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

Open Access
Publisher Website
Google Scholar
Abstract
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 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 GpIb and GpIbβ in heterologous cells and platelets. Truncations of GpIb at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3ζ. Deletion of GpIb from Trp570 to Ser590 eliminated 14-3-3ζ binding, and deletion of the sequence from Arg542-Trp570 enhanced binding of 14-3-3ζ to GpIb. All GpIb mutations that eliminated GpIb binding to the GST-14-3-3ζ fusion protein also eliminated GpIbβ binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIb/β/IX resulted in the phosphorylation of GpIbβ associated with enhanced binding of GpIbβ to GST-14-3-3ζ fusion protein and increased 14-3-3ζ coimmunoprecipitated with GpIb. When intact human platelets aggregated in response to 90 dynes/cm2 shear stress, 14-3-3ζ disassociated from GpIb. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3ζ from GpIb. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIb regulate 14-3-3ζ binding to GpIb/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.