THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 267, No. 18, Issue of June 25, PP. 12851-12859,1992 Printed in U. S. A.

Efficient Plasma Membrane Expression of a Functional Platelet Glycoprotein Ib-IX Complex Requires the Presence of Its Three Subunits*

(Received for publication, February 26, 1992)

Jose A. LopezSgll, Betty Leungs, Clifford C. Reynolds$, Chester Q. Lis, and Joan E. B. Fox$ll From the $Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, the §Department of Medicine, Division of Hematology, and the TDepartment of Pathology, University of California, Sun Francisco, California 94141-9100

The glycoprotein (GP) Ib-IX complex of the platelet plasma membrane mediates the adhesion of platelets to damaged blood vessel wall. The complex is composed of three membrane-spanning polypeptides, GP Ib,, GP IbB, and GP IX, all of which are absent from the plate- lets of patients with the hereditary bleeding disorder Bernard-Soulier syndrome. In this study we report stable expression of the recombinant receptor in three cell lines and demonstrate that the three subunits of the complex are necessary for its efficient expression on the plasma membrane. The expressed complex as- sociates with the cytoskeleton of the transfected cells through an interaction with actin-binding protein and binds its ligand, von Willebrand factor. These data suggest that the lack of plasma membrane GP Ib-IX complex in the Bernard-Soulier syndrome could poten- tially arise from mutations affecting any one of its three subunits.

Under physiological conditions, platelets circulate in the bloodstream without adhering to each other or to vascular endothelium. However, in response to disruptions in the in- tegrity of the endothelium, platelets adhere rapidly to the exposed subendothelial matrix, initiating a series of events: spreading of the platelets along the matrix, release of their granule contents, adhesion of platelets to one another, and acceleration of coagulation, which results in conversion of fibrinogen to insoluble fibrin. This complex process results within minutes in the formation of a tight platelet aggregate that seals the blood vessel and prevents further hemorrhage. Many of these events are adhesive in nature and involve distinct receptors on the platelet surface (Andrews and Fox, 1990; Kieffer and Phillips, 1990). The platelet receptor that plays the major role in mediating the initial contact with the subendothelial matrix is the glycoprotein (GP)’ Ib-IX com-

*This work was supported by Clinical Investigator Award HL02463 from the National Heart, Lung, and Blood Institute (to J. A. L.); Minority Medical Faculty Development Grant 15355 from the Robert Wood Johnson Foundation (to J. A. L.); and Grant HL30657 from the National Institutes of Health (to J. E. B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed Gladstone Insti- tute of Cardiovascular Disease, P.O. Box 419100, San Francisco, CA

’ The abbreviations used are: GP, glycoprotein; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; FACS, fluorescence- activated cell sorting; SDS, sodium dodecyl sulfate; PAGE, polyacryl- amide gel electrophoresis; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid vWf, von Willbrand factor; HEPES, 4-(2-hydroxy- ethyl)-1-piperazineethanesulfonic acid.

94141-9100.

plex (Tobelem et al., 1976). This complex is composed of three homologous membrane-spanning polypeptides (GP Ib,, GP Ib,, and GP IX) and mediates adhesion through an interaction with subendothelial von Willebrand factor (vWf) (Sakarias- sen et ai., 1979), a large multimeric glycoprotein that also circulates in the plasma. This ligand-receptor interaction does not normally occur in the circulation; it appears to require either that vWf first be bound to the subendothelial matrix or that a shear-induced conformational change of either the receptor or the ligand take place. The GP Ib-IX-vWf inter- action is especially critical for platelet adhesion in areas of rapid flow and high shear (Weiss et al., 1978).

Another important function of the GP Ib-IX complex is to serve as a site of attachment for the platelet membrane skeleton. This structure underlies the platelet plasma mem- brane and may be responsible for maintaining platelet shape and for regulating some of the functions of membrane glyco- proteins, including the ability of the GP Ib-IX complex to bind vWf (Fox, 1987). The attachment of the GP Ib-IX complex to the membrane skeleton is mediated through actin- binding protein (also known as filamin) (Fox, 1985a; Andrews and Fox, 1991).

The three polypeptides of the GP Ib-IX complex are mem- bers of a family of proteins with diverse functions that have in common the presence of tandem repeats of a 24-amino acid, leucine-rich motif (Lopez et aL, 1987, 1988; Hickey et al., 1989). Many members of the leucine-rich repeat family, including the polypeptides of the GP Ib-IX complex, also contain homologous sequences flanking the repeated motif. The largest of the three polypeptides that make up the GP Ib-IX complex is GP Ib,, with a M, of 135,000. Glycoprotein Ib, is associated with GP Ib, (MI z24,OOO) through a disulfide bond; the association of GP IX with GP Ib is noncovalent. Glycoprotein Ib, is composed of four major structural domains (Lopez et al., 1987): a globular domain at its amino terminus that contains seven leucine-rich repeats and the site of inter- action with vWf; a threonine-, proline-, and serine-rich region immediately extracellular to the platelet membrane that is highly 0-glycosylated; a transmembrane domain; and a cyto- plasmic domain of approximately 100 amino acids. In addition to the large number of 0-linked carbohydrate chains, GP Ib, contains four sites for N-glycosylation.

Glycoprotein Ib, and GP IX are of similar size and have a high degree of sequence similarity in their extracellular do- mains (approximately 60% of the positions in these domains contain identical or conserved amino acids) (Lopez et al., 1988; Hickey et al., 1989). Complete sequence divergence occurs within their putative cytoplasmic domains, that of GP IX consisting of only 6 to 8 amino acids and that of GP Ib, containing approximately 34 residues. Each subunit contains

12851

12852 Glycoprotein Ib-IX Complex Expression

one N-linked carbohydrate chain and no 0-linked carbohy- drate chains. The extracytoplasmic domains of both of these proteins contain one copy of the leucine-rich motif.

Defects in either the subendothelial component (vWf) or the platelet component (GP Ib-IX complex) of the platelet adhesion axis lead to bleeding disorders (von Willebrand’s disease and the Bernard-Soulier syndrome, respectively). The Bernard-Soulier syndrome is a rare autosomal recessive dis- order characterized by low platelet counts and giant platelets in which the three components of the GP Ib-IX complex are nearly or completely absent from the platelet surface (George et ~ l . , 1984). Another glycoprotein known to be a member of the leucine-rich family, GP V (Shimomura et al., 1990; Roth et al., 1990), is also absent in these platelets (Clemetson et ~ l . , 1982; Berndt et QL, 1983). Glycoprotein V apparently does not co-isolate with the GP Ib-IX complex from normal platelets (Berndt et al., 1985). Thus, the reason that this glycoprotein, in addition to the three polypeptides of the GP Ib-IX complex, is absent from the membrane of Bernard-Soulier syndrome platelets is not known. Whereas the functional disorder seen in the platelets of different individuals with the Bernard- Soulier syndrome appears to be rather uniform, the platelets of individual patients may contain residual amounts of any of the subunits (Clemetson et ~ l . , 1982; Drouin et ~ l . , 1988; Hourdill6 et QL, 1990).

An important question regarding the pathogenesis of the Bernard-Soulier syndrome is the nature of the defect or defects that lead to the absence of the GP Ib-IX complex from the platelet surface. One possibility is that mutations that reduce or abolish expression of any of the individual subunits lead to reduced levels or complete absence of the remaining two subunits. The second possibility is that the clinical syn- drome is due solely to the absence of GP Ib, (the subunit containing the vWf-binding domain), which itself may be required for the expression of GP I b p and GP IX. This scenario presupposes that mutations of GP I b p and GP IX do not affect the expression or vWf-binding function of GP Ib, and are thus clinically silent. Another issue that remains unresolved is what role, if any, GP V has in the pathogenesis of the disorder.

The aim of the present studies was to address these ques- tions. Our approach was to express the cDNAs for the glyco- proteins individually and in various combinations in cultured cells. We show that all three subunits of the GP Ib-IX complex are required for the efficient expression of a functional com- plex on the plasma membrane. The recombinant complex associated with the cytoskeleton of the transfected cells through an interaction with actin-binding protein and bound vWf in a ristocetin-dependent manner. Furthermore, the com- plex was successfully expressed on the cell surface in three distinct heterologous cell lines in the absence of GP V. These results suggest that the lack of a functional GP Ib-IX complex in the membrane of platelets from patients with the Bernard- Soulier syndrome could result from decreased synthesis of any one of the three subunits but is unlikely to result from the absence of GP V.

EXPERIMENTAL PROCEDURES

Antibodies-The anti-GP Ib-IX monoclonal antibodies AK-2, FMC-25, and WM-23 (Berndt et al., 1988) were a kind gift from Dr. Michael Berndt (Westmead Hospital, Sydney, Australia). Mono- clonal antibody AN-51 (McMichael et al., 1981) was obtained from Dako (Carpinteria, CA), and monoclonal antibody SZ-1 (Berndt et al., 1988) was purchased from AMAC (Westbrook, ME). Anti-GP V antiserum was generously provided by Dr. David Phillips (COR Therapeutics, South San Francisco, CA). Polyclonal antisera were raised in rabbits against human glycocalicin and human actin-binding protein (Fox, 1985a) and against a peptide encompassing the entire

cytoplasmic domain of human GP Ib,. These sera were used to detect the polypeptides on immunoblots.

Expression Vectors-Cloning of the cDNAs for GP Ib,, GP Iba, and GP IX has been reported previously (Lopez et al., 1987, 1988; Hickey et al., 1989). The cDNA encoding GP IX was a generous gift from Dr. Gerald Roth of the Seattle Veterans Administration Hos- pital. The three cDNAs (each containing the entire coding sequence and 3”untranslated region) were subcloned individually into the eukaryotic expression vector pDX (a kind gift from Dr. Kathleen Berkner of Zymogenetics, Seattle, WA) (Berkner et al., 1986) in which transcription is driven by the adenovirus major late promoter and the SV40 enhancer. We sequenced the purified expression plas- mids to ensure that no unwanted errors were introduced during their construction. The thymidine kinase plasmid pHSVlO6 was purchased from Bethesda Research Laboratories.

Cell Culture and Transfections-Chinese hamster ovary (CHO) cells were grown in a 1:l mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium with 10% fetal bovine serum. L- cells and HeLa cells were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Plasmid DNA for transfection was prepared by the alkaline-lysis method as described (Birnboim and Doly, 1979). Ten pg of each of the expression plasmids was used in each of the experiments in which surface expression of the complex was analyzed by the ability of the cells to bind AK-2 beads; thus, the total quantity of plasmid DNA in the transfection of the three plasmids was 30 pg. The total DNA content was kept constant in transfections with less than the full complement of expression vectors by the addition of sheared salmon sperm DNA. In the experiment that was analyzed by flow cytometry, the total amount of DNA was kept constant by adding a greater quantity of plasmid DNA. The resistance markers (pSV2neo for CHO and HeLa cells, pHSVlO6 for L-cells) were added in a 1 : lO ratio or a 1:20 ratio of resistance plasmid to individual GP Ib-IX complex plasmid. Transfection was performed by the calcium phosphate precipitation method of Graham and van der Eb (1973). Briefly, the plasmids were mixed in the appropriate ratios, ethanol-precipitated, and washed in ethanol and left to dry in a sterile hood. The DNA was resuspended in 50 pl of sterile water, and 50 r l of 2.5 M CaC12 was added. This mixture was added with mixing to 500 pl of 2 X HEPES-buffered saline. After 20-30 min, the mixture was added to a dish of cells that had reached approximately 30% confluence. The precipitate was allowed to remain on the cells for 4 h the medium was then removed, and the cells were shocked with 15% glycerol in phosphate-buffered saline (PBS) for 5 min. Cells were subjected to selection in G418 (CHO and HeLa) or hypoxan- thine/aminopterin/thymidine medium (L-cells) 2 days after transfec- tion. Glass cloning rings were used to isolate individual resistant clones. Cells were selected for high levels of GP Ib-IX complex expression by several rounds of fluorescence-activated cell sorting (FACS).

The CHO cells transfected with only GP Ib, and GP Ib, that expressed GP Ib on their surface were selected by several rounds of FACS. These cells were then transfected by electroporation (2 X lo6 cells/ml; voltage, 1.2 kV; capacitance, 1 pF) with a plasmid encoding GP IX and analyzed by flow cytometry after 72 h for surface expres- sion of GP Ib-IX.

Bead Assay-Monoclonal antibody-conjugated polyacrylamide im- munobeads (Bio-Rad) (average diameter, 10 pm) were kindly provided by Dr. Robert Andrews (Gladstone Institute of Cardiovascular Dis- ease). Primary transfectants were tested after colonies of (2418- resistant cells were visible on the culture dish. The cells were washed twice with Dulbecco’s modified Eagle’s medium/F-12 medium and incubated with a suspension of beads at a concentration of 10 p1 of beads/ml (from an original bead suspension in water at a 1:l ratio (v/v)). Cells were then washed at least five times with PBS. Micros- copy of the cells was performed on a Zeiss inverted microscope.

Fluorescence Microscopy-Cells were grown to confluence on glass slides, washed several times with PBS, and fixed with 4% paraform- aldehyde for 30 min at 25 “C. In experiments in which the intracellular location of proteins was examined, the cells were permeabilized with 0.1% Triton X-100 (Sigma). The cells were washed several times to remove the paraformaldehyde. They were then incubated with AK-2 (16 pg/ml in PBS, 15 mM sodium acetate, 1:50 sheep serum) overnight at 4 “C. The cells were then washed five times in PBS, 15 mM sodium acetate and incubated with a 1:300 dilution (in the same buffer) of biotinylated sheep anti-mouse immunoglobulin G (IgG) that had been preincubated in fetal bovine serum for 3 h in the dark. After incuba- tion, the cells were again washed several times with PBS, 15 mM sodium acetate, and incubated with a 1:300 dilution of streptavidin-

Glycoprotein Ib-IX Complex Expression 12853

Texas Red (Zymed, South San Francisco, CA) for 30 min, and washed twice more. Where the distribution of the GP Ib-IX complex and actin-binding protein in the same cells was evaluated, fixed, perme- abilized cells were first incubated with polyclonal anti-actin-binding protein serum and AK-2. The cells were washed, incubated with biotinylated sheep anti-rabbit IgG and fluorescein isothiocyanate- conjugated anti-mouse IgG, washed, and incubated with streptavidin- Texas Red. Microscopy was performed on a Zeiss universal micro- scope.

Flow Cytometry and Cell Sorting-Cells were harvested by treat- ment with 0.53 mM EDTA for 5 min and washed twice with PBS. They were resuspended in 1 ml of Dulbecco's modified Eagle's me- dium containing 5% calf serum at a concentration of 2 pg of the relevant monoclonal antibody/ml. After the cells were incubated for 30 min at 25 "C, they were centrifuged at 160 X g for 10 min, washed twice in PBS, and incubated in 2 pg/ml fluorescein isothiocyanate- conjugated rabbit anti-mouse IgG. They were then filtered through 70-pm nylon mesh. Flow cytometry was performed on a Becton- Dickinson (San Jose, CA) FACS 440 cytometer at an excitation wavelength of 488 nm from an argon-ion laser. The relative fluores- cence intensity and relative cell size (forward angle light scatter) were measured. Data were analyzed on a DEC 11-750 computer with Electronic Desk software (Stanford University, Palo Alto, CAI. Sort- ing was done with a 70-pm nozzle using an 11-drop delay and 3-drop deflection. In most cases the 5% of cells with the highest levels of fluorescence intensity were collected.

uon Willebrand Factor Binding-Glycoprotein Ib-IX-positive L- cells and control untransfected cells were detached with 0.53 mM EDTA, washed twice in PBS, and resuspended at a concentration of 7.5 X lo7 cells/ml in PBS, 0.1% bovine serum albumin containing '"I-labeled vWf (1 pg/ml) in the absence or presence of 0.75 mg of ristocetin/ml at 37 "C. The binding reactions were terminated by centrifugation (10,000 X g, 4 min, 4 "C) of 100-pl aliquots through 30% (w/v) sucrose in PBS/1% bovine serum albumin. Radioactivity associated with the pellet was counted in a gamma counter after aspiration of the supernatant. When vWf binding was evaluated by flow cytometry, cells were detached as described above, then fixed. The cells were then incubated in 2.0 pM biotinylated vWf in 0.75 mg/ ml ristocetin for 30 min at room temperature, washed four times with 0.5 X PBS, 0.1 M sodium acetate, then incubated for 45 min with a 1:300 dilution of streptavidin-Texas Red, and analyzed by cytometry.

Association of the Glycoprotein Ib-IX Complex with the Cytoskele- ton-Analysis of the association of the GP Ib-IX complex with the cytoskeleton was performed as described for platelets (Fox, 1985a), with minor modifications. Briefly, GP Ib-IX-expressing CHO cells, L-cells, and mock-transfected controls were lysed on tissue culture dishes in a buffer containing 1% Triton X-100, 5 mM EGTA, 58 mM sodium borate, pH 8.0 (10 min, 25 "C). Protease inhibitors included in the buffer were 2 mg of leupeptin/ml (Vega Biotechnologies, Tucson, AZ), 1 mM phenylmethylsulfonyl fluoride (Sigma), 10 mM benzarnidine (Sigma), 0.2 mg of soybean trypsin inhibitor/ml (Milli- pore, Freehold, NJ), and 0.2 mM diisopropyl fluorophosphate (Sigma). The Triton X-100-soluble supernatant was removed, and the super- natant and insoluble residue remaining on the plate were solubilized and analyzed on one-dimensional 5-15% sodium dodecyl sulfate (SDS)-polyacrylamide gels.

Immunoprecipitation of Actin-binding Protein with the Glycoprotein Ib-IX Complex-Two 75-cmZ plates of confluent cells were lysed in the Triton X-100-containing lysis buffer just described except that the EGTA was replaced with 5 mM CaClz to induce depolymerization of actin filaments. Remaining actin filaments were then sedimented by centrifugation at 15,600 X g for 4 min, and the pellet was discarded. The supernatant was incubated with 50 pl of AK-2 immunobeads for 2 h at 4 "C. The immunobeads were washed three times in the lysis buffer plus inhibitors, boiled 5 min in 50 p1 of Laemmli loading buffer, and electrophoresed on a 5-15% SDS-polyacrylamide gel. The gel was transferred to nitrocellulose, and the blot was probed with '''I- protein A. After autoradiography the blot was reprobed with anti- actin-binding protein serum and autoradiography was repeated.

Analytical Methods-SDS-PAGE (Laemmli, 1970), Western blot- ting (Towbin et al., 1979), and autoradiography (Fox, 1985a) were carried out as described.

RESULTS

Expression Constructs and Cell Lines-Our objective was to develop an expression system with which to study the func- tions of the GP Ib-IX complex and to determine the require-

ments for functional expression of the complex. We therefore chose to subclone individually each of the subunits into the same expression vector. This would allow direct comparison of membrane expression of G P Ib, in cells transfected with different combinations of the individual subunits. The cell lines chosen for heterologous expression were CHO, mouse thymidine kinase-deficient (tk-) L-cells, and the human ep- ithelial carcinoma cell line HeLa. These cell lines were chosen on the basis of their lack of immunologically detectable G P Ib and GP V polypeptides, and the presence of actin-binding protein that was immunologically cross-reactive with human platelet actin-binding protein. Expression of the polypeptides of the GP Ib-IX complex and of GP V was examined by both immunoblot analysis and immunofluorescence using antisera raised against the human polypeptides. None of the three cell lines expressed cross-reactive proteins of molecular weights similar to those of the human proteins (data not shown). Immunofluorescence of unpermeabilized cells with anti-GP V serum was similar to that seen with control serum, and no increase in surface fluorescence was detected after GP Ib-IX transfection (not shown). A polypeptide cross-reactive with anti-human actin-binding protein antibodies was detected in the three cell lines by both immunoblotting and immunoflu- orescence. On SDS-PAGE this polypeptide migrated with a molecular weight identical to that of human platelet actin- binding protein. Immunofluorescence with anti-actin-binding protein revealed a submembraneous distribution in the three lines (data not shown).

Efficient Plasma Membrane Expression of Glycoprotein Ib, Requires the Presence of the Three Subunits of the Glycopro- tein fb-IX Complex-We evaluated the requirements for sur- face-membrane expression of the GP Ib-IX complex by trans- fecting CHO cells with expression plasmids for GP Ib, alone or by cotransfecting with plasmids for G P Ib, and GP I b p , for GP Ib, and GP IX, or for all three subunits together. The cells were selected for stable expression by cotransfecting with the plasmid pSV2neo, which confers resistance to neo- mycin and its analogue G418 (Southern and Berg, 1982). Surface expression of the complex was evaluated initially by the ability of G418-resistant transfected cells to bind poly- acrylamide beads to which the anti-GP Ib, monoclonal anti- body AK-2 had been coupled (Fig. 1). Fig. lA shows G418- resistant CHO cells that bound AK-2-conjugated beads along- side cells that did not express the AK-2 epitope. Immunoflu- orescence of cells from the same transfection (Fig. 1B) dem- onstrated that the complex is distributed fairly uniformly on the plasma membrane.

Using the bead assay to evaluate surface expression of the complex, we found that only transfections with the full com- plement of subunits yielded colonies of cells that expressed the complex on the plasma membrane (Fig. 2 4 ) . Flow cytom- etry was used as an independent method to evaluate surface expression of the complex (Fig. 2B). Once again, transfections with the three subunits resulted in complex expression on the cell surface as indicated by a rightward shift in fluorescence. Plasma membrane expression of the epitope was never de- tected in transfections with GP Ib, alone or with GP Ib, plus G P IX. In some transfections using GP Ib, and GP Ibp, a small population of positive cells was detected. These cells expressing GP Ib (GP Ib, and GP Ibe) on their surface were subjected to four rounds of FACS, each time selecting the population of cells with the highest surface expression. We noted that after several days of growth, selected cells tended to revert to a surface-negative phenotype. We therefore chose to transfect these cells, which we had previously shown to express GP Ib on their surface, with the G P IX plasmid.

12854 Glycoprotein Ib-IX Complex Expression

I" bc

FIG. 1. Surface expression of re- combinant GP Ib-IX complex ana- lyzed by monoclonal antibody im- munobeads. CHO cells transfected with cDNAs encoding the subunits of the GP Ib-IX complex were incubated with im- munobeads coated with the anti-GP Ib, monoclonal antibody AK-2 and evalu- ated for surface binding of the beads directly on the tissue culture dishes by light microscopy. A, light micrograph of GP Ib-IX-expressing CHO cell clones alongside nonexpressing clones (arrow- head, upper right). B , immunofluores- cence of GP Ib-IX-expressing cells in- cubated with AK-2 followed by biotin- ylated sheep anti-mouse antiserum and streptavidin-Texas Red. Uniform sur- face distribution of the recombinant complex is apparent.

Transfection with GP IX resulted in a large increase in the level of the complex expressed on the surface in the majority of cells (Fig. 2C). We conclude from these results that surface membrane expression of GP Ib, absolutely requires the pres- ence of GP Ib, and is most efficient when all three subunits of the GP Ib-IX complex are present. The results also indicate that expression of the complex does not require GP V. The results were similar in all three cell lines (data not shown).

Characterization of the Recombinant Complex-To charac- terize the recombinant complex, we used CHO and L-cell lines selected for high levels of GP Ib-IX surface expression by cloning and by several rounds of FACS. Analysis of the expressed recombinant polypeptides by SDS-PAGE revealed that the molecular weights were similar to those of the poly- peptides from platelets. Glycoprotein Ib, expressed in both CHO and L-cells migrated at a somewhat lower molecular weight than the platelet control (Fig. 3A), although this may result partially from differences in the amount of protein loaded. Alternatively, the broad molecular weight band ob- served in the platelet control may result from the presence of

several polymorphic variants of GP Ib, in the pooled platelets from which the complex was purified (Moroi et aL, 1984). Recombinant GP Ib, migrated with a molecular weight similar to platelet GP Ib, (Fig. 3 B ) and at a higher molecular weight when run under nonreducing conditions, a finding compatible with it being disulfide-linked to GP Ib, (data not shown).

Berndt et al. (1985) developed a monoclonal antibody (SZ- 1) that recognizes neither GP Ib nor GP IX alone but binds its epitope only when the complex has been reconstituted. This observation, coupled with the finding that monoclonal antibodies against GP IX bind to the same number of sites as those with epitopes on GP Ib (Du et al., 1987), indicated that on the platelet membrane all available GP Ib is complexed to GP IX and none of the polypeptides are present in excess. To determine whether the GP Ib (GP Ib, and GP Ib,) expressed in CHO or L-cells was associated with GP IX, flow cytometric analysis was conducted using three monoclonal antibodies: AK-2 (anti-GP IbJ, WM-23 (directed against the carbohy- drate-rich region of GP Ib,), and SZ-1 (anti-GP Ib-IX com- plex) (Fig. 4). A comparable shift in fluorescence intensity

Glycoprotein Ib-IX Complex Expression 12855

Fluorescence

C m o r I

Fluorescence

FIG. 2. Surface expression of the complex requires its three subunits. A, the requirements for surface membrane expression of the complex were analyzed by counting G418-resistant cells that bound AK-2 immunobeads in transfections with different combina- tions of expression plasmids. All transfections included the neomycin resistance plasmid pSV2neo. 200 colonies from each transfection were analyzed. B, separate transfections were analyzed using flow cytom- etry with AK-2 followed by fluorescein isothiocyanate-labeled anti- mouse IgG. The G418-resistant cells were analyzed 4 weeks after transfection. A small number of cells expressing the epitope on their surface were found in the transfection with GP Ib, and GP Ibs, whereas the curves obtained from transfections with GP Ib, alone (not shown) and GP Ib, with GP IX were identical with that from a mock-transfected control (not shown). The transfection including the three subunits resulted in a rightward shift of the curve. C, the cells shown in Fig. 2B from the transfection with GP Ib, and GP Ibs that express GP Ib on the surface were selected by four rounds of FACS. Reversion of most of the cells to a surface-negative phenotype was noted within a few days of growth (identical to the control histogram). These cells were transfected with a control plasmid or with a plasmid encoding GP IX and surface expression of the AK-2 epitope was evaluated at 72 h. Transfection with GP IX resulted in a dramatic increase in surface expression of the complex (dashed l ine) , whereas transfection with a control plasmid did not change the surface phe- notype (solid l i n e ) .

was observed with each of the antibodies, indicating that all of the surface GP Ib was associated with GP IX. The absence of a population of cells expressing uncomplexed GP Ib is further evidence that membrane expression requires the three subunits of the complex.

Q m L 0

L e

B.

FIG. 3. Immunoblot analysis of recombinant GP Ib a- and @-chains. Cells expressing surface GP Ib-IX complex were lysed in Laemmli sample buffer, electrophoresed on a 5-15% gradient SDS- polyacrylamide gel, and electrophoretically transferred to nitrocellu- lose membrane. The blots were probed with polyclonal antisera against portions of the extracellular domains of either GP Ib, or GP Ib,, (see “Experimental Procedures”). A, immunoblot employing anti- serum against GP Ib,. Mock-transfected and GP Ib-IX-expressing CHO and L-cells were analyzed. Two GP Ib-IX complex-expressing L-cell clones (L3D and L2H) are shown. The arrowhead indicates the band corresponding to GP Ib,. The bands below GP Ib, represent degradation products, which appear more prominent because of over- loading of the sample. B, immunoblot analysis with antiserum against GP Ib,. The SDS-PAGE was performed under reducing conditions. An immunoreactive band migrating with the same molecular weight as platelet GP Ibs was detected (arrowhead), which shifted to a position compatible with being disulfide-linked to GP Ib, when run under nonreducing conditions (not shown).

Surface expression of the complex was evaluated using five anti-GP Ib-IX complex monoclonal antibodies. Along with the antibodies just described and shown in Fig. 4, FMC-25 (anti-GP IX) (Berndt et al., 1988) and AN-51 (epitope on GP Ib,) (McMichael et al., 1981) were also used. Two of these antibodies, SZ-1 and AN-51, require a native conformation for epitope recognition. All of the antibodies yielded identical histograms, indicating that the recombinant complex was in a conformation similar to that of the platelet complex.

Recombinant GP Ib-IX Complex Associates with the Cyto- skeleton-In platelets, the GP Ib-IX complex is attached to a submembranous actin-containing structure that functions as a membrane skeleton (Fox, 1985b). This association is me- diated by actin-binding protein. When cells are lysed with Triton X-100, the cytoskeleton remains insoluble and can be sedimented by centrifugation. Membrane proteins that re- main with the Triton-insoluble portion are assumed to be associated with the cytoskeleton. To determine whether the GP Ib-IX complex expressed in CHO cells or L-cells was attached to cytoskeletal structures, cells were lysed in Triton X-100-containing buffers, and the relative amounts of GP Ib- IX in the soluble and insoluble fractions were determined. Most of the GP Ib-IX complex remained associated with the Triton-insoluble fraction (Fig. 5A), indicating an association with the cytoskeleton. Two approaches were used to deter- mine whether this association was mediated by actin-binding protein. First, cells were lysed with Triton X-100 under con- ditions that favored depolymerization of actin filaments. The

12856 Glycoprotein Ib-IX Complex Expression

AK-2 WM-23

-.. 100

Fluorescence

sz-1

90 c

Fluorescence Fluorescence

FIG. 4. Glycoprotein Ib expressed on the cell surface is complexed with GP IX. Flow cytometry analysis was performed on parallel samples with either of three monoclonal antibodies that recognize distinct epitopes within the G P Ib-IX complex. The antibody SZ-1, which recognizes an epitope dependent upon the formation of a complex, showed an identical pattern of reactivity as did two antibodies with euitoues within GP Ib,. AK-2 (amino-terminal ligand-binding domain), and WM-23 (carbohydrate-rich region). No cells expressing G P Ib without GP IX were detected.

- -

GP Ib-IX complex that had been released into the supernatant as a result of actin depolymerization was immunoprecipitated with anti-GP Ib antibodies. The immunoprecipitate was sub- jected to SDS-PAGE and transferred to nitrocellulose. The blot was first probed with anti-glycocalicin (the extracyto- plasmic domain of GP Ib,) serum, then reprobed with anti- actin-binding protein antibodies. Fig. 5B shows that actin- binding protein coimmunoprecipitated with GP Ib, thus in- dicating an association between the two components in the transfected L-cells. Identical results were obtained with CHO cells. Second, we determined whether expressed GP Ib-IX and actin-binding protein colocalized in intact cells. Immu- nofluorescence studies were performed on permeabilized cells using both anti-GP Ib and anti-actin-binding protein anti- bodies. Fig. 5C shows that the two components colocalize on the platelet membrane in cells expressing the complex. To- gether, these results indicate that human GP Ib-IX expressed in rodent cells associates with heterologous actin-binding protein.

Expressed Glycoprotein Ib-IX Complex Binds von Wille- brand Factor in a Ristocetin-dependent Manner-To deter- mine whether expression of the three subunits of the GP Ib- IX complex is sufficient to confer function, we evaluated binding of the natural ligand for GP Ib-IX, vWf. Although both platelets and vWf circulate freely in the blood, the interaction of the GP Ib-IX complex with vWf under phys- iological conditions requires that a shear-induced conforma- tional change occurs in one of the components of the reaction or that the vWf first associate with the subendothelial matrix. One method used to effect binding of soluble vWf to GP Ib- IX in vitro is the addition of ristocetin, a peptide antibiotic that induces platelet agglutination in platelet-rich plasma (Howard and Firkin, 1971). When a defect in the ristocetin- induced association of GP Ib-IX with vWf is observed clini- cally, this is taken as evidence that the interaction between the two molecules is the basis of the observed bleeding disor- der (Coller, 1987). We therefore chose to study the function of the expressed complex by evaluating the ristocetin-induced binding of vWf. The specificity of the interaction was deter- mined by comparing vWf binding of GP Ib-IX-expressing L- cells with that of mock-transfected control cells. Ristocetin- dependent binding of vWf was evaluated by two means. First, detached cells were incubated with biotinylated vWf in the

presence of ristocetin, then labeled with streptavidin-Texas Red. The shift in fluorescence was evaluated by flow cytom- etry (Fig. 6A) . In the second approach, cells were incubated with '251-labeled vWf for different lengths of time. The reac- tion was stopped by centrifuging the cells through sucrose cushions, and the amount of bound '251-labeled vWf was measured (Fig. 6B) . Both of these methods demonstrated that transfected cells expressing the complex bound much more vWf than did mock-transfected control cells and that this binding was dependent on the presence of ristocetin. In the absence of ristocetin, GP Ib-IX-transfected and mock-trans- fected cells bound equivalent, low levels of vWf (Fig. 6B) . Ristocetin also induced some binding of vWf to mock-trans- fected cells (Fig. 6A) , indicating that an endogenous molecule on these cells may be able to bind this ligand. However, there was considerably more binding of vWf to those cells that expressed the GP Ib-IX complex on their surface.

DISCUSSION

We have expressed the GP Ib-IX complex of human plate- lets in three nonhematopoietic cell lines. The recombinant complex behaves like the platelet complex in that it associates with actin-binding protein in the cytoskeleton through its cytoplasmic domain and it binds vWf. Our results indicate that this membrane complex, analogous to other plasma mem- brane complexes such as those of the integrin superfamily and the T cell receptor-CD3 complex of T lymphocytes, requires the presence of several polypeptides for surface mem- brane expression. Both of the smaller subunits of the complex (GP Ibp and GP IX) are required for efficient expression of the ligand-binding subunit (GP Ib,) on the cell surface.

Much of our understanding of the functions of the GP Ib- IX complex results from observations made of platelets from patients congenitally deficient in the complex, i.e. those with the Bernard-Soulier syndrome. The original observation made by Nurden and Caen (1975) was that platelets from patients with the Bernard-Soulier syndrome were deficient in the major sialic acid-containing glycoprotein of the platelet mem- brane, GP I. More detailed studies later showed that in addition to GP Ib (so named to distinguish it from other membrane glycoproteins of similar molecular weight), two other glycoproteins, GP IX and GP V, were also absent. Until now, the specific requirements for the surface expression of

Glycoprotein Ib-IX Complex Expression 12857

A Platelets7 - cno

GPLb, - 1

Fluorescence

Triton X - 1 0 0 soluble

TotalJ J LTriton X-100 insoluble

B anti- glycocalicin

anti. ABP

*%Z Ir GPlb- c

91K- ‘w .* 100K- L

platelets’ LL2H platelets’ LL2H

FlTC AK-2 Texas Red AEP

FIG. 5. Recombinant GP Ib-IX complex associates with the cytoskeleton of transfected cells. A, CHO cells expressing the G P Ib-IX complex were lysed in a buffer containing 1% Triton X-100 (see “Experimental Procedures”). The amount of G P Ib-IX in the total lysate and that found in the Triton-soluble and Triton-insoluble fractions was determined by electrophoresis of equivalent amounts on 5-15% SDS-polyacrylamide gels, Western blotting, and probing the blot with anti-GP Ib, antisera. The majority of the complex was found in the Triton X-100 insoluble residue, indicative of an associ- ation with the cytoskeleton. B, coimmunoprecipitation of actin-bind- ing protein with GP Ib-IX. Cells were lysed in the Triton X-100- containing lysis buffer, this time excluding EGTA and including 5 mM calcium chloride and DNase I to induce depolymerization of actin filaments. The lysates were then cleared of residual actin filaments by ultracentrifugation, and the supernatant was immunoprecipitated with AK-2 immunobeads. A Western blot of the immunoprecipitate was first probed with antiserum to glycocalicin (extracellular domain of GP IbJ (left panel) followed by antiserum to actin-binding protein (right panel). Actin-binding protein precipitated from the superna- tant with G P Ib-IX. C, colocalization of G P Ib-IX and actin-binding protein in intact cells. CHO cells from a primary transfection were fixed, permeabilized, and probed with the cells that are expressing the GP Ib-IX complex stained with AK-2 and a fluorescein isothio- cyanate-conjugated second antibody. The right panel shows staining with anti-actin-binding protein followed by a Texas Red-labeled second antibody. In the cells that expressed the complex, the actin- binding protein colocalized with G P Ib-IX.

these platelet membrane polypeptides were not known. Based on data presented in this manuscript, we predict that muta- tions of any of the three subunits of the GP Ib-IX complex that affect expression of the individual subunits or that pre- clude formation of the complex will abolish or greatly diminish the surface membrane expression of the complex on circulat- ing platelets. Thus, mutations of any of the three subunits of

B Ristocetin added No ristocetin 40 I

Time (minutes)

FIG. 6. Ristocetin-dependent binding of vWf by GP Ib-IX complex-expressing cells. A, flow cytometry assessment of risto- cetin-dependent binding of G P Ib-IX complex-expressing L-cells. Biotinylated vWf was bound to detached cells in the presence of 0.75 mg of ristocetin/ml followed by streptavidin-Texas Red. The cells were then analyzed by cytometry. Two peaks were visualized in the G P Ib-IX-expressing cells, which paralleled the binding of the mono- clonal antibody AK-2. A small peak of cells that bound vWf was observed in mock-transfected cells. B, time course of vWf binding to G P Ib-IX-expressing cells. Mock-transfected or G P Ib-IX-expressing L-cells were incubated with ””I-labeled vWf in the presence or absence of ristocetin. At different time points the reaction was terminated by centrifugation through sucrose. The average of two determinations was plotted.

the complex could lead to similar clinical phenotypes. In addition, because a cDNA for G P V was not included in the transfections in the present studies, and because an endoge- nous protein cross-reacting with a polyclonal GP V antibody was not detectable on the surface of untransfected or trans- fected cells, our findings indicate that the GP Ib-IX complex does not require G P V for membrane expression. However, we cannot exclude the possibility that expression of GP V might further increase the surface expression of the complex. Conversely, the absence of G P V from the membrane of Bernard-Soulier platelets may indicate that GP V requires the GP Ib-IX complex for membrane expression.

In normal platelets, G P Ib,, GP IbO, and GP IX are present on the plasma membrane in equal amounts (Du et al., 1987). One mechanism that could account for these findings is coordinated synthesis of the three subunits. Against this notion, however, we demonstrated that, as in platelets, no uncomplexed G P Ib was detected on the surface of cells transfected with cDNAs for the three subunits. Because het- erologous promoters were used in the expression constructs, it is extremely unlikely that the absence of uncomplexed GP Ib on the surface of transfected cells would be due to coordi- nated synthesis of the subunits. In addition, we demonstrated that transfection of GP IX into cells that had previously been transfected with G P Ib, and GP Ib8 resulted in greatly in- creased surface expression of the complex. Thus, we show that rather than coordinated synthesis of the three subunits, the likely reason that GP Ib,, GP I b O , and GP IX are present in the platelet plasma membrane in equal amounts is that the

12858 Glycoprotein Ib-IX Complex Expression

membrane expression and stability of the complex require all three subunits.

The mechanisms by which one subunit affects the surface expression of an intact complex are not clear. In the case of the platelet GP IIb-IIIa complex and other integrins, the p- subunit (GP IIIa or p3 for the platelet integrin) is necessary for appropriate posttranslational processing of the cy-subunit, a prerequisite for the expression of the complex on the cell surface (O'Toole et al., 1989). However, it is uncertain whether the amounts of the cy- or of the @-subunit are limiting because conflicting data have been reported (Rosa and McEver, 1989; Duperray et al., 1989). The situation of the T-cell receptor- CD3 complex is more complex because there are seven poly- peptides associated in the membrane complex and several mechanisms operate for efficient surface expression of the complete complex. Some of the polypeptides appear to protect other members of the complex from proteolytic degradation (Wileman et al., 1990), whereas a t least one of the CD3 polypeptides (CD3C) (Minami et al., 1987) and both of the T- cell receptor polypeptides (Sancho et al., 1989) are necessary for transport of the entire complex to the T lymphocyte cell surface. The means by which GP Ibo and GP IX increase the surface expression of GP Ib, is not known and may involve more than one mechanism. Based on our findings that intra- cellular GP Ib, was not detectable in transfections that did not include GP Ibp: one possibility is that GP I b o prevents the intracellular degradation of GP Ib,. This possibility is also consistent with the preliminary finding that in COS cells transfected with only GP Ib,, GP Ib, did not appear on the cell surface, but a truncated amino-terminal fragment of the protein was detected in the tissue culture medium (Handin and Petersen, 1989).

We have demonstrated that the human GP Ib-IX complex expressed in two rodent cell lines associates with the cyto- skeleton of these cells as it does in platelets. In platelets this association is mediated by actin-binding protein (Fox, 1985a). In the cell types tested, actin-binding protein was present in a peripheral location, had a molecular weight similar to the human platelet molecule, and was recognized by antiserum raised against human actin-binding protein. Colocalization of actin-binding protein with the GP Ib-IX complex on the plasma membrane and its immunoprecipitation using anti- GP Ib antibodies strongly suggest that in transfected cells the association of the complex with the cytoskeleton is through actin-binding protein. Association of human platelet GP Ib- IX with rodent fibroblast actin-binding protein implies that the glycoprotein-binding domain of actin-binding protein has been conserved in mammals. Because actin-binding protein is present in many types of cells, it is conceivable that this domain may serve to link the cytoskeleton to as yet uniden- tified membrane proteins in other cell types. Such proteins would be predicted to contain sequences homologous to those found in the cytoplasmic domain of GP Ib-IX. The establish- ment of stable cell lines that express the GP Ib-IX complex thus provides a useful model system with which to study the role of the interaction between GP Ib-IX and actin-binding protein in regulating events on both sides of the plasma membrane.

That the recombinant complex is in a conformation similar to the platelet complex is suggested by the finding that five monoclonal antibodies that bind the receptor on the platelet surface also bind the recombinant receptor. Some of these antibodies do not recognize the denatured polypeptides, be- cause they require proper folding for epitope recognition.

J. A. Lbpez, B. Leung, C. C. Reynolds, C. Q. Li, and J. E. B. Fox, unpublished observation.

Furthermore, the complex binds vWf in a ristocetin-depend- ent manner, as in platelets. Thus, the expression system will allow us to study the interaction of vWf with the GP Ib-IX complex and the effects of mutations of the complex and alterations in its association with the cytoskeleton on this interaction.

Acknowledgments-We thank Drs. Robert Andrews, Chris Brown, David Farrell, and Janet Boyles for helpful discussions; Dr. Stanley C. Rall, Jr., for critical reading of the manuscript; Charles Benedict

tion; and Sally Gullatt Seehafer and A1 Averbach for editorial assist- and Tom Rolain for graphics; Tony Gridley for manuscript prepara-

ance. We are also grateful to Dr. Gerald Roth for providing the cDNA for GP IX, Drs. Michael Berndt and David Phillips for providing antibodies, Dr. Kathleen Berkner for the expression vector, Dr. Israel Charo for the biotinylated vWf, and Bill Huyn of the University of California, San Francisco, Laboratory for Cell Analysis, for perform- ing the cytometry analysis and cell sorting.

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