Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibα

Abstract

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.

A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function

“…FlnA binding facilitates GPIb surface expression in Chinese hamster ovary (CHO) cells (Feng et al, 2005), and the interaction between FlnA and GPIb has been reported to influence VWF receptor function, although conflicting effects are found in the literature. CHO cells transfected with GPIb mutants that lack the FlnA binding site have decreased VWF binding (Dong et al, 1997;Schade et al, 2003), VWF-induced cell aggregation (Mistry et al, 2000), and/or adhesion to a VWF matrix under high shear (Cranmer et al, 1999;Williamson et al, 2002;Cranmer et al, 2005). In contrast, another study has shown that FlnA binding to GPIb negatively regulates VWF binding to CHO cells and CHO cell adhesion under both static and flow conditions (Englund et al, 2001).…”

“…Whether FlnA influences the ligand binding properties of GPIb has been controversial in the literature. Some groups have reported that it enhances (Dong et al, 1997;Cranmer et al, 1999Cranmer et al, , 2005Mistry et al, 2000;Williamson et al, 2002;Schade et al, 2003) and others that it diminishes (Englund et al, 2001). Our studies directly confirm the former, pointing out the protein tyrosine kinase Syk.…”

The structure of the GPIb–filamin A complex

“…A GST fusion protein encompassing residues 515 to 610 of the cytoplasmic tail of GPIb␣ bound to FLNa. A synthetic 22-mer peptide (hereafter designated GPIb␣556-577), including the previously defined FLNa-binding site 11,28 ( Figure 1A), completely inhibited this binding ( Figure 1B) and also efficiently bound full-length FLNa when immobilized on Sepharose beads ( Figure 1C). Figure 1D shows that GST-fusion proteins representing IgFLNa17 and 19, but not 18, bound to GPIb␣556-577 beads.…”

“…It has been recently shown that the mutations of these residues to alanines abrogate GPIb␣ binding to FLNa. 28 There might also be some specificity in the regulation of FLNa-integrin and FLNa-GPIb␣ interactions. While the integrin cytoskeleton interaction may be regulated by phosphorylation of Tyr or Thr residues of integrin tails, 38,39 such regulation cannot take place in the case of the GPIb␣-FLNa interactions.…”

“…These findings are consistent with previous observations that mutations of Phe568 and Trp570 abolish the binding of GPIb␣ to FLNa. 28 Furthermore, the association between GPIb␣ and FLNa resists chaotropic solvents over a wide pH range, although sodium dodecyl sulfate can separate the complex. 30 This behavior is consistent with the many hydrophobic contacts in the FLNa-GPIb␣ interface.…”