Regulation of the dopamine transporter - Addiction Research ...

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Schmitt & Reith DAT Regulation transport, in addition to non–raft-associated DATs responding to PKC by a classical clathrin-dependent mechanism. On the basis of the preceding studies, it would be tempting to posit that trafficking of the DAT is mediated (at least in part) by PKC, with increased phosphorylation signaling that a given DAT protein is ready to be internalized. Recent work by Cervinski et al. 45 shows that d-methamphetamine increases DAT phosphorylation in addition to triggering internalization—both of which are prevented by PKC inhibitors. Taken together, these findings prompt a tantalizing hypothesis positing a link between substrate interaction, transporter phosphorylation by PKC, and subsequent membrane redistribution. Unfortunately, several lines of evidence suggest that the relationship between PKC, transporter phosphorylation, and membrane trafficking is far more complex. For example, removal of three classical PKC consensus sites from the DAT protein by mutagenesis of the target residues (Ser- 262, Ser-586, and Thr-613) to glycine prevents the phosphorylation normally observed after PKC activation with �-PMA but fails to prevent �-PMA– induced internalization and endosomal trafficking of the DAT. 62 Similarly, truncation of the transporter distal N terminus—which bears several serine residues implicated in phosphorylation after PKC activation—also eliminates �-PMA–induced DAT phosphorylation without hindering the typical endocytic response in HEK cells. 63 Furthermore, although removal of the serine-rich distal N terminus prevents methamphetamine-induced DAT phosphorylation, it does not affect internalization of the DAT in response to the substrate. 45 That is, although PKC clearly plays a role in the mechanism underlying the trafficking effects of amphetaminergic substrates, direct phosphorylation of the DAT is not part of that role. It is possible that activation of PKC results in DAT phosphorylation via a more circuitous route, involving thus far unidentified downstream kinases; however, the observation that substrateinduced trafficking can occur without appreciable phosphorylation of the DAT protein begs the question of whether DAT phosphorylation actually serves as a “proendocytosis” signal. Instead, it may be that PKC regulates the action of a DAT-associated scaffolding or cytoskeletal protein that is ultimately responsible for determining the trafficking fate of a given DAT. 49,51 Data suggest that scaffolding proteins can control the trafficking dynamics of the noradrenaline transporter (NET): the cytosolic scaffolding protein syntaxin 1A—a well-known mediator of plasmalemmal vesicle docking and fusion— directly interacts with the NET and is involved with regulation of surface NET expression levels. 64 Moreover, exposure to amphetamine results in cytosolic redistribution of plasmalemmal NETs with a concomitant increase in the association of the NET protein and syntaxin 1A at the plasma membrane. 65 It is not clear whether syntaxin 1A promotes the internalization of the NET or stabilizes its presence at the plasma membrane (classically, syntaxin 1A is considered an exocytosis-promoting vesicle fusion protein), but the mere interaction between the two indicates that scaffolding proteins involved in regulated vesicular neurotransmitter release may also play a role in regulating transmitter transporters. Although explicit syntaxin 1A-dependence in amphetamine-mediated trafficking has not yet been shown with the DAT, syntaxin 1A does interact with the N terminus of the DAT, 66 and this interaction has been recently shown to promote the efflux of intracellular dopamine by amphetamine. 67 Interestingly, PKC (the classical isoform, PKC-�) is known to promote amphetamine-stimulated dopamine efflux via interaction with a DAT-associated 68 protein complex; PKC-� is also necessary for the rapid short-term increase in surface DAT levels upon substrate exposure. 32 In the syntaxin study by Lee et al., 66 the authors also noted an interaction between the N terminus of the DAT and a protein known as the receptor for activated C kinases (RACK1). RACK1 can bind to activated PKC molecules and other intracellular signaling kinases and hence may help transduce increased intracellular PKC activity into a DAT proendocytosis signal without the need for a direct phosphotransferase interaction between the DAT and PKC. This protein–protein interaction might also contribute to amphetamine-induced dopamine efflux by attracting activated PKC to the transporter, promoting an inward-facing, “efflux-favoring” conformational state. 68,69 Furthermore, the conformational state of the DAT protein itself may influence its internalization rate, because disruption of the outward-facing conformational state by mutation of membrane-proximal N-terminal residues Arg-60 or Trp-63 to alanine results in increased DAT endocytosis. 70 Substrates could encourage an Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 323
DAT Regulation Schmitt & Reith inward-facing transporter conformation via the process of translocation, suggesting a potential link between high concentrations of substrate and eventual DAT endocytosis. Thus, even if the identity of the terminal protein mediator of substrate-mediated DAT internalization is obfuscated, PKC still appears to be a requisite middleman in the endocytic signaling cascade. However, another significant question remains: how might amphetaminergic substrates activate PKC in the first place? PKC activity is regulated by Ca 2+ and diacylglycerol, a phospholipid metabolite generated in concert with inositol triphosphate by the enzyme phospholipase C (PLC), which is also Ca 2+ dependent. Stimulation of PKC activity by diacylglycerol can be amplified by arachidonic acid (for references, see Zhang and Reith 71 ), which has been shown to affect DAT function (e.g., Refs. 71–73); however, the effect of amphetamine on endogenous arachidonic acid—potentially through regulation of phospholipase A2, releasing arachidonic acid—is not known. Usually, PLC is activated by agonist binding at GPCRs coupled to the Gq/11type �-subunit; however, compounds, such as amphetamine and methamphetamine, lack significant affinity for GPCRs other than the trace amine– associated receptor 1 (TAAR1), a Gs-coupled receptor. 74 It is conceivable that in certain experimental conditions and cell types, endogenous dopamine released by amphetamines could activate dopamine receptors, which can affect DAT function (see section on GPCR-mediated DAT regulation). However, Cervinski et al. 45 observed no differences in methamphetamine-induced DAT phosphorylation or internalization when cells were simultaneously pretreated with D1- andD2-like receptor antagonists. How, then, could amphetamine induce PLC activity? Although there have been few investigations into the non–transporter-mediated actions of amphetamines, research by Giambalvo indicates that treatment with 10 �M amphetamine enhances PLC activity in rat striatal synaptosomes and that pharmacological inhibition of PLC attenuates the usual activation of PKC by amphetamine. 75 Interestingly, inhibition of the Na + /Ca 2+ antiporter with amiloride also blocked amphetamine-induced PKC activation, suggesting that the ionic effects of DAT substrate translocation can directly influence activation of intracellular signaling cascades. When substrates are transported across the plasma membrane via the DAT, sodium ions are cotransported, and this increase in intracellular sodium concentration may cause Na + /Ca 2+ antiporters at mitochondrial and endoplasmic reticulum membranes to operate in reverse, favoring a net flux of Ca 2+ into the cytosolic compartment. Because PLC is also a Ca 2+ - dependent enzyme, the increase in intracellular calcium may catalyze PKC activation via stimulation of PLC in addition to its direct effect on PKC. 75 PI3K, MAPK, and other protein kinases Protein kinases other than PKC are also involved in DAT phosphorylation, but again, evidence of definitive phosphotransferase reactions has proven elusive. 76 As with PKC, both the PI3K and MAPK signaling pathways influence phosphorylation of DAT serine and threonine residues, 49 but their regulatory effects on DAT function appear to be opposite that of PKC, with kinase inhibition resulting in downregulation of and kinase activation resulting in upregulation of DAT activity, respectively. PI3K can be activated by GPCRs and by receptor tyrosine kinases, such as the insulin receptor. Although the classical isoform of PI3K itself is not considered a serine/threonine protein kinase, it is directly upstream of the serine/threonine kinase Akt (also known as protein kinase B). Inhibition of PI3K decreases the V max of dopamine uptake and causes loss of surface expressed DAT, which—like PKC- and substrateinduced downregulation—is dependent on endocytosis by clathrin-coated vesicles. 77,78 Conversely, treatment with insulin or expression of constitutively active forms of PI3K increases [ 3 H]dopamine uptake, a sign of putative surface DAT upregulation. 77 Unlike inhibition of PKC, inhibition of PI3K has been recently demonstrated to attenuate the dopamine-releasing effects of amphetamine; however, this is most likely due to reduction in cell surface DAT expression rather than a direct effect of PI3K on transporter function. 79 The MAPK pathway is a complex signaling network consisting of a three-tiered series of various serine/threonine protein kinases: primary members of the MAPK protein family (such as extracellular signal– regulated kinases-1 and -2 [ERK1/2]) are activated by secondary upstream kinases known as MAP/ERK kinases (MEKs) which are in turn activated by a tertiary group of further upstream kinases known as MEK kinases. 80 Reducing ERK1 and ERK2 324 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.
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