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.
Highlights
It has been demonstrated in several dopamine transporter (DAT)-transfected heterologous cell lines as well as in synaptosomes that activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), down-regulates dopamine transport (6 –13)
We investigated the postendocytic sorting of constitutively internalized DAT in both heterologous cells and cultured dopaminergic neurons
To enable the study of DAT trafficking with high specificity and to detect even small amounts of DAT endocytosis, we wanted to generate a DAT construct with a high affinity extracellular antibody epitope
Summary
Molecular Biology—TacDAT was generated by a two-step PCR procedure. First, FLAG-Tac was amplified from pcDNA3 FLAG-Tac [20] with primers generating an overhang identical to the N-terminal part of DAT. The cells were incubated with M1 (1 g/ml) in DMEM for 30 min at 4 °C, and medium was replaced with 37 °C DMEM and placed at 37 °C (or 4 °C for surface quantification) for various periods with different compounds to drive internalization. Surface ELISA—HEK293 cells, transfected with plasmids encoding HA-hDAT, TacDAT, FLAG-2-adrenergic receptor, or transferrin receptor-GFP, were seeded in 96-well plates (35,000 cells/well) the day before the experiment. For the co-localization experiments, cells were transfected with equal amounts of plasmid encoding TacDAT or FLAGtagged 2-adrenergic receptor and EGFP-Rab4, -Rab, or -Rab and seeded on coverslips treated with polyornithine. On the day of the experiment, the cells were incubated with Alexa Fluor 568-conjugated M1 antibody for 30 min at 4 °C, and the medium was replaced with 37 °C DMEM (ϩ10 M isoproterenol for experiments with the 2-adrenergic receptor) and placed at 37 °C for 1 h to allow internalization. JHC 1-64 was visualized using a 543 nm helium-neon laser line and a 585-nm long pass filter, EGFP was detected with a 488 nm argon-krypton laser line and a 505–550-nm band pass filter, and the distribution of JHC 1-64 on the neurons were analyzed with a Z-scan
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