From the Program in Molecular Cell Biology, Division
of Biology and Biomedical Sciences, Washington University School of
Medicine, St. Louis, Missouri 63110 and the § Program in
Host-Pathogen Interactions, University of California, San Francisco,
San Francisco, California 94143
Received for publication, August 11, 2000, and in revised form, January 23, 2001
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ABSTRACT |
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L-plastin (LPL) is a leukocyte actin binding
protein previously implicated in the activation of the integrin
A fundamental property of leukocyte integrins is the ability to
modulate their adhesive functions. As cells circulate through blood and
lymph, adhesion is minimal. In response to inflammatory signals,
integrin-mediated adhesion is markedly augmented. This property of the
integrins is physiologically critical, because it is required for
appropriate migration out of the vasculature into sites of inflammation
and at the same time limits the potentially host-damaging inflammatory
response to those sites alone. Because this activity of integrins is so
important to their role on leukocytes, much attention has been devoted
to the molecular mechanisms by which integrin adhesion is regulated.
Two distinct alterations in integrins are likely to be involved in
enhancement of adhesion. A rapid response to cell activation is an
increase in integrin diffusion, due to loss of cytoskeletal constraint
of integrin mobility. Increased diffusion may lead in turn to integrin
clustering at sites of cell interaction with ligand. A second response
to activation is a conformational change in the integrins, which often
reflects increased affinity for ligand. This conformational change may
require initial interaction with ligand and can be reflected in the
generation of new epitopes recognized by monoclonal antibodies, the
so-called ligand-induced binding sites
(LIBS1). Although a number of
signaling molecules, such as PKC and phosphatidylinositol 3-kinase, have been implicated in this process (1-3), these
enzymes may be many steps upstream from the actual change in integrin behavior. GTPases of the Ras and Rho families also have been implicated in regulation of integrin function (4-6), but their downstream targets
for this function have not been identified. Therefore, much remains to
be learned about the mechanisms involved in regulation of integrin avidity.
Our own studies have implicated the actin-binding protein
L-plastin (LPL) in regulation of integrin function (7, 35). Recently, we have shown that cell-permeant peptides from the N terminus
of LPL can rapidly activate To pursue these mechanistic questions, we turned to a
well-characterized model for studying integrin activation.
Cells and Reagents--
The human erythroleukemic cell line K562
transfected with cDNA encoding
Monoclonal antibodies L230 (anti-human
LPL peptides were described previously (7). The sequences of the
LPL-related peptides used in this study were LPL (ARGSVSDEEMMELREAFA), LPLtat (ARGSVSDEEMMELREAFAYGRKKRRQRRRG), tatLPL
(YGRKKRRQRRRGARGSVSDEEMMELREAFA), SCRtat
(AGDESEMEFVMASALRREYGRKKRRQRRRG), and TPLtat
(ATTQISKDELDELKEAFAYGRKKRRQRRRG).
Tat Fusion Protein Purification--
The cDNA containing the
LPL N-terminal Ca2+-binding domain (headpiece fragment,
amino acids 1-105) was cloned into pBluescript KS+ as an
XhoI/BamHI fragment. An myc tag
was fused in-frame with the C terminus of the headpiece sequence
(Headmyc). To obtain the headpiece fragment with the
tat sequence at the C terminus (Headtat), the
myc sequence was replaced by an oligonucleotide encoding the
12-amino acid tat peptide sequence. The Headmyc
fragment also was subcloned into the pTAT vector (a gift of Dr. Steven Dowdy, Washington University, St. Louis, MO) (16) to obtain a headpiece
with the tat sequence at the N terminus
(tatHead). The constructs were transformed into BL-21 LysS
cells (Novagen). Bacteria from 1-liter cultures were resuspended in 50 ml of PBS + 50 µg/ml lysozyme. The bacteria were lysed with three
freeze-thaw cycles and sonication, and lysates were clarified by
centrifugation. The supernatant was run through an immunoaffinity
column of LPL4A.1 mAb coupled to Sepharose 4B and eluted with PBS
containing 10 mM EDTA as LPL4A.1 binds to LPL in a
Ca2+-dependent manner. EDTA was removed by dialysis.
Adhesion Assay--
K FACS Analysis of Ligand-induced Binding Site (LIBS)
Expression--
Binding of mAb 7G2 (13) to
K F-actin Content--
F-actin content of
K LPL N-terminal Peptide Induces
To determine whether LPLtat could activate
The LPL Headpiece Induces LPLtat-induced Adhesion Does Not Require the LPLtat and RGD Act Synergistically to Increase Ligand-induced
Binding Site (LIBS) Expression--
Leukocyte integrin activation can
be achieved by several mechanisms. For many integrins, binding of
ligand or exposure to the divalent cation Mn2+ induces a
conformational change that results in increased adhesion because of
enhanced affinity for ligand (22). In the case of Inhibition of LPLtat-induced Adhesion Requires a Higher
Concentration of RGD Peptide than PMA- or Mn2+-induced
Adhesion--
As has been shown repeatedly, RGD peptide competes with
Vn for the ligand binding site in LPLtat-induced Adhesion Is Inhibited by Jasplakinolide, but Not
Cytochalasin D--
It is generally thought that integrins require
attachment to actin filaments for firm adhesion. However, adhesion
mediated by high affinity integrin receptors can be unaffected by
cytochalasin D (26), implying that new actin microfilament formation is
not required for this mechanism of attachment. Therefore, the
sensitivity of LPLtat-induced adhesion to cytochalasin D was
tested. As expected, PMA-induced adhesion, which requires the actin
cytoskeleton in post-receptor events, was completely inhibited by
cytochalasin D at 1 µg/ml. In contrast, cytochalasin D had no
inhibitory effect on either LPLtat or
Mn2+-induced
It is recognized that actin microfilaments can also negatively regulate
integrin activation by restricting integrin lateral mobility in resting
cells (10). As a result, low doses of cytochalasin D actually induces
LFA-1- and Mac-1-mediated adhesion by releasing integrins from
cytoskeletal constrains leading to integrin activation (10, 27-29). In
this study, low concentration of cytochalasin D (0.1 µg/ml) activated
Because actin depolymerization can induce
Because both actin depolymerization and increase in receptor affinity
are involved in LPLtat-induced adhesion, we asked whether actin depolymerization was required for the generation of high affinity
The term "integrin activation" is sometimes used to mean the
modulation of affinity that can be induced in many integrins by
Mn2+, activating antibodies, ligand, and sometimes other
physiologic or pharmacologic stimuli. If used in this way, integrin
activation is thought be related to a conformational change in the
extracytoplasmic domain of the integrins and can occur in a wide
variety of cell types. However, Despite the importance of this reversible activation of leukocyte
integrin-mediated adhesion, the mechanisms involved in regulation of
leukocyte integrin function remain obscure. Although a number of
signaling molecules have been identified that can be involved in the
signal transduction pathways of inside-out signaling, the effector
mechanisms leading to changes in integrin function have not been
elucidated. In general, two not mutually exclusive models have been
invoked, one involving receptor rearrangement on the plasma membrane
and another involving conformational change in the integrin itself (31,
32). Our previous work implicates LPL phosphorylation in a final common
pathway influencing integrin function after cell activation with
G-protein coupled, tyrosine kinase-mediated, and
PKC-dependent stimuli (7, 33-35). The fact that peptides
that mimic the phosphorylation site in the N terminus of LPL rapidly
stimulate integrin activation when introduced into PMN or monocyte
cytoplasm (7) presents an excellent opportunity to gain insight into
the effector mechanisms of inside-out signaling. Therefore, in this
work we have investigated the mechanism by which the LPL N terminus
activates integrin-mediated adhesion, using the well-characterized
model of A cell-permeant version of the LPL N-terminal domain (headpiece)
activated The requirements in the LPLtat acts synergistically with RGD ligand to induce the
expression of an LIBS epitope on Although the change in integrin conformation is essential for
LPLtat-induced adhesion, it is not sufficient, because
jasplakinolide blocks adhesion without significant inhibition of the
conformational change. It is now clear that integrin release from
cytoskeletal constraint is an early and essential aspect of activation
of adhesion (10-12). We hypothesize that, in addition to recruiting
new M
2 on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562
cell adhesion to vitronectin via
v
3, a
well-studied model for activable integrins, was examined. Cell permeant
versions of peptides based on the N-terminal sequence of LPL and the
LPL headpiece domain both activated
v
3-mediated adhesion. In contrast to
adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of
integrin
3 cytoplasmic domain tyrosines and was not
inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides
synergized with RGD ligand or Mn2+ for generation of a
conformational change in
v
3 associated with the high affinity state of the integrin, as determined by binding
of a ligand-induced binding site antibody. Although LPL and ligand
showed synergy for ligand-induced binding site expression when actin
depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin
depolymerization and ligand-induced integrin conformational change are
required for LPL peptide-induced adhesion. We hypothesize that the
critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
M
2
(Mac-1)-mediated adhesion in polymorphonuclear neutrophils (PMN). When
the peptides introduced into PMN cytosol contained phosphoserine at
position 5, which is the major if not exclusive site of phosphorylation
in LPL (7, 35), integrin activation was not inhibited by blockade of
PKC or phosphatidylinositol 3-kinase, suggesting that LPL
phosphorylation might be a mechanism by which these enzymes signal
changes in integrin function. Thus, LPL is likely a downstream effector
of multiple signaling pathways leading to integrin activation, and LPL
peptide induction of adhesion is a useful experimental system in which
to begin to understand regulation of integrin avidity.
v
3 is not normally expressed in
undifferentiated K562 cells, but when expressed through transfection of
v and
3 cDNAs,
v
3-mediated adhesion in K562 cells is
dependent on cell activation (8). Moreover, adhesion in response to PMA
or thrombin is dependent on phosphorylation of Tyr-747 in the
3 cytoplasmic tail, an event that is itself dependent on
the presence of the
v cytoplasmic tail (8, 9). Using
these cells, we have examined the requirements for LPL peptide-induced adhesion. We have found that a cell permeant version of the entire headpiece domain of LPL, as well as synthetic LPL peptides, can induce
adhesion. In contrast to PMA, LPL peptide-induced adhesion does not
require tyrosine phosphorylation of the
3 cytoplasmic tail, suggesting that LPL is downstream of the tyrosine signal in the
activation cascade. Nonetheless, LPL peptide-induced adhesion does
require actin depolymerization, presumably to induce integrin release
from cytoskeletal constraint (10-12), and LPL peptide does cooperate
with Arg-Gly-Asp (RGD) ligand to induce LIBS epitope expression. These
data suggest that the steps of increased integrin diffusion and
conformational change may be linked via modulation of the function of
the actin binding protein L-plastin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3
(K
v
3) and
v
3 in which the tyrosine residues at
position 747, 759, or both were mutated to phenylalanine (Y747F, Y759F, and Y747F/Y759F, respectively) were derived as described (8, 9). Cells were maintained in Iscove's modified Dulbecco's medium (Life Technologies, Inc., Gaithersburg, MD), containing 10%
heat-inactivated fetal calf serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 µg/ml penicillin, and
streptomycin under a 5% CO2 atmosphere. JY B
lymphoblastoid cells were from ATCC (Rockville, MD) and maintained in
RPMI 1640 medium (Life Technologies, Inc.) with the same supplements as
Iscove's modified Dulbecco's medium. Jasplakinolide was from
Molecular Probes (Eugene, OR).
v), 1A2.1
(anti-human
3), AP3 (anti-human
3), 7G2
(anti-human
3), W6/32 (anti-human HLA), P1F6
(anti-
v
5), mAb16 (anti-
5),
TS2/16 (anti-
1), and LPL4A.1 (anti-human LPL) have been
previously described (8, 13-15). Monoclonal mouse antibody against the
tat-(49-58) epitope (19) was a gift from Hybridolab, Institut Pasteur,
Paris, France. Calcein was purchased from Molecular Probes. 96-well
Immulon 2 plates were from Dynatech (Chantilly, VA). Vitronectin (Vn),
RGD (GRGDSP) and RAD (GRADSP) peptides, and phosphate-buffered saline (PBS) were purchased from Life Technologies, Inc. (Gaithersburg, MD).
Human serum albumin (HSA) was from Therapeutic Corp. (Los Angeles, CA).
Purified human recombinant osteopontin was a gift from Simon M. Blake,
SmithKline Beecham, King of Prussia, PA.
v
3 cells or
JY cells (1 × 107/ml) were washed once with HBSS (1×
Hanks' buffered salt solution with 20 mM Hepes and 8.9 mM sodium bicarbonate) and incubated with 2 µg/ml calcein
in HBSS for 30 min at RT. The cells were washed three times and
resuspended in HBSS2+ (HBSS with 1.0 mM
Mg2+ and 1.0 mM Ca2+) at 2 × 106/ml. Cells were subjected to various treatments as
detailed in respective figure legends. 1 × 105
cells/well were added to HSA-, Vn (10 µg/ml or as indicated in the
figures)-, or osteopontin (10 µg/ml)-coated 96-well Immulon 2 plates.
Cells were allowed to settle for 10 min before L-plastin peptides (50 µM), PMA (50 ng/ml), or MnCl2 (1 mM) were added to the wells. Cells were incubated at
37 °C for 1 h. After the incubation period, the fluorescence
(485-nm excitation and 530-nm emission wavelengths) was measured using
an fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA)
before and after washing four times with 180 µl of PBS. Percent
adhesion was calculated by dividing the fluorescence after washing by
the fluorescence before washing. The assays were performed in
triplicate, and data are presented as mean ± S.D. All experiments
were performed at least three times with similar results. In
preliminary experiments, fluorescence was shown to be linearly related
to cell number.
v
3 cells was assessed by fluorescence
flow cytometry (FACS). K
v
3 cells were
washed once with HBSS and resuspended in HBSS2+. Cells were
treated with control buffer, PMA (50 ng/ml), MnCl2 (1 mM), or 50 µM LPL peptides
(LPLtat, SCRtat, TPLtat,
tatLPL, or LPL) at 37 °C for 20 min. Cells were then
incubated with RGD or RAD peptides (100 µM or as
specified in the figures) together with the 7G2 antibody (5 µg/ml)
for 60 min at RT. After washing three times with FACS buffer (PBS + 200 µg/ml bovine serum albumin + 130 µg/ml NaN3), cells
were stained with fluorescein isothiocyanate-conjugated Fab-specific
anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) for 45 min on ice.
Cells were washed with FACS buffer and analyzed by flow cytometry. For
MnCl2-treated samples, 250 µM
MnCl2 was always present in the subsequent incubation and
washing buffers.
v
3 cells was determined as previously
described (41) with minor modifications. Briefly, cells were incubated
with buffer, 50 µM LPLtat, 1 µM
jasplakinolide, or both for 15 min at 37 °C and then solubilized by
incubation with an equal volume of Tris-buffered saline, pH 7.4, containing 4% Triton X-100, 4 mM EDTA, and 5 µg/ml
leupeptin. After centrifugation at 38,000 rpm for 30 min in a TL100.2
rotor (Beckman Optima TL Ultracentrifuge, Beckman Instruments,
Fullerton, CA), the supernatant was carefully removed, and the
gelatinous pellet was dissolved in SDS-polyacrylamide gel
electrophoresis sample buffer. After separation by SDS-polyacrylamide
gel electrophoresis, actin content of the Triton-insoluble pellet was
analyzed by Western blot with rabbit anti-actin (Sigma) and quantitated
by densitometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3-mediated Adhesion--
We previously
demonstrated that a cell permeant peptide containing LPL amino acids
2-19 fused at the C terminus with the HIV tat sequence
(LPLtat) induces
M
2-mediated
adhesion in PMN (7). LPLtat also causes
M
2-dependent adhesion to
C3bi, an
M
2-specific ligand, in Jurkat
cells transfected with
M
2
integrin.2 These data suggest
that LPL has a role in
M
2 activation in leukocytes. We next asked whether LPL was specifically involved in
M
2 activation or if it was involved in
the regulation of other integrins. The K562 cell, transfected with
exogenous integrins, is a well-established model system for studying
integrin functions in a myeloid background (8, 9, 14, 17), and our
laboratory has shown previously that cell activation is required for
adhesion mediated by
v
3 expressed in
these cells (8). Therefore, we tested the effects of LPLtat
on
v
3-mediated adhesion to Vn in stable
K562 transfectants (K
v
3).
LPLtat induced K
v
3 adhesion to
Vn but not to HSA (Fig. 1A).
Although LPLtat did not induce cell adhesion in the absence
of ligand, its effect was apparent even at a Vn coating concentration
of 0.1 µg/ml (Fig. 1B). This effect was specific to
LPLtat, because control peptides, including tatLPL, in which the tat sequence mediating
membrane permeability of the peptide was N-terminal to the LPL
sequence, SCRtat, in which the LPL sequence was scrambled,
and TPLtat, which contained amino acids 2-19 of the LPL
homologue T-plastin, did not increase adhesion (Fig. 1C).
LPLtat did not induce adhesion of the parental K562 cells,
which do not express
v
3, to Vn
(K562+LPLtat in Fig. 1C).
LPLtat-induced adhesion of K
v
3
was inhibited by the anti-
v antibody L230 and the
anti-
3 antibody 1A2.1 (Fig. 1D), further demonstrating the requirement for the
v
3
integrin. LPLtat, but not tatLPL,
SCRtat, or TPLtat, also induced
K
v
3 adhesion to osteopontin (data not
shown). Like adhesion to Vn, K
v
3 adhesion to osteopontin was inhibited by an anti-
3 mAb but not by
antibodies recognizing
5,
1, or HLA.
Thus, LPLtat activates
v
3- as
well as
M
2-mediated adhesion. In
contrast,
5
1-mediated adhesion to
fibronectin does not require activation in K562 cells (14), and
LPLtat had no significant effect on adhesion of
untransfected K562 to fibronectin (data not shown). Thus,
LPLtat affects integrin activation for adhesion, rather than
adhesion by already active integrins.
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Fig. 1.
LPLtat induces
v
3-mediated
adhesion to Vn in
K
v
3
cells. A, K
v
3 cells were
allowed to adhere to HSA (open bar)- or Vn (filled
bar)-coated surfaces, treated with buffer or 50 µM
LPLtat. B, K
v
3
cells were allowed to adhere to surfaces coated with various
concentrations of Vn as indicated and treated with buffer
(triangle) or 50 µM LPLtat
(circle). C, K
v
3
cells were allowed to adhere to Vn-coated surfaces and treated with
buffer or 50 µM peptides as specified. The bar
labeled K562+LPLtat depicts the binding of untransfected
K562 cells treated with LPLtat. D,
K
v
3 cells were incubated with 10 µg/ml
monoclonal antibodies W6/32 (HLA), L230
(
V), 1A2.1 (
3), P1F6
(complex-specific for
v
5), or
mAb16 (
5) for 30 min at RT before addition to
Vn-coated surfaces and activation with 50 µM
LPLtat. Adhesion assays were performed exactly as described
under "Experimental Procedures." Data are representative of at
least four independent experiments.
v
3 in a cell constitutively expressing
this integrin, we examined adhesion of the JY B lymphoma line, which is
known to have activable
v
3 (23, 37).
These cells also adhered to Vn when treated with LPLtat
(Fig. 2A), in a manner
dependent on Vn-coating concentration (Fig. 2B) and
3 integrin (Fig. 2C).
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Fig. 2.
LPLtat activates
v
3-mediated
adhesion in JY cells. A, JY cells were incubated with
Vn-coated surfaces and treated with buffer or 50 µM
peptides as specified. B, JY cells were allowed to adhere to
surfaces coated with the indicated concentrations of Vn and treated
with buffer (triangle) or 50 µM
LPLtat (circle). C, JY cells were
incubated with 10 µg/ml monoclonal antibodies W6/32 (HLA),
P1F6 (
V
5), 1A2.1
(
3), or mAb16 (
5) for
30 min at RT before addition to Vn-coated surfaces and activation with
50 µM LPLtat. Adhesion assays were performed
as described under "Experimental Procedures." Data are
representative of three independent experiments.
v
3-mediated
Adhesion in K
v
3 Cells--
In addition
to the site for phosphorylation, the N-terminal domain, or headpiece,
of LPL contains two EF-hand-type Ca2+ binding motifs (18),
and Ca2+ markedly affects the conformation of this domain.
To determine whether LPLtat induction of adhesion could be
recapitulated with the entire LPL N-terminal domain, we expressed an
LPL truncation mutant that contained the entire headpiece domain fused
to the tat peptide sequence either at the C terminus
(Headtat) or the N terminus (tatHead) (Fig.
3A). The purified proteins
were recognized by specific monoclonal antibodies directed toward the
tat-(49-58) epitope (19) or toward the LPL N terminus (Fig.
3B). As in the case of LPL peptides, Headtat, but
not tatHead induced K
v
3
adhesion to Vn. Headtat-induced adhesion was completely
inhibited by both anti-
v and anti-
3
antibodies (Fig. 3C). These data demonstrate that, in
addition to short peptides, the intact headpiece domain of LPL can
activate
v
3-dependent
adhesion in K
v
3 cells.
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Fig. 3.
LPL headpiece domain induces
v
3-mediated
adhesion in
K
v
3
cells. A, schematic representation of the recombinant
tat-expressing domains. TatHead had an
myc epitope at its C terminus, whereas Headtat
had the tat peptide in that position. B, 15 µl
of purified Headtat and tatHead proteins were
Western-blotted with anti-tat mAb (upper panel)
or the anti-LPL mAb LPL 4A.1 (lower panel). C,
K
v
3 cells were treated with W6/32
(control antibody), L230 (anti-alphaV), or 1A2.1
(anti-beta3) mAb as in Fig. 1 prior to measuring adhesion to
Vn in the presence of buffer (open bar), 2 µM
tatHead (hatched bar), or 2 µM
purified Headtat (filled bar). The bar labeled
K562+Headtat depicts adhesion to the Vn-coated surface of
parental untransfected K562 cells activated by Headtat.
Adhesion assays were performed as described under "Experimental
Procedures." Shown is a representative of three independent
experiments with similar results.
3
Cytoplasmic Tail Tyr-747 or Tyr-759--
PMA- and thrombin-activated
K
v
3 adhesion to Vn requires Tyr-747 of
the
3 cytoplasmic tail, because Y747F mutants fail to adhere in response to these stimuli (8). To determine whether LPLtat-induced adhesion was also dependent on Tyr-747, we
examined the effect of LPLtat on adhesion to Vn in K562
cells expressing
v
3 with single amino
acid mutations Y747F, Y759F, and the double mutant Y747F/Y759F. As
previously observed (8), the Y747F but not the Y759F mutation abolished
PMA-stimulated adhesion to Vn (Fig. 4).
However, K
v
3Y747F,
K
v
3Y759F, and
K
v
3Y747F/Y759F adhered equally well as
cells expressing wild-type
v
3 when
treated with LPLtat (Fig. 4). Thus, unlike PMA,
LPLtat-activated adhesion does not require
3
Tyr-747 or its phosphorylation. This demonstrates that the
LPLtat effect on
v
3 is either
downstream or independent of the Tyr-747-dependent
signaling cascade (20, 21) involved in PMA-induced adhesion.
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Fig. 4.
LPLtat-induced adhesion does
not require 3 cytoplasmic tail
Tyr-747. K562 cells transfected with
v and normal
3 (WT),
3 encoding a Y to F
mutation at Tyr-747 (Y747F),
3 encoding a Y
to F mutation at Tyr-759 (Y759F), or
3
expressing the Y747F/Y759F double mutation (Y747/759F) have
been described (8). Cells of each type with equal surface expression of
v
3 were treated with buffer control
(open bar), 50 ng/ml PMA (hatched bar), or 50 µM LPLtat (filled bar). Adhesion
assays were performed as described under "Experimental Procedures."
Data shown are representative of at least three independent
experiments.
v
3, RGD peptide or Mn2+
binding can induce this conformational change, which can be identified by the binding of monoclonal antibodies (mAb) that recognize LIBS epitope (8, 23, 24). This conformational change is associated with
increased affinity for Vn, apparently due to a decreased off-rate of
the receptor-ligand interaction (25). As shown in Fig.
5A, the anti-
3
mAb 7G2 (13) recognizes an LIBS epitope as its binding to
v
3 increased ~10-fold in response to
RGD peptide. Maximal 7G2 reactivity was achieved with 25 µM RGD peptide (Fig. 5A), and increasing the
RGD peptide concentration to as high as 2 mM did not induce
further 7G2 reactivity (data not shown). Control RAD peptide did not
enhance 7G2 binding (Fig. 5B). Treatment of K
v
3 with LPLtat alone only
minimally increased 7G2 binding (Fig. 5, A and
B). However, even at concentrations of RGD peptide that caused maximum 7G2 binding, 7G2 reactivity was further increased when
cells were treated with RGD in combination with LPLtat,
demonstrating marked synergy between the cytoplasmic peptide and the
ligand (Fig. 5A). Treatment of
K
v
3 with RGD together with control
peptides such as LPL (LPL aa 2-19 without associated tat),
or other peptides bearing the tat sequence,
TPLtat, tatLPL, and SCRtat, did not cause 7G2 reactivity to increase over the level induced by RGD alone
(Fig. 5B). Total
v
3 expression
on the surface of the transfected K562 cells was not altered by
LPLtat treatment as assessed by the (non-LIBS)
anti-
3 antibody AP3 (data not shown). LPLtat
synergized with RGD to induce LIBS expression in
v
3 with the Y747F and Y759F mutations
(Fig. 5C), consistent with the finding that
LPLtat-induced adhesion does not require these
3 cytoplasmic tail tyrosines. LPLtat also
synergized with Mn2+ to increase LIBS expression in wild
type and mutant receptors (Fig. 5D and data not shown). In
contrast, PMA treatment did not induce 7G2 binding either with or
without LPLtat (Fig. 5D). These data indicate
that LPLtat induces
v
3-mediated adhesion by cooperating with
ligand to induce expression of the high affinity conformation of the
integrin, independent of Tyr-747 or Tyr-759.
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Fig. 5.
LIBS recognition by mAb 7G2.
A, K v
3 cells were treated with
buffer control (triangle) or 50 µM
LPLtat (circle) for 20 min at 37 °C. The
expression of an LIBS epitope in the presence of indicated amounts of
RGD peptide was assessed by 7G2 staining and FACS analysis as described
under "Experimental Procedures." The mean fluorescence of each
histogram was taken for numerical comparison. B,
K
v
3 were treated with buffer
(None), 50 µM RGD or control RAD peptide,
and/or 50 µM LPL peptides, singly or in combinations as
in the figure. LIBS epitope expression was assessed by 7G2 binding as
in A. C, K
v
3Y747F
(open bar) and K
v
3Y759F
(filled bar) were treated with 50 µM RGD or
control RAD peptide, and/or 50 µM LPLtat
singly or in combination as in the figure. LIBS epitope expression was
assessed as in the previous panels. D,
K
v
3 were treated RGD (50 µM), MnCl2 (1 mM), PMA (50 ng/ml), and/or LPLtat (50 µM), alone and in
combinations as indicated in the figure, and LIBS epitope expression
was assessed as above. The data in each panel are representative of
three independent experiments with similar results.
v
3 and
inhibits adhesion to this protein. Because LPLtat cooperated
with RGD to induce an LIBS epitope and presumably the high affinity
state of the integrin, we asked whether adhesion induced by PMA,
Mn2+, and LPLtat had similar sensitivity to RGD
peptide inhibition. If LPLtat increased the number of high
affinity receptors, it should shift the ID50 for RGD to a
higher concentration. As this hypothesis predicted,
LPLtat-activated adhesion was much more resistant to RGD
peptide inhibition than either PMA or Mn2+ stimulation
(Fig. 6A). Mn2+-
or PMA-induced adhesion was completely inhibited by 50 µM
RGD peptides, with an ID50 at about 20 µM
(Fig. 6, B and C). LPLtat-induced adhesion, however, required 100-fold more RGD peptide for equivalent inhibition, with an ID50 of ~2.5 mM RGD (Fig.
6A). This result is consistent with the higher LIBS
expression induced by RGD and LPLtat than by RGD and
Mn2+ or RGD and PMA (Fig. 5D).
View larger version (10K):
[in a new window]
Fig. 6.
RGD peptide inhibition of
v
3-mediated
adhesion. A, K
v
3 cells
were incubated with buffer (the y intercepts for each curve)
or the indicated concentration of RGD peptide for 20 min at RT prior to
assessment of cell adhesion to Vn-coated surfaces upon treatment with
buffer (open circle), 50 ng/ml PMA (filled
circle), 1 mM MnCl2 (filled
triangle), or 50 µM LPLtat (filled
square). For B and C, cells were treated
with indicated amount of RGD (square) or RAD
(triangle) as control and then activated with 50 ng/ml PMA
(B) or 1 mM MnCl2 (C).
Adhesion assays were performed as in Fig. 1. Data are representative of
three separate experiments.
v
3-mediated
adhesion even at 10 µg/ml (Fig.
7A). These results demonstrate
that LPLtat induces adhesion mediated by high affinity
receptors, independent of actin microfilaments.
View larger version (13K):
[in a new window]
Fig. 7.
Effects of cytochalasin D on
v
3-mediated
adhesion. A, LPLtat-induced adhesion is not inhibited
by cytochalasin D. K
v
3 were treated with
Me2SO (the y intercepts for each curve) or
indicated concentrations of cytochalasin D for 30 min at 37 °C
before measurement of adhesion to Vn upon incubation with buffer
(open circle), 50 ng/ml PMA (filled circle), 50 µM LPLtat (filled square), or 1 mM MnCl2 (filled triangle). Adhesion
was assessed as described under "Experimental Procedures."
B, low concentration cytochalasin D stimulates
v
3-mediated adhesion.
K
v
3 were treated with varying
concentrations cytochalasin D as indicated prior to measurement of
adhesion to Vn-coated surfaces upon exposure to buffer (open
circle) or 50 ng/ml PMA (filled circle). Representative
of three independent experiments.
v
3-mediated adhesion as well (Fig.
7B). Apparently, as in the case of LFA-1 and Mac-1, release
of unactivated integrins from cytoskeletal constraint can lead to
v
3-mediated adhesion.
v
3-mediated adhesion, the requirement for
actin depolymerization in
v
3-mediated adhesion was examined. Jasplakinolide stabilizes pre-existing F-actin,
prevents actin depolymerization, and induces a net increase in actin
polymerization (30). It was previously shown to inhibit LFA-1-dependent adhesion (12). Similarly, jasplakinolide
inhibited
v
3-mediated adhesion induced by
LPLtat, Mn2+, or PMA in
K
v
3 (Fig.
8A) and in JY cells (data not
shown). Taken together, the cytochalasin D and jasplakinolide data
suggest that, although actin polymerization is not required for
LPLtat-initiated
v
3-mediated
adhesion, actin depolymerization is. F-actin content of cells treated
with LPLtat and jasplakinolide was determined (Fig.
8B). Although LPLtat caused about a 50% decrease
in total F-actin, jasplakinolide treatment more than doubled cellular F actin. Jasplakinolide also prevented the LPLtat-induced
decrease in F-actin.
View larger version (14K):
[in a new window]
Fig. 8.
Effect of jasplakinolide on
v
3-mediated
adhesion, F-actin, and LIBS expression. A,
K
v
3 were treated with 1 µM
jasplakinolide (filled bar) or an equivalent amount of
Me2SO vehicle as the control (open bar) for 45 min at 37 °C and incubated with buffer, 50 µM
LPLtat, 1 mM MnCl2, or 50 ng/ml PMA
as indicated. Adhesion assays were performed exactly as described under
"Experimental Procedures." B, F-actin content of
K
v
3 cells treated with buffer (control),
50 µM LPLtat, 1 µM
jasplakinolide, or the combination of LPLtat and
jasplakinolide was determined as described under "Experimental
Procedures." The ordinate is in arbitrary densitometric
units. Shown is the average of two independent experiments.
C, K
v
3 cells were treated with
Me2SO (open bar) or 1 µM
jasplakinolide (filled bar) for 45 min at 37 °C. Cells
were then incubated with 50 µM RGD peptide, 50 µM LPLtat, or 1 mM
MnCl2 singly or in combinations as indicated in the figure,
and 7G2 LIBS expression assessed by FACS analysis as described in Fig.
4. Shown are data representative of three independent
experiments.
v
3. Surprisingly, jasplakinolide had
little effect on synergistic expression of the 7G2 epitope by RGD and
LPLtat (Fig. 8C). In addition, cytochalasin D had
no effect on the ability of RGD, LPLtat, or their
combination to induce the LIBS epitope recognized by 7G2 (data not
shown). Thus, although actin depolymerization is required for
v
3-mediated adhesion, it is neither
necessary nor sufficient for achieving a high affinity conformation of
the receptor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and
3
integrins on bone marrow-derived cells, including leukocytes and
platelets, are different from integrins on other cell types, because
without physiologic or pharmacologic activation, they do not mediate
adhesion. Thus, for these integrins, cell activation is an absolute
requirement for function. Activation of adhesion likely results from
integrin clustering within the membrane as well as changes in affinity
of individual integrins (10-12). Moreover, leukocyte integrins can be
distinguished from platelet integrin
IIb
3
because, although
IIb
3 activation for ligand binding is irreversible, activation of leukocyte integrins is a
reversible process. Indeed, reversible activation of integrin-mediated adhesion is thought to be important in a variety of critical leukocyte functions, including migration, phagocytosis, and cell-mediated cytotoxicity.
v
3-mediated adhesion in K562 cells.
v
3-mediated adhesion as well as
the 18-amino acid LPL peptides previously shown to activate
M
2-mediated adhesion. As previously
discussed (7), integrin activation by the LPL N-terminal peptides
requires their entry into the cytoplasm, because peptides of identical
sequence without the tat addition have no effect on integrin
function. Placing the tat sequence at the N-terminal end of
either the peptide or the headpiece domain abrogates function even
though this placement does not decrease entry into the
cytoplasm.3 This suggests the
possibility that a free LPL N terminus is essential for its integrin
regulatory function and that addition of the 12-amino acid
tat peptide blocks some essential function of this region of
the protein. However, this domain binds neither actin nor vimentin
(36), and there are no known cytoplasmic proteins that interact with
the LPL headpiece. Although the headpiece does bind Ca2+
through its tandem EF-hand domains, the active peptides do not, and
modulation of intracytoplasmic Ca2+ by itself is not known
to affect integrin function. Thus, the molecular interactions by which
the LPL N terminus activates integrin avidity are unknown. Nonetheless,
both the headpiece domain and the N-terminal peptides induce
v
3-mediated adhesion, and the peptides
clearly synergize with RGD ligand to activate a conformational change
in
v
3 associated with increased ligand
affinity. We hypothesize that this occurs as the result of an
interaction between the N-terminal domain of LPL and an as yet unknown
target, which could be the integrin itself.
3 cytoplasmic domain for
LPLtat induction of adhesion are distinct from PMA or
thrombin, because the
3 Y747F mutant, which fails to
support adhesion stimulated by PMA, supports normal adhesion induced by
the LPL peptides. Because the Y747F mutant also supports adhesion in
cells with constitutively active integrins (8), it is clear that
phosphorylation of this tyrosine is not required for
v
3-mediated adhesion. Rather, Tyr-747
phosphorylation is likely required in a signal transduction pathway
involving the integrin, perhaps by recruiting an SH2- or PTB-containing
protein, that leads to a final common pathway of integrin adhesion that
does not itself require Tyr-747. If this is the case, then LPL
functions downstream of Tyr-747 phosphorylation.
3 recognized by mAb
7G2. The RGD peptide can induce 7G2 binding in a
dose-dependent manner, as is expected for a LIBS mAb; in
contrast, at optimal concentration, LPLtat induces little
7G2 binding. Thus, LPLtat does not act like an activating
antibody or certain
IIb
3 chimeras, for
which LIBS expression becomes independent of ligand. Instead,
LPLtat appears to augment the conformational change induced
by RGD. In K562, as in other cells, only a minority of integrins
expresses the LIBS epitope in the presence of ligand. Virtually nothing
is known about what distinguishes receptors undergoing conformational
change from those that do not. LPLtat seems to increase the
fraction of receptors capable of achieving this conformational change
when exposed to ligand. In studying purified
v
3, Orlando and Cheresh (25) noted that
prior exposure to RGD markedly decreased the off-rate of its binding to
Vn and suggested that this was the mechanism by which the
ligand-induced conformational change in the integrin induced stable
cell adhesion. It is likely that, in the context of an intact cell,
LPLtat exaggerates this normal response, leading to marked
strengthening of adhesion. It is noteworthy that
LPLtat-mediated adhesion is insensitive to cytoskeleton
disruption with cytochalasin D, suggesting that the LPL effect on
integrin interaction with ligand dominates any potential indirect
effect of adhesion strengthening through modulation of actin-integrin interactions.
v
3 to respond to a ligand-induced
conformational change, LPLtat induces integrin release from
cytoskeletal constraint. Consistent with this, treatment of cells with
LPLtat diminishes total F-actin content and simultaneous
treatment with jasplakinolide inhibits this effect of the peptide.
Effects on both cytoskeleton and integrin conformation are required for
stable adhesion induced by LPLtat, even though cytoskeletal
release is not required for the peptide effects on integrin
conformation. Moreover, cytoskeletal release occurs in the absence of
ligand (10), so conformational change is not a prerequisite for
release. In platelets, alterations in integrin-cytoskeleton interaction
are important for inside-out signaling (38), and an increase in
intracytoplasmic Ca2+ can activate
IIb
3 by releasing it from cytoskeletal
constraints (39). Release of integrins from cytoskeletal constraint may require phosphorylation of the myristoylated alanine-rich protein kinase C substrate (40) as well as LPL. Thus, LPL peptide appears to
induce two necessary but potentially independent events in control of
integrin function. It is intriguing to speculate that the peptide may
interact directly with integrin cytoplasmic tails to achieve these
effects. Because phosphorylation of LPL may activate its release from
actin filaments,2 it is possible that signal transduction
leading to LPL phosphorylation frees the N terminus to interact with
integrins, leading in turn to their release from cytoskeleton and
diffusion to sites of ligand concentration, where a conformational
change leading to high affinity interaction occurs upon ligand binding.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Terry Woodford-Thomas and the late Dr. Matthew Thomas for many useful discussions during the course of these experiments, and Dr. Woodford-Thomas for her careful critique of the paper. We also thank Dr. F. Patrick Ross for providing the osteopontin.
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FOOTNOTES |
---|
* This work was supported by Grant AI35811 from the National Institutes of Health (to E. J. B.).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: Program in Host Pathogen Interactions, Campus Box 0654, University of California at San Francisco, 513 Parnassus Ave., San Francisco, CA 94143-0654. Tel.: 415-514-0167; Fax: 415-514-0169; E-mail: ebrown@medicine.ucsf.edu.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M007324200
2 J. Wang and E. J. Brown, unpublished data.
3 S. L. Jones and E. J. Brown, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
LIBS, ligand-induced
binding site;
LPL, L-plastin;
Headtat, a
recombinant protein containing amino acids 1-105 of
L-plastin fused at the C terminus to 12 amino acids of the
HIV tat sequence;
HSA, human serum albumin;
Kv
3, K562 cells transfected with
v
3 integrin;
LPLtat, a
synthetic peptide based on amino acids 2-19 of L-plastin
followed by 12 amino acids of the HIV tat sequence;
PKC, protein kinase C;
PMA, phorbol 12-myristate 13-acetate;
PMN, polymorphonuclear neutrophils;
RAD, Arg-Ala-Asp peptide;
RGD, Arg-Gly-Asp peptide;
SCRtat, a synthetic peptide in which
amino acids 2-19 of L-plastin have been scrambled,
followed by 12 amino acids of the HIV tat sequence;
tatHead, a recombinant protein containing amino acids 1-105
of L-plastin fused at the N terminus to 12 amino acids of
the HIV tat sequence;
tatLPL, a synthetic peptide
based on amino acids 2-19 of L-plastin fused at the N
terminus to 12 amino acids of the HIV tat sequence;
TPLtat, a synthetic peptide based on amino acids 2-19 of
T-plastin followed by 12 amino acids of the HIV tat
sequence;
Vn, vitronectin;
FACS, fluorescence flow cytometry;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody;
HBSS, Hanks'
balanced salt solution;
RT, room temperature;
HIV, human
immunodeficiency virus.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Laudanna, C.,
Mochly-Rosen, D.,
Liron, T.,
Constantin, G.,
and Butcher, E. C.
(1998)
J. Biol. Chem.
273,
30306-30315 |
2. | Shimizu, Y., Mobley, J. L., Finkelstein, L. D., and Chan, A. S. H. (1995) J. Cell Biol. 131, 1867-1880[Abstract] |
3. |
Pacifici, R.,
Roman, J.,
Kimble, R.,
Civitelli, R.,
Brownfield, C. M.,
and Bizzarri, C.
(1994)
J. Immunol.
153,
2222-2233 |
4. |
Reedquist, K. A.,
Ross, E.,
Koop, E. A.,
Wolthuis, R. M. F.,
Zwartkruis, F. J. T.,
van Kooyk, Y.,
Salmon, M.,
Buckley, C. D.,
and Bos, J. L.
(2000)
J. Cell Biol.
148,
1151-1158 |
5. |
Katagiri, K.,
Hattori, M.,
Minato, N.,
Irie, S.,
Takatsu, K.,
and Kinashi, T.
(2000)
Mol. Cell. Biol.
20,
1956-1969 |
6. | Caron, E., and Hall, A. (1998) Science 272, 1717-1721[CrossRef] |
7. |
Jones, S. L.,
Wang, J.,
Turck, C. W.,
and Brown, E. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9331-9336 |
8. |
Blystone, S. D.,
Williams, M. P.,
Slater, S. E.,
and Brown, E. J.
(1997)
J. Biol. Chem.
272,
28757-28761 |
9. |
Blystone, S. D.,
Lindberg, F. P.,
Williams, M. P.,
McHugh, K. P.,
and Brown, E. J.
(1996)
J. Biol. Chem.
271,
31458-31462 |
10. |
Kucik, D. F.,
Dustin, M. L.,
Miller, J. M.,
and Brown, E. J.
(1997)
J. Clin. Invest.
97,
2139-2144 |
11. | Lub, M., van Kooyk, Y., van Vliet, S., and Figdor, C. (1997) Mol. Biol. Cell 8, 341-351[Abstract] |
12. |
Stewart, M. P.,
McDowall, A.,
and Hogg, N.
(1998)
J. Cell Biol.
140,
699-707 |
13. | Gresham, H. D., Goodwin, J. L., Anderson, D. C., and Brown, E. J. (1989) J. Cell Biol. 108, 1935-1943[Abstract] |
14. | Blystone, S. D., Graham, I. L., Lindberg, F. P., and Brown, E. J. (1994) J. Cell Biol. 127, 1129-1137[Abstract] |
15. | Rosales, C., Jones, S. L., McCourt, D., and Brown, E. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3534-3538[Abstract] |
16. |
Ezhevsky, S. A.,
Nagahara, H.,
Vocero-Akbani, A.,
Gius, D. R.,
Wei, M.,
and Dowdy, S. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10699-10704 |
17. | Blystone, S. D., Lindberg, F. P., LaFlamme, S. E., and Brown, E. J. (1995) J. Cell Biol. 130, 745-754[Abstract] |
18. | Lin, C. S., Aebersold, R. H., and Leavitt, J. (1990) Mol. Cell. Biol. 10, 1818-1821[Medline] [Order article via Infotrieve] |
19. |
Vives, E.,
Brodin, P.,
and Lebleu, B.
(1997)
J. Biol. Chem.
272,
16010-16017 |
20. |
Jenkins, A. L.,
Nannizzi-Alaimo, L.,
Silver, D.,
Sellers, J. R.,
Ginsberg, M. H.,
Law, D. A.,
and Phillips, D. R.
(1998)
J. Biol. Chem.
273,
13878-13885 |
21. | Law, D. A., DeGuzman, F. R., Heiser, P., Ministri-Madrid, K., Killeen, N., and Phillips, D. R. (1999) Nature 401, 808-811[CrossRef][Medline] [Order article via Infotrieve] |
22. | Ginsberg, M. H. (1995) Biochem. Soc. Trans. 23, 439-446[Medline] [Order article via Infotrieve] |
23. |
Pampori, N.,
Hato, T.,
Stupack, D. G.,
Aidoudi, S.,
Cheresh, D. A.,
Nemerow, G. R.,
and Shattil, S. J.
(1999)
J. Biol. Chem.
274,
21609-21616 |
24. | Filardo, E. J., Brooks, P. C., Deming, S. L., Damsky, C., and Cheresh, D. A. (1995) J. Cell Biol. 130, 441-450[Abstract] |
25. |
Orlando, R. A.,
and Cheresh, D. A.
(1991)
J. Biol. Chem.
266,
19543-19550 |
26. | Faull, R. J., Kovach, N. L., Harlan, J. M., and Ginsberg, M. H. (1994) J. Exp. Med. 179, 1307-1316[Abstract] |
27. | Lub, M., van Vliet, S., Oomen, S. P. M. A., Pieters, R. A., Robinson, M., Figdor, C. G., and van Kooyk, Y. (1997) Mol. Biol. Cell 8, 719-728[Abstract] |
28. |
van Kooyk, Y.,
van Vliet, S.,
and Figdor, C. G.
(1999)
J. Biol. Chem.
274,
26869-26877 |
29. |
Elemer, G. S.,
and Edgington, T. S.
(1994)
J. Biol. Chem.
269,
3159-3166 |
30. |
Bubb, M. R.,
Senderowicz, A. M. J.,
Sausville, E. A.,
Duncan, K. L. K.,
and Korn, E. D.
(1994)
J. Biol. Chem.
269,
14869-14871 |
31. | Lub, M., van Kooyk, Y., and Figdor, C. G. (1995) Immunol. Today 16, 479-483[CrossRef][Medline] [Order article via Infotrieve] |
32. | Shimizu, Y., Rose, D. M., and Ginsberg, M. H. (1999) Adv. Immunol. 72, 325-380[Medline] [Order article via Infotrieve] |
33. |
Jones, S. L.,
Knaus, U. G.,
Bokoch, G. M.,
and Brown, E. J.
(1998)
J. Biol. Chem.
273,
10556-10566 |
34. |
Jones, S. L.,
and Brown, E. J.
(1996)
J. Biol. Chem.
271,
14623-14630 |
35. |
Wang, J.,
and Brown, E. J.
(1999)
J. Biol. Chem.
274,
24349-24356 |
36. |
Correia, I.,
Chu, D.,
Chou, Y.-H.,
Goldman, R. D.,
and Matsudaira, P.
(1999)
J. Cell Biol.
146,
831-842 |
37. |
Sadhu, C.,
Masinovsky, B.,
and Staunton, D. E.
(1998)
J. Immunol.
160,
5622-5628 |
38. |
Calderwood, D. A.,
Shattil, S. J.,
and Ginsberg, M. H.
(2000)
J. Biol. Chem.
275,
22607-22610 |
39. |
Bennett, J. S.,
Zigmond, S.,
Vilaire, G.,
Cunningham, M. E.,
and Bednar, B.
(1999)
J. Biol. Chem.
274,
25301-25307 |
40. |
Zhou, X.,
and Li, J.
(2000)
J. Biol. Chem.
275,
20217-20222 |
41. | Painter, R. G., and Ginsberg, M. (1982) J. Cell. Biol. 92, 565-573[Abstract] |