The NH2-terminus of Norepinephrine Transporter Contains a Basolateral Localization Signal for Epithelial Cells

When expressed in epithelial cells, dopamine transporter (DAT) was detected predominantly in the apical plasma membrane, whereas norepinephrine transporter (NET) was found in the basolateral membrane, despite 67% overall amino acid sequence identity. To identify possible localization signals responsible for this difference, DAT–NET chimeras were expressed in MDCK cells and localized by immunocytochemistry and transport assays. The results suggested that localization of these transporters in MDCK cells depends on their highly divergent NH2-terminal regions. Deletion of the first 58 amino acids of DAT (preceding TM1) did not change its apical localization. However, the replacement of that region with corresponding sequence from NET resulted in localization of the chimeric protein to the basolateral membrane, suggesting that the NH2-terminus of NET, which contains two dileucine motifs, contains a basolateral localization signal. Mutation of these leucines to alanines in the context of a basolaterally localized NET/DAT chimera restored transporter localization to the apical membrane, indicating that the dileucine motifs are critical to the basolateral localization signal embodied within the NET NH2-terminal region. However, the same mutation in the context of wild-type NET did not disrupt basolateral localization, indicating the presence of additional signals in NET directing its basolateral localization within the plasma membrane.