The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive-and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4 -2 (neural precursor cell expressed, developmentally downregulated 4 -2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4 -2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4 -2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4 -2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

Eps15, a phosphorylation substrate of the epidermal growth factor (EGF) receptor kinase, has been shown to bind to the ␣-subunit of the clathrin-associated protein complex AP-2. Here we report that in cells, virtually all Eps15 interacts with the cytosol and membrane-bound forms of AP-2. This association is not affected by the treatment of cells with EGF. Immunofluorescence microscopy reveals nearly absolute co-localization of Eps15 with AP-2 and clathrin, and analysis by immunoelectron microscopy shows that the localization of membrane-associated Eps15 is restricted to the profiles corresponding to endocytic coated pits and vesicles. Unexpectedly, Eps15 was found at the edge of forming coated pits and at the rim of budding coated vesicles. This asymmetric distribution is in sharp contrast to the localization of AP-2 that shows an even distribution along the same types of clathrin-coated structures. These findings suggest several possible regulatory roles of Eps15 during the formation of coated pits.

Plasma membrane transporters belonging to the family of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters play an important role in terminating the activity of the monoamine neurotransmitters and of ␥-aminobutyric acid (see Ref. 1). Thus, reuptake of dopamine (DA) 1 from the synaptic cleft by the dopamine transporter (DAT) serves as the major mechanism for terminating dopaminergic neurotransmission in the brain (2). Because the efficiency of DA removal depends on the number of DAT molecules expressed at the plasma membrane, trafficking processes that control transporter distribution in the cell represent a potentially important mechanism by which neurotransmission could be regulated. Newly synthesized DAT acquires glycosylation in the endoplasmic reticulum (ER) and Golgi complex and is then trafficked to the plasma membrane. Typically, a large pool of mature DAT molecules are found at the cell surface of dopaminergic neurons and when DAT is heterologously expressed in tissue culture cells. However, DAT localization can be altered rapidly. Acute exposure of cells to either phorbol esters or substrates reduces the number of plasma membrane DATs and thus DAT function, and this reduction is due to acceleration of DAT endocytosis through a dynamin-dependent mechanism (3-6). Recently, it has been shown that DAT can interact with the anchoring protein PICK 1 and the adaptor protein 8). However, in general, the molecular mechanisms controlling DAT trafficking are not yet well understood.The DAT molecule is predicted to have 12 membrane-spanning sequences with both amino and carboxyl termini oriented intracellularly. The specific function of DAT transmembrane (TM) motifs and termini are not well established. TM domains may play a role in intra-and intermolecular interactions. Many membrane receptors and other integral membrane proteins require dimerization or higher oligomerization for their activity. Several lines of evidence have suggested that monoamine transporters are also dimers or oligomers. Results with dominant negative forms of the serotonin transporter (SERT) and norepinephrine transporter were consistent with this idea (9, 10). Oligomerization of SERT was demonstrated directly by co-immunoprecipitation (11). The results of fluorescence resonance energy transfer (FRET) studies further confirmed that SERT and a ␥-aminobutyric acid transporter (GAT-1) are homo-oligomers (12, 13). Early radiation inactivation studies also suggested that DAT exists as a dimer or oligomer (14,15). Recently, the potential for DAT to exist as dimer or higherorder oligomer in the plasma membrane has been demonstrated using chemical cross-linking (16). More recently, Torres and co-workers (17) reported detection of DAT oligomerization by co-immunoprecipitation. In general, the mechanisms and functional roles of oligomerization of monoamine transporters remain to be defined. However, it has been proposed that SERT oligomerization may be important for its transport activity (9). Oligomerization may be also required for proper traffic...

The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587-597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms. The plasma membrane dopamine transporter (DAT) belongs to the family of plasma membrane Na þ /Cl -dependent neurotransmitter transporters, which includes transporters for norepinephrine, serotonin, glycine and GABA (1). The re-uptake of dopamine by DAT is the primary mechanism of termination of dopaminergic signaling in the brain. DAT is also a target of psychostimulants. In the mammalian central nervous system, DAT is expressed exclusively in a small subset of neurons, called dopaminergic neurons, the most prominent of which arise from the substantia nigra and ventral tegmental area and project to the striatum and cerebral cortex. Electron microscopy studies revealed DAT localization in the plasma membrane near the active zone in synapses located along the axonal processes, as well as in various intracellular membrane compartments in the soma and dendrites (2-4).Several lines of evidence, largely generated in heterologous expression cell systems, suggest that DAT expression at the cell surface can be rapidly regulated by endocytosis. For instance, protein kinase C (PKC)-dependent endocytosis of DAT has been observed in various mammalian cell lines (5-9). Phorbol esters also reduce dopamine uptake capacity, DAT transport-associated currents and capacitance measurements in Xenopus oocytes expressing the human DAT (10). Direct evidence for regulated DAT trafficking in neuronal cells is limited to data obtained in vitro using rat brain synaptosomes (11). Down-regulation of surface DAT and the accumulation of DAT in endosomes can be also triggered by amphetamine and other DAT su...

Although interactions between the μ2 subunit of the clathrin adaptor protein complex AP-2 and tyrosinebased internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of μ2-receptor interactions, we constructed an epitope-tagged μ2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of μ2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant μ2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous μ2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a μ2-dependent and -independent manner.