Filamin A Binding Stabilizes Nascent Glycoprotein Ibα Trafficking and Thereby Enhances Its Surface Expression

Abstract

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIb␣ possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIb␣ directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIb␣ biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wildtype or mutant GpIb␣ with GpIb␤, GpIX with and without filamin A. We observed that surface GpIb␣ expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIb␣, GpIb␤, and GpIX (CHO-GpIb␣/␤IX), lysates from CHO-GpIb␣/␤IX ؉ filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIb␣, but lesser amounts of the ϳ15-kDa C-terminal peptide released when the extracellular domain of GpIb␣ is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIb␣ at C-terminal residue 557 or by a deletion between amino acids 560 -570, the decreased synthesis of mature GpIb␣ is accompanied by decreased immature GpIb␣ and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIb␣ in CHO-GpIb␣/␤IX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIb␣ binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIb␣ to the cell surface.

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).…”

The structure of the GPIb–filamin A complex

“…18 After transfection (72 hours) the cells were rinsed with phosphate-buffered saline (PBS) and lysed in PBS containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM EGTA, 1 mM EDTA, 1 g mL Ϫ1 pepstatin A, 1 g mL Ϫ1 aprotinin, and 1 g mL Ϫ1 leupeptin. Cell lysates were centrifuged at 15 000g for 10 minutes.…”

“…NMR spectroscopy confirmed that the double mutant caused no major defects in folding of the domain (data not shown). We also expressed GFP-tagged full-length human FLNa in the CHO-GPIb␣␤/IX cells that stably express GPIb␣␤/IX complex, 18 and were able to bring down FLNa with the GPIb␣␤/IX complex ( Figure 3C). Double-mutant (G1879D and C1912D) GFP-FLNa constructs expressed at levels equivalent to endogenous FLNa in CHO cells did not associate at all with GPIb␣␤/IX ( Figure 3C), confirming that IgFLNa17 is also the major GPIb␣-binding site in vivo.…”

“…7 The cytoplasmic tails of GPIb␣ are attached to actin filaments by FLNa. 2,8,9 Previous work showed that the GPIb␣ cytoplasmic domain interacted with C-terminal domains of FLNa (domains [17][18][19] 10 and delimited the GPIb␣ binding site for FLNa to a short span of residues 557 to 575. 11 The GPIb␣-FLNa interaction is essential for the platelet adhesion to VWF that binds extracellular domain of the GPIb-V-IX receptor, for maintaining normal platelet integrity and shape, and for normal signal transduction reactions involved in platelet activation.…”

Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

“…16 The intracellular properties of the receptor have also been incriminated in defective platelet biogenesis in relation to the linkage of GPIb-V-IX to the submembranous actin cytoskeleton, which is mediated by filamin. 17 Actin reorganization, with the participation of PKCα, appears to be important for the formation of proplatelet extensions, but the involvement of a GPIb-actin link in this process has not been established. 18,19 Finally, as GPIb-V-IX is expressed at high density on the cell surface (24,000 copies/platelet), it could represent a membrane structuring element in the course of proplatelet formation.…”