Constitutive and Protein Kinase C‐Induced Internalization of the Dopamine Transporter is Mediated by a Clathrin‐Dependent Mechanism

Abstract

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...

Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

“…SERT expression in depleted cell lysate CHO-SERT CHO-(SERT؉Rab4) CHO-SERT CHO-(SERT؉Rab4) Co-localization of Rab4 and SERT Is Dependent on 5HT-Like the other plasma membrane proteins (48), SERT recycles through the endosomal system (34 -36). We next investigated whether SERT traversed through Rab4-containing endosomes (34,36).…”

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

“…Numerous studies from both our laboratory (Melikian and Buckley, 1999;Loder and Melikian, 2003;Holton et al, 2005) and others (Zhu et al, 1997;Granas et al, 2003;Sorkina et al, 2005) have demonstrated a clear role for endocytic trafficking in maintaining basal DAT surface levels and mediating PKC-induced DAT downregulation. Previous results from our laboratory revealed that DAT carboxy terminal residues L591, Y593 and I595 are necessary for basal DAT endocytosis, whereas residues 587-591 (FREKL) are required for PKC-mediated DAT downregulation (Holton et al, 2005).…”

“…In PC12 and HEK cell lines, DAT basally internalizes at rates of 2-3% per minute, which corresponding to a surface t 1/2 ~13 minutes (Loder and Melikian, 2003;Li et al, 2004;Boudanova, 2008). Acute PKC activation decreases DAT surface levels by increasing DAT endocytic rates (Loder and Melikian, 2003;Sorkina et al, 2005) and, in parallel, decreasing DAT recycling rates (Loder and Melikian, 2003). Recent siRNA studies suggest that both constitutive and PKC-stimulated DAT endocytosis are clathrin-mediated , and that Nedd 4-2-dependent DAT ubiquitination on amino terminal residues is also required for PKC-stimulated DAT internalization (Sorkina et al, 2006;Miranda et al, 2007).…”

Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

“…In presynaptic axon terminals, CME is required for the retrieval of synaptic vesicle proteins following neurotransmitter release, and for the recycling of vesicles back to the reserve pool 7 . Also, in presynaptic terminals, protein kinase C (PKC)-induced internalization of DAT from the synapse is clathrin-and dynamin-dependent 76 . The dopamine transporter DAT is located in presynaptic terminals and facilitates dopamine re-uptake from the synaptic cleft, thereby regulating signal strength (for review see 77 ).…”