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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Pathophysiology and Human
Molecular Genetics, Centro de Investigaciones Biológicas (CSIC),
Madrid, Spain.
This work aimed at investigating the function of the [C674R]
mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide
bridge. Individuals heterozygous for this mutation show a platelet
GPIIb-IIIa content approximately 30% of normal controls, which is less
than expected from one normal functioning allele. Coexpression of
normal [674C]GPIIb and mutant [674R]GPIIb with normal
GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary
(CHO) cells, suggesting that [674R]GPIIb interferes with the
association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the
surface exposure of GPIIb-IIIa. However, substitution of 674C for A
produced a much lesser inhibition than R, suggesting that a
positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in
transiently transfected CHO cells. BiP was also found associated with
[674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or
normal heterodimers. Overexpression of BiP did not increase the surface
exposure of [674R]GPIIb-IIIa complexes, indicating that its
availability was not a limiting step. Platelets from the thrombasthenic
patient expressing [674R]GPIIb-IIIa were found to bind soluble
fibrinogen in response to physiologic agonists or dithiothreitol
treatment. Thus, the [674R]GPIIb mutation leads to a retardation
of the secretory pathway, most likely related to its binding to the
molecular chaperone BiP, with the result of a defective number of
functional GPIIb-IIIa receptors in the cell surface.
(Blood. 2001;97:2640-2647) Glanzmann thrombasthenia (GT)1 is a
recessively inherited bleeding disorder characterized by appearance of
symptoms immediately after birth, normal platelet count, prolonged
bleeding time, and low to absent clot retraction. A distinct feature of
GT is the lack of platelet aggregation both spontaneously or in
response to physiologic agonists and a normal ristocetin-induced
aggregation. GT is a rather heterogeneous entity,2 but, in
all cases studied, the thrombasthenic phenotype is associated with
either a deficient or defective platelet fibrinogen (Fg)
receptor.3,4 GT is currently classified as type I or type
II.5 In type I GT there is absence of platelet GPIIb-IIIa
and Fg and lack of clot retraction; in type II GT the platelet content
may be around 10%, Fg may be detectable, and weak clot retraction may
be observed. The platelet from the so-called GT variants may contain
normal or near normal dysfunctional GPIIb-IIIa
complexes.4,6 The calcium-dependent heterodimer formed by
the glycoproteins GPIIb and GPIIIa, also known as integrin
Construction of expression vectors with normal or mutant GPIIb
complementary DNAs
Cell culture and transfection
Flow cytometry analysis Transfected cells were harvested, using 0.5 mM EDTA in phosphate-buffered saline (PBS), washed with PBS, resuspended at a density of 106 cells/100 µL, and incubated for 20 minutes at 4°C with moAbs specific to GPIIb (M3) or GPIIIa (P37). Next, cells were washed and exposed to fluorescein isothiocyanate-conjugated (FITC) F(ab')2 fragment of rabbit antimouse immunoglobulin (DAKO A/S, Denmark) at 4°C for 20 minutes, and the surface fluorescence was analyzed in a Coulter flow cytometer, model EPICS XL.Immunoprecipitation of GPIIb-IIIa and Western analysis of BiP Immunoprecipitation of GPIIb-IIIa from CHO cells was performed as previously described.17,18 Briefly, transfected cells were treated with lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM phenylmethyl sulfonyl fluoride, 1% Triton X-100, 0.05% Tween 20, and 0.03% sodium azide) for 30 minutes at 4° C, and then precleared lysates were immunoprecipitated with either moAb M3 or moAb P37. The immunoprecipitates were then bound to protein A-Sepharose CL-4B beads, washed, and eluted by incubating 10 minutes at 100°C in 50 µL reducing loading buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 5% -mercaptoethanol, 4% sodium dodecyl sulfate [SDS], and 0.002%
bromophenol blue). To examine the association of GPIIb and/or GPIIIa
with the chaperone BiP (GRP78), immunoprecipitates obtained in the
presence of 10 U/mL apyrase (Sigma, St Louis, MO) were electrophoresed
in 0.1% SDS-7.5% polyacrylamide gels and transferred to
nitrocellulose membranes. The membrane was incubated with a polyclonal
antibody to rodent BiP and then with a secondary goat antirabbit
immunoglobulin (Ig)G-peroxidase conjugated (Sigma). The membrane was
developed by incubation in PBS containing 0.015%
H2O2 and 0.5 mg/mL 4-chloro-1-naphtol.
Pulse-chase analysis of GPIIb-IIIa synthesis Pulse-chase analysis of CHO cells transfected with wild-GPIIb and/or [674R]GPIIb was performed 48 hours after transfection. The cells were incubated in DMEM without methionine for 30 minutes and then pulsed for 30 minutes with [35S]methionine (200 µCi/mL). The plates were washed 3 times with PBS containing 1 mg/mL cold methionine and incubated with DMEM containing unlabeled methionine for 0, 0.5, and 2 hours. Cells were washed with PBS containing cold methionine and extracted in lysis buffer. Immunoprecipitation analysis was performed as described above. The immunoprecipitates were electrophoresed at 50 V in 0.1% SDS-7.5% polyacrylamide slab gels. The gels were vacuum dried and exposed to hypersensitive x-ray film. The immunoreactive bands were digitized with a high-resolution scanner and analyzed on a Macintosh computer, using the public domain software NIH Image (http: //rsb.info.nih.gov/nih-image).Preparation of FITC-Fg Human Fg was incubated with 10% celite-FITC (Calbiochem, San Diego, CA) in PBS at room temperature for 60 minutes. The pH was adjusted to 8.5 with 5% Na2CO3. The celite-FITC was separated from the labeled Fg by centrifugation, and the unreacted free FITC was cleaned from FITC-Fg by passing through a PD-10 column (Pharmacia, Uppsala, Sweden) in PBS, pH 7.4, and exhaustive dialysis at 4°C. The fluorescein-to-protein ratio was determined by measuring absorbance at 495 nm and 280 nm. Aliquots of FITC-Fg are stored at 80°C and thawed for experiments on a single
day only.
Binding of Fg to activated platelets Blood was collected in 1/10 volume of 3.8% sodium citrate, and platelet-rich plasma (PRP) was obtained by centrifugation at 180g for 20 minutes at room temperature. The PRP was then centrifuged at 800g for 10 minutes, and the platelet pellet was resuspended in Tyrode buffer, pH 7.4 (5 mM HEPES, 2 mM MgCl2, 0.3 mM NaH2PO4, 3 mM KCl, 134 mM NaCl, 12 mM NaHCO3, 0.1% glucose, 0.1% bovine serum albumin, and 1 mM Cl2Ca.) at a final concentration of 4 × 107/mL. Platelets (50 µL) were stimulated for 5 minutes at room temperature with one or more of the following activating agents: 20 mM dithiothreitol (DTT) (BRL, Life Technologies), 20 nmol/L phorbol 12-myristate 13-acetate (PMA) (Sigma), 200 µM adenosine 5'-diphosphate (Sigma), 1 mM ()epinephrine (Sigma), 1.5 U/mL human plasma thrombin (Sigma), 100 µM thrombin receptor agonist
peptide (SFLLRN), 2 µM platelet activating factor-16 (Calbiochem). To
prevent thrombin-induced fibrin polymerization, GPRP peptide (Bachem,
Bubendorf, Switzerland) was added to the incubation medium. Next, 15 µg FITC-Fg was added to activated platelets and incubated 15 minutes
at room temperature. After washing, the platelets were resuspended in
Tyrode buffer for flow cytometry analysis. We examined also the binding
of FITC-labeled PAC-1, antibody specific for the active conformation of
GPIIb-IIIa, in PMA-treated platelets.
Materials Restriction enzymes were obtained from Boehringer (Mannheim, Germany). The pcDNA3 expression vector was from Invitrogen (San Diego, CA). Most other reagents were purchased from Sigma Chemical Company (St. Louis, MO), Merck (Darmstadt, Germany), or Calbiochem-Novabiochem Corporation (San Diego, CA). [35S]-methionine (SA 1000 Ci/mmol) was from Amersham Ibérica (Madrid, Spain). moAbs specific for GPIIIa (P37) and GPIIb (M3) were a gift of Drs J. González and M. V. Alvarez from the Institute Rocasolano (CSIC; Madrid, Spain). Polyclonal antirodent BiP and pMT/hamster wild BiP cDNA were a generous gift from Dr Linda Hendershot at St. Jude's Research Hospital (Memphis, TN). FITC-PAC1 was obtained from Dr J. Lopez (VA Medical Center, Houston, TX).
Pulse-chase analysis of stability of [674R]GPIIb-IIIa complexes We have reported a case of compound heterozygosity for GPIIb associated with type II GT.14 The mutation inherited from the father [IVS5(+2)CA] renders the mRNA unstable and, therefore, the only mRNA-GPIIb found in the platelets of this patient was the one encoding the mutated [C674R]GPIIb inherited from the mother. The patient showed very low platelet GPIIb-IIIa content and elevated ratio of proGPIIb to GPIIbH. Moreover, coexpression of [674R]GPIIb and GPIIIa in CHO cells led to a marked reduction in the surface exposure of GPIIb-IIIa complexes. Although the latter observation indicates that subunit dimerization is not prevented, the reason for the low rate of surface expression of GPIIb-IIIa is not apparent. In principle, it could be the result of either instability of the [674R]GPIIb-IIIa complexes and/or retardation in the intracellular transit along the secretory pathway. To investigate this point, cells transiently cotransfected with normal GPIIIa and either mutant [674R]GPIIb or normal GPIIb were pulse-labeled for 30 minutes with [35S]methionine and then chased with medium containing unlabeled methionine for 0.5 or 2 hours before GPIIb-IIIa complexes were immunoprecipitated with an anti-GPIIb moAb. As expected, reciprocal changes in the labeling of proGPIIb, and of GPIIbH and GPIIIa, as a function of the chasing time were observed in cells coexpressing normal GPIIb and GPIIIa proteins (Figure 1). This observation implies stability of GPIIb-IIIa complexes and their transport into the Golgi apparatus. In cells cotransfected with GPIIIa and mutant [674R]GPIIb, proGPIIb, GPIIbH, and GPIIIa subunits were coprecipitated with anti-GPIIb at all times, but the labeling flow was considerably slower than in cells coexpressing both normal subunits. The decay of labeled proGPIIb was retarded (Figure 1), suggesting that the number of subunits entering the secretory pathway may be diminished. After 2 hours of chase, the pattern of labeling of cells expressing [674R]GPIIb was similar to that observed in the control cells at zero time. In CHO cells, the majority (99%) of GPIIb-IIIa is located on the plasma membrane.19 According to this, in a pulse-chase experiment the labeling intensities of GPIIb and GPIIIa should be proportional to the amount of GPIIb-IIIa complexes that have reached the cell surface. The reduced rate of labeling of [674R]GPIIb-IIIa complexes could be caused by impairment of the association of [674R]GPIIb with GPIIIa, by retardation of the metabolic flow along the maturation and trafficking pathway, or by the concurrent action of both mechanisms. Whatever the mechanism, it seems that the [674R]GPIIb mutation either changes the kinetics of a preexisting step or imposes a new limiting step to the normal processing pathway of GPIIb-IIIa complexes.
Association of abnormal GPIIb or GPIIIa subunits with the endoplasmic reticulum chaperone BiP (GRP78) The pulse-chase analysis seems to suggest that an early step in the surface expression pathway of [674R]GPIIb-IIIa may be impaired. Since the substitution of 674C in GPIIb implies the disruption of an intramolecular disulfide bridge and subsequent conformational changes, we found of interest to analyze the interaction of normal or [674R]GPIIb with the chaperone BiP. For this purpose, total cell lysates from CHO cells coexpressing GPIIIa and either normal or mutant [674R]GPIIb, or expressing only normal GPIIb, mutant [674R]GPIIb, or GPIIIa, were immunoprecipitated with an anti-GPIIb moAb and the precipitates used for Western analysis using a polyclonal antibody against the hamster BiP chaperone. Figure 2 indicates that the chaperone BiP was coprecipitated with [674R]GPIIb but not with normal GPIIb. To elucidate whether the immunoprecipitated BiP was associated with mutant monomeric pro[674R]GPIIb or it was also bound to heterodimers, we performed the same type of analysis precipitating portions of the cell lysates with an anti-GPIIIa antibody reacting with monodimeric and heterodimeric GPIIIa. Figure 3 shows that anti-GPIIIa coprecipitated BiP only in cells coexpressing [674R]GPIIb and normal GPIIIa, suggesting that, at least under our experimental conditions, BiP associated with abnormal conformations of monomeric GPIIb as well as with mutant heterodimeric complexes. The observation that BiP immunoprecipitates with a mutant form of GPIIb (E324K) (Figure 3) unable to form heterodimers with GPIIIa20 clearly indicates that the BiP binding is independent of the type of GPIIb mutation and its consequence on assembly; it also indicates that assembly is not a prerequisite for BiP binding.
The apparently high-affinity binding of BiP to mutated [674R]GPIIb suggested that availability of BiP could become limiting, preventing, therefore, the access of new proGPIIb peptides into the maturation pathway. To investigate this possibility, we cotransfected 2 different amounts of mutant [674R]GPIIb with normal GPIIIa and BiP expression plasmids. No significant effect of BiP was detected in the rate of surface expression of [674R]GPIIb-IIIa complexes (results not shown), making unlikely that it could have been limiting. Mutation analysis of the 674 and 687 residues of GPIIb The 674C of GPIIb forms an intramolecular disulfide bridge with 687C. To ascertain whether the [674R]GPIIb dysfunction was associated with the loss of the disulfide bridge, or with the specific need for a C at position 674, we analyzed the functional repercussion of loosing the 674-687 disulfide bridge by changing the Cs 674 or 687 for A. Figure 4 (upper panel) depicts the surface expression of GPIIb-IIIa in CHO-IIIa cells transfected with normal or mutated forms of GPIIb. The mutation C674R found in the proband decreased by approximately 70% the surface expression of transfected GPIIb-IIIa. The C674A or C687A mutations produced smaller (approximately 40%-50%) inhibitions. The mutation of either 674C or 687C produced similar inhibition; however, the additive result of mutating both residues suggests the appearance of effects unrelated to disulfide disruption (Figure 4). Total cell lysates from transfected cells were immunoprecipitated with anti-GPIIb, and the presence of BiP in the precipitates was analyzed by Western blotting. The results so obtained indicate (Figure 4, bottom) that BiP was tightly associated not only with natural-occurring mutations of GPIIb but also with mutated forms of GPIIb created in the laboratory.
Physiologic responsiveness of platelets carrying the [674R]GPIIb mutation The reduced level of expression of [674R]GPIIb-IIIa justifies the thrombasthenic phenotype of the patient. However, we found of interest from the academic and prognostic standpoints to determine the ability of the mutated [674R]GPIIb-IIIa complexes to undergo an activated state in response to stimulation by agonists. Platelets from the proband and normal individuals were incubated in the presence of physiologic agonists, DTT, or PMA, and the binding of soluble FITC-Fg determined by flow cytometric analysis. The fluorescence of the proband's platelets was about 10% of the control values (results not shown). Thus, the enhanced fluorescence intensity after platelet activation was reasonably proportional to the number of surface receptors, suggesting that the mutated platelet [674R]GPIIb-IIIa complexes undergo normal in-out signaling-mediated conformational changes. This conclusion is also supported by the enhanced binding of the activation-dependent IgM PAC1 to the proband's platelets after PMA-induced postreceptor stimulation (Figure 5).
Competition between normal and [674R]GPIIb for surface expression of GPIIb-IIIa complexes The heterozygous states for the [674R]GPIIb mutation expressed less than 50% of platelet GPIIb-IIIa of normal individuals. Since this mutated GPIIb allele yields normal amounts of full-length transcripts, we hypothesized that a reason for the decreased surface exposure of GPIIb-IIIa complexes could be a competition between the translational products of the normal and mutated alleles at some limiting step(s). To elucidate this point, we cotransfected CHO-IIIa cells with progressively increasing ratios of normal and/or mutated GPIIb cDNAs. Figure 6A depicts the surface GPIIb-IIIa content as a function of the amount of transfected expression plasmid(s). This process shows a saturation-type kinetics, and the maximal value with the mutated GPIIb plasmid was reached at much lower concentration than with normal GPIIb. Figure 6B shows the measured and estimated surface exposure of GPIIb-IIIa in cells coexpressing molar ratios of normal and mutated GPIIb from 0.2 to 5. In estimating the surface GPIIb-IIIa we assumed, according to results on panel A (Figure 6), that in this range of plasmid concentration transfection is not a limiting step and that surface expression from both cDNAs should be additive. Unexpectedly, instead of additivity, the results show a progressively increasing inhibition of surface exposure of normal GPIIb-IIIa complexes as the normal [674C]GPIIb-to-mutant [674R]GPIIb ratio decreased from 5 to 0.2 (Figure 6C). It should be remarked that cotransfection of equimolar amounts (2 µg of each expression plasmid) of normal and mutant GPIIb, presumably the expected physiologic condition, inhibited the surface exposure of GPIIb-IIIa by approximately 35%. Moreover, pulse-chase analysis of cells cotransfected with equimolar amounts of normal and mutant GPIIb clearly shows a prolonged retention of labeling in proGPIIb and decreased labeling of GPIIb-IIIa (Figure 7). Densitometric analysis of data shown in Figure 7 was consistent with that obtained by flow cytometry (Figure 6). The immunoprecipitation with anti-GPIIIa of heavily labeled GPIIIa in all conditions (Figure 7, bottom panel) rules out that availability of GPIIIa could have been limiting for the surface expression of GPIIb-IIIa.
Since the mutated [674R]GPIIb is bound to BiP, a limited
availability of this chaperone could explain the observed competition between the normal and mutant forms of GPIIb. We investigated this
possibility coexpressing BiP with variable amounts of normal or mutant
GPIIb so that the mutant-to-normal GPIIb ratio varied from 1 to 3. The
data in Figure 8 clearly indicate that
overexpression of BiP did not overcome the inhibitory effects of the
mutant [674R]GPIIb on the surface exposure of the normal subunit,
but, on the contrary, a consistent inhibitory effect was observed at
all mutant-to-normal GPIIb ratios analyzed. Finally, the possibility
should be considered that binding of BiP to [674R]GPIIb-IIIa
complexes would make limiting the availability of GPIIIa subunits.
However, it is unlikely that this mechanism might be quantitatively
important in view of the scarce amounts of mutant heterodimers
immunoprecipitated with antibodies capable of identifying each subunit
as either monomer or heterodimer.
Role of the 674-687 disulfide bond of GPIIb on the biological function of the GPIIb-IIIa complex The C674R GPIIb mutation disrupts the intrachain 674-687 disulfide bond. This mutation has been found in a thrombasthenic phenotype, heterozygote compound for GPIIb, whose platelet content of GPIIb-IIIa was about 10% of the normal control individuals and that only platelet mRNA-GPIIb was the one encoding [674R]GPIIb. Coexpression of normal GPIIIa and [674R]GPIIb in CHO cells confirmed the reduced rate of surface expression of [674R]GPIIb-IIIa complexes. However, from this type of experiment it cannot be ascertained whether the loss of the 674-687 disulfide bond impairs the function of the cell surface receptors. The platelets from the proband respond to a battery of activating agents by increasing the binding of soluble Fg with intensity proportional to the number of exposed receptors. This observation clearly indicates that loss of the 647-687 intrachain disulfide bond does not compromise the agonist-mediated responsiveness of the exposed [674R]GPIIb-IIIa complexes. Thus, considering that the [674R]GPIIb allele is transcribed at normal rates and this mutation does not preclude a normal receptor responsiveness, the knowledge of the limiting step(s) in the expression of mutant heterodimers could eventually help to define a therapeutic target(s).In secreted proteins, disulfide bonds are important for stabilization
of the tertiary structure as well as for their assembly into multimeric
complexes.21 Disruption of intrachain disulfide bonds in
GPIIb or GPIIIa produces variable degrees of functional impairment.
Deletion of 106A-111Q, including 107C, disrupts the 107-130 intrachain
disulfide bond in GPIIb,22 preventing heterodimerization of GPIIb-IIIa.23 Similar results were observed with the
129S-161S deletion comprising the 130C and 146C
residues.24 In GPIIIa, disruption of disulfide bonds by
the C374Y and C560F mutations are associated with type II
thrombasthenia,25,26 and C542R and C457Y substitutions are
involved in type I GT.27,28 However, removal of C655
GPIIIa by site-directed mutagenesis does not affect the
GPIIb-IIIa.29 The Cs 674 and 687 in GPIIb are conserved in
all the The nascent polypeptide chains would tend to aggregate in the
high-protein-containing intracellular medium unless it was impeded by
the assistance of molecular chaperones. The chaperones are important in
polypeptide folding, protein assembly, and intracellular translocation.31-33 They bind to hydrophobic domains on
the surface of unfolded polypeptides. As the disulfide bonds are formed
and the protein folding proceeds, most likely, different chaperones act
sequentially until a competent native protein is
secreted.34-36 The BiP is a molecular chaperone that
targets irreversibly misfolded proteins for their degradation in the
endoplasmic reticulum (ER). It is generally accepted that misfolded
proteins are never secreted and remain sequestered in the ER until
their degradation is completed. On these grounds, we found of interest
to investigate the interaction of [674R]GPIIb with BiP and
whether this chaperone was involved in determining the slow rate of
surface exposure of [674R]GPIIb-IIIa complexes. In agreement with
a previous observation on the mutagenesis of G273 of
GPIIb,37 BiP bound mutated forms of residues 674 or 687 of
GPIIb but not to normal [674C]GPIIb. It should be remarked that,
although some of the introduced mutations improved the exposure of
GPIIb-IIIa, none of them approached values above 50% of the normal
rates. Thus, it seems reasonable to conclude that, at least under our
experimental conditions, a relationship exists between the association
of GPIIb with BiP and reduced surface expression of GPIIb-IIIa. It is
generally accepted that the fate of misfolded proteins is their
complete degradation in the ER. However, in our case, in agreement with
an observation made on some mutated forms of the human chorionic
gonadotropin- It is worth noting that replacement of 674C for R appeared to have a more deleterious effect in terms of surface expression than A, which might be due to constraints imposed by the side-chain on the BiP binding. It should also be mentioned that, although mutation of either 674C or 687C produced similar effects, the additive effect of both mutations suggests their effect may not be due only to disulfide disruption. A likely interpretation for this observation is an altered polypeptide conformation independent from disulfide disruption. Alternatively, the nonmutated C may engage in disulfide bridging with another C, sufficient to provide a conformation improving the trafficking or retaining BiP less efficiently. Competition between normal and mutant [674R]GPIIb subunits The heterozygous states for mutations in GPIIb or GPIIIa are known to contain subnormal amounts of platelet GPIIb-IIIa, although near normal amounts have also been reported.18 In principle, the finding of subnormal platelet GPIIb-IIIa content suggests that transcripts from a single allele are not sufficient to maintain normal translational rates and, therefore, assembly and surface exposure. The amount of mutated platelet mRNA-GPIIb in the heterozygote state for [674R]GPIIb was within the expected normal range; nevertheless, the amount of platelet GPIIb-IIIa was approximately 30% of the normal values or, what is the same, about half of the estimated content, assuming that the contribution of both, normal (50%) and mutant allele (10%), was additive. The lack of additivity implies that some type of negative interaction must be taking place between the mutant and normal subunits. Under our experimental conditions, coexpression of normal GPIIIa and progressively increasing ratios of mutant to normal GPIIb led to progressive inhibition of the surface expression of GPIIb-IIIa in CHO cells. The physiologic significance of this finding is supported by the fact that transfection of CHO cells with GPIIIa and equimolar amounts of normal and mutant GPIIb inhibited the exposure of normal GPIIb-IIIa to a similar extent to that observed in the platelets of the heterozygote individual. The inhibitory effect of a mutant protein on the wild-type gene product is known as "dominant-negative" effect.39,40 Dominant-negative effect of mutated proteins seems to be involved in the etiopathogenesis of several human diseases, such as the thyroid hormone resistance syndrome41,42 and those originated by mutant p53 tumor suppressor proteins43 and mutations in the tyrosine kinase domain of the insulin receptor.44We have reported competition between other mutated forms of GPIIb and normal subunits.19 The competition between normal and mutant subunits may be exerted in different ways, depending for instance on whether they form homodimers or heterodimers that may compete for the binding of other subunits or ligands. On the basis of the strong binding of BiP to monomeric as well as to heterodimeric mutant [674R]GPIIb-IIIa complexes, we investigated whether the availability of BiP for the assisted folding of normal subunits could have been limiting. This possibility can be ruled out since coexpression of BiP enhanced rather than diminished the inhibitory effects of mutant [674R]GPIIb. The increased availability of BiP could result in increasing steady-state levels of [674R]GPIIb-BiP complexes. Thus, the inhibitory effect of overexpressing BiP adds further support to the postulate that the mutant [674R]GPIIb-BiP complex perturbs the secretory pathway of normal subunits by competing at some limiting step. To conclude, the lack of the 674-687 disulfide bond in [674R]GPIIb does not preclude a normal agonist-induced binding of Fg or of the activation-dependent IgM PAC1 by [674R]GPIIb-IIIa receptors. BiP forms very stable complexes with either pro[674R]GPIIb and [674R]GPIIb-IIIa heterodimers but not with normal GPIIb or GPIIb-IIIa. Mutation analysis of residue 674 of GPIIb indicates that noncharged or no-polar residues improved the functional performance of the subunit. However, none of the analyzed mutants reached the normal values when coexpressed with normal GPIIIa, and all of them were tightly bound to BiP. There is a dose-dependent competition between normal and [674R]GPIIb-IIIa complexes for cell surface expression, and this effect is enhanced by coexpression of BiP. These observations suggest that the tight binding of BiP to [674R]GPIIb may be related to the slow rate of progression of [674R]GPIIb-IIIa along the secretory pathway and the competition between normal and mutant heterodimers.
Antibodies P37 and M3 directed against GPIIIa and GPIIb, respectively, were a gift of Drs J. González and M. V. Alvarez from the Instituto Rocasolano of the Spanish Council of Research (CSIC). We are greatly indebted to Dr Linda Hendershot at St Jude Children's Research Hospital, Memphis, TN, for the generous gift of antibody against rodent BiP and the BiP expression plasmid.
Submitted June 1, 2000; accepted December 20, 2000.
Supported in part by grants from the Dirección General de Investigación Científica y Técnica (DGICYT PB97-1240 and DGICYT PM97-0016), Fondo de Investigaciones Sanitarias (96/2014), and Comunidad Autónoma de Madrid 08.4/0031/1998, and by a grant-in-aid from the Agencia Española de Cooperación Internacional (AECI, n/ref 99CN0009). E.G.A.-S. received a fellowship from the Fundación Areces.
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 U.S.C. section 1734.
Reprints: Roberto Parrilla, Centro de Investigaciones Biológicas (CSIC), Velázquez 144, Madrid-28006, Spain; e-mail: rparrilla{at}cib.csic.es.
1. Glanzmann E. Hereditare hemorrhagische thrombasthenie: ein beitrag zur pathologie der blut plattchen. J Kinderkr. 1918;88:113-141.
2.
George JN, Caen JP, Nurden AT.
Glanzmann's thrombasthenia: the spectrum of clinical disease.
Blood.
1990;75:1383-1395 3. Phillips DR, Agin PP. Platelet membrane defects in Glanzmann's thrombasthenia: evidence for decreased amounts of two major glycoproteins. J Clin Invest. 1977;60:535-545. 4. Nurden AT, Rosa JP, Fournier D, et al. A variant of Glanzmann's thrombasthenia with abnormal GPIIb-IIIa complexes in the platelet membrane. J Clin Invest. 1987;79:962-969. 5. Caen JP. Glanzmann's thrombasthenia. Clin Haematol. 1972;1:383-392. 6. Fournier DJ, Kabral A, Castaldi PA, Berndt MC. A variant of Glanzmann's thrombasthenia characterized by abnormal glycoprotein IIb/IIIa complex formation. Thromb Haemost. 1989;62:977-983[Medline] [Order article via Infotrieve]. 7. Heidenreich R, Eisman R, Delgrosso K, et al. The organization of the gene for platelet glycoprotein IIb. Biochemistry. 1990;29:1232-1244[CrossRef][Medline] [Order article via Infotrieve].
8.
Fitzgerald LA, Steiner B, Rall SC, Lo S-S, Phillips DR.
Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone: identity with platelet glycoprotein IIIa and similarities to integrin.
J Biol Chem.
1987;262:3936-3939 9. Rosa J-P, Bray PF, Gayet O, et al. Cloning of glycoprotein IIIa cDNA from human erythroleukemic cells and localization of the gene to chromosome 17. Blood. 1988;71:593-600.
10.
Lanza F, Kieffer N, Phillips DR, Fitzgerald LA.
Characterization of the human platelet glycoprotein IIIa gene: comparison with the fibronectin receptor
11.
Zimrin AB, Giditz S, Lord S, et al.
The genomic organization of platelet glycoprotein IIIa.
J Biol Chem.
1990;265:8590-8595 12. French DL, Coller DS. Hematological important mutations: Glanzmann thrombasthenia. Blood Cells Mol Dis. 1997;23:39-51[CrossRef][Medline] [Order article via Infotrieve]. 13. French DL. The molecular genetics of Glanzmann's thrombasthenia. Platelets. 1998;9:5-20.
14.
González-Manchón C, Fernández-Pinel M, Arias-Salgado EG, et al.
Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann's thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes.
Blood.
1999;93:866-875 15. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989;77:61-68[CrossRef][Medline] [Order article via Infotrieve]. 16. Gulick T. Transfection using DEAE-dextran. In: Ausubel FM,Brent R,Kingston RE,et al., eds. Current Protocols in Molecular Biology. Vol 1. New York, NY: Wiley; 1997:9.2.1. 17. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
18.
Ferrer M, Tao J, Iruín G, et al.
Truncation of GPIIIa (
19.
Ferrer M, Fernández M, González-Manchón C, González J, Ayuso MS, Parrilla R.
A mutant (Arg327
20.
Tao J, Arias-Salgado EG, González-Manchón C, et al.
A 1063G 21. Seckler R, Jaenicke R. Protein folding and protein refolding. FASEB J. 1992;6:2545-2552[Abstract]. 22. Calvete JJ, Alvarez MV, Rivas G, Hew C-L, Henschen A, González-Rodríguez J. Interchain and intrachain disulfide bonds and in human platelet glycoprotein IIb: localization of epitopes for several monoclonal antibodies. Biochem J. 1989;261:551-560[Medline] [Order article via Infotrieve].
23.
Newman PJ, Seligsohn U, Lyman S, Coller BS.
The molecular genetic basis of Glanzmann thrombasthenia in the Iraqi-Jewish and Arab populations in Israel.
Proc Natl Acad Sci U S A.
1991;88:3160-3164 24. French DL, Chen F, Xu L, Coller BS. A common Arg584Stop mutation in glycoprotein (GP)IIb in 2 additional patients with Glanzmann thrombasthenia [abstract]. Blood. 1993;88:30a.
25.
Grimaldi CM, Chen F, Scudder LE, Coller BS, French DL.
A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa ( 26. Ambo H, Kamata T, Handa M, et al. Three novel integrin beta3 subunit missense mutations (H280P, C560F, and G579S) in thrombasthenia, including one (H280P) prevalent in Japanese patients. Biochem Biophys Res Commun. 1998;251:763-768[CrossRef][Medline] [Order article via Infotrieve].
27.
Ruan J, Schmugge M, Clemetson KJ, et al.
Homozygous Cys542
28.
Milet S, Bourre F, Peyruchaud O, et al.
Amino acid substitution Cys457 to Tyr in the
29.
Wang R, Peterson J, Aster RH, Newman PJ.
Disruption of a long-range disulfide bond between residues Cys406 and Cys655 in glycoprotein IIIa does not affect the function of platelet glycoprotein IIb-IIIa [letter].
Blood.
1997;90:1718-1719 30. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11-25[CrossRef][Medline] [Order article via Infotrieve]. 31. Gaut JR, Hendershot LM. The modification and assembly of proteins in the endoplasmic reticulum. Curr Opin Cell Biol. 1993;5:589-595[CrossRef][Medline] [Order article via Infotrieve]. 32. Hartl FU. Molecular chaperones in cellular protein folding. Nature. 1996;381:571-580[CrossRef][Medline] [Order article via Infotrieve].
33.
Ruddon RW, Bedows E.
Assisted protein folding.
J Biol Chem.
1997;272:3125-3128 34. Frydman J, Hartl FU. Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms. Science. 1996;272:1497-1502[Abstract].
35.
Hammond C, Helenius A.
Folding of VSV G protein: sequential interaction with BiP and calnexin.
Science.
1994;266:456-458
36.
Kim PS, Arvan P.
Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum.
J Cell Biol.
1995;128:29-38 37. Khan MJ, Kieber-Emmons T, Vilaire G, Murali R, Poncz M, Bennett JS. Effect of mutagenesis of GPIIb amino acid 273 on the expression and conformation of the platelet integrin GPIIb-IIIa. Biochemistry. 1996;35:14304-14311[CrossRef][Medline] [Order article via Infotrieve].
38.
Bedows E, Norton SE, Huth JR, Suganuma N, Boime I, Ruddon RW.
Misfolded human chorionic gonadotropin beta subunits are secreted from transfected Chinese hamster ovary cells.
J Biol Chem.
1994;269:10574-10580 39. Hershkowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987;329:219-222[CrossRef][Medline] [Order article via Infotrieve]. 40. Sheppard D. Dominant negative mutants: tools for the study of protein function in vitro and in vivo. Am J Respir Cell Mol Biol. 1994;11:1-6[Abstract].
41.
Parrilla R, Mixson J, McPherson J, McClaskey J.
Characterization of seven novel mutations of the c-erbA-
42.
Nagaya T, Jameson JL.
Thyroid hormone receptor dimerization is required for dominant negative inhibition by mutations that cause thyroid hormone resistance.
J Biol Chem.
1993;268:15766-15771
43.
Unger T, Mietz JA, Scheffiner M, Yee CL, Howley PM.
Functional domains of wild type and mutant p53 proteins involved in transcriptional regulation transdominant inhibition and transformation suppression.
Mol Cell Biol.
1993;13:5186-5194 44. Frattali AL, Treadway JL, Pessin JE. Insulin/IGF-1 hybrid receptors: implications for the dominant negative phenotype in syndromes of insulin resistance. J Cell Biochem. 1992;48:43-50[CrossRef][Medline] [Order article via Infotrieve].
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  N. Butta, E. G. Arias-Salgado, C. Gonzalez-Manchon, M. Ferrer, S. Larrucea, M. S. Ayuso, and R. Parrilla Disruption of the {beta}3 663-687 disulfide bridge confers constitutive activity to {beta}3 integrins Blood, October 1, 2003; 102(7): 2491 - 2497. [Abstract] [Full Text] [PDF]
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S. Larrucea, C. Gonzalez-Manchon, N. Butta, E. G. Arias-Salgado, L. Shen, M. S. Ayuso, and R. Parrilla Agonist-induced aggregation of Chinese hamster ovary cells coexpressing the human receptors for fibrinogen (integrin alpha IIbbeta 3) and the platelet-activating factor: dissociation between adhesion and aggregation Blood, April 15, 2002; 99(8): 2819 - 2827. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
IIb
) or mutant (
) [674R]GPIIb cDNAs. The surface
exposure of GPIIb-IIIa heterodimers was analyzed by flow cytometry with
anti-GPIIb. The results are the means ± SEM of at least 3 independent experiments in duplicate. (B) CHO-IIIa cells were
cotransfected with normal to mutant [674R]GPIIb molar ratios from
0.2 to 5, and the surface expression of GPIIb-IIIa complexes was
determined by flow cytometry (
). At the normal to mutant
ratio of 1, 2 µg of each plasmid was cotransfected. The estimated
values (
) were obtained, assuming that, according to results on
panel A, the surface expression from the translational product of both
cDNAs should be additive. (C) The percentage of inhibition of surface
exposure of GPIIb-IIIa was calculated from the differences between the
estimated and the measured values shown in panel B.
616-762) prevents complex formation with GPIIb: novel mutation in exon 11 of GPIIIa associated with thrombasthenia.
Blood.
1998;92:4712-4720
His) GPIIb associated to thrombasthenia exerts a dominant negative effect in stably transfected CHO cells.
Thromb Haemost.
1996;76:292-301