|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 January 2007, Vol. 109, No. 2, pp. 603-609. Prepublished online as a Blood First Edition Paper on September 28, 2006; DOI 10.1182/blood-2006-05-024091.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Glycoprotein Ib
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
and one Ibß subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ib, C484 or C485, forms the disulfide bond with Ibß. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibß. In the context of isolated peptides containing the Ib or Ibß transmembrane domain and nearby Cys residue, C484 and C485 in the Ib peptide were both capable of forming a disulfide bond with the Ibß peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibß subunits but only 1 Ib and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ib, not the number of Ibß subunits therein. Overall, these results demonstrate that Ib is covalently connected to 2 Ibß subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The ß2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
subunit in the complex also transmits a signal to the platelet that leads to platelet activation and aggregation.1,2 In addition to mediating outside-in signals, the GP Ib-IX-V complex is also capable of transmitting intracellular signals to the outside.3–7 How this receptor complex mediates signaling in both directions is not clear, but recent work has suggested the likely interaction between the Ib and Ibß subunits as a potential key element of the mechanism.8–10 Thus, understanding the interaction between GP Ib and GP Ibß, and in general the organizing principle of the GP Ib-IX-V complex, will help to unveil the mechanism underlying transmembrane signaling mediated by the complex.
It is widely accepted that the GP Ib-IX-V complex contains 7 subunits composed of 4 different polypeptides: Ib, Ibß, IX, and V with a respective stoichiometry of 2:2:2:1.11–15 Each subunit is a type I transmembrane protein. A disulfide bond links GP Ib and GP Ibß, forming a complex known as GP Ib, that in turn interacts noncovalently with GP IX and GP V to generate the Ib-IX-V complex. The Ib extracellular domain contains 9 Cys residues, 7 of which reside in the N-terminal domain that contains the binding site for VWF. The N-terminal domain contains 3 disulfides, C4-C17, C209-C248, and C211-C264.16 C65 is buried in the hydrophobic core of the N-terminal domain and is therefore unpaired.17,18 The other 2 Cys residues in GP Ib, C484 and C485, are located near the transmembrane (TM) domain and are conserved across species. The Ib/Ibß ratio of 1:1 in the receptor complex predicts that only 1 of the 2 Cys residues forms a disulfide bond with the membrane-proximal C122 in GP Ibß (Figure 1). However, it is not clear which one is paired with C122, and, more intriguingly, what role the free thiol group of the other Cys residue plays. It seems paradoxic that 2 neighboring Cys residues are exposed to presumably similar, if not the same, oxidizing environments, yet they have entirely different fates.
In the present study, we sought to assign the disulfide between GP Ib and GP Ibß. To our surprise, we found that both C484 and C485 are linked to GP Ibß by disulfide bonds. In other words, contrary to the currently prevailing model, 1 Ib subunit is linked through disulfide bonds to 2 Ibß subunits.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The vector pDX19,20 was used in the expression of GP Ib-IX complex in Chinese hamster ovary (CHO) cells. The CHO K1 cell line was obtained from ATCC (Manassas, VA). The expression vector pGEX-4T-3 was purchased from Amersham Biosciences (Piscataway, NJ). WM23, an anti-Ib monoclonal antibody, was kindly provided by Dr M. Berndt. Other antibodies against individual subunits of GP Ib-IX complex, including FMC25, Gi27, SZ2, and AK2, were purchased from Axxora (San Diego, CA), Beckman Coulter (Fullerton, CA), Chemicon (Temecula, CA), or Santa Cruz Biotechnology (Santa Cruz, CA). An anti-myc antibody was purchased from Sigma (St Louis, MO), and an HRP-conjugated anti-HA antibody was from Roche (Indianapolis, IN).
Effect of Cys mutations on the Ib-Ibß disulfide in transfected CHO cells
Mutations of the target cysteine residue to serine were carried out in the pDX vectors containing the indicated cDNA19,20 using a Quikchange mutagenesis kit (Stratagene, La Jolla, CA). The mutant gene sequences were confirmed by DNA sequencing (SeqWright, Houston, TX).
Constructs containing the Ib, Ibß, and IX cDNAs were transiently cotransfected into CHO K1 cells as previously described.21 At 2 days after transfection, cells from each transfection (
5 x 105 cells) were harvested and lysed in 60 µL cell-lysis buffer (1% Triton X-100, 5 mM CaCl2, 5 mM N-ethylmaleimide, 58 mM sodium borate, pH 8.0) containing 10% (vol/vol) protease inhibitor cocktail for mammalian tissues (Sigma). The supernatant from the cell lysate was mixed with standard Tris-glycine SDS sample buffer (pH 6.8) and resolved using a precast 4-12% Bis-Tris NuPAGE SDS gel with MOPS running buffer (Invitrogen, Carlsbad, CA) or, if necessary, a 5% Tris-glycine SDS gel with standard Tris-glycine running buffer. MagicMark protein standard (Invitrogen) was loaded on each gel as molecular weight markers. After electrophoresis, proteins were transferred to a PVDF membrane, immunoblotted with appropriate antibodies, and detected by chemiluminescence. In parallel, some transfected cells were stained using anti-Ib or anti-IX antibodies for fluorescence-activated cell sorting (FACS) analysis of surface expression levels of the GP Ib-IX complex.21
Production of Ib and Ibß TM peptides
A 10 x His sequence was inserted following the N-terminal Met residue of the glutathione S-transferase (GST) domain encoded in the pGEX-4T-3 vector (Amersham Biosciences), generating an expression vector termed pHex. The genes encoding residues S473 to R542 in GP Ib and L121 to S181 in GP Ibß were amplified by polymerase chain reaction (PCR) from the respective cDNAs and inserted into the pHex vector as a BamHI-XhoI fragment. The His-GST-TM fusion protein was overexpressed in the inclusion body of Escherichia coli and purified according to published protocols.22,23 Briefly, the fusion protein was first separated from soluble cytosolic proteins and membrane fractions22; solubilized in 50 mM Tris·HCl, pH 8.0, 100 mM NaCl, 0.2% N-lauroyl sarcosine, and 5 mM ß-mercaptoethanol; and purified by Ni-affinity chromatography. The purified protein was then cleaved by thrombin (10-20 U/mg protein) overnight at room temperature in 50 mM Tris·HCl, pH 8.0 buffer containing 0.1% N-lauroyl sarcosine and 200 mM imidazole. The TM peptides were separated from the His-GST fragment by preparative reverse-phase high-performance liquid chromatography (HPLC).23 The purity and identity of the Ib and Ibß TM peptides was confirmed by SDS–polyacrylamide gel electrophoresis (PAGE), analytic HPLC and MALDI-TOF mass spectrometry (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Extinction coefficients of these peptides were calculated according to Pace et al.24
In vitro thiol-disulfide exchange assay
Purified TM peptides were mixed with dodecylphosphocholine (DPC) (Avanti Polar Lipids, Alabaster, AL) in ethanol at a peptide-to-detergent molar ratio of 1:200. The solution was dried by argon stream to a thin film and placed under vacuum overnight. The dried protein-to-detergent mixture was hydrated in 40 µL reaction buffer (100 mM Tris·HCl, pH 8.0, 100 mM KCl, 2 mM EDTA, 1 mM oxidized glutathione, 1 mM reduced glutathione) purged with helium gas, topped with argon gas, sealed, and incubated at room temperature for 6 hours to allow the thiol-disulfide exchange reaction to reach equilibrium. In the control reaction, 2.5 mM freshly prepared dithiothreitol (DTT) was included. The final peptide concentration in the reaction was 25 µM. To quench the reaction, 6.7 µL 0.5 M HCl, followed by 33.3 µL organic solvent containing 0.1% TFA, was added to the glass vial. The reaction products were analyzed by reverse-phase HPLC in a C4 or phenyl analytic column (Grace Vydac, Hesperia, CA) using a linear gradient of buffer A (0.1% TFA) and buffer B (6:3:1 vol/vol 2-propanol-to-acetonitrile-to-water). The assignment of the eluted peaks was assisted by mass determination (Protein Core Facility, Baylor College of Medicine).
C-terminal epitope tagging of GP Ib, GP Ibß, and GP IX
The DNA fragment encoding the HA tag (YPYDVPDYA) or myc tag (EQKLISEEDL) was attached, in a step-wise fashion, to the C-terminal end of the Ib, Ibß, or IX cDNA using a Quikchange mutagenesis kit (Stratagene). The mutant gene sequences were confirmed by DNA sequencing (SeqWright). For transient transfection, the total amount of DNA encoding the tagged subunits (single tag or 2 tags) was the same as the other 2 subunits. Lysates from transfected cells were pretreated with 40 µL 50% Protein G-agarose beads (Sigma) at 4°C for 1 hour, then immunoprecipitated with 1 µg indicated antibody and 80 µL 50% Protein G-agarose beads at 4°C overnight. The proteins bound to the beads were eluted in Tris-glycine SDS sample buffer, separated in a 4-12% Bis-Tris SDS gel under nonreducing conditions, and immunoblotted using an HRP-conjugated anti-HA antibody.
Preparation of human platelets and Ib VNTR genotyping
Washed human platelets were obtained from blood freshly donated by healthy persons and prepared as described before.25 The protocol was approved by the Institutional Review Boards of The University of Texas Health Science Center at Houston. Both donors signed informed consent, in accordance with the Declaration of Helsinki, before 10 mL blood was drawn. The platelets were pelleted by centrifugation at 1500g for 10 minutes; washed twice with 10 mL of 30 mM glucose, 120 mM NaCl, 10 mM EDTA, 5 mM sodium citrate, pH 6.5; washed once with 10 mL of 134 mM NaCl, 10 mM EDTA, 10 mM Tris·HCl, pH 7.4; and then lysed in cell-lysis buffer to 1 x 109 platelets/mL. Finally, 1 to 2 µL supernatant was used for SDS-PAGE and Western blot.
Genomic DNA samples that had been identified to contain type A, B, C, or D VNTR in previous studies were used as the templates to amplify the gene fragments containing the Ib VNTR encoding sequence (V.A.-K., unpublished results, January 2001). The 2 primers used were 5'-TCCACTGCTTCTCTAGACAG and 5'-TTCTCTCAAGGTCCCCAAAC. The PCR fragments were digested with XbaI and XmaI and inserted, respectively, into the Ib cDNA gene that had been cloned into the pcDNA3.1 vector (Invitrogen). Expression vectors containing various types of VNTR were confirmed by DNA sequencing.
Genomic DNA of the 2 donors in this study was prepared from the buffy coat using the QIAamp DNA blood mini kit (Qiagen, Chatworth, CA), and used as the template for PCR. The 2 primers used for PCR were 5'-CTATTCTACTCATGGTCCACTGCTTCTCTAGAC and 5'-GGAAGCGAACCTCGAAGGAAGAGCAGCCAGGC. The PCR products were analyzed by agarose gel electrophoresis and subsequently sequenced (SeqWright).
Deglycosylation of GP Ib from platelet and CHO cells
The lysates of either human platelets (50 µL) or transiently transfected CHO cells (500 µL from a 10-cm culture dish) was immunoprecipitated with anti-Ib antibody SZ2 (30 µg/mL) and protein G-agarose for 2 hours at 4°C. After 3 times of washing by PBS containing 0.1% Triton X-100, GP Ib bound to the agarose beads was eluted by boiling for 10 minutes in 40 to 50 µL 50 mM sodium phosphate, pH 7.0, containing 0.1% SDS. The eluted GP Ib was then treated with a mixture of PNGase F, sialidase, ß-galactosidase, glucosaminidase, and O-glycosidase (supplied in the Enzymatic CarboRelease Kit; QA-Bio, San Mateo, CA) at 37°C for 3 hours. Afterward, the treated proteins were analyzed by SDS-PAGE and blotted with WM23.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-Ibß disulfide
To determine which membrane-proximal Cys residue in GP Ib forms the disulfide bond with GP Ibß (Figure 1), 3 Ib mutant cDNAs, which contain mutation C484S (termed SC), C485S (CS), and the double mutation C484S/C485S (SS), respectively, were constructed (Figure 2A). Each mutant cDNA was transiently cotransfected with the wild-type Ibß (ßC) and IX cDNAs into CHO cells. The expression levels of the GP Ib-IX complex, assayed by FACS to assess surface expression (Figure S2) and by Western blot to measure overall expression (Figure 2B), were not significantly affected by replacement of the Cys residues.
|
|
-Ibß disulfide were initially analyzed after resolution by SDS-PAGE under nonreducing conditions (Figure 2B). In CHO cells expressing the wild-type Ib-IX complex (CCßCIX cell), the majority of GP Ib was in complex with GP Ibß, resulting in a dominant GP Ib band in the gel. Replacing both Cys residues with Ser rendered GP Ib unable to form any intermolecular disulfide bond in SSßCIX cells. When either one, but only one, of the 2 Cys residues was replaced with Ser, GP Ib was linked to another protein by a disulfide bond to generate a complex that had an apparent molecular weight approximately averaging those of GP Ib and GP Ib.
To test whether GP Ib was still covalently linked to GP Ibß in this new complex, a mutant Ibß cDNA containing the mutation C122S (ßS) was constructed, and a series of transiently transfected CHO cells was analyzed. In CCßSIX cells, GP Ib was absent, and the majority of GP Ib was not covalently linked to any proteins, confirming that C122 is required for the Ib-Ibß disulfide (Figure 2C). A small portion of GP Ib formed disulfide bond(s) with not-yet-identified proteins to produce complexes of apparent molecular weights around 220 kDa (Figure 2C). A similar distribution of GP Ib was observed in SCßSIX cells, indicating that C485 is still linked with C122 of GP Ibß when C484 was replaced by Ser. In CSßSIX cells, the majority of GP Ib was present in protein complexes of approximately 220 kDa. An additional band was observed, but it was not the same as in SCßCIX or CSßCIX cells (Figure 2D), indicating that C122 was involved in an intermolecular disulfide with C484 in CSßCIX cells.
Overall, these results showed that, in transfected CHO cells, both C484 and C485 of GP Ib form a disulfide bond with C122 of GP Ibß. Mutating C484 or C485 did not affect the ability of the other residue to form a disulfide bond with C122. In the absence of C122, the Cys residues in GP Ib, especially C484, formed disulfide bond(s) with not-yet-identified proteins. In the GP Ib-IX complex, the only explanation for both C484 and C485 to form disulfide bonds with C122 is that GP Ib forms covalent links with 2 copies of GP Ibß.
In vitro formation of the Ib-Ibß disulfide bond
We next used the thiol-disulfide exchange assay to test whether we could replicate formation of the Ib-Ibß disulfide bond(s) between model peptides in vitro. In an earlier study we showed that replacing either the Ib or Ibß TM domain with an unrelated sequence affected formation of the Ib-Ibß disulfide bond in transfected CHO cells.21 Given the close proximity of the Ib-Ibß disulfide to the TM domains (Figure 1), it is possible that the interaction between the Ib and Ibß TM domains favorably positions the membrane-proximal Cys residues for disulfide bond formation.
The thiol-disulfide exchange reaction has been used qualitatively and quantitatively to measure protein interactions, protein folding, and stability.26–29 It was adapted to monitor association of TM helices in detergents and lipid bilayers.30,31 In this assay, Cys-containing peptides or proteins were placed in an environment in which the thiol-disulfide exchange occurs reversibly and quickly. Thus, disulfide formation depends largely on protein-protein association to bring 2 Cys residues into proximity and the intrinsic propensity of the Cys residue to form a disulfide bond. The extent of disulfide formation, in the form of new covalent structure, can be readily monitored by HPLC.
Three Ib TM peptides and 1 Ibß TM peptide were obtained (Figure S1). They contained the membrane-proximal Cys residue(s) in question, the TM domain, and a part of or the entire cytoplasmic domain of the respective subunit (Figure 3A). The simplest reaction scheme, consisting of a TM peptide with one Cys residue, contains thermodynamically coupled dimerization and disulfide formation steps (Figure 3B). In DPC micelles, greater than 80% of the Ibß TM peptide formed a disulfide-linked homodimer; by contrast, a significantly lower portion of the Ib TM peptide formed a homodimer (Figure 3C). Because the dimerization step was the same for both SC and CS peptides, more CS homodimer products indicated that C484 is more likely than C485 to form a disulfide bond between 2 Ib peptides.
|
TM peptide containing only one Cys residue, either C484 or C485, the dominant product in the reaction, was the disulfide-linked ß heterodimer (Figure 3C). In other words, both SC and CS TM peptides were able to out-compete with the Ibß TM peptide for association with the Ibß TM peptide, suggesting that the TM-TM interaction between the Ib and Ibß TM domains and their neighboring residues is strong and specific. The reactivities of C484 and C485 with C122 to form the intermolecular disulfide bond were similar, judged by the extent of heterodimer formation in each reaction (Table S1).
Because of its 2 Cys residues, the CC TM peptide generated more products in the thiol-disulfide exchange reaction (Figure 3D). Consistent with the self-oligomerizing ability of the SC and CS peptides, the majority of the CC peptide, when incubated by itself, was in the oxidized monomeric form (with an intramolecular disulfide). A number of products were generated when the CC peptide was mixed with the Ibß TM peptide. An ß2 heterotrimeric complex containing 2 intermolecular disulfides was a major product (Figure 3D). Note that a significant amount of oxidized CC monomer and a disulfide-linked ßß homodimer were also present. Together, these products are consistent with the ß2 model, albeit with a different disulfide pattern among the 3 peptides.
Overall, these results matched those obtained by cell transfection remarkably well. Both C484 and C485 were capable of forming a disulfide bond with Ibß; C484 was more likely than C485 to form a disulfide bond with other proteins. C122 in GP Ibß prefers to make a disulfide bond with Ib. Finally, the association between the Ib and Ibß TM domains and their flanking regions supports the ß2 configuration, facilitating formation of the intermolecular disulfides.
GP Ib-IX complex expressed in CHO cells contains 2 copies of GP Ibß but only 1 copy of GP Ib and IX
As an alternative test of whether 2 copies of GP Ibß are present in GP Ib, coimmunoprecipitation (co-IP) experiments were used to determine whether HA-tagged GP Ibß (ßHA) was in the same protein complex with myc-tagged GP Ibß (ßmyc) in transfected CHO cells (Figure 4A). Although addition of the HA or myc tag to GP Ibß led to a moderate decrease in surface expression of the GP Ib-IX complex (Figure S2), its expression level was sufficiently high to permit co-IP studies. In transfected CHO cells, a significant amount of GP Ibß was in a presumably denatured form, resulting in a "smearing" background in the nonreducing SDS gel (Figure 4A). To block out false-positive co-IP signals that may result from nonspecific interactions or simple aggregation of misfolded GP Ibß, only the Ibß subunits that had been properly assembled into the Ib-IX complex were monitored. Thus, both co-IP and SDS-PAGE steps were carried out under nonreducing conditions so that GP Ib could be kept intact and subsequently visualized by Western blot.
|
ßHAIX cells by blotting for the HA tag amid the smearing background (Figure 4A). Further, HA-containing GP Ib coprecipitated with anti-Ib (AK2) and anti-IX (FMC25) antibodies (Figure 4A), indicating that attachment of the HA tag to GP Ibß did not affect assembly of the GP Ib-IX complex or formation of the Ib-Ibß disulfide bonds. The same results were observed after the addition of the myc tag (data not shown).
In the CHO cells transfected with both tagged Ibß subunits as well as the wild-type Ib and IX subunits (ßHAßmycIX cells), the HA-containing GP Ib coprecipitated with anti-myc antibody but not with nonspecific IgG antibody, indicating that both ßHA and ßmyc subunits were in the same Ib-IX complex (Figure 4A). Several unidentified HA-containing protein complexes larger than GP Ib also coprecipitated with anti-myc antibody, which may be due to the nonspecific interactions of misfolded GP Ibß. In contrast to the cocomplexation of ßHA and ßmyc subunits, HA-tagged GP Ib (HA) did not coprecipitate with anti-myc antibody in HAmycßIX cells (Figure 4B), nor did HA-tagged GP IX with anti-myc antibody in ßIXHAIXmyc cells (Figure 4C). Therefore, there are only 1 Ib, 1 IX, and at least 2 Ibß subunits in the GP Ib-IX complex expressed in CHO cells.
GP Ib from human platelets and CHO cells are equivalent
The absence of a nucleus precludes any simple transfection studies on the platelet. To shed light on the Ib/Ibß ratio in GP Ib expressed in the platelet, we compared the apparent molecular weights of GP Ib, GP Ib, and GP Ibß derived from transfected CHO cells and freshly prepared resting human platelets. We obtained human platelets from 2 healthy donors, according to the protocol approved by the Institutional Review Board. Lysates from platelets and from CHO cells expressing the wild-type Ib-IX complex were resolved by SDS-PAGE and then probed with anti-Ib (WM23) or anti-Ibß (Gi27) antibodies.
The apparent molecular weights of GP Ib and GP Ib from platelets were higher than those from CHO cells, respectively (Figure 5A). Because GP Ibß from CHO cells and platelets had the same apparent molecular weights (Figure 5B), the molecular weight difference in GP Ib was due to that in GP Ib. To pinpoint the difference in GP Ib from platelets and CHO cells, we first analyzed the number of 13-residue tandem nucleotide repeat sequence (VNTR) in the Ib genes from the 2 donors. These repeats in the macroglycopeptide region of GP Ib contain multiple glycosylation sites, and the VNTR polymorphism contributes to variation in the Ib molecular weight.32,33 As shown in Figure 5C and later confirmed by DNA sequencing (not shown), the Ib cDNA gene that had been used in this study had type D VNTR (1 repeat), whereas donor no. 1 had heterozygous type C/D VNTR alleles and donor no. 2 type C. Subsequent direct comparison of type C VNTR-containing GP Ib from platelets and CHO cells showed that the VNTR polymorphism did not account for all the difference in the Ib molecular weight. Further, after being partially deglycosylated under the same condition, GP Ib from donor no. 2 and CHO cells had the same molecular weights (Figure 5D). Thus, the size difference between GP Ib from human platelets and transfected CHO cells was due to a combination of VNTR polymorphism and glycosylation difference in GP Ib, not the number of Ibß subunits therein. We conclude that in the resting platelet GP Ib contains 1 copy of GP Ib along with 2 copies of GP Ibß, just as in CHO cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
is connected by disulfide linkages to 2 Ibß subunits in the GP Ib-IX-V complex in the resting platelet. This finding differs from the widely accepted stoichiometry of the complex, in which the Ib/Ibß ratio was thought to be 1:1. The stoichiometry of the GP Ib-IX-V complex was previously estimated primarily by quantitating the number of subunits present on the platelet surface. Using subunit-specific antibodies, a ratio of 2:2:1 was estimated for the Ib, IX, and V subunits in the complex.12–14 GP Ibß was not included in these estimates because of the lack of suitable Ibß-specific antibodies. Instead, a Ib/Ibß ratio of 1:1 was deduced from the apparent molecular weights determined by SDS-PAGE. An apparent molecular weight of 170 kDa for GP Ib, 135 kDa for GP Ib, and 25 kDa for GP Ibß is most consistent with a ß heterodimeric model.11,12 As far as we are aware, this is the only evidence supporting a Ib/Ibß ratio of 1:1.
Results presented here support a Ib/Ibß ratio of 1:2. First, mutating either C484 or C485 in GP Ib resulted in the same Ib- and Ibß-containing complex in CHO cells with an apparent molecular weight averaging those of GP Ib and GP Ib. Furthermore, both C484 and C485 consistently formed disulfide bonds with GP Ibß in the context of isolated TM peptides. Finally, co-IP of differently tagged subunits showed the presence of at least 2 Ibß subunits but only 1 copy of Ib and IX in the GP Ib-IX complex. Therefore, we propose that the molar ratio among the Ib, Ibß, IX, and V subunits in the complex is 2:4:2:1 (Figure 6).
|
- or IX-specific antibodies.13,34 These bands were attributed to possible "intramolecular disulfide cleavages in the -chain of glycoprotein Ib"34 and/or "partial reduction of GP Ib."13 However, reduction of intramolecular disulfides in GP Ib did not affect its mobility in the SDS gel (Figure 2B, compare Ib from SSßCIX cells in the nonreducing condition with the reducing condition). Generation of 2 products (Ib-Ibß and Ib) from partial reduction of GP Ib can be readily explained by a 1:2 ratio but not by a 1:1 ratio. Indeed, the lower band of the 2 minor bands seemed to match well with GP Ib under reducing conditions.
The revised stoichiometry of the GP Ib-IX-V complex will require reexamination of some data related to GP Ib. More importantly, it will help shed light on the mechanism by which this receptor complex carries out its function. For instance, the cytoplasmic signaling protein 14-3-3
can interact with both Ib and Ibß cytoplasmic domains.35–39 Both interactions are important to the function of the GP Ib-IX-V complex.3–5,8,9,40 14-3-3 is a dimer, and disrupting 14-3-3 dimerization decreases its affinity for the GP Ib-IX complex but not the isolated peptide containing the Ib cytoplasmic domain.41 On the basis of the ß heterodimeric model for GP Ib, it was thought that 2 monomers in the 14-3-3 dimer interact with the Ib and Ibß cytoplasmic domains, respectively, with the implicit assumption that one 14-3-3 dimer interacts with one GP Ib complex.41 Now that the ß2 heterotrimeric model posits an additional Ibß cytoplasmic domain and consequently a total of four 14-3-3–binding sites in GP Ib, the Ib-14-3-3 interaction and its likely alteration in response to intracellular signals may be more complicated than previously thought.
The ß2 heterotrimeric model of GP Ib also opens a door for possible disulfide rearrangement in the GP Ib-IX-V complex. There are 2 theoretically possible disulfide arrangements among the 4 membrane-proximal Cys residues in GP Ib: C122-C484/C485-C122 and C122-C122/C484-C485. The former is the de facto one in the resting platelet. Our characterization of the TM peptides suggests, however, that the latter could be achieved if given favorable conditions (Figure 3D). Moreover, when GP Ib is free of disulfides with GP Ibß (as in CCßSIX cells), it could form a disulfide-linked complex with other proteins through C484, C485, or both (Figure 2C). Although we have not determined all the components of the Ib-containing complex of more than 220 kDa that is present in CCßSIX, SCßSIX, and CSßSIX cells, its apparent molecular weight is consistent with that of a Ib homodimer. We also note that a Ib-containing polypeptide of 210 kDa was found as the target of an autoantibody in a patient with prolonged bleeding time.42 It remains to be seen whether GP Ib actually forms a disulfide with a protein other than GP Ibß in the platelet and, more importantly, if it does, whether it correlates with ligand binding as well as downstream signaling.
|
Acknowledgments |
|---|
This work was supported by grants from the National Institutes of Health (HL082808), the American Heart Association-Texas Affiliate (0565078Y), and by the Welch Foundation (AU-1581) (R.L.) and the Howard Temin Award (CA096706) from the National Cancer Institute (R.L.).
|
Footnotes |
|---|
Prepublished online as Blood First Edition Paper, September 28, 2006
DOI: 10.1182/blood-2006-05-024091
Contribution: S.-Z.L., X.M., and R.L. designed and performed the research, analyzed the data, and wrote the paper; V.A.-K., S.S. and J.A.L. contributed vital new reagents.
S.-Z.L. and X.M. contributed equally to this study.
The online version of this article contains a data supplement.
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Conflict-of-interest disclosure: the authors declare no competing financial interests.
Correspondence: Renhao Li,Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, MSB 6.130, 6431 Fannin St, Houston, TX 77030; e-mail: renhao.li{at}uth.tmc.edu.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
IIbß3) mediated by fibrinogen and glycoprotein Ib-von Willebrand factor. J Biol Chem 1992; 267:11300–11306. constrains the lateral diffusion of the GP Ib-IX complex and modulates von Willebrand factor binding. Biochemistry 1997; 36:12421–12427.[CrossRef][Medline]
[Order article via Infotrieve] weakens its interaction with von Willebrand factor and impairs cell adhesion. Biochemistry 2003; 42:2245–2251.[CrossRef][Medline]
[Order article via Infotrieve] and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear. J Biol Chem 2002; 277:2151–2159. regulates von Willebrand factor-induced platelet activation. Blood 2003; 102:2122–2129. and its complex with von Willebrand factor A1 domain. Science 2002; 297:1176–1179. N-terminal domain reveals an unmasking mechanism for receptor activation. J Biol Chem 2002; 277:35657–35663.IIbß3: roles of the transmembrane and cytoplasmic domains. Proc Natl Acad Sci U S A 2001; 98:12462–12467. results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region: structure/function implications. J Biol Chem 1992; 267:10055–10061. and risk of coronary heart disease. Blood 2004; 103:963–965.. J Biol Chem 1996; 271:7362–7367. interacts with platelet glycoprotein Ib subunits Ib and Ibß. Blood 1998; 91:1295–1303. signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex. Biochemistry 1998; 37:638–647.[CrossRef][Medline]
[Order article via Infotrieve] and GpIbß regulate 14-3-3 binding to GpIb/IX/V. Blood 2000; 95:551–557. binding site within the cytoplasmic tail of platelet glycoprotein Ib. Blood 2004; 104:420–427. protein in regulating the VWF binding function of platelet glycoprotein Ib-IX and its therapeutic implications. Blood 2005; 106:1975–1981.-form 14-3-3 protein recognizing the platelet glycoprotein Ib and distinct from the c-Raf-binding site. J Biol Chem 1998; 273:33465–33471. in forming a GP Ib-IX-V complex on the cell surface. J Biol Chem 1995; 270:16302–16307.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
Related Article in Blood Online:
This article has been cited by other articles:
|
|
 |
|
  J. Rivera, M. L. Lozano, L. Navarro-Nunez, and V. Vicente Platelet receptors and signaling in the dynamics of thrombus formation Haematologica, May 1, 2009; 94(5): 700 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, J. Lu, and E. V. Prochownik Modularity of the Oncoprotein-like Properties of Platelet Glycoprotein Ib{alpha} J. Biol. Chem., January 16, 2009; 284(3): 1410 - 1418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Varga-Szabo, I. Pleines, and B. Nieswandt Cell Adhesion Mechanisms in Platelets Arterioscler. Thromb. Vasc. Biol., March 1, 2008; 28(3): 403 - 412. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2007 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
forms disulfide bonds with 2 glycoprotein Ibß subunits in the resting platelet
