Cytoplasmic domains of GpIbα and GpIbβ regulate 14-3-3ζ binding to GpIb/IX/V

Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIb alpha binds directly to a dimeric 14-3-3 adapter protein zeta isoform, To explore structural determinants of GpIb/IX/V binding to 14-3-3 zeta, the authors examined 14-3-3 zeta interactions with GpIb alpha and GpIb beta in heterologous cells and platelets. Truncations of GpIb alpha at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3 zeta, Deletion of GpIb alpha from Trp(570) to Ser(590) eliminated 14-3-3 zeta binding, and deletion of the sequence from Arg(542)-Trp(570) enhanced binding of 14-3-3 zeta to GpIb alpha. All GpIb alpha mutations that eliminated GpIb alpha binding to the GST-14-3-3 zeta fusion protein also eliminated GpIb beta binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIb alpha/beta/IX resulted in the phosphorylation of GpIb beta associated with enhanced binding of GpIb beta to GST-14-3-3 zeta fusion protein and increased 14-3-3 zeta coimmunoprecipitated with GpIb alpha,When intact human platelets aggregated in response to 90 dynes/cm(2) shear stress, 14-3-3 zeta disassociated from GpIb alpha. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3 zeta from GpIb alpha, These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIb alpha regulate 14-3-3 zeta binding to GpIb alpha/beta/IX, and suggest that protein kinase A-dependent phosphorylation of GpIb beta enhances 14-3-3 zeta binding to the GpIb/IX/V complex in human platelets. (Blood. 2000; 95:551-557) (C) 2000 by The American Society of Hematology.