Affiliation:
1. From the Veterans Administration Medical Center, Baylor College of Medicine and Rice University, Houston, TX, and the Baker Medical Research Institute, Prahran, Victoria, Australia.
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.
Publisher
American Society of Hematology
Subject
Cell Biology,Hematology,Immunology,Biochemistry
Cited by
63 articles.
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