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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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Quantitation of the effects of truncation mutations on cell spreading. The 123, Δ591/2b3a, and Δ559/2b3a cells were incubated for 1 h at 37°C in vWF-coated chamber slides, washed, fixed, and permeabilized. The cells were then stained with rhodamine-labeled phalloidin, and photographed under a Nikon fluorescence microscope using a cooled CCD camera. To estimate the surface area of round cells, RGD-treated 123 cells were allowed to adhere to vWF and stained with rhodamine-labeled phalloidin as above. Three randomly selected fields (for each cell line) were then analyzed for surface area of adherent cells using Image-Pro Plus software. Results were expressed as mean ± SD (for 123 cells, n = 200; for Δ591/2b3a cells, n = 180; for Δ559/2b3a cells, n = 192; for RGD-treated 123 cells, n = 60). The difference between 123 cell and Δ591/2b3a cells is highly significant (P < 0.00001).
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Figure 8: Quantitation of the effects of truncation mutations on cell spreading. The 123, Δ591/2b3a, and Δ559/2b3a cells were incubated for 1 h at 37°C in vWF-coated chamber slides, washed, fixed, and permeabilized. The cells were then stained with rhodamine-labeled phalloidin, and photographed under a Nikon fluorescence microscope using a cooled CCD camera. To estimate the surface area of round cells, RGD-treated 123 cells were allowed to adhere to vWF and stained with rhodamine-labeled phalloidin as above. Three randomly selected fields (for each cell line) were then analyzed for surface area of adherent cells using Image-Pro Plus software. Results were expressed as mean ± SD (for 123 cells, n = 200; for Δ591/2b3a cells, n = 180; for Δ559/2b3a cells, n = 192; for RGD-treated 123 cells, n = 60). The difference between 123 cell and Δ591/2b3a cells is highly significant (P < 0.00001).

Mentions: To investigate whether 14-3-3 binding plays a role in GPIb-IX–induced integrin-vWF interaction and integrin-dependent cell spreading on vWF, the 123 cells and Δ591/2b3a cells were allowed to adhere to vWF-coated microtiter wells. As examined under the microscope, ∼70% of the 123 cells were spread on both vWF- and fibrinogen-coated microtiter wells. In contrast, only a small percentage (∼30%) of Δ591/2b3a cells appeared spreading on vWF, indicating that GPIb-IX–induced integrin-vWF interaction was inhibited (Fig. 7). To quantitate the cell spreading objectively, cells adherent to vWF were permeabilized and stained with rhodamine-labeled phalloidin. Fluorescently stained cells in randomly selected fields were quantitated for cell surface area using Image-Pro Plus software (Media Cybernetics). As shown in Fig. 8, the average surface area of Δ591/2b3a cells were about half of that of 123 cells, indicating that the spreading of the mutant cell line was significantly reduced but not totally abolished. Since Δ591/2b3a cells adhered and spread on fibrinogen in a manner similar to 123 cells, the ligand-binding function of αIIbβ3 and the integrin-mediated spreading process was not impaired in the Δ591/2b3a cell line. Thus, inhibition of GPIb-IX– and integrin-dependent spreading on vWF in this cell line is unlikely to be caused by a defect in ligand-binding function of αIIbβ3 or in the integrin's post-ligand occupancy events. These data suggest that 14-3-3ζ binding to the COOH-terminal region of GPIbα plays an important role in GPIb-IX–mediated activation of integrin αIIbβ3. Spreading of a small percentage of mutant cells reflects a background level of αIIbβ3-vWF interaction or the interaction of vWF with the endogenous CHO cell integrin (see Fig. 2 C).


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)

Quantitation of the effects of truncation mutations on cell spreading. The 123, Δ591/2b3a, and Δ559/2b3a cells were incubated for 1 h at 37°C in vWF-coated chamber slides, washed, fixed, and permeabilized. The cells were then stained with rhodamine-labeled phalloidin, and photographed under a Nikon fluorescence microscope using a cooled CCD camera. To estimate the surface area of round cells, RGD-treated 123 cells were allowed to adhere to vWF and stained with rhodamine-labeled phalloidin as above. Three randomly selected fields (for each cell line) were then analyzed for surface area of adherent cells using Image-Pro Plus software. Results were expressed as mean ± SD (for 123 cells, n = 200; for Δ591/2b3a cells, n = 180; for Δ559/2b3a cells, n = 192; for RGD-treated 123 cells, n = 60). The difference between 123 cell and Δ591/2b3a cells is highly significant (P < 0.00001).
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Related In: Results  -  Collection

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Figure 8: Quantitation of the effects of truncation mutations on cell spreading. The 123, Δ591/2b3a, and Δ559/2b3a cells were incubated for 1 h at 37°C in vWF-coated chamber slides, washed, fixed, and permeabilized. The cells were then stained with rhodamine-labeled phalloidin, and photographed under a Nikon fluorescence microscope using a cooled CCD camera. To estimate the surface area of round cells, RGD-treated 123 cells were allowed to adhere to vWF and stained with rhodamine-labeled phalloidin as above. Three randomly selected fields (for each cell line) were then analyzed for surface area of adherent cells using Image-Pro Plus software. Results were expressed as mean ± SD (for 123 cells, n = 200; for Δ591/2b3a cells, n = 180; for Δ559/2b3a cells, n = 192; for RGD-treated 123 cells, n = 60). The difference between 123 cell and Δ591/2b3a cells is highly significant (P < 0.00001).
Mentions: To investigate whether 14-3-3 binding plays a role in GPIb-IX–induced integrin-vWF interaction and integrin-dependent cell spreading on vWF, the 123 cells and Δ591/2b3a cells were allowed to adhere to vWF-coated microtiter wells. As examined under the microscope, ∼70% of the 123 cells were spread on both vWF- and fibrinogen-coated microtiter wells. In contrast, only a small percentage (∼30%) of Δ591/2b3a cells appeared spreading on vWF, indicating that GPIb-IX–induced integrin-vWF interaction was inhibited (Fig. 7). To quantitate the cell spreading objectively, cells adherent to vWF were permeabilized and stained with rhodamine-labeled phalloidin. Fluorescently stained cells in randomly selected fields were quantitated for cell surface area using Image-Pro Plus software (Media Cybernetics). As shown in Fig. 8, the average surface area of Δ591/2b3a cells were about half of that of 123 cells, indicating that the spreading of the mutant cell line was significantly reduced but not totally abolished. Since Δ591/2b3a cells adhered and spread on fibrinogen in a manner similar to 123 cells, the ligand-binding function of αIIbβ3 and the integrin-mediated spreading process was not impaired in the Δ591/2b3a cell line. Thus, inhibition of GPIb-IX– and integrin-dependent spreading on vWF in this cell line is unlikely to be caused by a defect in ligand-binding function of αIIbβ3 or in the integrin's post-ligand occupancy events. These data suggest that 14-3-3ζ binding to the COOH-terminal region of GPIbα plays an important role in GPIb-IX–mediated activation of integrin αIIbβ3. Spreading of a small percentage of mutant cells reflects a background level of αIIbβ3-vWF interaction or the interaction of vWF with the endogenous CHO cell integrin (see Fig. 2 C).

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