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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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Coimmunoprecipitation of endogenous CHO cell 14-3-3 with GPIb-IX. Cells were stably transfected with integrin αIIbβ3 together with wild-type GPIb-IX (123) or GPIb-IX mutants with GPIbα cytoplasmic domain truncated at residue 591 to delete 14-3-3–binding site (Δ591/2b3a). The 123 cells and Δ591/2b3a cells were solubilized and immunoprecipitated with the monoclonal antibody, WM23, against GPIbα (Ib) or control mouse IgG (Ig). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with a rabbit antibody against 14-3-3ζ or a rabbit anti-GPIbα antibody. Note that 14-3-3ζ was coimmunoprecipitated with wild-type GPIb-IX but not Δ591.
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Figure 4: Coimmunoprecipitation of endogenous CHO cell 14-3-3 with GPIb-IX. Cells were stably transfected with integrin αIIbβ3 together with wild-type GPIb-IX (123) or GPIb-IX mutants with GPIbα cytoplasmic domain truncated at residue 591 to delete 14-3-3–binding site (Δ591/2b3a). The 123 cells and Δ591/2b3a cells were solubilized and immunoprecipitated with the monoclonal antibody, WM23, against GPIbα (Ib) or control mouse IgG (Ig). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with a rabbit antibody against 14-3-3ζ or a rabbit anti-GPIbα antibody. Note that 14-3-3ζ was coimmunoprecipitated with wild-type GPIb-IX but not Δ591.

Mentions: We showed previously (Du et al. 1996) that the intracellular signaling molecule 14-3-3ζ binds to a site in the COOH-terminal 15 residues (residues 595–610) of the cytoplasmic domain of GPIbα. To investigate the role of 14-3-3 in GPIb-IX–mediated activation of αIIbβ3 in the CHO cell model, we established a CHO cell line (Δ591/2b3a cells) that coexpresses αIIbβ3 and a mutant GPIb-IX, Δ591, that lacks the 14-3-3–binding site (18 residues) at the COOH terminus of GPIbα, but retains the functional filamin-binding domain in GPIbα (Cunningham et al. 1996; Du et al. 1996). As shown in Fig. 4, wild-type GPIb-IX expressed in CHO cells (123 cells) coimmunoprecipitates with an endogenous CHO cell 14-3-3 protein reactive with anti–14-3-3ζ antibodies (Fig. 4). The mutant GPIb-IX (Δ591), however, failed to coimmunoprecipitate endogenous CHO cell 14-3-3 (Fig. 4). As a control, we also immunoblotted the same immunoprecipitates with an anti-GPIbα antibody, and observed that similar amounts of GPIbα were immunoprecipitated from both the Δ591/2b3a cells and 123 cells (expressing wild-type GPIb-IX; Fig. 4). Thus, the Δ591 mutant GPIb-IX is defective in binding to endogenous 14-3-3.


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)

Coimmunoprecipitation of endogenous CHO cell 14-3-3 with GPIb-IX. Cells were stably transfected with integrin αIIbβ3 together with wild-type GPIb-IX (123) or GPIb-IX mutants with GPIbα cytoplasmic domain truncated at residue 591 to delete 14-3-3–binding site (Δ591/2b3a). The 123 cells and Δ591/2b3a cells were solubilized and immunoprecipitated with the monoclonal antibody, WM23, against GPIbα (Ib) or control mouse IgG (Ig). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with a rabbit antibody against 14-3-3ζ or a rabbit anti-GPIbα antibody. Note that 14-3-3ζ was coimmunoprecipitated with wild-type GPIb-IX but not Δ591.
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Related In: Results  -  Collection

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Figure 4: Coimmunoprecipitation of endogenous CHO cell 14-3-3 with GPIb-IX. Cells were stably transfected with integrin αIIbβ3 together with wild-type GPIb-IX (123) or GPIb-IX mutants with GPIbα cytoplasmic domain truncated at residue 591 to delete 14-3-3–binding site (Δ591/2b3a). The 123 cells and Δ591/2b3a cells were solubilized and immunoprecipitated with the monoclonal antibody, WM23, against GPIbα (Ib) or control mouse IgG (Ig). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with a rabbit antibody against 14-3-3ζ or a rabbit anti-GPIbα antibody. Note that 14-3-3ζ was coimmunoprecipitated with wild-type GPIb-IX but not Δ591.
Mentions: We showed previously (Du et al. 1996) that the intracellular signaling molecule 14-3-3ζ binds to a site in the COOH-terminal 15 residues (residues 595–610) of the cytoplasmic domain of GPIbα. To investigate the role of 14-3-3 in GPIb-IX–mediated activation of αIIbβ3 in the CHO cell model, we established a CHO cell line (Δ591/2b3a cells) that coexpresses αIIbβ3 and a mutant GPIb-IX, Δ591, that lacks the 14-3-3–binding site (18 residues) at the COOH terminus of GPIbα, but retains the functional filamin-binding domain in GPIbα (Cunningham et al. 1996; Du et al. 1996). As shown in Fig. 4, wild-type GPIb-IX expressed in CHO cells (123 cells) coimmunoprecipitates with an endogenous CHO cell 14-3-3 protein reactive with anti–14-3-3ζ antibodies (Fig. 4). The mutant GPIb-IX (Δ591), however, failed to coimmunoprecipitate endogenous CHO cell 14-3-3 (Fig. 4). As a control, we also immunoblotted the same immunoprecipitates with an anti-GPIbα antibody, and observed that similar amounts of GPIbα were immunoprecipitated from both the Δ591/2b3a cells and 123 cells (expressing wild-type GPIb-IX; Fig. 4). Thus, the Δ591 mutant GPIb-IX is defective in binding to endogenous 14-3-3.

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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