Mast Cell-specific Gangliosides and Fc∊RI Follow the Same Endocytic Pathway From Lipid Rafts in RBL-2H3 Cells

Abstract

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in Fc∊RI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with Fc∊RI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against Fc∊RI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as Fc∊RI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against Fc∊RI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and Fc∊RI are internalized in the same coated vesicles. At 5 min, the gangliosides and Fc∊RI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as Fc∊RI. This study contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.