Role of Rab Proteins In Endocytosis and Trafficking of Factor VIIa and Endothelial Cell Protein C Receptor In Endothelial Cells.

Abstract 1145

Recent studies from our laboratory and others showed that endothelial cell protein C receptor (EPCR), the cellular receptor for protein C and activated protein C (APC), also serves as a receptor for factor VII (FVII) and activated factor VII (FVIIa). At present, the physiological importance of FVII/FVIIa binding to EPCR is largely unknown, but this interaction may play a role in the clearance or transport of FVII/FVIIa from circulation to tissues. Our recent studies showed that FVIIa (or APC) binding to EPCR promoted the endocytosis of EPCR via dynamin and caveolar-dependent pathways, and the endocytosed receptor-ligand complexes were accumulated in the recycling compartment (REC) before being targeted back to the cell surface (Blood 2009;114:1974-1986). Rab GTPases (Rab 4, Rab 5, Rab 7 and Rab 11 etc.), which localize to specific endosomal structures, have been shown to play crucial roles in the endocytic and exocytic pathways of receptor or receptor/ligand complexes. The role of these Ras-like small GTPases is unknown in endocytosis and trafficking of EPCR and EPCR/FVIIa complexes. The present study was undertaken in order to investigate the role of different Rab GTPases (Rab 4A, Rab 5 and Rab11) in the intracellular trafficking of EPCR and internalized FVIIa in endothelial cells. For this, we examined the effect of expressing wild-type (wt) or mutant Rab proteins on the intracellular distribution of FVIIa in human umbilical vein endothelial cells (HUVEC). The wild-type, constitutively active and dominant negative mutants of Rab 4A, Rab 5 and Rab 11 were cloned in adenoviral shuttle vector pacAd5 K-N pA CMV and the recombinant adenoviruses expressing these Rab GTPase variants were generated in human embryonic kidney (HEK) cells. HUVEC were infected with recombinant adenoviruses encoding for the wild-type, active or dominant negative mutant of Rab 4A, Rab 5 and Rab 11 (25 moi/cell). After culturing the cells for 24 h, they were incubated with recombinant FVIIa conjugated with Alexa fluor 488 fluorescent dye (AF488-FVIIa) for 1 h at 37°C. The intracellular distribution of FVIIa was analyzed by monitoring the fluorescence of AF488-FVIIa by confocal microscopy. The intracellular distribution of EPCR and Rab proteins was evaluated by confocal microscopy after immunofluorescence staining. Expression of Rab 4A wt or constitutively active Rab 4A (Q67L) forms led to accumulation of AF488-FVIIa within the Rab 4A positive early/sorting endosomes, whereas FVIIa accumulation in the REC was inhibited. In cells expressing Rab 4A dominant negative form (S22N), FVIIa was trafficked normally and accumulated in the REC. Rab 4A is known to regulate fusion of early and sorting endosomes, as well as recycling of the internalized receptor or receptor/ligand complexes from early/sorting endosomes back to the cell surface. Increased accumulation of FVIIa in early/sorting endosomes but a decrease in REC in HUVEC transduced to express wt and constitutively active Rab 4A, suggests that Rab 4A plays a role in the transport of internalized FVIIa and FVIIa-EPCR complexes from sorting endosomes back to the cell surface. HUVEC expressing Rab 5 wt or active mutant (Q79L) showed larger endosomal structures beneath the plasma membrane where EPCR and FVIIa were accumulated; very little FVIIa entered the REC. The trafficking of internalized FVIIa remained unaffected in HUVEC expressing Rab 5A dominant negative form (S34N). As Rab 5 is known to induce receptor internalization and fusion between early endosomes, the large endosomal structures containing AF488-FVIIa found in HUVEC expressing wt or constitutively active form but not in cells expressing the dominant negative form suggests that Rab 5 facilitates internalization of FVIIa-EPCR complexes. In contrast to the data obtained in HUVEC expressing Rab 4A and Rab 5, the intracellular trafficking of AF488-FVIIa remained unaffected in HUVEC expressing either wt or constitutively active Rab 11 mutant. Rab 11 dominant negative mutant (S34N) prevented the entry of AF488-FVIIa into REC. The observation that the dominant negative form of Rab 11 inhibits the entry of internalized FVIIa to the REC indicates that the activation of Rab 11 by GTP is required for the transport of FVIIa from sorting endosomes toward the recycling compartment. Overall our present data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR and internalized FVIIa in endothelial cells.