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. Gplb/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 GpIbα binds directly to a dimeric 14- 3-3 adapter protein ζ isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3ζ, the authors examined 14-3-3ζ interactions with GpIbα and GpIbβ in heterologous cells and platelets. Truncations of GpIbα at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3ζ. Deletion of GpIbα from Trp⁵⁷⁰ to Ser⁵⁹⁰ eliminated 14-3-3ζ binding, and deletion of the sequence from Arg⁵⁴²- Trp⁵⁷⁰ enhanced binding of 14-3-3ζ to GpIbα. All GpIbα mutations that eliminated GpIbα binding to the GST-14-3-3ζ fusion protein also eliminated GpIbα binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIbα/β/IX resulted in the phosphorylation of GpIbβ associated with enhanced binding of GpIbα to GST-14,3-3ζ fusion protein and increased 14-3-3ζ coimmunoprecipitated with GpIbα. When intact human platelets aggregated in response to 90 dynes/cm² shear stress, 14-3- 3ζ disassociated from GpIbα. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3ζ from GpIbα. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIbα regulate 14-3-3ζ binding to GpIbα/β/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbβ enhances 14-3-3ζ binding to the GpIb/IX/V complex in human platelets.