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 ␤ 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.

Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway

“…Molecular Biology-The generation of plasmids encoding FLAG-tagged TacDAT (pcDNA3 TacDAT) and TacDAT C24 (pcDNA3 TacDAT C24) and mycPICK1 (pCMV mycPICK1) was described previously (43,44). The eGFP-tagged Rab constructs (pEGFP-C1 Rab7 and pEGFP-C1 Rab11) were a kind gift from Dr. Katherine W. Roche, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD (45).…”

“…1b, upper panel). However, when we fused the entire DAT sequence to Tac and coexpressed the resulting construct (TacDAT) (43) (Fig. 1a) in the Flp-In T-REx 293 eYFP-PICK1 cells, we did not see any signs of eYFP-PICK1 clustering (Fig.…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…Accordingly, it was proposed that the intracellular transporters represented a recruitable transporter pool permitting rapid mobilization of transporter to the surface during periods of high signaling activity. However, for DAT, it has recently been suggested that a substantial fraction of the constitutively internalized DAT is sorted to a lysosomal/degradative pathway with probably only a rather small fraction sorted to Rab4-positive "short-loop" recycling pathways (Eriksen et al, 2010a).…”

Determination of spatial proximity between the N-terminus and cocaine binding site of the dopamine transporter by FRET.

“…JHC 1-064 was previously shown to preferentially label surface expressed dopamine transporter [10]. To correct for spectral bleed-through, background measurements were taken in the donor channel (DD; excitation and emission wavelengths for YFP are 514 and 527 nm respectively) and in the FRET channel (DA; excitation wavelength for YFP is 514 where emission wavelength for rhodamine is 585 nm) separately and all values were corrected with respect to background.…”