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Identification of a novel 14-3-3zeta binding site within the cytoplasmic domain of platelet glycoprotein Ibalpha that plays a key role in regulating the von Willebrand factor binding function of glycoprotein Ib-IX.
- Source :
-
Circulation research [Circ Res] 2009 Dec 04; Vol. 105 (12), pp. 1177-85. Date of Electronic Publication: 2009 Oct 29. - Publication Year :
- 2009
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Abstract
- Rationale: The interaction between platelet glycoprotein (GP) Ib-IX and von Willebrand factor (VWF) is initiated by conformational changes in immobilized VWF and is also regulated by the intraplatelet proteins 14-3-3zeta and filamin A. Both 14-3-3zeta and filamin A associate with the cytoplasmic domain of GPIbalpha, whereas little is known about their relationship in regulating the VWF binding function of GPIb-IX.<br />Objective: To explore the mechanism underlying the roles of 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX.<br />Methods and Results: A truncation mutant of GPIbalpha (Delta565) deleting the C-terminal 14-3-3zeta binding sites retained 14-3-3zeta binding function, in contrast, deletion of the C-terminal residues 551 to 610 of GPIbalpha totally abolished 14-3-3zeta binding, indicating that the residues 551 to 564 of GPIbalpha are important in the interaction between 14-3-3zeta and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), R557GpSLP561 corresponding to residues 557 to 561 of GPIbalpha eliminated the association of 14-3-3zeta with Delta565. MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding, and diminished ability to mediate cell adhesion to VWF under flow conditions.<br />Conclusions: These data suggest a phosphorylation-dependent binding of 14-3-3zeta to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3zeta binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.
- Subjects :
- 14-3-3 Proteins genetics
Animals
Binding Sites
Blood Platelets drug effects
CHO Cells
Cell Membrane Permeability
Contractile Proteins genetics
Cricetinae
Cricetulus
Cytoplasm metabolism
Cytoskeleton metabolism
Filamins
Humans
Microfilament Proteins genetics
Mutation
Peptides pharmacology
Phosphorylation
Platelet Glycoprotein GPIb-IX Complex genetics
Protein Binding
Protein Structure, Tertiary
Recombinant Proteins metabolism
Ristocetin pharmacology
Transfection
von Willebrand Factor genetics
14-3-3 Proteins metabolism
Blood Platelets metabolism
Contractile Proteins metabolism
Microfilament Proteins metabolism
Platelet Adhesiveness drug effects
Platelet Glycoprotein GPIb-IX Complex metabolism
von Willebrand Factor metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1524-4571
- Volume :
- 105
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Circulation research
- Publication Type :
- Academic Journal
- Accession number :
- 19875727
- Full Text :
- https://doi.org/10.1161/CIRCRESAHA.109.204669