The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIb␣ and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIb␣ intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569 -579 of the GPIb␣ cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant ⌬557-568 and ⌬569 -579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIb␣ for interaction with filamin-1.

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin ␣ IIb ␤ 3 -dependent stable adhesion and spreading. Interaction of the 14-3-3 adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIb␣ subunit has been implicated in the control of ␣ IIb ␤ 3 activation and cell spreading. In this study, we have examined potentially novel 14-3-3 binding sites by expressing mutant forms of GPIb␣ in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIb␣-14-3-3 interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3 from GPIb␣. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIb␣-14-3-3 binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3 binding site within the cytoplasmic tail of GPIb␣ that has an important functional role in regulating integrin-dependent cell spreading. IntroductionThe 14-3-3 protein family consists of ubiquitous homodimeric or heterodimeric intracellular adaptor proteins that take part in the signaling pathways of numerous biologic responses. 1 There have been 5 isoforms identified in human platelets, the ⑀ and isoforms being weakly expressed relative to the more abundant ␥, , and ␤ isoforms. 2 The crystal structure of 14-3-3 revealed the association of 2 monomers, each composed of a bundle of 9 antiparallel helices, to form a large negatively charged groove that could be implicated in interactions with other proteins. 3 The 14-3-3 proteins bind to specific phosphoserine-containing motifs, 4 and their dimeric nature allows them to act as intramolecular and intermolecular phosphorylation-dependent bridges. 5 The functions of the different isoforms in platelets are, however, still poorly understood.In a seminal study, it was reported that 14-3-3 interacted with the platelet von Willebrand factor (VWF) receptor, the glycoprotein Ib-V-IX (GPIb-V-IX) complex. 6 A binding site for 14-3-3 was found at the C-terminus of GPIb␣ within a serine-rich 606-SGHSL-610 sequence bearing similarities to phosphoserine containing 14-3-3 binding motifs. 1,7 Further work showed that the serine at position 609 was predominantly phosphorylated in resting platelets and that phosphorylation was required for 14-3-3 binding. 8 Glutathione S-transferase (GST)-14-3-3 pull-down and immunoprecipitation studies of GPIb-IX-transfected cells pointed to the existence of a separate binding or regulatory site in the GPIb␣ 570-590 ...

Glycoprotein (GP)Ib

Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbα is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbα, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbα. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561–Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbα binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbα in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbα Trp570→Ala and Phe568→Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbα, which is critically dependent on both Trp570 and Phe568.