The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.

Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.