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.
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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Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ilinois 60612, USA.
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Englund
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Berndt
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D048948:14-3-3 Proteins; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D003285:Contractile Proteins / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D064448:Filamins; D005786:Gene Expression Regulation; D008840:Microfilament Proteins / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000096:biosynthesis* / Q000378:metabolism* / Q000502:physiology; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000502:physiology*; D011506:Proteins / Q000502:physiology*; D017384:Sequence Deletion / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase; D014841:von Willebrand Factor / Q000378:metabolism
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D010455:Peptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D011506:Proteins; D014841:von Willebrand Factor; D005340:Fibrinogen; D014446:Tyrosine 3-Monooxygenase