title: Postendocytic sorting of constitutively internalized dopamine transporter in cell lines and dopaminergic neurons.
abstract: The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the "short loop" recycling marker Rab4, and little co-localization with the marker of "long loop" recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the beta(2)-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.
authors.affiliation: Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Jacob
authors.initials: J
authors.lastname: Eriksen
authors.affiliation: Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Walden Emil
authors.initials: WE
authors.lastname: Bjørn-Yoshimoto
authors.affiliation: Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Trine Nygaard
authors.initials: TN
authors.lastname: Jørgensen
authors.affiliation: Medicinal Chemistry Section, Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland 21224.
authors.forename: Amy Hauck
authors.initials: AH
authors.lastname: Newman
authors.affiliation: Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address: gether@sund.ku.dk.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D002460:Cell Line; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D008636:Mesencephalon / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D010447:Peptide Hydrolases; D018343:Receptors, Adrenergic, beta-2; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human