The antibody to GD3 ganglioside, R24, is rapidly endocytosed and recycled to the plasma membrane via the endocytic recycling compartment. Inhibitory effect of brefeldin A and monensin.
Gangliosides are sialic acid-containing glycosphingolipids present on mammalian plasma membranes, where they participate in cell-surface events such as modulation of growth factor receptors and cell-to-cell and cell-to-matrix interactions. Antibodies to gangliosides have been associated with a wide range of clinically identifiable acute and chronic neuropathy syndromes. In addition, antibodies to tumor-associated gangliosides are being used as therapeutic agents. Their binding to and release from cell membranes and intracellular destinations have not so far been extensively examined. In this study, we characterized in both GD3 ganglioside-expressing Chinese hamster ovary (CHO)-K1 and SK-Mel 28 melanoma cells the intracellular trafficking and subcellular localization of the mouse monoclonal antibody to GD3, R24. By biochemical techniques and detailed confocal microscopic analysis, we demonstrate that the GD3-R24 antibody complex is rapidly and specifically internalized by a dynamin 2-independent pathway and then accumulates in the endocytic recycling compartment. In addition, we show that the R24 antibody exits the recycling compartment en route to the plasma membrane by a dynamin 2-dependent pathway sensitive to brefeldin A and monensin. Taken together, our results indicate that the GD3-R24 complex is endocytosed in GD3-expressing cells, accumulates in the recycling endosome, and is transported back to the plasma membrane via a route that involves clathrin-coated vesicles.
Iglesias-Bartolomé
Ramiro
R
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica, Universidad Nacional de Córdoba, Argentina.
Crespo
Pilar M
PM
Gomez
Guillermo A
GA
Daniotti
Jose L
JL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology*; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D006224:Cricetinae; D034285:Dynamin II / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005732:Gangliosides / Q000276:immunology*; D006801:Humans; D008545:Melanoma / Q000188:drug therapy / Q000378:metabolism; D018613:Microscopy, Confocal; D008985:Monensin / Q000494:pharmacology*; D021381:Protein Transport; D013347:Subcellular Fractions
D000911:Antibodies, Monoclonal; D005732:Gangliosides; D020126:Brefeldin A; C026226:ganglioside, GD3; D008985:Monensin; D034285:Dynamin II