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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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GPIb-IX activates integrin-vWF interaction and integrin-dependent cell spreading on vWF. (A) Three CHO cell lines, 1b9 (expressing GPIb-IX), 2b3a (expressing αIIbβ3), and 123 (expressing GPIb-IX and integrin αIIbβ3) were incubated in the microtiter plates coated with 10 μg/ml vWF for 30 min at 37°C in the presence of 5 μg/ml botrocetin. 123 cells were also incubated in the vWF-coated wells in the presence of 2 mM of the integrin antagonist peptide, RGDS (123+RGDS), a monoclonal antibody specific for integrin αIIbβ3 complex (123+4F10) and a monoclonal antibody against human β3 (123+SZ21) at 100 μg/ml, 500 ng/ml prostaglandin E1 (PGE1), 25 nM calphostin C, and 0.1 μM Wortmannin. (B) To compare αIIbβ3 function, 2b3a cells and 123 cells were incubated in microtiter wells coated with 10 μg/ml fibrinogen. After washing, the adherent cells were photographed under a phase contrast microscope (20× objective lens). (C) Cells expressing GPIb-IX (1b9) and coexpressing both αIIbβ3 and GPIb-IX (123) were allowed to adhere to botrocetin-treated vWF-coated glass slides in the absence or presence of RGDS peptide. After fixation and permeabilization, cells were stained with rhodamine-labeled phalloidin, and photographed under a fluorescence microscope (40× objective lens).
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Figure 2: GPIb-IX activates integrin-vWF interaction and integrin-dependent cell spreading on vWF. (A) Three CHO cell lines, 1b9 (expressing GPIb-IX), 2b3a (expressing αIIbβ3), and 123 (expressing GPIb-IX and integrin αIIbβ3) were incubated in the microtiter plates coated with 10 μg/ml vWF for 30 min at 37°C in the presence of 5 μg/ml botrocetin. 123 cells were also incubated in the vWF-coated wells in the presence of 2 mM of the integrin antagonist peptide, RGDS (123+RGDS), a monoclonal antibody specific for integrin αIIbβ3 complex (123+4F10) and a monoclonal antibody against human β3 (123+SZ21) at 100 μg/ml, 500 ng/ml prostaglandin E1 (PGE1), 25 nM calphostin C, and 0.1 μM Wortmannin. (B) To compare αIIbβ3 function, 2b3a cells and 123 cells were incubated in microtiter wells coated with 10 μg/ml fibrinogen. After washing, the adherent cells were photographed under a phase contrast microscope (20× objective lens). (C) Cells expressing GPIb-IX (1b9) and coexpressing both αIIbβ3 and GPIb-IX (123) were allowed to adhere to botrocetin-treated vWF-coated glass slides in the absence or presence of RGDS peptide. After fixation and permeabilization, cells were stained with rhodamine-labeled phalloidin, and photographed under a fluorescence microscope (40× objective lens).

Mentions: Under a microscope, most adherent 1b9 cells (expressing GPIb-IX only) on vWF showed a rounded morphology similar to nonadherent cells (Fig. 2 A). In contrast, 123 cells (coexpressing GPIb-IX and αIIbβ3) spread on the vWF-coated surface (Fig. 2 A). Spreading of 123 cells was abolished by RGDS peptide (Fig. 2 A), indicating that spreading was mediated by integrins. Spreading of 123 cells was also inhibited by the monoclonal antibody 4F10, against human αIIbβ3 complex, and by anti-human β3 antibody SZ21 (Fig. 2 A). These data indicated that spreading was mainly mediated by integrin αIIbβ3 and that endogenous integrins were unlikely to play a major role. It is unlikely that coexpression of GPIb-IX with αIIbβ3 in the 123 cell line resulted in constitutively active integrin αIIbβ3, as 123 cells did not bind to soluble fibrinogen without prior activation (data not shown, see Fig. 3). Thus, vWF binding to GPIb-IX induces integrin-vWF interaction and integrin-mediated cell spreading. To examine whether GPIb-IX–mediated signaling pathway in CHO cells mimics that in platelets, we examined the effects of platelet activation inhibitors. We found that the PGE1, which elevates intracellular cAMP, wortmannin, and calphostin C, which inhibit PI-3 kinase and PKC, respectively, also inhibited GPIb-IX and integrin-dependent CHO cell spreading on vWF. Thus, GPIb-IX expressed in CHO cells induced integrin interaction with vWF in a manner similar to that in platelets.


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)

GPIb-IX activates integrin-vWF interaction and integrin-dependent cell spreading on vWF. (A) Three CHO cell lines, 1b9 (expressing GPIb-IX), 2b3a (expressing αIIbβ3), and 123 (expressing GPIb-IX and integrin αIIbβ3) were incubated in the microtiter plates coated with 10 μg/ml vWF for 30 min at 37°C in the presence of 5 μg/ml botrocetin. 123 cells were also incubated in the vWF-coated wells in the presence of 2 mM of the integrin antagonist peptide, RGDS (123+RGDS), a monoclonal antibody specific for integrin αIIbβ3 complex (123+4F10) and a monoclonal antibody against human β3 (123+SZ21) at 100 μg/ml, 500 ng/ml prostaglandin E1 (PGE1), 25 nM calphostin C, and 0.1 μM Wortmannin. (B) To compare αIIbβ3 function, 2b3a cells and 123 cells were incubated in microtiter wells coated with 10 μg/ml fibrinogen. After washing, the adherent cells were photographed under a phase contrast microscope (20× objective lens). (C) Cells expressing GPIb-IX (1b9) and coexpressing both αIIbβ3 and GPIb-IX (123) were allowed to adhere to botrocetin-treated vWF-coated glass slides in the absence or presence of RGDS peptide. After fixation and permeabilization, cells were stained with rhodamine-labeled phalloidin, and photographed under a fluorescence microscope (40× objective lens).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169335&req=5

Figure 2: GPIb-IX activates integrin-vWF interaction and integrin-dependent cell spreading on vWF. (A) Three CHO cell lines, 1b9 (expressing GPIb-IX), 2b3a (expressing αIIbβ3), and 123 (expressing GPIb-IX and integrin αIIbβ3) were incubated in the microtiter plates coated with 10 μg/ml vWF for 30 min at 37°C in the presence of 5 μg/ml botrocetin. 123 cells were also incubated in the vWF-coated wells in the presence of 2 mM of the integrin antagonist peptide, RGDS (123+RGDS), a monoclonal antibody specific for integrin αIIbβ3 complex (123+4F10) and a monoclonal antibody against human β3 (123+SZ21) at 100 μg/ml, 500 ng/ml prostaglandin E1 (PGE1), 25 nM calphostin C, and 0.1 μM Wortmannin. (B) To compare αIIbβ3 function, 2b3a cells and 123 cells were incubated in microtiter wells coated with 10 μg/ml fibrinogen. After washing, the adherent cells were photographed under a phase contrast microscope (20× objective lens). (C) Cells expressing GPIb-IX (1b9) and coexpressing both αIIbβ3 and GPIb-IX (123) were allowed to adhere to botrocetin-treated vWF-coated glass slides in the absence or presence of RGDS peptide. After fixation and permeabilization, cells were stained with rhodamine-labeled phalloidin, and photographed under a fluorescence microscope (40× objective lens).
Mentions: Under a microscope, most adherent 1b9 cells (expressing GPIb-IX only) on vWF showed a rounded morphology similar to nonadherent cells (Fig. 2 A). In contrast, 123 cells (coexpressing GPIb-IX and αIIbβ3) spread on the vWF-coated surface (Fig. 2 A). Spreading of 123 cells was abolished by RGDS peptide (Fig. 2 A), indicating that spreading was mediated by integrins. Spreading of 123 cells was also inhibited by the monoclonal antibody 4F10, against human αIIbβ3 complex, and by anti-human β3 antibody SZ21 (Fig. 2 A). These data indicated that spreading was mainly mediated by integrin αIIbβ3 and that endogenous integrins were unlikely to play a major role. It is unlikely that coexpression of GPIb-IX with αIIbβ3 in the 123 cell line resulted in constitutively active integrin αIIbβ3, as 123 cells did not bind to soluble fibrinogen without prior activation (data not shown, see Fig. 3). Thus, vWF binding to GPIb-IX induces integrin-vWF interaction and integrin-mediated cell spreading. To examine whether GPIb-IX–mediated signaling pathway in CHO cells mimics that in platelets, we examined the effects of platelet activation inhibitors. We found that the PGE1, which elevates intracellular cAMP, wortmannin, and calphostin C, which inhibit PI-3 kinase and PKC, respectively, also inhibited GPIb-IX and integrin-dependent CHO cell spreading on vWF. Thus, GPIb-IX expressed in CHO cells induced integrin interaction with vWF in a manner similar to that in platelets.

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.

Show MeSH
Related in: MedlinePlus