously described an Ab, CBR LFA-1/2, that modulates
increased binding of LFA-1 bearing cells to ligands through a specific
interaction with the ß subunit of LFA-1 and mimics the effect seen by
activation of intracellular signal transduction pathways (30). To
define the molecular steps required for activation of LFA-1, we have
compared the mode of activation by CBR LFA-1/2 to that produced by
phorbol ester stimulation. The rate of activation of binding to
immobilized ICAM-1 of LFA-1-bearing cells by CBR LFA-1/2 mAb was
compared with that induced by phorbol esters. CBR LFA-1/2 mAb induced
binding of JY cells to ICAM-1 at a rate comparable to that seen with
phorbol ester stimulation (Fig. 1). As
demonstrated previously with cross linking of CD3 or phorbol ester
treatment of T cells (18), maximal levels of binding with both stimuli
were seen by 10 min. Binding to ICAM-1 was specific under both
conditions of stimulation as shown by inhibition with mAb TS1/22 to
LFA-1 (Fig. 1).
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We compared activation of LFA-1 adhesiveness by
intracellular signaling pathways to activation by CBR LFA-1/2 mAb. Two
assays were used, binding of JY cells to ICAM-1 adsorbed to plastic
substrates and ICAM-1-dependent homotypic aggregation of JY cells.
Since protein kinase activity has been implicated in the
activation of LFA-1, we examined whether inhibition of its activity by
staurosporine affected binding to ICAM-1. Pretreatment with
staurosporine of the B cell line JY abolished PMA-stimulated binding to
ICAM-1 (Fig. 2). In contrast,
staurosporine did not inhibit CBR LFA-1/2-stimulated binding to ICAM-1
substrates. Similar results were obtained in homotypic aggregation of
JY cells, which is dependent on interaction of LFA-1 with ICAM-1
(46). PMA-stimulated, but not CBR LFA-1/2 mAb-stimulated aggregation,
was inhibited by staurosporine (Table I
). Aggregation was dependent on
ICAM-1 as shown by inhibition with RR1/1 mAb. Since staurosporine has
been reported to have an effect on a broad range of protein kinases,
the effect on PMA-induced binding to ICAM-1 and aggregation may be due
to inhibition of not only protein kinase C but other kinases in the
pathway required for inside-out activation of LFA-1. However, its
activity is not required for mAb-induced binding to ICAM-1 or homotypic
aggregation.
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Protein phosphatases play a critical role in the modulation
of signal transduction events (47). To determine whether preservation
of phosphatase activity was essential for activation of LFA-1
adhesiveness by CBR LFA-1/2 or protein kinase C, we examined the effect
of treatment of cells with the protein phosphatase inhibitor, okadaic
acid. Okadaic acid markedly diminished activation by phorbol esters,
but not by CBR LFA-1/2 mAb (Fig. 3).
Titration revealed that half maximal inhibition occurred between 0.4
and 1 µM of okadaic acid (not shown). Concentrations of less than 0.1
µM okadaic acid had no effect on control mAb-, PMA-, or mAb CBR
LFA-1/2-stimulated binding to ICAM-1 (data not shown). Studies on
homotypic adhesion revealed an interesting contrast. Okadaic acid
completely inhibited both CBR LFA-1/2 mAb-stimulated and PMA-stimulated
homotypic aggregation. These results suggest that CBR LFA-1/2 mAb is
able to activate binding of LFA-1 independently of phosphatase 1 and/or
2A activity, whereas protein kinase C-mediated activation requires
preservation of this activity and implicates phosphatase activity in
the pathway of activation by protein kinase C. However, LFA-1-dependent
aggregation is more complex, and an additional step distal to CBR
LFA-1/2 mAb-stimulated activation that is phosphatase dependent appears
required for aggregation.
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Protein tyrosine kinase activity has been shown to play a role in
intracellular signaling following binding of integrin family members to
ligand, and stimulation of a protein tyrosine kinase cascade has been
demonstrated upon activation of both ß1 and
ß2 integrin family members. Since activation of LFA-1 by
PMA stimulation does not occur through direct phosphorylation, we
examined whether tyrosine kinase activity was a component of the
pathway to activation or whether it was a component of the signaling
cascade required to induce cell-cell aggregation. We used the protein
tyrosine kinase inhibitor genistein to examine whether this activity
was required to mediate both LFA-1 binding to ICAM-1 and cell
aggregation. Genistein had little effect in JY cells on CBR LFA-1/2 mAb
or PMA-induced adhesiveness to ICAM-1 (Fig. 4). In contrast, aggregation by both
stimuli was completely blocked by treatment of the cells with the
inhibitor (Table I).
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C3 exoenzyme, which covalently modifies and inactivates the small
G protein RhoA, has previously been shown to inhibit aggregation of JY
cells (9). We examined the effect of this agent on ICAM-1 binding to
determine whether RhoA-stimulated functions, such as cytoskeletal
reorganization, are critical for activation of LFA-1 by either PMA or
direct activation with mAb (Fig. 5).
After 16 h of treatment with the C3 exoenzyme, binding to ICAM-1
in response to both phorbol esters or CBR LFA-1/2 was preserved (Fig. 5). These conditions impaired the ability of cells to aggregate as
previously reported (9). C3 exoenzyme inhibited both PMA- and CBR
LFA-1/2 mAb-stimulated homotypic aggregation (Table I). The lack of
effect on activation of LFA-1 binding to ICAM-1 on substrates suggests
that the activity of the Rho family of GTP-binding proteins is not
required for the formation of a stable LFA-1/ICAM complex, but for a
later step. This was consistent with the observation that the cells,
once bound to ICAM-1 substrates, failed to spread (data not shown).
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It has previously been shown that binding of T
lymphoblasts to ICAM-1 in response to phorbol ester stimulation can be
inhibited by depletion of intracellular energy pools (4). We compared
whether activation of binding by CBR LFA-1/2 or PMA-mediated binding to
purified ligand requires intact energy pools (Fig. 6). Cells that were pretreated with
2-deoxyglucose and sodium azide exhibited little binding to ICAM-1 in
response to CBR LFA-1/2 or phorbol esters. Cell-cell aggregation is
inhibited by treatment of cells with sodium azide (29). Thus, although
activation through CBR LFA-1/2 appears to occur independently of
several known signal transduction pathways, it still requires cellular
energy to achieve the high avidity conformation.
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The cytoplasmic domain of the LFA-1 ß subunit is critical
for avidity regulation through intracellular signaling pathways. Since
CBR LFA-1/2 is able to modulate binding to ICAM-1 despite inhibition of
various signal transduction pathways, we examined whether the Ab was
able to induce binding to ICAM-1 through the extracellular domain,
independently of an intact ß cytoplasmic domain. We first examined
the effect of truncation of the cytoplasmic domain of the ß subunit
of LFA-1 on binding to ICAM-1 under our standard assay conditions.
Deletion of the ß2 cytoplasmic tail of LFA-1 (731*)
resulted in loss of binding to ICAM-1 in response to activation of
intracellular signaling pathways by PMA or through CBR LFA-1/2 (Fig. 7A).
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B). We examined binding to ICAM-1 under conditions
of low-stringency washing to accentuate any subtle differences in the
activation pattern. When cells were activated by phorbol ester
stimulation, ICAM-1 binding was markedly diminished if the critical
region between amino acids 756–762 was deleted or the C-terminal
phenylalanine (766A) was changed to alanine (Fig. 7C). In parallel, activation by CBR LFA-1/2 was also
diminished. Mutation of Phe 766 to tyrosine had no effect, as
previously shown (37). Effect of Mn2+ on ß cytoplasmic mutant binding to ICAM-1
The inability of Abs and phorbol esters to stimulate binding
to ICAM-1 on cells bearing ß cytoplasmic mutations raised the
question of whether the mutations resulted in molecular changes that
made LFA-1 incapable of assuming a high avidity conformation. To
address this, we tested whether Mn2+ was able to enhance
binding of LFA-1 independently of the other activation pathways. Stable
transfected cell lines expressing either the wild-type ß subunit or
the mutations outlined in Figure 8 were
placed in Mn2+ containing media and assessed for binding to
ICAM-1. As shown in Figure 8, all the mutants were capable of binding
to ICAM-1, indicating that the phenotypic behavior of these mutants is
related to disruption of specific interactions or modifications in the
cytoplasmic region.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of LFA-1 by the CBR LFA-1/2 Ab occurs at a comparable rate to that seen with stimulation of inside-out signaling pathways. This rapid induction of ligand binding has been demonstrated with other anti LFA-1- Abs (KIM127, KIM 185, and NKL16) (28, 49).
Activation through mAb CBR LFA-1/2, in contrast to PMA, does not
require preservation of protein kinase activity. Inhibition of protein
kinase activity by staurosporine does not interfere with cell binding
to ICAM-1 brought about by the activating Ab, CBR LFA-1/2. This is
similar to what is observed through the subunit-specific Ab,
NKL-16, which was also not affected by treatment of cells
with staurosporine(29), but is in contrast to the effects of
staurosporine on B cell binding to ICAM-1 previously reported
(39).
We extended these studies and demonstrated that inhibition of protein phosphatase activity by okadaic acid blocks the phorbol ester-mediated stimulation of LFA-1 binding to ICAM-1 but did not affect ICAM-1 binding in response to CBR LFA-1/2 mAb. This not only demonstrated that activation through CBR LFA-1/2 mAb does not require preservation of protein phosphatase PPI and PP2A activity, but also implicates this phosphatase activity in the pathway that leads to activation of LFA-1 by protein kinase C as previously shown in purified B cells (39). In contrast, binding to ICAM-1 substrates stimulated by protein kinase C or the mAb CBR LFA-1/2 was affected by inhibition of protein tyrosine kinase activity with genistein. Experiments with C3 exoenzyme suggest that activation of LFA-1 binding to ICAM-1 by both the Ab and protein kinase C does not depend upon activity of the small GTP-binding protein RhoA, which has been implicated in rearrangement of the actin network and disrupts cell-cell aggregation (9, 50). These results suggested that serine/threonine protein kinase activity, but not phosphatase activities, are required for full activation via inside-out signaling, but not by external activation achieved through CBR LFA-1/2 mAb. Neither tyrosine kinase nor RhoA activity is required for ICAM-1 substrate binding induced by inside-out signaling or the activating Ab.
When we analyzed the effect of these inhibitors on LFA-1-mediated cell-cell aggregation, we observed that C3 exoenzyme impaired the ability of both PMA and CBR LFA-1/2 mAb to induce cell aggregation, suggesting that the LFA-1/ICAM interaction initiates a signaling cascade that feeds into the RhoA family of GTP-binding proteins or that multiple pathways acting in parallel are required for LFA-1-dependent homotypic aggregation. Homotypic aggregation was previously reported to be inhibited by C3 exoenzyme (9). The cell aggregation assay also revealed that a staurosporine-sensitive step is required for aggregation stimulated by PMA, but not by CBR LFA-1/2 mAb. The pathway leading to cell aggregation is also sensitive to protein tyrosine kinase and phosphatase inhibition under either condition of stimulation. It is an important observation that intact cellular energy sources are required for both ligand binding and cell aggregation in response to either Ab or PMA activation. This suggests that specific interactions and/or modifications are required for LFA-1 to attain full activation.
Previous studies have demonstrated that specific regions of the ß2 subunit are required for PMA-mediated activation (37, 48); thus, it was critical to test whether the regions of the ß subunit that were critical for phorbol ester activation also were involved in inducing activation by this ß subunit Ab. The regions between residues 756–762 and the phenylalanine at position 766, were found to be necessary not only for activation by phorbol esters but also for activation by CBR LFA-1/2 mAb. A central question was whether mutations in this region rendered the molecules incapable of achieving the high avidity conformation associated with activation of LFA-1 and enhanced binding to ICAM-1. Therefore, we studied the effect of Mn2+ on the ability of these mutants to bind to ICAM-1. Mn2+ has been shown to stimulate binding of LFA-1 to ICAM-1 directly in several cell lines (8, 30). All of the LFA-1 mutants exhibited enhanced binding to ICAM-1 in response to Mn2+, demonstrating that the mutants are fully competent to recognize the ligand.
Critical regions in the ß subunit are required for ligand binding stimulated by both Ab and inside-out signaling. Our data support the model that the ß cytoplasmic domain plays a key role in LFA-1-dependent ligand binding. Since both depletion of intracellular energy stores and critical regions of the ß subunit are required for activation by both the Ab and inside-out signaling pathways, one model is that a key energy-dependent interaction must be maintained to mediate ligand binding that is independent of activation of protein kinase C, protein tyrosine kinase, and phosphatase activities. A specific protein-protein interaction, such as the recently described cytohesin-1, is a candidate for modulating the activity of LFA-1 (51). Enhanced ligand binding by PMA stimulation has been proposed to occur through post receptor signaling events that strengthen cytoskeletal interaction (52). By disrupting the ß subunit, it is possible that interactions with the cytoskeleton that are critical for enhanced binding are lost. Furthermore, our results showed that the LFA-1/ICAM interaction is not sufficient to induce cell aggregation. Intracellular pathways, as well as specific regions of the LFA-1 molecule, are required for cellular aggregation, consistent with the idea that the LFA-1/ICAM pair is a signaling complex. This is supported by the observation that LFA-1-mediated cell adhesion induces the tyrosine phosphorylation of p130cas and its association with the adapter protein C-crk (53).
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lilli Petruzzelli, Division of Hematology/Oncology, University of Michigan Medical Center, 1150 West Medical Center Dr., MSRB III, Room 5301, Ann Arbor, MI 48109. E-mail address:
3 Abbreviations used in this paper: LFA-1, lymphocyte function-associated Ag; LAD, leukocyte adhesion deficiency; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxy-fluorescein acetoxymethyl ester.
Received for publication May 29, 1997. Accepted for publication December 30, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit: an integrin with an embedded domain defining a protein superfamily. J. Cell Biol. 108:703.-chain (CDw49d) induces homotypic lymphocyte aggregation. J. Immunol. 144:777.[Abstract]
subunits. EMBO J. 8:3759.[Medline]
IIbß3 through cytoskeletal reorganization and tyrosine phosphorylation in human platelets. J. Biol. Chem. 270:11927.Lß2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86:233.[Medline]
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E. S. Harris, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman The Leukocyte Integrins J. Biol. Chem., July 28, 2000; 275(31): 23409 - 23412. [Full Text] [PDF]
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