Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.
BACKGROUND
Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway.


OBJECTIVE
The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX.


RESULTS
In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain.


CONCLUSIONS
Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.
Mangin
P H
PH
INSERM UMR_S 949, EFS-Alsace, Université de Strasbourg, Strasbourg, France. pierre.mangin@efs-alsace.fr
Receveur
N
N
Wurtz
V
V
David
T
T
Gachet
C
C
Lanza
F
F
D048948:14-3-3 Proteins / Q000737:chemistry*; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D006487:Hemostasis; D006801:Humans; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000096:biosynthesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis* / Q000235:genetics*; D011485:Protein Binding; D020033:Protein Isoforms; D013927:Thrombosis / Q000097:blood / Q000235:genetics
D048948:14-3-3 Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D020033:Protein Isoforms