Neural cell adhesion molecule is endocytosed via a clathrin-dependent pathway

Neural cell adhesion molecule (NCAM) constitutes a group of cell surface glycoproteins that regulate cell-cell interactions in the developing and adult brain. Endocytosis is a mechanism which dynamically controls the amount of cell surface NCAM expression and may involve the rapid changes occurring in NCAM expression under certain physiological or pathological conditions. However, the endocytic pathway of NCAM is presently unknown. Using astrocytes in culture and immunofluorescence we show that NCAM is internalized and that the immunolabelling presents a high degree of colocalization with clathrin, alpha-adaptin and transferrin, suggesting that NCAM is endocytosed by a clathrin-dependent pathway. Potassium depletion which disrupts clathrin-mediated endocytosis, inhibited internalization of NCAM. Electron microscopy and immunogold studies also demonstrate that the surface of clathrin-coated vesicles are also immunolabelled for both alpha-adaptin and PSA-NCAM, the highly sialylated isoform of NCAM. Furthermore, immunoprecipation studies demonstrate that NCAM is associated with both clathrin and alpha-adaptin, a component of adaptor complex AP-2, in brain, neurons and astrocytes. These findings indicate that NCAM is mainly endocytosed via clathrin-coated vesicles, suggesting a possible mechanism that may contribute to the rapid changes in NCAM expression at the cell surface.