* Department of Vascular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La
Jolla, California 92037; and § Becton-Dickinson Immunocytometry Systems, San Jose, California 95131
Platelet agonists increase the affinity state of
integrin IIb
3, a prerequisite for fibrinogen binding
and platelet aggregation. This process may be triggered
by a regulatory molecule(s) that binds to the integrin
cytoplasmic tails, causing a structural change in the receptor.
3-Endonexin is a novel 111-amino acid protein
that binds selectively to the
3 tail. Since
3-endonexin is present in platelets, we asked whether it can affect
IIb
3 function. When
3-endonexin was fused to green
fluorescent protein (GFP) and transfected into CHO
cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to
monitor
IIb
3 affinity state in transfected cells by flow
cytometry. Cells transfected with GFP and
IIb
3 bound
little or no PAC1. However, those transfected with
GFP/
3-endonexin and
IIb
3 bound PAC1 specifically
in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/
3-endonexin
did not affect levels of surface expression of
IIb
3 nor
did it modulate the affinity of an
IIb
3 mutant that is
defective in binding to
3-endonexin. Affinity modulation of
IIb
3 by GFP/
3-endonexin was inhibited by coexpression of either a monomeric
3 cytoplasmic tail
chimera or an activated form of H-Ras. These results
demonstrate that
3-endonexin can modulate the affinity state of
IIb
3 in a manner that is structurally specific
and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such
protein-protein interactions at the level of the cytoplasmic tails of
IIb
3.
Integrins are A good example of the pathophysiological significance
of rapid integrin regulation involves platelet Studies with intact and permeabilized platelets indicate
that specific intracellular mediators promote rapid increases or decreases in ligand binding to Using a yeast two-hybrid screening strategy, we recently
discovered a novel 111-amino acid polypeptide called Reagents
Mammalian expression vectors for green fluorescent protein (GFP)1
(pS65T-C1 and pEGFP-C1) were obtained from Clontech (Palo Alto, CA).
Monoclonal antibodies PAC1, A2A9, D57, anti-LIBS1, and anti-LIBS6 were obtained from ascites and purified as described (30). PAC1 was conjugated to phycoerythrin (PE) by first derivatizing it with N-succinimidyl
S-acetylthioacetate (Pierce Chemical Co., Rockford, IL). SH groups were
deprotected with hydroxylamine, and the antibody was then coupled to PE
that had been derivatized with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce Chemical Co.). PE:PAC1 conjugates (1:1
mol/mol) were isolated by sizing on a Superose 6 column (Pharmacia Fine
Chemicals, Piscataway, NJ). In one experiment, PAC1 IgM DNA Constructs
To express Plasmid DNA encoding the cytoskeletal protein, VASP, was a gift from
Ulrich Walter and Thomas Jarchau (25) (Medizinische Universitatsklinik,
Wurzburg, Germany). The coding sequence of VASP was amplified with
Pfu polymerase (Stratagene, La Jolla, CA) using a sense primer containing
an XhoI site and an antisense primer with an HindIII site. The digested
PCR fragment was subcloned into XhoI- and HindIII-cut pEGFP-C1 so
that VASP would be expressed in-frame at the carboxy terminus of GFP.
Plasmid DNA encoding FRNK, an autonomously expressed carboxy-terminal segment of pp125FAK, was a gift from Michael Schaller (University
of North Carolina, Chapel Hill, NC) (57). FRNK was amplified with Pfu
polymerase with a sense primer containing a BglII site and an antisense
primer containing an EcoRI site. The digested PCR fragment was subcloned into BglII- and EcoRI-cut pEGFP-C1 so that FRNK would be expressed in-frame at the carboxy terminus of GFP.
pCDM8 expression vectors encoding wild-type Transient Protein Expression in CHO Cells
cDNAs were transfected into CHO-K1 cells with Lipofectamine (GIBCO
BRL, Gaithersburg, MD). A total of 5 µg of plasmid DNA and 20 µl of
Lipofectamine solution was incubated for 10 min in 200 µl of DME and
then diluted with 3.8 ml of DME. Unless otherwise indicated, the amount
of DNA per transfection included 0.5 µg each of Evaluation of GFP/ Expression of GFP/ To study the binding of GFP/ To evaluate whether GFP/ Evaluation of 48 h after transfection, CHO cells were resuspended at 1-2 × 106 cells per
ml in Tyrode's buffer containing 2 mM CaCl2 and MgCl2 (49). Cells were
then incubated in the dark in 50-µl aliquots with 20 µg/ml PE-PAC1 for 30 min at room temperature. Some samples also contained an anti- After electronic compensation of the FL1, FL2, and FL3 fluorescence
channels, PE-PAC1 binding (FL2) was analyzed on the gated subset of
live cells (propidium iodide-negative, FL3 channel) that were positive for
GFP expression (FL1 channel). PAC1 binding was expressed as an "activation index" calculated from median fluorescence intensity measurements (49). The activation index is defined as 100 × (Fx-Fi)/(Fm-Fi),
where Fx is PAC1 fluorescence in the absence and Fi is PAC1 fluorescence in the presence of Ro 43-5054 or Integrilin. Fm is PAC1 fluorescence in the presence of anti-LIBS6.
Fibrinogen Binding Assay
Fibrinogen binding to GFP-positive cells was determined by flow cytometry using biotinylated anti-LIBS1, which recognizes a fibrinogen-sensitive
epitope on the CHO Cell Aggregation Assay
Fibrinogen-dependent aggregation of CHO cells was quantitated by flow
cytometry as described (16), with minor modifications. First, CHO cells
stably expressing Subcellular Localization of GFP/ 48 h after transfection, CHO cells were cultured on fibrinogen-coated coverslips for 2 h at 37°C, and then processed and analyzed by fluorescence
microscopy for expression of GFP, Expression of CHO cells provide a useful model system for characterizing the adhesive and signaling functions of ectopically expressed
Previous studies have shown that
48 h after cotransfection of CHO cells with expression
plasmids encoding Fig. 3 shows the results of a representative experiment.
PAC1 binding to CHO cells transfected with GFP/
This impression was confirmed by the series of experiments summarized in Fig. 4. Compared with cells expressing GFP, those expressing GFP/
Platelets containing Functional Consequences of Integrin Affinity
Modulation by To determine whether the changes in PAC1 binding induced by GFP/
When fibrinogen binds to activated
In the experiment shown in Fig. 6 B, it can be seen that
GFP/ Factors That Influence Integrin Activation
by Additional experiments were conducted to clarify the mechanism of action of GFP/ Next, GFP/
Affinity modulation of In platelets, heterotrimeric GTP-binding proteins have
been implicated in affinity modulation. On the other hand,
the role of the small GTPase, H-Ras, which is also present
in these cells, has not been examined. Recently, Hughes
and co-workers found that a constitutively active form of
H-Ras (G12V) acts as a general suppressor of integrin adhesive function in CHO cells (33). Similarly, we found that
the expression of H-Ras (G12V) inhibited the effects of
GFP/ Subcellular Localization of In order for
When CHO cells containing
These studies demonstrate that: (a) in CHO cells, expression of We interpret the effect of GFP/ The present results were obtained by expressing Additional studies will be required to determine how
cellular energy depletion or coexpression of activated
H-Ras inhibits affinity modulation by Suppression of integrin activation in CHO cells by activated H-Ras involves a Raf-1-initiated MAP kinase pathway and is transcription independent (33). In contrast, an
activated variant of R-Ras was recently implicated in the
promotion of integrin-mediated cell adhesion (75). Since
the reported opposite actions of activated R-Ras and H-Ras
affect both Based on the present results, we propose that the interaction of A complete understanding of the proximate events in
inside-out signaling will require identification of all relevant integrin-binding proteins and a more refined knowledge of integrin structure. Progress is beginning to be made
in both of these areas (11, 42, 55). Several proteins have
been described that bind directly to integrin cytoplasmic
tails, at least in vitro. These include structural proteins of
the cytoskeleton, such as F-actin (specific for the While not relevant to platelets, the localization of Focal adhesions are dynamic structures containing integrins, cytoskeletal elements, and signaling molecules that
form on the basal surfaces of many types of cells in culture
and in platelets during spreading on fibrinogen (47). These
macromolecular assemblies may function to optimize traction during cell motility and to promote information flow
from the extracellular matrix to the nucleus (5, 17, 18).
The lack of consistent and strong localization of These studies provide the first clues about the functions
of heterodimers and each subunit contains a relatively large extracellular domain, a membrane-spanning domain, and a 20-70-amino acid cytoplasmic tail. They function in cell adhesion and signaling
by interacting with extracellular matrix proteins or cellular
counter-receptors on the one hand, and with intracellular
proteins on the other (8, 34, 59). The adhesive function of
many integrins is subject to rapid regulation by two processes collectively referred to as "inside-out" signaling: (a)
a structural change intrinsic to the heterodimer, and (b) clustering of heterodimers within the plane of the plasma
membrane. The former modulates the affinity of the
ligand-receptor interaction and thus is often referred to as
"affinity modulation." The latter increases the valency
and, therefore, the avidity of the interaction. These two
types of regulation are not mutually exclusive, and their
relative contributions probably vary with the integrin and the cell type (12, 20, 62, 71).
IIb
3. Circulating platelets ordinarily do not interact with each other
or with the blood vessel wall. However, when the vessel is
damaged by trauma or disease, platelets become activated
and
IIb
3 is converted within seconds into a functional receptor for several Arg-Gly-Asp-containing ligands, including fibrinogen and von Willebrand factor. Since ligand
binding is required for platelet aggregation, inside-out signaling is a prerequisite for primary hemostasis and for formation of occlusive platelet thrombi in vascular diseases
(9, 27). Affinity modulation is thought to be responsible
for the initial, reversible phase of fibrinogen binding to
platelets, while integrin clustering may be involved in stabilizing the interaction (14, 52).
IIb
3. Excitatory
platelet agonists, such as thrombin, increase ligand binding
by a process that involves heterotrimeric G proteins and
protein and lipid kinases (38, 61, 69, 74). On the other
hand, substances such as prostacyclin and nitric oxide,
which stimulate protein kinase A and protein kinase G, respectively, inhibit or reverse ligand binding (22, 28). In addition to intracellular mediators, the cytoplasmic tails of
IIb
3 appear to participate in the regulation of fibrinogen binding. Platelets from patients with variant forms of
Glanzmann thrombasthenia due to a deletion or mutation
in the
3 cytoplasmic tail fail to aggregate in response to
agonists despite near normal levels of
IIb
3 (6; Wang, R.,
D.R. Ambruso, and P.J. Newman. 1994. Blood. 84:244a).
However, it is not clear how intracellular signals affect the
cytoplasmic tails of
IIb
3 or how changes at the level of
these tails regulate ligand binding. One hypothesis is that
specific intracellular proteins bind to the tails and promote
a structural change that is propagated across the plasma membrane to the extracellular face of the receptor. Accordingly, recent efforts have focused on identifying proteins that interact with integrin cytoplasmic tails (11).
3endonexin, which is capable of binding to the cytoplasmic
tail of the
3 integrin subunit, both in yeast and in vitro
(63). However, it fails to bind to other integrin tails, including those of
1,
2, and
IIb. Since
3-endonexin is expressed in platelets, the present studies were carried out to
determine whether this protein can modulate the ligandbinding function of
IIb
3. Using a CHO cell model system
to transiently express
IIb
3 and
3-endonexin, we now report that this protein can increase the affinity state and the
adhesive function of
IIb
3. Moreover, these effects are structurally specific and subject to metabolic regulation.
Materials and Methods
was first reduced to the 185-kD monomer at pH 8.6 with 20 mM cysteine before conjugating to PE (46).
3-endonexin as a protein fused to the carboxy terminus of
GFP,
3-endonexin cDNA was excised from a yeast expression vector
with XbaI and BamHI (63), and the recessed 3
termini were filled in using Klenow (Boehringer Mannheim Biochemicals, Indianapolis, IN). The
GFP vectors, pS65T-C1 and pEGFP-C1, were cut with XhoI, blunt-ended
with Klenow, and ligated to
3-endonexin with T4 DNA ligase (Boehringer Mannheim Biochemicals). After transformation of DH5
, clones in
the correct orientation were selected by PCR using a sense primer in GFP
and an antisense primer in
3-endonexin.
IIb,
3, a mutant form
of
3 containing a single amino acid substitution (S752P), and H-Ras
(G12V) have been described (33, 49). Tac-
3 and Tac-
5 chimeras were in
the vector, CMV-IL2R (7). All expression plasmids were amplified in Escherichia coli and purified (Plasmid Maxi Kit; Qiagen, Inc., Chatsworth,
CA). Before use in transfection experiments, each plasmid was sequenced
in the Scripps Research Institute DNA Core Facility to confirm the authenticity of the coding sequences.
IIb and
3 and varying
amounts of the GFP plasmids to obtain equivalent degrees of GFP expression (e.g., 4 µg of pS65T, 4 µg of pS65T/
3-endonexin, 0.02 µg of pEGFP,
0.2 µg of pEGFP/
3-endonexin, or 0.05 µg of pEGFP/VASP). When necessary, an empty vector (pcDNA3; Invitrogen, San Diego, CA) was included to equalize the amount of DNA transfected. In some experiments,
2 µg of the Tac-
3, Tac-
5, or H-Ras (G12V) plasmid was cotransfected
along with pEGFP/
3-endonexin and the plasmids for
IIb and
3. DNA/
Lipofectamine mixtures were added to CHO cells at 30-50% confluence
in a 100-mm tissue-culture plate. 6 h later, the medium was changed to
DME containing 10% FBS, 1% nonessential amino acids, 2 mM l-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin. 48 h after transfection, the
cells were evaluated biochemically and by flow cytometry.
3-Endonexin Expression in
CHO Cells
3-endonexin fusion proteins was confirmed by immunoblotting. 48 h after transfection, the cells were lysed for 30 min at 4°C in
a lysis buffer containing 1% Triton X-100, 0.9% NaCl, 1 mM CaCl2, 50 mM
Tris, pH 7.2, and protease inhibitors (100 U/ml aprotinin, 0.5 mM leupeptin, 4 mM Pefabloc) (63). After clarification of the lysate in a microfuge,
protein concentration was determined with a bicinchoninic acid reagent
(BCA; Pierce Chemical Co.). 30 µg of each sample was then electrophoresed
in 14% SDS-polyacrylamide gels under reducing conditions. After transfer to nitrocellulose, immunoblotting was performed with a rabbit polyclonal
antibody reactive with GFP (Clontech) or rabbit antibodies reactive with
3-endonexin (63). After the addition of affinity-purified, HRP-conjugated goat anti-rabbit IgG, the blots were developed for 0.1-1 min using the
enhanced chemiluminescence reaction (ECL; Amersham Corp., Arlington Heights, IL).
3-endonexin to the
3 integrin cytoplasmic tail, the 47-amino acid
3 tail was expressed in bacteria with a (His)6
tag at its amino terminus (pET His Tag System; Novagen, Inc., Madison,
WI), and then immobilized on a nickel-agarose matrix. Transiently transfected CHO cells expressing equivalent amounts of GFP/
3-endonexin or
GFP were lysed in 0.4 ml of the Triton X-100 lysis buffer. After clarification, 0.35 ml was diluted with an equal volume of lysis buffer containing
no Triton, and each sample was batch-incubated with 0.1 ml packed volume of the His-
3 tail affinity matrix for 12 h at 4°C while shaking. After
washing the matrices five times with 10 vol of lysis buffer, bound proteins were eluted into 0.7 ml of lysis buffer by addition of 1 M imidazole. Samples were electrophoresed on 12% SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose. Immunoblotting was performed with the polyclonal anti-GFP antibody.
3-endonexin could be specifically coimmunoprecipitated with the
3 integrin subunit from CHO cells, immunoprecipitation studies were carried out in the Trition X-100 lysis buffer essentially as described (30). The
3 integrin subunit was immunoprecipitated
from 2-mg aliquots of cell lysate using 10 µg/ml of the murine mAb, SSA6,
and protein A-Sepharose (1). Control immunoprecipitations were carried
out with affinity-purified mouse IgG (Zymed Laboratories, Inc., South
San Francisco, CA) and with an isotype-matched mAb against von Willebrand factor, RG 7 (a gift from Zaverio Ruggeri; Scripps Research Institute, La Jolla, CA). Samples were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions and subsequent Western blots were probed with the polyclonal anti-GFP antibody. To demonstrate equivalent recovery of the
3 integrin subunit in the immunoprecipitates, blots
were stripped and reprobed with Ab 8035, a rabbit polyclonal antibody
specific for
3.
IIb
3 Affinity State
3 antibody (2% anti-LIBS6 ascites) that stabilizes
IIb
3 in a high affinity state
(30). Others contained an
IIb
3-selective inhibitor of ligand binding (either 2 µM Ro 43-5054 or 10 µM Integrilin) (2, 56). Samples were then diluted with 0.5 ml Tyrode's buffer containing 10 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO) and analyzed on a FACS®can or
FACS®Calibur flow cytometer (Becton Dickinson, San Jose, CA). In one set of experiments, the cells were preincubated for 30 min at room temperature with 4 mg/ml of 2-deoxy-d-glucose (Sigma Chemical Co.) and
0.2% sodium azide before incubation with PE-PAC1.
3 subunit (19). Cells were prepared as for the PAC1 binding studies and incubated for 30 min in the dark at room temperature with
fibrinogen (250 µg/ml; Enzyme Research Laboratories, South Bend, IN),
biotin-LIBS1 (20 µg/ml), and phycoerythrin-streptavidin (4% final dilution; Molecular Probes, Inc., Eugene, OR). To calculate the activation index, some aliquots were incubated with anti-LIBS6 to induce maximal fibrinogen binding, while others were incubated with the function-blocking anti-
IIb
3
ntibody, A2A9, to determine nonspecific fibrinogen binding.
Cells were then diluted with Tyrode's buffer containing 10 µg/ml propidium iodide, and analyzed by flow cytometry.
IIb
3 (49) were labeled with a red fluorescent tracer, hydroxyethidine (Polysciences Inc., Junction City, OR). Then 250 µl of these
cells (4 × 106/ml) were added to siliconized glass cuvettes containing 250 µl of cells (2 × 106/ml) that had been transfected with GFP/
3-endonexin (or
GFP) and
IIb
3. After addition of 300 µg/ml fibrinogen, the cells were
stirred with a magnetic stir bar at 1,000 rpm for 20 min at room temperature. In some cases, the incubations with fibrinogen were also carried out
in the presence of 20 µg/ml A2A9 or 10 µM Integrilin to inhibit fibrinogen
binding. Incubations were stopped by addition of 0.25% formaldehyde,
and the samples were kept on ice for 30 min before flow cytometric detection of mixed red-green cellular aggregates.
3-Endonexin
IIb, and
3 as described (32). HMEC-1
human endothelial cells, which express
V
3, were similarly cultured on fibrinogen-coated coverslips, and then microinjected with plasmid DNA
(0.5 µg/µl) encoding various GFP proteins (45). After 4 h at 37°C, the cells
were processed and analyzed by fluorescence microscopy for expression
of GFP,
V, and
3.
Results
3-Endonexin in CHO Cells
IIb
3 (43, 49, 50). Therefore,
3-endonexin was
transiently coexpressed with
IIb
3 in these cells to study
its effects on the ligand-binding function of this integrin.
3-Endonexin cDNA was fused in-frame to the 3
end of
two different versions of GFP in a mammalian expression
plasmid. One form (S65T) is red-shifted and the other (EGFP) is both red-shifted and codon-optimized for mammalian expression. 48 h after transfection, expression of
recombinant proteins was assessed by Western blotting of
cell lysates. GFP/
3-endonexin was detectable using an
anti-GFP antibody, and the codon-optimized plasmid provided higher levels of protein expression for a given amount of DNA transfected (Fig. 1). Subsequently, therefore, the amount of each plasmid used was adjusted to obtain roughly equivalent amounts of GFP/
3-endonexin expression, and the plasmids were used interchangeably in
the following experiments. GFP/
3-endonexin was also detectable with polyclonal antibodies raised against either
recombinant human
3-endonexin or a synthetic peptide
consisting of the carboxy-terminal 17 residues of the protein (Fig. 1). No hamster protein cross-reactive with these
antibodies was detected in CHO cells. These results indicate that full-length GFP/
3-endonexin can be expressed
in CHO cells.
Fig. 1.
Expression of GFP
and GFP/3-endonexin in
CHO cells. CHO cells were
either mock transfected or
transfected with 4 µg of the indicated plasmids as described in Materials and
Methods. 48 h later, the cells
were lysed in a buffer containing Triton X-100, and 30 µg
of each sample was probed
on Western blots with the indicated polyclonal antibodies. (Upper arrow) Position
of GFP/
3-endonexin; (lower
arrow) position of GFP. The
band in the "mock" lane
stained with anti-
3-endonexin
carboxy-terminal antibody
migrated more slowly than
GFP/
3-endonexin and was
nonspecific.
[View Larger Version of this Image (39K GIF file)]
3-endonexin binds in
vitro to the
3 integrin subunit from detergent-solubilized
platelets and CHO cells (13, 63). To determine if
3-endonexin retains its ability to bind to the
3 integrin subunit
after its fusion to GFP, lysates from CHO cells expressing
GFP/
3-endonexin were passed over an affinity matrix
containing the bacterially expressed
3 cytoplasmic tail.
GFP/
3-endonexin, but not GFP, was specifically retained
by and eluted from this affinity matrix (Fig. 2 A). Moreover, GFP/
3-endonexin and the
3 integrin subunit could
be specifically coprecipitated from CHO cell lysates (Fig. 2
B). Finally, CHO cells containing GFP/
3-endonexin were
strongly fluorescent in the FL1 channel of a flow cytometer
(see below). Thus, fusion of
3-endonexin to the carboxy
terminus of GFP abrogates neither the integrin-binding function of
3-endonexin nor the fluorescent properties of GFP.
Fig. 2.
Interactions of GFP/3-endonexin with the
3 integrin
cytoplasmic tail. (A) CHO cells were transfected with 4 µg of
pS65T-GFP/
3-endonexin or pS65T-GFP. 48 h later, the cells
were lysed in a Triton X-100-containing buffer, and equal volumes of the lysates were batch-incubated with a His-
3 cytoplasmic tail affinity matrix. After extensive washing, proteins were
eluted with 1 M imidazole, and equal volumes of the indicated
fractions were transferred to nitrocellulose and probed with a
polyclonal antibody to GFP. (Upper arrow) Position of GFP/
3endonexin; (lower arrow) position of GFP. Previous studies of
this kind have already documented that
3-endonexin interacts with the
3 but not the
1 cytoplasmic tail (63). (B) CHO cells expressing both
IIb
3 and GFP/
3-endonexin (+ GFP/
3-EN) or
IIb
3 alone (
GFP/
3-EN) were lysed in Triton X-100 lysis
buffer, and clarified lysates were immunoprecipitated either with
a monoclonal anti-
3 antibody (SSA6), an isotype control antibody (RG 7), or mouse IgG (mIgG). Western blotting was performed initially with a polyclonal antibody to GFP and blots were
then reprobed with a polyclonal anti-
3 antibody. The first two
lanes represent 30 µg of lysate. Lys, lysate; FT, flow-through; W1,
first wash; WL, last wash; E, eluate.
[View Larger Version of this Image (67K GIF file)]
3-Endonexin Increases the Affinity State of
Integrin
IIb
3
IIb
3
nd GFP/
3-endonexin, the affinity state of
IIb
3 was determined by flow cytometry using a PE conjugate of the fibrinogen-mimetic mAb, PAC1.
Since transfection efficiencies varied from 15-45%, data
acquisition included only live cells positive for GFP fluorescence. About 75% of these cells were also positive for
IIb
3, as assessed by staining with an antibody specific for
the
IIb
3 complex (D57). To standardize the results of
PAC1 binding from experiment to experiment, binding
was expressed as an activation index calculated from median fluorescence values (49). To obtain this index, nonspecific PAC1 binding was determined in the presence of a
selective inhibitor of ligand binding to
IIb
3 (either Ro
43-5054 or Integrilin). Maximal PAC1 binding was determined in the presence of an activating anti-
3 antibody (anti-LIBS6) (49). After subtraction of nonspecific binding, this maximal binding was assigned an activation index
of 100. Consequently, the activation index for PAC1 binding can range from 0 to 100.
3-
endonexin and
IIb
3 exhibited a relatively high activation
index of 44 (Fig. 3 A). In contrast, PAC1 binding to cells
transfected with GFP and
IIb
3 exhibited a lower activation index of 18 (Fig. 3 D), a value similar to that observed
previously for
IIb
3 transfectants in the absence of GFP
(31, 49). Thus, expression of
3-endonexin appears to activate
IIb
3 and increase its affinity for a cognate ligand.
Fig. 3.
Effect of GFP/3endonexin on PAC1 binding
to
IIb
3 in CHO cells. CHO
cells were transfected with
IIb
3 and either GFP/
3-endonexin (contour plots A, B, and
C) or GFP (plots D, E, and
F). 48 h later, binding of PEPAC1 (x-axis) to green fluorescent-positive cells (y-axis)
was analyzed by flow cytometry. Each contour plot represents 10,000 cells. Plots B
and E represent nonspecific
PAC1 binding determined in
the presence of Ro 43-5054. Plots C and F represent maximal PAC1 binding in the
presence of an activating
anti-
3 antibody, anti-LIBS6.
Note that there was more
PAC1 binding to GFP/
3-
endonexin cells (plot A) than
to GFP cells (plot D), a conclusion supported by the calculated activation indices (plot A, activation index = 44%;
plot D, activation index = 18%).
[View Larger Version of this Image (63K GIF file)]
3-endonexin consistently
showed an increase in PAC1 binding, and the difference
was statistically significant (P < 0.03). In contrast, PAC1
binding to cells expressing an unrelated GFP fusion protein, GFP/VASP, was not increased despite similar levels
of recombinant protein expression. VASP was chosen because it is present in platelets and localizes to integrin-rich
focal adhesions (25). Although not shown, the PAC1 activation index for GFP/
3-endonexin cells (44 ± 5) began to
approach that for cells expressing a constitutively active
form of
IIb
3 (
IIb
6A
3; 61 ± 6; n = 3) (49). Expression
of GFP/
3-endonexin or the other GFP proteins did not
affect levels of surface expression of
IIb
3, as determined
by the binding of antibody D57. All together, these results
indicate that expression of
3-endonexin can increase the
affinity state of
IIb
3.
Fig. 4.
GFP/3-endonexin causes activation of
IIb
3 in a structurally specific manner. PAC1 binding to transfected CHO cells
was analyzed by flow cytometry as described in Materials and
Methods and in the legend to Fig. 3. (Left) CHO cells were
cotransfected with wild-type
IIb
3 and either GFP (open bar),
GFP/
3-endonexin (black bar), or GFP/VASP (shaded bar). PAC1
binding was expressed as an activation index, and the data represent means ± SEM for 15 experiments with GFP and GFP/
3-
endonexin and three experiments with GFP/VASP. (Right) CHO
cells were cotransfected with the signaling-deficient integrin mutant,
IIb
3 (S752P), and either GFP (open bar) or GFP/
3-
endonexin (black bar). Data represent the means ± SEM of
three experiments.
[View Larger Version of this Image (24K GIF file)]
IIb
3 with a specific point mutation
in the
3 cytoplasmic tail at position 752 (S
P) fail to bind fibrinogen or aggregate (6). Furthermore, the binding of
3-endonexin to this mutant
3 integrin subunit is markedly reduced (63). When
IIb
3 (S752P) was coexpressed
with GFP/
3-endonexin in CHO cells, no increase in
PAC1 binding was observed (Fig. 4). This suggests that
3endonexin modulates the affinity state of
IIb
3 in a structurally specific manner.
3-Endonexin
3-endonexin translate into increased binding of a physiological ligand, the binding of fibrinogen to
CHO cells was studied by flow cytometry. Bound fibrinogen was detected with a biotinylated mAb (anti-LIBS1)
specific for a fibrinogen-sensitive epitope on the
3 subunit (19). Specific fibrinogen binding was defined as that
inhibitable by a function-blocking anti-
IIb
3 antibody, A2A9. CHO cells expressing wild-type
IIb
3 bind little or
no fibrinogen at a saturating concentration of ligand (250 µg/ml) (48). The same was true for cells expressing GFP
and
IIb
3. However, those expressing GFP/
3-endonexin
and
IIb
3 bound increased amounts of fibrinogen (Fig. 5).
Similar results were obtained when fibrinogen binding
was measured directly with biotinylated fibrinogen (not
shown). Thus, expression of GFP/
3-endonexin can lead
to an increase in fibrinogen binding to
IIb
3.
Fig. 5.
GFP/3-endonexin induces fibrinogen binding to
IIb
3.
48 h after transfection of CHO cells with
IIb
3 and either GFP
(open bar) or GFP/
3-endonexin (black bar), the cells were resuspended in Ca2+- and Mg2+-containing Tyrode's buffer and incubated for 30 min at room temperature in the presence of 250 µg/ml
fibrinogen. Then fibrinogen binding to the transfected cells was
assessed by flow cytometry using phycoerythrin-streptavidin and
a biotinylated anti-
3 antibody (anti-LIBS 1) sensitive to the
presence of bound fibrinogen. Specific binding was defined as
that blocked by antibody A2A9 (20 µg/ml), and it was expressed
as an activation index. Data represent the means ± SEM of two
experiments, each performed in triplicate.
[View Larger Version of this Image (19K GIF file)]
IIb
3 on the surface
of platelets or CHO cells under stirring conditions, the
cells aggregate (4, 16). To determine whether GFP/
3-endonexin can trigger this aggregation response, CHO cells expressing GFP/
3-endonexin and
IIb
3 were mixed with
cells containing
IIb
3 and a red fluorescent tracer, hydroxyethidine. After stirring for 20 min in the presence of
300 µg/ml fibrinogen, the formation of mixed, red-green
cellular aggregates was monitored by flow cytometry. The
rationale for this experimental design is that if fibrinogen
first becomes bound to activated
IIb
3 on the GFP/
3-
endonexin cells, this cell-bound fibrinogen should then be
able to recruit the red fluorescent cells into mixed aggregates, even though the
IIb
3 on the red fluorescent cells is
initially in a low affinity state (Fig. 6 A) (16).
Fig. 6.
GFP/3-endonexin
causes fibrinogen-dependent
aggregation of CHO cells. A
illustrates the rationale for
this aggregation protocol, which is discussed in the text.
In B, as detailed in Materials and Methods, CHO cells that
had been transfected with
IIb
3 and GFP (left) or with
IIb
3 and GFP/
3-endonexin
(center and right) were mixed
with CHO cells that had
been stably transfected with
IIb
3, and then stained with
the red fluorescent dye, hydroxyethidine. After stirring
for 20 min in the presence of
300 µg/ml fibrinogen, the
cells were fixed with formaldehyde, and 10,000 propidium iodide-negative and
GFP-positive cells (y-axis)
were analyzed by flow cytometry. (B, right) The incubation with fibrinogen was
carried out in the presence of
20 µg/ml antibody A2A9 to
inhibit fibrinogen binding. Mixed red-green cellular aggregates appear to the right
of the vertical line on the
FL2 axis.
[View Larger Version of this Image (51K GIF file)]
3-endonexin promoted the formation of mixed aggregates (center), an effect that could be inhibited by the
function-blocking antibody, A2A9 (right), or the cyclic
peptide, Integrilin (not shown). In three such experiments,
an average of 7.0 ± 1.6% of the cells expressing GFP/
3endonexin were engaged in red-green aggregates, compared with 3.5 ± 1.9% of cells expressing GFP. While this
effect may seem small, it was statistically significant (P < 0.01). Moreover, it should be emphasized that the extent
of mixed aggregation was limited by the required use of
red fluorescent cells expressing low affinity
IIb
3. These
results indicate that affinity modulation of
IIb
3 by
3-
endonexin can cause fibrinogen-dependent cell aggregation.
3-Endonexin
3-endonexin. Although PAC1 is a
multimeric IgM antibody, GFP/
3-endonexin was also found
to increase the binding of a monomeric form of PAC1 obtained by enzyme digestion. In addition, PAC1 binding because of GFP/
3-endonexin was not affected by preincubation of the cells with 10 µM cytochalasin D, an inhibitor
of actin polymerization (data not shown). Since actin polymerization promotes integrin clustering (12, 71), which
would be expected to influence preferentially the binding of multivalent ligands, these results suggest that GFP/
3endonexin is primarily a modulator of
IIb
3 affinity rather
than avidity.
3-endonexin was studied in CHO cells expressing both
IIb
3 and a
3 cytoplasmic tail chimera containing the extracellular and transmembrane domains of
the Tac subunit of the IL-2 receptor. We reasoned that the
chimera, which does not dimerize with
IIb (7, 40), would
compete intracellularly with
IIb
3 for
3-endonexin. If so,
it should prevent
3-endonexin from binding to and modulating the function of
IIb
3. Indeed, expression of the Tac/
3 chimera prevented GFP/
3-endonexin from activating
IIb
3 (Fig. 7). In contrast, a Tac chimera containing the
structurally unrelated
5 cytoplasmic tail exhibited no such
effect. This is consistent with the idea that
3-endonexin
modulates integrin affinity through an interaction with the
3 cytoplasmic tail.
Fig. 7.
Factors influencing integrin activation by GFP/3-endonexin. CHO cells were transfected with
IIb
3
nd either GFP or
GFP/
3-endonexin. As detailed in Materials and Methods, some
transfectants were subjected to additional treatments before determination of PAC1 binding. These included (a) cotransfection
with a Tac/
3 tail chimera; (b) cotransfection with a Tac/
5 tail chimera; (c) cotransfection with a constitutively active form of H-Ras
(G12V); or (d) energy depletion by preincubation for 30 min with
0.2% sodium azide and 4 mg/ml 2-deoxy-d-glucose. Data are the
means ± SEM of three experiments.
[View Larger Version of this Image (17K GIF file)]
IIb
3 by platelet agonists requires metabolic energy (68). In CHO cells, PAC1 binding
induced by GFP/
3-endonexin was not observed if the
cells were pretreated with sodium azide and 2-deoxy-dglucose to inhibit oxidative metabolism (Fig. 7). In this respect, the effect of
3-endonexin in the CHO cell system is
similar to that of excitatory agonists in the platelet system.
3-endonexin on PAC1 binding (Fig. 7). Taken together with the energy depletion experiments, this indicates that the function of GFP/
3-endonexin is subject to
metabolic regulation.
3-Endonexin
3-endonexin to directly influence the function of the
3 integrin cytoplasmic tail, these proteins must
be located together in the cell. To address this question,
HMEC-1 human endothelial cells, which attach and spread
on immobilized fibrinogen through
V
3, were microinjected with DNA encoding GFP/
3-endonexin or GFP. 4 h
later, specific green fluorescence could be observed diffusely in the cytoplasm and the nucleus. The degree of
nuclear fluorescence was much greater in the case of GFP/
3-endonexin (Fig. 8). An identical pattern of GFP/
3-
endonexin localization was observed in CHO cells that
had been allowed to spread on fibrinogen through
IIb
3
(not shown). These results are consistent with a generalized cytoplasmic distribution of GFP/
3-endonexin and with a nuclear localization that may be promoted by a consensus nuclear localization signal in
3-endonexin (see Discussion).
Fig. 8.
Expression of GFP, GFP/3-endonexin, and GFP/VASP
in HMEC-1 cells. Cells were allowed to spread for 2 h on fibrinogencoated coverslips, and then were microinjected with the indicated
expression plasmids. 4 h later, green fluorescence was visualized in a
fluorescence microscope using an FITC filter set. Uninjected cells
were not visible under these conditions. Bar, 10 µm.
[View Larger Version of this Image (45K GIF file)]
V
3 or
IIb
3 are allowed
to spread on fibrinogen, the
3 cytoplasmic tail is necessary and sufficient for localization of the
3 integrins to focal adhesions (40, 72). Immunostaining of HMEC-1 cells
revealed that
V and
3 were localized both in a diffuse
pattern consistent with a generalized plasma membrane
distribution and in discrete foci characteristic of focal adhesions (Fig. 9). There was no strong or consistent localization of GFP/
3-endonexin to these focal adhesions, excluding the possibility that
3-endonexin might associate
tightly with the
3 cytoplasmic tail during cytoskeletal assembly. However, some weak staining of
3-endonexin in
focal adhesions was observed, suggesting that a weaker or
more transient association may occur (Fig. 9, arrowheads).
No localization of GFP to focal adhesions was detected.
As a positive control, GFP was fused to FRNK, an autonomously expressed segment of pp125FAK that contains a focal adhesion targeting sequence (57). After microinjection, GFP/FRNK significantly localized to focal adhesions, demonstrating that a GFP fusion protein can target to
these structures under the experimental conditions used
here (Fig. 8). Thus, GFP/
3-endonexin is not strongly or
consistently concentrated in focal adhesions.
Fig. 9.
Subcellular localization of GFP/3-endonexin and
V
3 in HMEC-1 cells. Cells were allowed to spread for 2 h on fibrinogen and then were microinjected with GFP/
3-endonexin. 4 h later, the cells were fixed, stained with rhodamine-labeled antibodies to
V or
3, and then examined by microscopy for GFP fluorescence (top) and rhodamine fluorescence (bottom). Two different cells are shown,
one in the lefthand panels, the other in the righthand panels. Arrowheads denote the occasional coalignment of GFP/
3-endonexin and
V
3 in focal adhesions. Bar, 5 µm.
[View Larger Version of this Image (100K GIF file)]
Discussion
3-endonexin as a fusion protein with GFP is associated with an increase in the affinity state of integrin
IIb
3.
This affinity change enables the cells to undergo fibrinogen-dependent aggregation. (b) Affinity modulation of
IIb
3 by GFP/
3-endonexin is structurally specific in that
other GFP proteins (GFP; GFP/VASP) do not promote this response. Furthermore, GFP/
3-endonexin does not
affect the function of
IIb
3 (S752P), a mutant integrin that
is defective in binding to
3-endonexin and in integrin signaling. (c) Affinity modulation by
3-endonexin may be
the consequence of its direct interaction with
IIb
3 since it
is prevented by coexpression of a Tac-
3 cytoplasmic tail
chimera. (d) The effect of
3-endonexin on
IIb
3 may be
subject to metabolic regulation since it is not observed if
cellular energy is depleted or if the cells are cotransfected with an activated form of H-Ras. (e) GFP/
3-endonexin is
found in both the nuclear and cytoplasmic compartments
after CHO cells or HMEC-1 cells have spread on a fibrinogen matrix via a
3 integrin. Taken together, these results
indicate that
3-endonexin may play a significant role in
cell adhesion and signaling through integrin
IIb
3.
3-endonexin on PAC1
and fibrinogen binding to CHO cells to represent an example of inside-out signaling in which
3-endonexin increases the affinity of individual
IIb
3 heterodimers for
specific ligands. An alternative interpretation that
3-endonexin triggers oligomerization of
IIb
3 complexes and
therefore increases receptor avidity cannot be excluded,
but it seems less likely for several reasons. First, changes
within
IIb
3 that enable the binding of RGD-containing
macromolecular ligands have been detected with both a
monovalent Fab fragment of PAC1 as well as with the native, multivalent antibody (1). This indicates that regulated ligand binding to
IIb
3 is not absolutely dependent
on the valency of the ligand or, presumably, the receptor.
Second, the effect of GFP/
3-endonexin on
IIb
3 was detected using either native PAC1 or a monomeric fragment of the antibody. Third, agonist-induced clustering of
2 integrins in leukocytes and possibly
IIb
3 in platelets is facilitated by polymerization of F-actin (12, 14, 71). However, cytochalasin D, an inhibitor of actin polymerization,
had no effect on PAC1 binding induced by GFP/
3-endonexin. While it is not possible to quantitate precisely the
relative contributions of affinity and avidity regulation,
based on the above considerations, we speculate that
3-endonexin can regulate reversible fibrinogen binding through
affinity modulation. Other factors, including actin polymerization and cytoskeletal reorganization, may enhance cell adhesion by promoting receptor clustering and irreversible ligand binding. Consistent with this idea, cytochalasin D has been reported to inhibit primarily the later, irreversible phase of fibrinogen and PAC1 binding to
platelets and CHO cells (14, 53, 54).
3-
endonexin ectopically in CHO cells. Therefore, it is possible that the function of the endogenous protein in platelets
or other cells differs quantitatively or qualitatively from
that described here. Despite this caveat, a number of observations indicate that affinity modulation may result directly from the interaction of
3-endonexin with the cytoplasmic tail of the
3 integrin subunit. A mutational analysis of the
3 tail has shown that membrane-distal residues near the carboxy terminus of the tail (N756ITY) are
required for the interaction with
3-endonexin (13). Mutation or deletion of these same residues also disrupts insideout integrin signaling in platelets and CHO cells (50;
Wang, R., D.R. Ambruso, and P.J. Newman. 1994. Blood.
84:244a). Moreover, coexpression of a
3 tail chimera, but
not an
5 tail chimera, prevented affinity modulation by
3-endonexin, possibly because the former chimera but
not the latter could compete with
IIb
3 for binding to
3endonexin. Finally, when other recombinant GFP proteins
such as GFP and GFP/VASP were expressed in CHO
cells, they failed to increase
IIb
3 affinity.
3-endonexin.
Nonetheless, these results imply that this function of
3endonexin is subject to metabolic regulation. In this context, studies with platelets have suggested that serine-threonine kinases (61), tyrosine kinases (23), and PI 3-kinase
(38, 74; Kovacsovics, T.J., J.H. Hartwig, L.C. Cantley, and
A. Toker. 1995. Blood. 86:454a) are involved in promoting fibrinogen binding to
IIb
3. In contrast, compounds that
activate protein kinase A or G inhibit fibrinogen binding
(22, 28). Perhaps
3-endonexin is a direct substrate of specific kinases or phosphatases or is a target of downstream
effectors of these enzymes. For example,
3-endonexin
contains several serine and threonine residues in favorable
contexts for phosphorylation by protein kinase C and A
(63).
1 and
3 integrins, it seems unlikely that the
pathways triggered by these GTPases converge directly on
3-endonexin. Although platelets contain both H-Ras and
R-Ras, and platelet stimulation by thrombin activates H-Ras,
the functions of these GTPases in this terminally differentiated cell are unknown (64).
3-endonexin with the
3 cytoplasmic tail triggers
a structural change in the integrin to reconfigure the extracellular face of the receptor so that it can engage fibrinogen. While the nature of this propagated change is unknown, one possibility is that there is a reorientation of the
3 subunit relative to the
IIb subunit. This is plausible
given the biophysical evidence for interactions between
the cytoplasmic tails of
IIb and
3 (24) and for agonist-
induced structural changes in the extracellular domains of
IIb
3 in platelets (65). In a similar manner, relatively subtle changes within preexisting dimers may play a role in
ligand-triggered, "outside-in" signaling across other plasma
membrane receptors, including the bacterial aspartate receptor (66) and the mammalian EGF receptor (15).
2 tail)
(36),
-actinin (
tails) (51), talin (
) (29), and filamin (
2)
(60), and potential signaling molecules, such as calreticulin
(
tails) (10), pp125FAK (
) (58), integrin-linked kinase (
)
(26), and cytohesin-1 (
2) (37). Of note, the expression of
calreticulin and cytohesin-1 appears to stimulate or stabilize a high affinity state of integrins
2
1 and
L
2, respectively (10, 37). There is no sequence similarity between
either of these proteins and
3-endonexin. Thus, a structurally diverse group of cytoplasmic tail-binding proteins
may function to regulate integrins. Some like cytohesin-1 and
3-endonexin may be restricted in their action because
of their binding specificities, while others like calreticulin,
which recognizes a conserved motif in all integrin
tails,
may be less specific.
3-
endonexin to the cytoplasm and nucleus of HMEC-1 and
CHO cells suggests that this protein may have more than one
function. The nuclear localization may be explained, in
part, by the presence of a consensus nuclear localization
signal in
3-endonexin (K62RKK) (35, 63). Interestingly,
several proteins implicated in cell adhesion and adhesive
signaling, including ZO-1,
-catenin, zyxin, c-Abl, and
HEF1, either exhibit cytoplasmic and nuclear localization or
shuttle between the cytoplasm and the nucleus depending
on the adhesive state of the cell (Nix, D.A., and M.C. Beckerle. 1995. Mol. Biol. Cell. 6:366a; 3, 21, 41, 44). The identification of other proteins that can bind to
3-endonexin
should help to explain its pattern of subcellular localization.
3-endonexin to
3-rich focal adhesions suggests that it may interact most strongly with the
3 cytoplasmic tail while cells are
in suspension or are in the early phases of adhesion. Thus,
it is attractive to speculate that
3-endonexin may participate in integrin activation but may dissociate at later times
to permit cytoskeletal interactions with the integrin tails.
3-endonexin, but they leave several questions unanswered. Does
3-endonexin influence outside-in signaling
events, such as protein tyrosine phosphorylation (8)? Is
3endonexin subject to posttranslational modifications in
vivo, and does this affect its subcellular localization or
function? Does
3-endonexin modulate the adhesive function of
V
3, which like
IIb
3 appears to be subject to rapid
regulation in some cell types (67, 73)? Does
3-endonexin
regulate
IIb
3 in platelets?
Received for publication 23 December 1996 and in revised form 24 March 1997.
1. Abbreviations used in this paper: GFP, green fluorescent protein; PE, phycoerythrin.We thank David Phillips for providing Integrilin, Michael Schaller for FRNK cDNA, Beat Steiner for Ro 43-5054, Zaverio Ruggeri for antibody RG 7, and Ulrich Walter and Thomas Jarchau for VASP cDNA.
These studies were supported by research grants from the National Institutes of Health (HL 56595, HL 48728, AR 27214) and from Cor Therapeutics, Inc. H. Kashiwagi is the recipient of a Banyu Fellowship in Lipid Metabolism and Atherosclerosis sponsored by Banyu Pharmaceutical Co., Ltd. and the Merck Company Foundation. M. Eigenthaler is the recipient of a fellowship from the American Heart Association (CA Chapter). This is manuscript 10536-VB from the Scripps Research Institute.
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