Filamin A binding stabilizes nascent glycoprotein Ibalpha 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 GpIbalpha possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbalpha directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbalpha biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbalpha with GpIbbeta, GpIX with and without filamin A. We observed that surface GpIbalpha expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbalpha, GpIbbeta, and GpIX (CHO-GpIbalpha/betaIX), lysates from CHO-GpIbalpha/betaIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbalpha, but lesser amounts of the approximately 15-kDa C-terminal peptide released when the extracellular domain of GpIbalpha is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbalpha at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbalpha is accompanied by decreased immature GpIbalpha and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbalpha in CHO-GpIbalpha/betaIX 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 GpIbalpha binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbalpha to the cell surface.
Feng
Shuju
S
Michael E. DeBakey Veterans Affairs Medical Center, Baylor College of Medicine and Rice University, Houston, Texas 77030, USA.
Lu
Xin
X
Kroll
Michael H
MH
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism* / Q000502:physiology; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism; D064448:Filamins; D008840:Microfilament Proteins / Q000378:metabolism* / Q000502:physiology; D046912:Multiprotein Complexes / Q000096:biosynthesis; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis* / Q000378:metabolism*; D021381:Protein Transport
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D019038:Platelet Glycoprotein GPIb-IX Complex; C067713:lactacystin; D020126:Brefeldin A; D000111:Acetylcysteine