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

Efficient plasma membrane expression of a functional platelet glycoprotein Ib-IX complex requires the presence of its three subunits.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
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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…