Role of Actin-binding Protein in Insertion of Adhesion Receptors into the Membrane*

Sylvie C. MeyerDagger §, David A. Sanan, and Joan E. B. FoxDagger §par

From the Dagger  Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the § Children's Hospital Oakland Research Institute, Oakland, California 94609, and the  Gladstone Institute of Cardiovascular Disease, San Francisco, California 94141

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 Ibalpha , GP Ibbeta , 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 Ibalpha (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 beta 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 Ibalpha , GP Ibbeta , and GP IX as described previously (21). In some experiments, as indicated below, a cDNA coding for a truncated form of GP Ibalpha 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 Ibalpha (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 Ibalpha (mAb Ib-23; generously provided by Dr. B. Steiner, Hoffmann-La Roche, Basel, Switzerland) (24) or against beta 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 Ibalpha 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 Ibalpha , GP Ibbeta , 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 Ibalpha 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 Ibalpha 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 Ibalpha , 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 Ibalpha . 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 Ibalpha was present in the ABP-containing cells compared with the ABP-deficient cells.

Examination of fixed permeabilized cells by confocal microscopy showed that in the ABP-deficient cells, most of the GP Ibalpha was found intracellularly, and little was on the cell surface (Fig. 2A). In contrast, in the ABP-containing cells, the majority of the GP Ibalpha was present on the cell surface (Fig. 2B). There was no evidence of increased loss of GP Ibalpha 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 Ibalpha 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 Ibalpha 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.

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 Ibalpha 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 Ibalpha expressed in the membrane was determined by flow cytometry (A), and the total amount of GP Ibalpha 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.

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 Ibalpha that lacked the domain that interacts with ABP (17). The truncated GP Ibalpha 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 Ibalpha (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 Ibalpha than in those expressing truncated GP Ibalpha . 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 Ibalpha (solid line). In five experiments, the mean of fluorescence in the cells expressing full-length GP Ibalpha was 1.4-1.7-fold higher than in the cells expressing truncated GP Ibalpha .


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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 Ibbeta , GP IX, and either full-length GP Ibalpha or a truncated form of GP Ibalpha 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 Ibalpha expressed in comparable numbers of cells. Lane 1, nontransfected cells; lane 2, truncated GP Ibalpha ; lane 3, full-length GP Ibalpha .

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 Ibalpha 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 Ibalpha 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.

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.

Role of ABP in Regulating Expression of beta 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 beta 1 integrin inserted into the plasma membrane of ABP-deficient and ABP-containing cells. As shown by flow cytometry (Fig. 7), beta 1 integrin was incorporated into the membrane of the ABP-deficient cells (dashed line). However, the amount of beta 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 beta 1 integrin with an antibody against actin-binding protein (Fig. 8) or to immunoprecipitate actin-binding protein with an anti-beta 1 integrin antibody (data not shown).


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Fig. 7.   Expression of beta 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-beta 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.   beta 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 beta 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.

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 Ibalpha 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.

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.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta 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 beta 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 beta 2-containing integrins (12), under our experimental conditions, we were unable to detect an interaction between beta 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, alpha -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 beta 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.

    ACKNOWLEDGEMENTS

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 Ibalpha , S. Zuerbig for technical assistance and graphics, B. Zhao for technical assistance, and Gene Lazuta for editorial assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Research Grant HL30657 (to J. E. B. F.).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.

par To whom correspondence should be addressed: Joseph J. Jacobs Center for Thrombosis and Vascular Biology (FF 20), Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-3874; Fax: 216-445-2051.

1 The abbreviations used are: ABP, actin-binding protein; GP, glycoprotein; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Hartwig, J. H., and Stossel, T. P. (1981) J. Mol. Biol. 145, 563-581[Medline] [Order article via Infotrieve]
  2. Weihing, R. R. (1988) Biochemistry 27, 1865-1869[Medline] [Order article via Infotrieve]
  3. Gorlin, J. B., Yamin, R., Egan, S., Stewart, M., Stossel, T. P., Kwiatkowski, D. J., Hartwig, J. H. (1990) J. Cell Biol. 111, 1089-1105[Abstract]
  4. Solum, N. O., Olsen, T. M., Gogstad, G. O., Hagen, I., Brosstad, F. (1983) Biochim. Biophys. Acta 729, 53-61[Medline] [Order article via Infotrieve]
  5. Fox, J. E. B. (1985) J. Clin. Invest. 76, 1673-1683[Medline] [Order article via Infotrieve]
  6. Fox, J. E. B. (1985) J. Biol. Chem. 260, 11970-11977[Abstract/Free Full Text]
  7. Okita, J. R., Pidard, D., Newman, P. J., Montgomery, R. R., Kunicki, T. J. (1985) J. Cell Biol. 100, 317-321[Abstract]
  8. Ezzell, R. M., Kenney, D. M., Egan, S., Stossel, T. P., Hartwig, J. H. (1988) J. Biol. Chem. 263, 13303-13309[Abstract/Free Full Text]
  9. Andrews, R. K., and Fox, J. E. B. (1991) J. Biol. Chem. 266, 7144-7147[Abstract/Free Full Text]
  10. Andrews, R. K., and Fox, J. E. B. (1992) J. Biol. Chem. 267, 18605-18611[Abstract/Free Full Text]
  11. Ohta, Y., Stossel, T. P., and Hartwig, J. H. (1991) Cell 67, 275-282[Medline] [Order article via Infotrieve]
  12. Sharma, C. P., Ezzell, R. M., and Arnaout, M. A. (1995) J. Immunol. 154, 3461-3470[Abstract/Free Full Text]
  13. Cunningham, C. C., Gorlin, J. B., Kwiatkowski, D. J., Hartwig, J. H., Janmey, P. A., Byers, H. R., Stossel, T. P. (1992) Science 255, 325-327[Medline] [Order article via Infotrieve]
  14. George, J. N., Nurden, A. T., and Phillips, D. R. (1984) N. Engl. J. Med. 311, 1084-1098[Abstract]
  15. Berndt, M. C., and Caen, J. P. (1984) Prog. Hemostasis Thromb. 7, 111-150[Medline] [Order article via Infotrieve]
  16. Berndt, M. C., Gregory, C., Kabral, A., Zola, H., Fournier, D., and Castaldi, P. A. (1985) Eur. J. Biochem. 151, 637-649[Abstract]
  17. Cunningham, J. G., Meyer, S. C., and Fox, J. E. B. (1996) J. Biol. Chem. 271, 11581-11587[Abstract/Free Full Text]
  18. Aakhus, A. M., Wilkinson, J. M., and Solum, N. O. (1992) Thromb. Haemostasis 67, 252-257[Medline] [Order article via Infotrieve]
  19. Meyer, S. C., Zuerbig, S., Cunningham, C. C., Hartwig, J. H., Bissell, T., Gardner, K., Fox, J. E. B. (1997) J. Biol. Chem. 272, 2914-2919[Abstract/Free Full Text]
  20. Truglia, J. A., and Stracher, A. (1981) Biochem. Biophys. Res. Commun. 100, 814-822[Medline] [Order article via Infotrieve]
  21. Meyer, S. C., and Fox, J. E. B. (1995) J. Biol. Chem. 270, 14693-14699[Abstract/Free Full Text]
  22. Graham, F. L., and van der Eb, A. J. (1973) Virology 52, 456-467[Medline] [Order article via Infotrieve]
  23. Seed, B. (1987) Nature 329, 840-842[CrossRef][Medline] [Order article via Infotrieve]
  24. Meyer, S., Kresbach, G., Haring, P., Schumpp-Vonach, B., Clemetson, K. J., Hadvary, P., Steiner, B. (1993) J. Biol. Chem. 268, 20555-20562[Abstract/Free Full Text]
  25. Stupack, D. G., Stewart, S., Carter, W. G., Wayner, E. A., Wilkins, J. A. (1991) Scand. J. Immunol. 34, 761-769[Medline] [Order article via Infotrieve]
  26. Lopez, J. A., Leung, B., Reynolds, C. C., Li, C. Q., Fox, J. E. B. (1992) J. Biol. Chem. 267, 12851-12859[Abstract/Free Full Text]
  27. McLean, I. W., and Nakane, P. K. (1974) J. Histochem. Cytochem. 22, 1077-1083[Medline] [Order article via Infotrieve]
  28. Fox, J. E. B., Boyles, J. K., Berndt, M. C., Steffen, P. K., Anderson, L. K. (1988) J. Cell Biol. 106, 1525-1538[Abstract]
  29. Cunningham, C. C. (1995) J. Cell Biol. 129, 1589-1599[Abstract]
  30. Fox, J. E. B., Lipfert, L., Clark, E. A., Reynolds, C. C., Austin, C. D., Brugge, J. S. (1993) J. Biol. Chem. 268, 25973-25984[Abstract/Free Full Text]
  31. Zhang, J., Fry, M. J., Waterfield, M. D., Jaken, S., Liao, L., Fox, J. E. B., Rittenhouse, S. E. (1992) J. Biol. Chem. 267, 4686-4692[Abstract/Free Full Text]
  32. Grondin, P., Plantavid, M., Sultan, C., Breton, M., Mauco, G., and Chap, H. (1991) J. Biol. Chem. 266, 15705-15709[Abstract/Free Full Text]
  33. Horvath, A. R., Muszbek, L., and Kellie, S. (1992) EMBO J. 11, 855-861[Abstract]
  34. Alloisio, N., Morle, L., Bachir, D., Guetarni, D., Colonna, P., and Delaunay, J. (1985) Biochim. Biophys. Acta 816, 57-62[Medline] [Order article via Infotrieve]
  35. Sondag, D., Alloisio, N., Blanchard, D., Ducluzeau, M.-T., Colonna, P., Bachir, D., Bloy, C., Cartron, J.-P., and Delaunay, J. (1987) Br. J. Haematol. 65, 43-50[Medline] [Order article via Infotrieve]
  36. Reid, M. E., Takakuwa, Y., Conboy, J., Tchernia, G., and Mohandas, N. (1990) Blood 75, 2229-2234[Abstract]
  37. Dalla Venezia, N., Gilsanz, F., Alloisio, N., Ducluzeau, M.-T., Benz, E. J., Delaunay, J. (1992) J. Clin. Invest. 90, 1713-1717N[Medline] [Order article via Infotrieve]. D.
  38. Alloisio, N., Dalla Venezia, N., Rana, A., Andrabi, K., Texier, P., Gilsanz, F., Cartron, J.-P., Delaunay, J., and Chishti, A. H. (1993) Blood 82, 1323-1327[Abstract]
  39. Reid, M. E., Takakuwa, Y., Tchernia, G., Jensen, R. H., Chasis, J. A., Mohandas, N. (1989) The Red Cell: Seventh Ann Arbor Conference, pp. 553-573, Alan R. Liss, Ann Arbor, MI
  40. Lombardo, C. R., Willardson, B. M., Low, P. S. (1992) J. Biol. Chem. 267, 9540-9546[Abstract/Free Full Text]
  41. Marfatia, S. M., Lue, R. A., Branton, D., and Chishti, A. H. (1994) J. Biol. Chem. 269, 8631-8634[Abstract/Free Full Text]
  42. Marfatia, S. M., Lue, R. A., Branton, D., and Chishti, A. H. (1995) J. Biol. Chem. 270, 715-719[Abstract/Free Full Text]
  43. Matsumura, K., and Campbell, K. P. (1994) Muscle & Nerve 17, 2-15[Medline] [Order article via Infotrieve]
  44. Campbell, K. P. (1995) Cell 80, 675-679[Medline] [Order article via Infotrieve]
  45. Ohlendieck, K., and Campbell, K. P. (1991) J. Cell Biol. 115, 1685-1694[Abstract]
  46. Ohlendieck, K., Matsumura, K., Ionasescu, V. V., Towbin, J. A., Bosch, E. P., Weinstein, S. L., Sernett, S. W., Campbell, K. P. (1993) Neurology 43, 795-800[Abstract]
  47. Ibraghimov-Beskrovnay, O., Ervasti, J. M., Leveille, C. J., Slaughter, C. A., Sernett, S. W., Campbell, K. P. (1992) Nature 355, 696-702[CrossRef][Medline] [Order article via Infotrieve]
  48. Matsumura, K., Lee, C. C., Caskey, C. T., Campbell, K. P. (1993) FEBS Lett. 320, 276-280[CrossRef][Medline] [Order article via Infotrieve]


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