Proteins of the Calnexin Cycle Regulate αIIbβ3 Surface Expression in Human Megakaryocyte-Lineage Cells.

The αIIbβ3 integrin receptor mediates platelet aggregation, and individuals without functional αIIbβ3 manifest the mucocutaneous bleeding disorder Glanzmann thrombasthenia. Since platelets also participate in the pathophysiology of thrombosis, anti-αIIbβ3 agents have been successful in reducing mortality after percutaneous coronary interventions and from platelet-mediated coronary thrombosis. Accordingly, much research has focused on the structure, activation and signaling of the mature receptor on the cell surface. There is much to be gained medically and scientifically by understanding the intracellular processes by which integrins are assembled and expressed. Some forms of Glanzmann thrombasthenia result from mutations that disrupt normal integrin folding and assembly, causing the integrin subunits to be retained in the ER and degraded. These cases indicate the existence of a quality control mechanism for αIIbβ3 by which misfolded subunits are recognized, retained and degraded. Previous studies in HEK293 cells and murine megakaryocytes have identified the calnexin cycle as part of this quality control mechanism. We have expanded upon these findings and studied their functional implications by determining the effect of siRNA-induced knock down of proteins from six families of post-translational processing proteins on αIIbβ3 surface expression on human megakaryocyte-lineage cells derived from umbilical cord blood (UCB): lectins, glucosyltransferases, mannosidases, HSP90s, HSP70s, BiP, protein disulfide isomerases, and tetraspanins. We report that siRNA against two proteins in the calnexin cycle decreased surface expression of natively-folded αIIbβ3 on UCB-derived megakaryocytes as measured by binding of 10E5, an αIIbβ3 complex-dependent mAb. siRNA against UDP-glucose ceramide glucosyltransferase-like 1(UGGT, a glucosyltransferase) decreased binding by 36 +/− 9%, and siRNA against EDEM1 (a mannosidase) decreased binding by 15 +/− 14%, both p < 0.05. Additionally, siRNA against CD9 increased binding by 17 +/− 8%, p < 0.05, suggesting that CD9 may be a negative regulator of αIIbβ3 surface expression. UGGT is a glucosyltransferase protein and folding sensor of the calnexin cycle that recognizes and binds to glycoproteins that are in nearly-native folding conformations, but does not bind to fully folded glycoproteins. Glycoproteins that are glucosylated by UGGT may be bound by calnexin and retained in the ER, increasing their time for folding. Fully folded proteins cannot be reglucosylated by UGGT and escape the calnexin cycle, exiting the ER. EDEM1 is a mannosidase that interacts with calnexin to accept terminally misfolded proteins and direct them to degradation. Together, these data suggest a model in which the level of expression of native-folded αIIbβ3 on the cell surface is regulated in the ER by the calnexin cycle.