Regulated Trafficking of the Human Dopamine Transporter

Plasma membrane neurotransmitter transporters rapidly traffic to and from the cell surface in neurons. This trafficking may be important in regulating neuronal signaling. Such regulation will be subject to the number of trafficking transporters and their trafficking rates. In the present study, we define an acutely recycling pool of endogenous ␥-aminobutyric acid transporters (GAT1) in cortical neurons that comprises approximately one-third of total cellular GAT1. Kinetic analysis of this pool estimates exocytosis and endocytosis time constants of 1.6 and 0.9 min, respectively, and thus approximately one-third of the recycling pool is plasma membrane resident in the basal state. Recent evidence shows that GAT1 substrates, second messengers, and interacting proteins regulate GAT1 trafficking. These triggers could act by altering trafficking rates or by changing the recycling pool size. In the present study we examine three GAT1 modulators. Calcium depletion decreases GAT1 surface expression by diminishing the recycling pool size. Sucrose increases GAT1 surface expression by blocking clathrin-and dynamin-dependent endocytosis, but it does not change the recycling pool size. Protein kinase C decreases surface GAT1 expression by increasing the endocytosis rate, but it does not change the exocytosis rate or the recycling pool size. Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that ϳ1000 transporters comprise the acutely recycling pool, of which 300 are on the surface in the basal state, and five transporters insert into the plasma membrane every second. This insertion could represent the fusion of one transporter-containing vesicle.

Section: Pkc-mediated Decreases In Gat1 Surface Expression Are Due Tomentioning

confidence: 99%

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1

Wang¹,

Quick

2005

“…Polyubiquitin chains can be formed by conjugation of ubiquitin to any of seven lysines in the ubiquitin molecule, although chains linked through Lys We have recently demonstrated that ubiquitination mediates protein kinase C (PKC) 2 -dependent endocytosis of the human dopamine transporter (DAT) (13-15). Upon PKC activation by phorbol esters, heterologously expressed DAT is internalized via a clathrin-dependent endocytic pathway and degraded in lysosomes (16,17). Ubiquitination of three lysines in the aminoterminal domain of DAT is critical for PKC-dependent DAT endocytosis.…”

mentioning

confidence: 99%

“…Upon PKC activation by phorbol esters, heterologously expressed DAT is internalized via a clathrin-dependent endocytic pathway and degraded in lysosomes (16,17). Ubiquitination of three lysines in the aminoterminal domain of DAT is critical for PKC-dependent DAT endocytosis.…”

mentioning

confidence: 99%

Lysine 63-linked Polyubiquitination of the Dopamine Transporter Requires WW3 and WW4 Domains of Nedd4-2 and UBE2D Ubiquitin-conjugating Enzymes

Viña-Vilaseca

Sorkin

2010

RNA interference screen previously revealed that a HECTdomain E3 ubiquitin ligase, neuronal precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), is necessary for ubiquitination and endocytosis of the dopamine transporter (DAT) induced by the activation of protein kinase C (PKC). To further confirm the role of Nedd4-2 in DAT ubiquitination and endocytosis, we demonstrated that the depletion of Nedd4-2 by two different small interfering RNA (siRNA) duplexes suppressed PKC-dependent ubiquitination and endocytosis of DAT in human and porcine cells, whereas knock-down of a highly homologous E3 ligase, Nedd4-1, had no effect on DAT. The abolished DAT ubiquitination in Nedd4-2-depleted cells was rescued by expression of recombinant Nedd4-2. Moreover, overexpression of Nedd4-2 resulted in increased PKC-dependent ubiquitination of DAT. Mutational inactivation of the HECT domain of Nedd4-2 inhibited DAT ubiquitination and endocytosis. Structure-function analysis of Nedd4-2-mediated DAT ubiquitination revealed that the intact WW4 domain and to a lesser extent WW3 domain are necessary for PKC-dependent DAT ubiquitination. Moreover, a fragment of the Nedd4-2 molecule containing WW3, WW4, and HECT domains was sufficient for fully potentiating PKC-dependent ubiquitination of DAT. Analysis of DAT ubiquitination using polyubiquitin chain-specific antibodies showed that DAT is mainly conjugated with Lys 63 -linked ubiquitin chains. siRNA analysis demonstrated that this polyubiquitination is mediated by Nedd4-2 cooperation with UBE2D and UBE2L3 E2 ubiquitin-conjugating enzymes. The model is proposed whereby each ubiquitinated DAT molecule is modified by a single four-ubiquitin Lys 63 -linked chain that can be conjugated to various lysine residues of DAT.Post-translational modification of proteins by ubiquitination has recently emerged as an important mechanism governing membrane trafficking processes. Ubiquitin moieties attached to integral membrane proteins serve to target these proteins for rapid internalization at the cell surface and, after internalization, to lysosomes for degradation (1-3). In addition, ubiquitination of the components of internalization and endosomal sorting machineries regulate interactions and turnover of these proteins (1, 4 -5).Ubiquitination is accomplished through sequential actions of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase, the latter typically determining the substrate specificity of the reaction (6 -7). Proteins can be mono-and polyubiquitinated. Polyubiquitin chains can be formed by conjugation of ubiquitin to any of seven lysines in the ubiquitin molecule, although chains linked through Lys We have recently demonstrated that ubiquitination mediates protein kinase C (PKC) 2 -dependent endocytosis of the human dopamine transporter (DAT) (13-15). Upon PKC activation by phorbol esters, heterologously expressed DAT is internalized via a clathrin-dependent endocytic pathway and degraded in lysosomes (16,17). Ubiquitination of thre...

“…18). In addition to downregulation of the plasma membrane pool of DAT, PKC activation caused degradation of DAT in Madin-Darby canine kidney cells (14). In contrast, it has been suggested that internalized DAT is mostly sorted to the recycling pathway in PC12 cells stimulated with PMA (15).…”

mentioning

confidence: 99%

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

Miranda

Sorkina

et al. 2005

120

133

Dopamine transporter (DAT) localization in dopaminergic neurons plays an important role in regulating dopamine signaling. However, the mechanisms of DAT trafficking that control DAT localization are still poorly understood. To gain insight into these mechanisms, human DAT was purified in large amounts using a two-step affinity chromatography procedure from untreated HeLa cells or cells treated with phorbol 12-myristate 13-acetate (PMA). Mass spectrometric analysis of purified DAT complexes revealed the presence of several proteins, among which ubiquitin was particularly abundant in the PMA-treated sample. Western blotting of highly purified DAT protein confirmed constitutive ubiquitylation of DAT and a dramatic increase in DAT ubiquitylation in cells treated with PMA. This increase was blocked by pretreatment with the protein kinase C (PKC) inhibitor bis-indolylmaleimide. DAT ubiquitylation by ectopically expressed ubiquitin was demonstrated in cells transiently transfected with yellow fluorescent proteintagged ubiquitin. In addition, fluorescence resonance energy transfer was detected between cyan fluorescent protein-tagged DAT and yellow fluorescent protein-tagged ubiquitin, indicative of DATubiquitin conjugation. Interestingly, the largest fluorescence resonance energy transfer signals were observed in endosomes. Ubiquitylated DAT was detected in the plasma membrane using cell surface biotinylation as well as in intracellular compartments, suggesting that ubiquitylation begins at the plasma membrane and is maintained in endosomes. In both porcine aortic endothelial and HeLa cells, where PKC-dependent DAT ubiquitylation was observed, PKC activation resulted in rapid degradation of DAT (t1 ⁄ 2 ‫؍‬ 1-2 h). Altogether, these data suggest that PKC-induced DAT ubiquitylation may target DAT to lysosomal degradation. The dopamine transporter (DAT)2 is a member of the family of Na ϩ / Cl Ϫ -dependent plasma membrane transporters (SLC6 gene family) that are responsible for rapid clearance of neurotransmitters from the extracellular space. This family also includes norepinephrine, serotonin, ␥-aminobutyric acid, and glycine transporters (1). Members of the SLC6 family share similar predicted topology of a single polypeptide that contains twelve transmembrane segments, a large second extracellular glycosylated loop, and cytoplasmic amino-and carboxyl-terminal tails (2, 3). DAT and several other transporters of this family have been found to be constitutively oligomerized in vitro and in cells (4 -8).The amount of DAT at the plasma membrane and, therefore, dopamine uptake capacity are determined by trafficking of the DAT protein, which is regulated by several signaling cascades including signaling through protein kinase C (PKC). Activation of PKC by 4␣-phorbol 12-myristate 13-acetate (PMA) leads to a reduction in the V max of dopamine transport without a change in the substrate affinity (K m ) as well as in down-regulation of surface DAT protein (9 -15). This downregulation of DAT activity and levels has been shown to be ...