Negative Regulation of Dopamine Transporter Endocytosis by Membrane-Proximal N-Terminal Residues

Abstract

The plasma membrane dopamine transporter (DAT) takes extracellular dopamine back up into dopaminergic neurons. Although the number of DATs at the cell surface is regulated by endocytosis and recycling, the molecular mechanisms that control this endocytic trafficking of DAT are not defined. To map the sequence motifs that are involved in constitutive DAT endocytosis, mutagenesis of human DAT tagged with yellow fluorescent protein (YFP) and an extracellular HA epitope was performed. Removal of the entire N terminus of DAT resulted in accumulation of the resulting DAT mutant (YFP-HA-⌬N-DAT) in early and recycling endosomes in HeLa and PAE cells, and in primary rat mesencephalic-striatal neuronal cocultures. This endosomal accumulation was due to rapid constitutive internalization of YFP-HA-⌬N-DAT by the clathrin-dependent pathway. Small deletions and multialanine substitutions in the N terminus revealed two molecular determinants within the membrane proximal residues 60 -65 that are important for preventing rapid internalization of DAT. First, mutations of Arg60 or Trp63, leading to disruption of the "outward facing" DAT conformation, correlated with an increased pool of mobile DATs in the plasma membrane and accelerated constitutive internalization of the DAT mutants. Second, mutation of Lys65 also correlated with elevated endocytosis. While none of these mutations alone recapitulated the marked endocytic phenotype of YFP-HA-⌬N-DAT, simultaneous elimination of both the outward conformation of DAT and Lys65 resulted in DAT mutants that were rapidly internalized. Thus, our studies reveal a new link between DAT endocytosis and conformation-dependent uptake activity that represents a novel mode for regulating DAT function.

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

“…dopaminergic neurons in culture (19,22,41). The present Tac-DAT construct represents an alternative way of generating an extracellular antibody epitope and carries several properties that complement already existing approaches for studying DAT trafficking.…”

“…Subsequent mutational analyses suggested that this constitutive internalization is promoted by a non-conventional trafficking motif in the DAT C terminus (10,18) and possibly negatively regulated by residues residing in the membraneproximal DAT N terminus (19). Recently, we demonstrated by using the fluorescent cocaine analogue JHC 1-64 as label that DAT endogenously expressed in cultured midbrain dopaminergic neurons undergoes constitutive internalization (8).…”

Protein Kinase C-dependent Ubiquitination and Clathrin-mediated Endocytosis of the Cationic Amino Acid Transporter CAT-1

“…To obtain high resolution three-dimensional images of the cells, a Z-stack of images was acquired through Cy5, Cy3, and FITC filter channels using a Marianas Imaging workstation and SlideBook 4.2 software (Intelligent Imaging Innovation, Denver, CO) as described previously (39). Typically, 15-20 serial two-dimensional images were recorded at 300-nm intervals.…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…The molecular mechanisms underlying substrate and inhibitor induced surface regulation is still enigmatic; recently, however, it has been suggested that an inwardfacing conformation of DAT is more prone to internalization than an outward-facing conformation of DAT (Sorkina et al, 2009). Thus, substrate might promote THE SLC6 NEUROTRANSMITTER TRANSPORTERS internalization simply by increasing the fraction of transporter molecules residing in an inward-facing conformation, which might alter the interaction of the transporter with as-yet-unknown proteins.…”