Filamin A binding stabilizes nascent glycoprotein Ibα trafficking and thereby enhances its surface expression

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbα possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbα directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbα biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbα with GpIbβ, GpIX with and without filamin A. We observed that surface GpIbα expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbα, GpIbβ, and GpIX (CHO-GpIbα/βIX), lysates from CHO-GpIbα/βIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbα, but lesser amounts of the ∼15-kDa C-terminal peptide released when the extracellular domain of GpIbα is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbα at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbα is accompanied by decreased immature GpIbα 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 GpIbα to the cell surface.