Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin αIIbβ3 using a reconstituted mammalian cell expression model

被引:107
作者
Gu, MY
Xi, XD
Englund, GD
Berndt, MC
Du, XP
机构
[1] Univ Illinois, Coll Med, Dept Pharmacol, Chicago, IL 60612 USA
[2] Baker Med Res Inst, Prahran, Vic 3181, Australia
关键词
platelet; glycoprotein Ib-IX; integrin; 14-3-3; von Willebrand factor;
D O I
10.1083/jcb.147.5.1085
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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 GPIb alpha 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 GPIb alpha 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 GPIb alpha.
引用
收藏
页码:1085 / 1096
页数:12
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