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© The Rockefeller University Press, 0021-9525/1999//1085 $5.00
The Journal of Cell Biology, Volume 147, Number 5, , 1999 1085-1096

Original Article

Analysis of the Roles of 14-3-3 in the Platelet Glycoprotein Ib-IX–Mediated Activation of Integrin {alpha}IIbβ3 Using a Reconstituted Mammalian Cell Expression Model



Minyi Gua, Xiaodong Xia, Graham D. Englunda, Michael C. Berndtb, and Xiaoping Dua

a Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ilinois 60612
b Baker Medical Research Institute, Prahran, VIC 3181, Australia
Department of Pharmacology, the University of Illinois, College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612.(312) 996-1225(312) 355-0237

xdu{at}uic.edu

We have reconstituted the platelet glycoprotein (GP) Ib-IX–mediated activation of the integrin {alpha}IIbβ3 in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing {alpha}IIbβ3 adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin {alpha}IIbβ3 and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against {alpha}IIbβ3. vWF binding to GPIb-IX also activates soluble fibrinogen binding to {alpha}IIbβ3 indicating that GPIb-IX mediates a cellular signal leading to {alpha}IIbβ3 activation. Deletion of the 14-3-3–binding site in GPIb{alpha} inhibited GPIb-IX–mediated fibrinogen binding to {alpha}IIbβ3 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 GPIb{alpha} induced an endogenous integrin-dependent cell spreading on vWF without requiring {alpha}IIbβ3, but inhibited vWF-induced fibrinogen binding to {alpha}IIbβ3. Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX–mediated activation of {alpha}IIbβ3 requires 14-3-3 interaction with GPIb{alpha}.

Key Words: platelet • glycoprotein Ib-IX • integrin • 14-3-3 • von Willebrand factor

© 1999 The Rockefeller University Press

Abbreviations used in this paper: GFP, green fluorescent protein; GP, glycoprotein; PGE1, prostaglandin E1; PKA, protein kinase A; vWF, von Willebrand factor.


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