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Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

Gu M, Xi X, Englund GD, Berndt MC, Du X - J. Cell Biol. (1999)

Bottom Line: Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3.Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3).Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ilinois 60612, USA.

ABSTRACT
We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

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Effects of truncation mutations on ristocetin-induced vWF binding to GPIb-IX. The 2b3a cells (expressing αIIbβ3 only), 123 cells, Δ591/2b3a cells and Δ559/2b3a cells were incubated with (+vWF) or without (No vWF) vWF in the presence of 1 mg/ml ristocetin. The cells were then stained with a monoclonal antibody, SZ29, specific for human vWF, and analyzed by flow cytometry to estimate vWF binding.
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Figure 6: Effects of truncation mutations on ristocetin-induced vWF binding to GPIb-IX. The 2b3a cells (expressing αIIbβ3 only), 123 cells, Δ591/2b3a cells and Δ559/2b3a cells were incubated with (+vWF) or without (No vWF) vWF in the presence of 1 mg/ml ristocetin. The cells were then stained with a monoclonal antibody, SZ29, specific for human vWF, and analyzed by flow cytometry to estimate vWF binding.

Mentions: To determine whether deletion of the 14-3-3–binding site in GPIbα affects GPIb-IX–mediated activation of the integrin αIIbβ3, we examined whether ristocetin-induced vWF binding to the mutant GPIb-IX stimulates the binding of FITC-labeled fibrinogen to Δ591/2b3a cells. Fig. 5 A shows that vWF induces soluble fibrinogen binding to 123 cells which is inhibited by RGDS peptide. In contrast, vWF-induced fibrinogen binding to Δ591/2b3a cells is absent. It is unlikely that the defect in fibrinogen binding to Δ591/2b3a cells results from naturally occurring mutations developed in the CHO cells during selection as the Δ591/2b3a cells are established by mass sorting of cells reactive with both antibodies against αIIbβ3 and GPIb-IX and not by single cell cloning. It is also unlikely that the inhibition of integrin activation results from defective binding of vWF as vWF binding to 591/2b3a cells is not negatively affected (Fig. 6). As Δ591/2b3a cells adhered and spread on fibrinogen (Fig. 7), the possibility of a defective integrin function can be further excluded. Thus, our data indicate that the 14-3-3–binding site of GPIbα plays an important role in GPIb-IX–mediated integrin activation.


Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model.

Gu M, Xi X, Englund GD, Berndt MC, Du X - J. Cell Biol. (1999)

Effects of truncation mutations on ristocetin-induced vWF binding to GPIb-IX. The 2b3a cells (expressing αIIbβ3 only), 123 cells, Δ591/2b3a cells and Δ559/2b3a cells were incubated with (+vWF) or without (No vWF) vWF in the presence of 1 mg/ml ristocetin. The cells were then stained with a monoclonal antibody, SZ29, specific for human vWF, and analyzed by flow cytometry to estimate vWF binding.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169335&req=5

Figure 6: Effects of truncation mutations on ristocetin-induced vWF binding to GPIb-IX. The 2b3a cells (expressing αIIbβ3 only), 123 cells, Δ591/2b3a cells and Δ559/2b3a cells were incubated with (+vWF) or without (No vWF) vWF in the presence of 1 mg/ml ristocetin. The cells were then stained with a monoclonal antibody, SZ29, specific for human vWF, and analyzed by flow cytometry to estimate vWF binding.
Mentions: To determine whether deletion of the 14-3-3–binding site in GPIbα affects GPIb-IX–mediated activation of the integrin αIIbβ3, we examined whether ristocetin-induced vWF binding to the mutant GPIb-IX stimulates the binding of FITC-labeled fibrinogen to Δ591/2b3a cells. Fig. 5 A shows that vWF induces soluble fibrinogen binding to 123 cells which is inhibited by RGDS peptide. In contrast, vWF-induced fibrinogen binding to Δ591/2b3a cells is absent. It is unlikely that the defect in fibrinogen binding to Δ591/2b3a cells results from naturally occurring mutations developed in the CHO cells during selection as the Δ591/2b3a cells are established by mass sorting of cells reactive with both antibodies against αIIbβ3 and GPIb-IX and not by single cell cloning. It is also unlikely that the inhibition of integrin activation results from defective binding of vWF as vWF binding to 591/2b3a cells is not negatively affected (Fig. 6). As Δ591/2b3a cells adhered and spread on fibrinogen (Fig. 7), the possibility of a defective integrin function can be further excluded. Thus, our data indicate that the 14-3-3–binding site of GPIbα plays an important role in GPIb-IX–mediated integrin activation.

Bottom Line: Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3.Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3).Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ilinois 60612, USA.

ABSTRACT
We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.

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