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.
BACKGROUND
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.


OBJECTIVE
To explore the mechanism underlying the roles of 14-3-3zeta and filamin A in regulating the VWF binding function of GPIb-IX.


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.


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.
Yuan
Yanhong
Y
School of Biological Science and Medical Engineering, Beijing University of Aeronautics and Astronautics, 37 Xueyuan Road, Haidian district, Beijing, China.
Zhang
Weilin
W
Yan
Rong
R
Liao
Yi
Y
Zhao
Lili
L
Ruan
Changgeng
C
Du
Xiaoping
X
Dai
Kesheng
K
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D016466:CHO Cells; D002463:Cell Membrane Permeability; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D010973:Platelet Adhesiveness / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012310:Ristocetin / Q000494:pharmacology; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin