Plasma membrane dopamine (DA) transporter (DAT) is expressed exclusively in dopaminergic neurons and serves for the reuptake of extracellular DA into these neurons to limit the duration and amplitude of DA signaling in the brain. DAT is a primary target of addictive psychostimulants such as cocaine and amphetamines. DAT expression levels at the plasma membrane of synaptic areas of striatal DA axons is the major determinant of extracellular DA concentrations, and these levels are defined by membrane trafficking. However, mechanisms by which proper distribution of DAT within the neuron and its membrane compartments is controlled by trafficking, how intracellular signaling regulates this trafficking and how abused psychostimulants affect DAT traffic in vivo remain poorly understood. The fundamental goal of our research is to define how intracellular trafficking regulates DAT function. During the last cycle of this program, we described the kinetics and mechanisms of the “long-distance” DAT traffic between midbrain and striatum through the medial forebrain bundle axons using HA-epitope tagged DAT (HA-DAT) knock-in mice that we have previously generated. Analysis of HA-DAT trafficking in intact mouse brain was enabled by the development of a novel HA-DAT endocytosis assay using stereotactic injections of HA antibodies. We have also discovered dramatic down-regulation of DAT in the striatum of amphetamine-sensitized male but not female mice, with the strongest amphetamine effects observed in nucleus accumbens. We identified small-molecule compounds that stabilize DAT oligomers and cause robust DAT endocytosis with remarkable specificity to DAT, which led to the demonstration of the coupling of DAT molecular mechanics and substrate- transport activity with its oligomerization and endocytosis. Based on these studies, we hypothesize that an inward-facing (IF) conformation of DAT induced by amphetamine favors DAT endocytosis. Generation of new DAT mutants led to hypothesis that a PDZ domain protein mediates protein kinase C dependent ubiquitination and endocytosis of DAT. Development of the DAT fusion protein with ascorbate peroxidase (APEX2) and demonstration of the feasibility of tracking the temporally resolved proximity proteome of APEX2-DAT opened new avenues for defining DAT interactome in specific brain regions and in animals treated with amphetamine, which will be indispensable for studying mechanisms involved in DAT regulation in vivo. We will pursue our comprehensive analysis of DAT trafficking and function by: 1) defining the molecular mechanisms of drug- and stimuli-induced endocytosis of DAT using heterologous cells and DA neurons; 2) elucidating the mechanisms underlying down-regulation of DAT in response to amphetamine challenge of amphetamine-sensitized mice and sex-dependence of this response; 3) defining the brain-region-specific proximity proteome of DAT in mouse brain using APEX2-based biotin labeling combined with quantitative mass-sp...