The goal of this study was to determine whether
actin-binding protein (ABP) regulates membrane composition.
ABP-deficient and ABP-containing cells were transfected with the
cDNAs coding for glycoprotein (GP) Ib-IX, a platelet receptor that
interacts with ABP. Most of the GP Ib-IX remained inside the
ABP-deficient cells. When ABP was present, functional GP Ib-IX was
inserted into the membrane. GP Ib-IX lacking the domain that interacts with ABP also showed increased membrane insertion in ABP-expressing cells. Furthermore, a fragment of ABP that lacks the dimerization and
GP Ib-IX-binding sites restored the spreading of the cells and
increased the amount of GP Ib-IX in the membrane. Finally, expression
of ABP also increased endogenous
1 integrin in the membrane. These results indicate that 1) ABP maintains the properties of the cell such that adhesion receptors can be efficiently expressed in the membrane; 2) increased receptor expression is accompanied by
increased ability of the cell to spread; and 3) ABP exerts its effect
by a mechanism that does not appear to involve direct cross-linking of
actin filaments or direct interaction with receptors.
 |
INTRODUCTION |
The membrane of mammalian cells is lined by a network of actin
filaments cross-linked by a variety of proteins. One of the cross-linking proteins is actin-binding protein
(ABP),1 a dimer composed of
two identical subunits (270 kDa) associated via a self-association site
(1) in the carboxyl-terminal part of the molecule (2). The amino
terminus contains the actin-binding domain (2, 3). The remaining
sequence is formed by 24 repeats. In addition to cross-linking actin
filaments, ABP can interact with certain membrane glycoproteins
(4-12).
Recently, the availability of a melanoma cell line that lacks
actin-binding protein (13) has provided evidence that ABP is important
in regulating the morphology and motility of the cell. Thus, the
surface of ABP-deficient cells is covered with large blebs, and the
cells display a decreased ability to extend the membrane projections
required for normal spreading and motility. After transfection of the
ABP-deficient cells with cDNA encoding ABP, the cells regained
their ability to extend membrane projections, spread, and migrate.
Extension of membrane processes requires both a reorganization of the
cytoskeleton and an increased availability of membrane at the site of
the extension. Because ABP is known to be a component of a specialized
part of the cytoskeleton that is in close apposition to the membrane
and associates with specific membrane glycoproteins (4-12), we
wondered whether the reason that ABP-deficient cells are unable to
extend normal projections might be because ABP is normally involved in
regulating membrane properties. The best characterized interaction of
ABP with a membrane protein is with GP Ib-IX (4-10), the platelet von
Willebrand factor receptor that mediates the initial attachment of
platelets at a site of injury (14, 15). This receptor consists of three
transmembrane subunits (GP Ib
, GP Ib
, and
GP IX) (16). Interaction of the receptor complex with ABP is mediated
by a region in the central portion of the cytoplasmic domain of GP
Ib
(9, 10, 17); this region interacts with a region in
the carboxyl-terminal third of ABP (8, 18, 19). In this study, we
examined the role of ABP in regulating expression of this receptor.
ABP-deficient cells or cells that had been stably transfected with the
cDNA for ABP were transfected with the cDNA for the three
subunits of the GP Ib-IX complex. In the absence of ABP, most of the GP
Ib-IX remained inside the cell. The presence of ABP resulted in
increased expression of GP Ib-IX in the membrane and increased ability
of the cells to bind von Willebrand factor. The increased expression of
GP Ib-IX in the membrane of the ABP-containing cells did not result
from association of GP Ib-IX with ABP because a truncated form of GP
Ib-IX that lacked the domain that interacts with ABP also showed
increased expression in the membrane. Furthermore, a fragment of ABP
that lacks the carboxyl-terminal 548 amino acids and therefore cannot
bind to GP Ib-IX (19) or directly cross-link actin filaments (20) was
just as effective as full-length ABP in increasing the surface
expression of GP Ib-IX and also in restoring the ability of the cells
to extend projections and to spread. Finally, expression of ABP also
increased the amount of endogenous
1 integrin in the
membrane, even though there is no evidence that this receptor interacts
with ABP. These studies suggest that 1) a previously unrecognized
function of ABP is to maintain the properties of the cell such that
adhesion receptors can be efficiently expressed in the membrane; 2)
increased receptor expression is accompanied by restoration of the
ability of the membrane to extend projections and to spread; and 3) ABP
exerts its effect by a mechanism that does not involve direct
cross-linking of actin filaments or direct interaction with the
adhesion receptors.
 |
MATERIALS AND METHODS |
Cell Culture and Transfections--
The melanoma cells used in
these studies were derived from a human malignant melanoma lacking
actin-binding protein (13). To develop a cell line that expressed
actin-binding protein, cells were transfected with the cDNA coding
for actin-binding protein (13). Both ABP-deficient and ABP-containing
cells (kindly provided by Dr. C. Cunningham, Brigham Women's Hospital,
Boston) were stably transfected with the cDNAs coding for GP
Ib
, GP Ib
, and GP IX as described
previously (21). In some experiments, as indicated below, a cDNA
coding for a truncated form of GP Ib
that cannot
interact with ABP was used. The truncated form was generated by
introducing a stop codon at position 545 of the cytoplasmic domain of
GP Ib
(17). In other experiments, the ABP-deficient cells were transiently transfected by the calcium phosphate method (22)
with the cDNA coding for full-length or truncated forms of ABP. The
transfection efficiencies obtained with this method ranged from 30 to
80%. Full-length ABP inserted into pCDM8 (pCDM8-ABP) (23) was obtained
from Dr. C. Cunningham. A truncated form (ABP-(1-2099)) that lacks
only the carboxyl-terminal 548 amino acids has been previously
described (19). A 1.5-kilobase pair DNA fragment (nucleotides
6581-8116) coding for only the carboxyl-terminal end of ABP
(ABP-(2136-2647)) was generated by polymerase chain reaction and
inserted into pCDM8. The primers used to amplify the fragment
corresponded to nucleotides 6581-6601 preceded by an ATG sequence and
to nucleotides 8093-8116. The sequences were verified by DNA
sequencing analysis.
Flow Cytometry--
Cells were stained with a monoclonal
antibody against GP Ib
(mAb Ib-23; generously provided
by Dr. B. Steiner, Hoffmann-La Roche, Basel, Switzerland) (24) or
against
1 integrin (mAb 1965; Chemicon International,
Inc., Temecula, CA) (25) and with fluorescein isothiocyanate-conjugated
anti-mouse IgG (Amersham International, Buckinghamshire, United
Kingdom) and analyzed with a FACScan flow cytometer (Becton Dickinson
Advanced Cellular Biology, San Jose, CA) as described previously
(21).
Fluorescence Microscopy--
Cells were allowed to settle for
16 h onto glass slides, fixed, permeabilized with 0.5% Triton
X-100 (Sigma), and stained as described previously (21, 26). The
samples were labeled with 5 µg/ml mAb Ib-23 or mAb ABP-4 (generously
provided by Dr. J. Hartwig, Brigham Women's Hospital) (8), washed,
incubated with biotinylated goat anti-mouse IgG (Amersham
International), washed, and incubated with fluorescein
isothiocyanate-conjugated streptavidin (Amersham International). In
some experiments, actin filaments were stained with 2 µg/ml
tetramethylrhodamine isothiocyanate-labeled phalloidin (Sigma).
Fluorescence microscopy was performed with an inverted microscope
(DIAPHOT-TMD, Nikon). Confocal microscopy images were collected with a
Leica TCS-NT laser scanning confocal microscope. Images were collected
through the cells at a step size of ~0.3 µm.
Phase-contrast Microscopy--
To assess the ability of the
melanoma cells to extend membrane projections and to spread, cells were
plated on 60-mm tissue culture dishes and were examined after 24 h
under a phase-contrast microscope (Olympus CK2).
Immunogold Staining for Electron Microscopy--
The cells were
plated on 60-mm tissue culture dishes. After 24 h, cells were
fixed for 1 h in pyridoxal phosphate fixative containing 4%
paraformaldehyde, 75 mM lysine, 10 mM sodium
meta-periodate, and 37.5 mM sodium phosphate, pH
7.4 (27). The samples were stained with 2 µg/ml mAb Ib-23, washed,
and incubated with a 1:20 dilution of goat anti-mouse IgG antibodies
conjugated with 10-nm gold particles (Amersham International). Samples
were washed, fixed in 2.5% glutaraldehyde, processed for thin
section electron microscopy, and examined in a JEM 100 CX II microscope
(Jeol Ltd., Tokyo, Japan) (28).
Analysis of Cell Proteins--
For detection of GP Ib-IX or ABP
in transfected cells, cells were solubilized in an SDS-containing
buffer, and solubilized proteins were electrophoresed through
SDS-polyacrylamide gels and transferred to nitrocellulose. GP
Ib
was detected with mAb Ib-4 (generously provided by
Dr. B. Steiner) (24), and ABP was detected with either mAb ABP-4 or an
affinity-purified polyclonal antibody raised against purified
full-length ABP. Antigen-antibody complexes were detected by
chemiluminescence (Amersham International). The intensity of the bands
was quantitated by densitometry on a Macintosh computer using NIH Image
software. Immunoprecipitation experiments were performed as described
previously (19).
 |
RESULTS |
Role of ABP in Regulating Expression of GP Ib-IX in the Plasma
Membrane--
To determine whether ABP is important in allowing a
receptor with which it interacts to be expressed in the membrane, the three cDNAs encoding GP Ib
, GP Ib
,
and GP IX were transfected into ABP-deficient melanoma cells and into
cells that had been stably transfected with the cDNA encoding ABP
(13). Even in the absence of ABP, GP Ib-IX was expressed on the cell
surface (Fig. 1A). However,
the amount of GP Ib-IX expressed on the surface of the ABP-deficient
cells was significantly lower than on the surface of the ABP-containing
cells (compare dashed and solid lines). In five
different experiments, the mean fluorescence obtained with the
ABP-deficient cells was 7-12-fold lower than with the ABP-containing
cells. Western blot analysis (Fig. 1B) revealed that
although there was less GP Ib
expressed in the melanoma cells in the absence of ABP than in its presence, it did not account for the 7-12-fold difference in the membrane (in five independent experiments, the total amount of GP Ib
expressed in the
ABP-deficient cells was 1.4-3-fold lower than in the ABP-containing
cells).

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Fig. 1.
Expression of GP Ib-IX in ABP-deficient or
ABP-containing melanoma cells. GP Ib-IX was stably transfected
into ABP-transfected or ABP-deficient melanoma cells. The amount of GP
Ib-IX inserted into the membrane was detected by flow cytometry
(A). The dashed line represents ABP-deficient
melanoma cells; the solid line represents cells stably
transfected with ABP; and the dotted line represents nontransfected melanoma cells. In the experiment shown, the cells were
stained with an antibody against GP Ib , but similar results were obtained using an antibody against GP IX. The total amount
of GP Ib-IX expressed in the cells was determined by Western blot
analysis (B). Different concentrations (2.5 × 104 to 105) of ABP-containing or ABP-deficient
cells were solubilized in an SDS-containing buffer and analyzed by
Western blotting using an antibody against GP Ib . The
intensity of the bands was quantitated by densitometry. The
inset shows the intensity of the bands obtained when 5 × 104 ABP-containing (lane 1) or ABP-deficient
(lane 2) cells were loaded. At this concentration, the bands
were within the linear range of the chemiluminescence reaction and
showed that 2.5-fold more GP Ib was present in the
ABP-containing cells compared with the ABP-deficient cells.
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|
Examination of fixed permeabilized cells by confocal microscopy
showed that in the ABP-deficient cells, most of the GP
Ib
was found intracellularly, and little was on the cell
surface (Fig. 2A). In
contrast, in the ABP-containing cells, the majority of the GP
Ib
was present on the cell surface (Fig. 2B).
There was no evidence of increased loss of GP Ib
in the
cell culture medium in either case (data not shown).

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Fig. 2.
Confocal microscopy images showing the
distribution of GP Ib in ABP-deficient and
ABP-containing cells. ABP-deficient cells or cells stably
transfected with ABP were transfected with the cDNA encoding GP
Ib-IX. Cells were harvested, allowed to settle onto glass slides,
fixed, and permeabilized, and the distribution of GP Ib
was detected. The images represent projections of four confocal slices
representing 1.1-1.3 µm through the middle of the cells.
A, ABP-deficient, GP Ib-IX-expressing cells; B, ABP-containing, GP Ib-IX-expressing cells. The bar
represents 10 µm.
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|
To determine whether the increased insertion of GP Ib-IX into the
membrane of ABP-containing cells was a direct consequence of the
presence of ABP, the ABP-deficient cells expressing GP Ib-IX were
transiently transfected with the cDNA encoding ABP and analyzed
48 h later. Flow cytometry analysis showed that transient expression of ABP increased the amount of GP Ib
incorporated into the membrane (Fig.
3A). In four separate
experiments, the amount of GP Ib-IX incorporated into the membrane was
increased 4-11-fold. However, expression of ABP had little effect on
the total amount of GP Ib-IX expressed in the cells (Fig.
3B, compare lanes 1 and 2).
Transfection of the vector alone did not increase the surface
expression of GP Ib-IX (data not shown).

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Fig. 3.
Expression of GP Ib-IX in the membrane is
increased following transient expression of full-length actin-binding
protein. ABP-deficient melanoma cells stably expressing GP Ib-IX
were transiently transfected with the cDNA encoding full-length
ABP. 48 h after transfection, the amount of GP Ib
expressed in the membrane was determined by flow cytometry
(A), and the total amount of GP Ib expressed
was detected by Western blotting (B). C is a
Western blot using a polyclonal antibody raised against full-length ABP
and shows the amount of ABP expressed. Lane 1, ABP-deficient cells; lane 2, cells transfected with full-length ABP.
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|
Mechanism by Which ABP Increases the Surface Expression of GP
Ib-IX--
To determine whether the increased insertion of GP Ib-IX
into the membrane of ABP-containing cells resulted from association of
GP Ib-IX with the cytoskeleton, we used cells that contained ABP and
stably transfected them with either full-length GP Ib-IX complex or GP
Ib-IX complex containing a truncated form of GP Ib
that
lacked the domain that interacts with ABP (17). The truncated GP
Ib
has been characterized previously and shown to be
incapable of interacting with ABP (17). Western blot analysis of total
cell lysates showed the presence of similar amounts of GP Ib-IX in
cells transfected with truncated or full-length GP Ib
(Fig. 4B, compare lanes
2 and 3; a difference in mobility is not detected on
the 7.5% SDS gel because the molecular mass only changes from 135 to
129 kDa (17)). In four independent experiments, there was 1.1-fold
(S.D. = 0.1) more GP Ib-IX in the cells expressing full-length GP
Ib
than in those expressing truncated GP
Ib
. FACS analysis (Fig. 4A) showed that
truncated GP Ib-IX (dashed line) was inserted into the cell
membrane almost as efficiently as full-length GP Ib
(solid line). In five experiments, the mean of fluorescence
in the cells expressing full-length GP Ib
was
1.4-1.7-fold higher than in the cells expressing truncated GP
Ib
.

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Fig. 4.
FACS analysis showing the expression of
full-length or truncated GP Ib-IX in the membrane of ABP-containing
cells. Melanoma cells stably transfected with ABP were stably
transfected with the cDNAs encoding GP Ib , GP IX,
and either full-length GP Ib or a truncated form of GP
Ib lacking the region in the cytoplasmic domain that
interacts with ABP. The amount of GP Ib-IX expressed in the membrane
was quantitated by flow cytometry (A). The dotted
line shows cells not transfected with GP Ib-IX; the dashed
line shows cells expressing truncated GP Ib-IX; and the
solid line shows cells expressing full-length GP Ib-IX.
B is a Western blot showing the amount of GP
Ib expressed in comparable numbers of cells. Lane
1, nontransfected cells; lane 2, truncated GP
Ib ; lane 3, full-length GP
Ib .
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|
The finding that truncated GP Ib-IX was efficiently expressed in the
membrane suggested that while the presence of ABP is important in
allowing insertion of GP Ib-IX into the membrane, the association
between the proteins is not. Previously, we have shown that the binding
site for GP Ib
is located between amino acids 1850 and
2136 and that deletion of residues 2099-2136 abolishes the binding to
GP Ib-IX (19). Thus, as a further test of the possibility that
interaction of GP Ib-IX with actin-binding protein is not the reason
for the increased surface expression of GP Ib-IX in ABP-containing
cells, we expressed a truncated ABP (ABP-(1-2099)) that does not
interact with GP Ib-IX in ABP-deficient cells expressing GP Ib-IX.
Analysis of the transfected cells confirmed that the protein was
expressed and that it had the appropriate molecular mass (Fig.
5B, left inset,
compare lanes 2 and 3). The amount of truncated
ABP expressed in the transiently transfected cells was comparable to
the amount of full-length ABP expressed in the permanently transfected
cell line used for the experiments shown in Figs. 1 and 2 (Fig.
5B, left inset, compare lanes 2 and 3). Flow cytometry analysis showed that the presence of the
truncated form of ABP (ABP-(1-2099)) induced a 6-7-fold increase in
the amount of GP Ib-IX present on the cell surface (Fig.
5B). Similar increases were seen in three independent
experiments. However, there was little difference in the total amount
of GP Ib-IX expressed in the cells (Fig. 5B, right
inset). Transfection of a construct encoding only the
carboxyl-terminal end of ABP (ABP-(2136-2647)) did not increase the
surface expression of GP Ib-IX (Fig. 5C).

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Fig. 5.
FACS analysis showing that truncated ABP
(ABP-(1-2099)) that does not bind to GP Ib-IX increases the surface
expression of GP Ib-IX, but the carboxyl-terminal end
(ABP-(2136-2647)) does not. A represents a schematic
representation of ABP. ABP-deficient melanoma cells expressing GP Ib-IX
were transiently transfected with the cDNA encoding ABP that lacks
part of the GP Ib-IX-binding site (ABP-(1-2099)) or with the cDNA
encoding only the carboxyl-terminal end (ABP-(2136-2647)). 48 h
after transfection, the amount of GP Ib on the surface
of the cells was quantitated by flow cytometry (B and
C). The signal obtained with nontransfected cells is
represented by a dotted line, that with ABP-deficient cells by a dashed line, and that with cells transiently
transfected with the cDNA encoding truncated ABP by a solid
line. The left insets are Western blots using a
monoclonal (B) or polyclonal (C) antibody against
ABP and show the amount of ABP expressed in the cells. The right
insets are Western blots using a monoclonal antibody against GP
Ib-IX and show that the total amount of GP Ib-IX expressed in the cells
does not increase after expression of ABP. Lane 1,
ABP-deficient cells; lane 2, cells transiently transfected
with truncated ABP. To demonstrate that the transiently transfected
cells were expressing only the truncated form of ABP and to demonstrate
that the amount of truncated ABP expressed was comparable to the amount
of full-length ABP expressed, the amount of full-length ABP in a
comparable number of cells is shown in lane 3.
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|
When the distribution of the truncated form of ABP was examined by
immunofluorescence and compared with that of full-length ABP in the
stably transfected cells, no difference could be detected. Thus, both
full-length ABP (Fig. 6A) and
the truncated form of ABP (Fig. 6C) colocalized with actin
filaments and were concentrated toward the periphery of the cell (Fig.
6, B and D).

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Fig. 6.
Immunofluorescence microscopy of cells
expressing full-length or truncated ABP. ABP-deficient cells
expressing GP Ib-IX were transiently transfected with the cDNA
encoding ABP lacking the carboxyl-terminal 548 amino acids
(ABP-(1-2099)). 72 h later, the cells were harvested, allowed to
settle onto glass slides, fixed, and permeabilized, and the
distribution of actin and actin-binding protein (C and
D) was compared with that in cells stably expressing GP
Ib-IX and full-length ABP (A and B). In this
experiment, the transfection efficiency was at least 35% (all the
cells stained for actin (D) and ~35% stained for ABP
(C)). Actin-binding protein staining is shown in
A and C, and actin filaments in B and
D. The bar represents 15 µm.
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Role of ABP in Regulating Expression of
1 Integrin
in the Plasma Membrane--
To determine if the cell-surface
expression of proteins normally present in melanoma cells was regulated
by actin-binding protein, we compared the amount of
1
integrin inserted into the plasma membrane of ABP-deficient and
ABP-containing cells. As shown by flow cytometry (Fig.
7),
1 integrin was
incorporated into the membrane of the ABP-deficient cells (dashed
line). However, the amount of
1 integrin present on
the surface of the ABP-containing cells (solid line) was
markedly increased (in three experiments, the mean of fluorescence in
the ABP-containing cells was 3-4.5-fold higher than in the
ABP-deficient cells). The increased insertion of endogenous integrin in
the ABP-containing cells did not appear to result from an interaction
with ABP since we were unable to immunoprecipitate
1
integrin with an antibody against actin-binding protein (Fig.
8) or to immunoprecipitate actin-binding
protein with an anti-
1 integrin antibody (data not
shown).

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Fig. 7.
Expression of 1 integrin in
the membrane is regulated by actin-binding protein. Melanoma cells
deficient in ABP (dashed line) or containing ABP
(solid line) were labeled with an anti- 1 integrin antibody and analyzed by flow cytometry. The dotted
line represents the signal obtained from ABP-containing cells
stained with irrelevant ascites.
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Fig. 8.
1 integrin does not
co-immunoprecipitate with actin-binding protein. Cells were lysed
in a Triton X-100-containing lysis buffer that induced depolymerization
of the cytoskeleton. Actin-binding protein was immunoprecipitated,
electrophoresed through an SDS-polyacrylamide gel, and transferred to
nitrocellulose. Blots were probed with a monoclonal antibody against
1 integrin (left panel) or against ABP
(right panel). Lanes 1, cell lysate (8-fold fewer
cells than the amount used in the immunoprecipitations); lanes
2, ABP-containing cell lysate immunoprecipitated with mouse IgG;
lanes 3, ABP-containing cell lysate immunoprecipitated with anti-ABP antibody.
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Mechanism by Which ABP Alters Cell Morphology--
Previously,
others have shown that the ABP-deficient cells are covered with large
blebs and do not spread well, whereas the stably transfected cells do
not bleb and are able to extend membrane projections and to spread
(13). To examine the effects of ABP on the membrane surface in more
detail, thin sections of the two cell lines were examined by electron
microscopy. While blebs could be seen by electron microscopy (for
example, in Fig. 9A, two blebs can be seen in the right-hand half of the portion of membrane shown),
the most striking feature of the membrane was the smoothness of the
surface (Fig. 9A). In contrast, the surface of the
ABP-containing cells not only lacked blebs, but was covered with
projections, indicative of a much more motile cell and of the restored
ability of the cells to induce the cytoskeletal reorganizations and
membrane reorganizations needed for migration (Fig. 9B). The
samples shown in Fig. 9 were immunolabeled with GP Ib-IX antibodies
followed by gold-conjugated secondary antibodies. The density of gold
label on the membranes confirmed that very little GP Ib-IX was
expressed in the membrane when ABP was missing (Fig. 9A),
whereas the amount in the membrane was markedly increased when ABP was
present. Moreover, it showed that the microprocesses present in the
ABP-containing cells contained considerable amounts of GP Ib-IX (Fig.
9B). In both cell lines, GP Ib-IX appeared to be randomly
distributed: there was no evidence of clustering.

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Fig. 9.
Electron micrographs showing the surface
morphology and expression of GP Ib-IX in the membrane of ABP-deficient
and ABP-containing melanoma cells. Melanoma cells deficient in ABP
(A) or containing ABP (B) were stably transfected
with the cDNAs for GP Ib-IX. Cells were grown on tissue culture
plates for 24 h, fixed, and labeled with antibody against GP
Ib and then with anti-mouse IgG antibody coupled to
10-nm gold particles. Cells were processed for thin section electron
microscopy. The bar represents 380 nm.
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The carboxyl-terminal end of ABP contains not only the GP Ib-IX-binding
site, but also the dimerization site (2, 3). So truncated ABP lacking
the carboxyl-terminal 548 residues cannot directly cross-link actin. If
the importance of ABP is in directly cross-linking the submembranous
cytoskeleton, we predicted that expression of the truncated form of ABP
in the ABP-deficient cells would not restore the ability of the cells
to extend membrane projections and to spread. In contrast, since the
truncated form increased the expression of GP Ib-IX in the membrane, we
predicted that if the importance of ABP was in allowing appropriate
reorganization of the membrane, the truncated form would result in
normal morphology and spreading. Thus, to gain insight into the
mechanism by which ABP exerts its effects, cells were transiently
transfected with either full-length or truncated forms of ABP, allowed
to spread on tissue culture plates for 24 h, and examined by
phase-contrast microscopy. As described previously (13) and shown in
Fig. 10A, phase-contrast
microscopy revealed blebs all over the surface of the ABP-deficient
cells; moreover, the cells remained rounded and were unable to spread.
Similar morphologies were observed with cells transfected with the
vector only (Fig. 10B) or the cDNA encoding only the
carboxyl-terminal 511 amino acids (ABP-(2136-2647)) (Fig.
10C). In contrast, cells expressing the truncated form of ABP lacking only the carboxyl-terminal 548 amino acids (ABP-(1-2099)) (Fig. 10D) had few blebs and were able to extend projections
and to spread. The appearance of the transfected cells was very similar to that of cells transfected with full-length ABP (Fig.
10E).

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Fig. 10.
Phase-contrast microscopy showing that
expression of ABP-(1-2099) restores the ability of melanoma cells to
spread. ABP-deficient melanoma cells expressing GP Ib-IX
(A) were transiently transfected with pCDM8 without insert
(B) or with pCDM8 containing a cDNA encoding
ABP-(2136-2647) (C), ABP-(1-2099) (D), or
full-length ABP (E). 24 h after transfection, the cells
were harvested and plated in tissue culture dishes. The cells were
observed with a phase-contrast microscope 24 h later. The cells
shown in D and E are not all transfected: the
nontransfected cells are round and show blebs; in contrast, the
transfected cells are well spread. The bar represents 75 µm.
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 |
DISCUSSION |
Actin-binding protein plays an important role in regulating cell
morphology and motility (13, 29). Actin-binding protein also interacts
with membrane receptors (4-12), but the functional importance of such
interactions is not known. In this report, we describe a previously
unrecognized function of ABP. Using an ABP-deficient cell line, we show
that ABP regulates the expression of adhesion receptors in the
membrane. ABP increases the surface expression of receptors by a
mechanism that does not involve direct interaction with the receptors,
does not appear to involve direct cross-linking of actin filaments, and
correlates with restoration of the ability of cells to extend
projections and to spread.
The conclusion that ABP regulates the composition of the plasma
membrane came from the study of two membrane receptors, the endogenous
1 integrin subunit and expressed GP Ib-IX complex. Both
receptors were inserted into the membrane of an ABP-deficient melanoma
cell line. However, the amount of GP Ib-IX and
1
integrin incorporated into the membrane increased as much as 12-fold
when ABP cDNA was transfected into the cells. Several lines of
evidence indicated that the increased expression of adhesion receptors resulted from some function of ABP other than its interaction with
receptor. Thus, although there has been a report that ABP interacts
with
2-containing integrins (12), under our experimental conditions, we were unable to detect an interaction between
1 integrin and ABP. Furthermore, by studying GP Ib-IX, a
platelet adhesion receptor that interacts with ABP by a well
characterized mechanism (4-10), we were able to demonstrate that GP
Ib-IX lacking the region in the cytoplasmic domain that interacts with
ABP also showed increased expression in the ABP-containing cells.
Finally, a truncated form of ABP lacking the region that interacts with GP Ib-IX was just as effective as full-length ABP in increasing the
expression of GP Ib-IX in the membrane of the cells. Taken together,
these findings indicate that the importance of ABP is not in attaching
to a receptor and anchoring it in the membrane, but rather in
maintaining a more general organization of the submembranous region
that allows the appropriate protein composition of the membrane to be
maintained.
The increased expression of adhesion receptors in the ABP-containing
cells could conceivably occur because ABP must be present in the
submembranous cytoskeleton for membrane vesicles to be efficiently
inserted into the plasma membrane. Alternatively, in the absence of
submembranous ABP, membrane receptors might be inserted normally, but
subsequently removed from the membrane more rapidly than in the
ABP-containing cells. In considering the mechanism by which ABP exerts
its effects, it is of interest that ABP induces not only an altered
expression of membrane receptors, but also a marked difference in the
ability of the cell to extend projections and to spread. To extend
projections and to spread, cells must reorganize both the cytoskeleton
and the membrane at specific locations. There are no indications of
marked differences in the cytoskeleton of the ABP-deficient cells
compared with the ABP-containing cells. For example, the amounts of
gelsolin,
-actinin, profilin, and fodrin are similar in the two
cases (13). Moreover, the total actin content is the same, and at least
until the ABP-containing cells have spread, there is not a detectable
difference in the amount of actin that is polymerized into filaments in
the two cell lines (29).
This study shows that whatever the mechanism by which ABP exerts its
effects, it is induced equally well by full-length ABP and ABP that
cannot bind to GP Ib-IX because it lacks the carboxyl-terminal 548 amino acids. The truncated form of ABP contains its actin-binding domain, and immunofluorescence microscopy indicated that it interacted with submembranous actin. Because the dimerization site is in the
carboxyl-terminal end, it appears unlikely that truncated ABP could
directly cross-link actin filaments, although we cannot exclude the
possibility that it can do this by other unidentified mechanisms in the
intact cell. It is also conceivable that truncated ABP could regulate
the organization of the submembranous network of cytoskeleton proteins
through a mechanism that does not require it to dimerize. Another
possibility is that the truncated form of ABP interacts with
unidentified membrane proteins that play a role in maintaining the
membrane such that GP Ib-IX and
1 integrin expression in
the membrane can be regulated appropriately. It is also becoming
apparent that the membrane skeleton of cells can bind a variety of
signaling molecules including pp60c-src (30-33)
and phosphoinositide 3-kinase (31), so yet another possibility is that
ABP is required to maintain the appropriate submembranous location of
such molecules. It is conceivable that by regulating actin
polymerization, organization of the cytoskeleton, or generation of
lipid products, signaling molecules could maintain the organization of
the membrane or of the submembranous cytoskeleton. Such an organization
could be essential for appropriate insertion or removal of membrane
proteins, maintenance of cell morphology, or effective function of
signal transduction mechanisms involved in membrane projection
extension and cell spreading.
This study was performed in a melanoma cell line. However, ABP is
present in a large variety of cell types, and it appears probable that
it could play an important role in regulating the insertion of membrane
proteins and the properties of the membrane in these cells also. ABP is
a component of a submembranous skeleton, so conceivably, defects in
additional proteins in this skeleton could have similar effects on the
composition and function of cell membranes. There are several examples
of cytoskeletal proteins whose absence results in decreased expression
of a membrane protein with which they normally interact. For example,
human erythrocytes deficient in protein 4.1 have a decreased content of
glycophorin C (34-38), the membrane glycoprotein with which it
interacts (38-42). Another well characterized interaction of a
cytoskeletal protein with a plasma membrane glycoprotein complex is
that of dystrophin with the dystrophin-associated glycoprotein complex
(for review, see Refs. 43 and 44). The absence of dystrophin causes all the dystrophin-associated proteins to be drastically reduced (45-48). With these examples, it is assumed that the absence of the membrane glycoproteins results from the absence of the cytoskeletal protein with
which their cytoplasmic domain interacts. However, the absence of these
proteins causes additional changes in membrane stability, ion fluxes,
or integrity. This study raises the possibility that the absence of the
cytoskeletal proteins may cause a more generalized abnormality in the
submembranous region that in turn affects the expression of membrane
glycoproteins. Future studies will be needed to determine whether, like
ABP, other submembranous cytoskeletal proteins play a primary role in
maintaining membrane properties or directed migration such that the
appropriate expression of membrane glycoproteins can be achieved.
We are grateful to Dr. C. Cunningham for
providing the melanoma cells, Dr. J. Hartwig for the cDNA coding
for ABP and for anti-ABP antibodies, Drs. J. López and G. Roth
for the cDNAs coding for GP Ib-IX, Dr. B. Steiner for anti-GP Ib-IX
antibodies, Dr. J. Cunningham for the construct coding for the
truncated form of GP Ib
, S. Zuerbig for technical
assistance and graphics, B. Zhao for technical assistance, and Gene
Lazuta for editorial assistance.