The NH(2)-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 NH(2)-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 NH(2)-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 NH(2)-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.
Gu
H H
HH
Department of Pharmacology, Yale University, New Haven, CT 06510, USA.
Wu
X
X
Giros
B
B
Caron
M G
MG
Caplan
M J
MJ
Rudnick
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D050483:Dopamine Plasma Membrane Transport Proteins; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D021382:Protein Sorting Signals / Q000502:physiology*; D016415:Sequence Alignment; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D021382:Protein Sorting Signals; C493227:SLC6A2 protein, human; C493214:SLC6A3 protein, human; C493228:Slc6a2 protein, mouse; C493215:Slc6a3 protein, mouse; D027981:Symporters