Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrindependent
pathway. We found that binding of transferrin to the receptor induced rapid generation of cell
surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset
of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their
basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme
expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these
conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K+ depletion
of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin
much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity
led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon
transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions
by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin
receptor complex by clathrin-independent pathway.