Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis

The targeting of membrane proteins into axons and dendrites is of critical importance for directional signal transmission within specific neural circuits. Many dendritic proteins have been shown to reach the somatodendritic membrane based on selective sorting and transport of carrier vesicles. Using rat hippocampal neurons in culture, we investigated the trafficking pathways of Delta/Notch-like EGF-related receptor (DNER), a transmembrane Notch ligand which is specifically expressed in CNS dendrites. Mutations in the cytoplasmic domain of DNER that abolished somatodendritic localization also increased its surface expression. Furthermore, inhibition of endocytosis resulted in disruption of the somatodendritic localization of DNER, indicating that the somatodendritic targeting of DNER is dependent on endocytosis. The DNER cytoplasmic domain binds to a clathrin adaptor protein complex-2 via a proximal tyrosine motif and a 40 amino acid stretch in the mid-domain, but not by the C-terminal tail. Molecular and pharmacological inhibition revealed that the surface expression of DNER is regulated by clathrin-dependent and -independent endocytosis. In contrast, the somatodendritic targeting of DNER is predominantly regulated by clathrin- and adaptor protein complex-2-independent endocytosis via the C-terminal tail of DNER. Our data suggest that clathrin-independent endocytosis is critical for the polarized targeting of somatodendritic proteins.