Regulation of the dopamine transporter - Addiction Research ...

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Schmitt & Reith DAT Regulation phosphorylation by using small-molecule inhibitors of MEK affects DAT function and trafficking similarly to PI3K inhibition, decreasing the V max of dopamine uptake without altering the Km and inducing cytosolic redistribution of plasmalemmal DAT into endosomal compartments. 78,81 The MAPK and PI3K signaling cascades are probably involved in the regulation of DAT activity by various classes of transmembrane receptors, such as GPCRs (discussed in the following section) and receptor tyrosine kinases. For example, in addition to insulin—which has been shown to increase plasmalemmal DAT function—activation of the neurotrophin receptor tyrosine kinase TrkB with brainderived neurotrophic factor also upregulates DAT activity, an effect that hinges on both the MAPK and PI3K pathways. 52 MAPK family members ERK1 and ERK2 have also been implicated in cocaine addiction and relapse after cocaine withdrawal, as central mediators of long-term sensitization to cocaine associated cues (for review, see Lu et al. 82 ). Ubiquitination and other direct posttranslational modifications Covalent attachment of the small soluble protein ubiquitin to the ε-amino moiety of lysine residues in target cellular proteins is known as ubiquitination. The ubiquitination reaction is catalyzed by a multistep enzymatic system, with the final covalent attachment mediated by members of a family of proteins known as E3 ubiquitin ligases (the multifarious functions of ubiquitin are reviewed in Welchman et al. 83 ). The most widely recognized function of ubiquitination is polyubiquitination, which occurs when several ubiquitin subunits are attached to one lysine, forming a chain. Polyubiquitin chains serve as a signaling motif, indicating that a target protein is to be trafficked to the 26S proteasome for proteolytic destruction. However, attachment of one ubiquitin subunit to a target protein (monoubiquitination) is also a signaling motif and is thought to encourage retention of target proteins in sorting endosomes for eventual trafficking to the lysosomal degradation pathway. 84 Ubiquitination can fulfill roles other than those directly serving protein breakdown. For example, ubiquitination of the yeast �-factor receptor Ste2 promotes endocytosis of the receptor–ligand complex prior to degradation in the vacuole. 85 The yeast multidrug transporter Pdr5, 86 and the Ste6 �-factor pheromone transporter, 87 both members of the ATP-binding cassette multidrug transporter family, appear to be prepared by ubiquitination for endocytic delivery to the vacuole for proteolytic turnover. In these cases, ubiquitination may serve as a signal for protein trafficking rather than a signal for protein degradation itself. Work by Hicke’s group 88 has provided more information on ubiquitin as a signal for endocytosis of the plasma membrane protein Ste2p, the mating pheromone �-factor receptor. Unlike the ubiquitin proteasome recognition signal, the internalization signal does not require polyubiquitin formation through Lys-18 but rather relies on monoubiquitination of one lysine residue of the Ste2p. There is evidence that, like phosphorylation, ubiquitination of the DAT affects cell surface expression and membrane trafficking of the transporter. Interestingly, many of the same intracellular signaling kinases involved in DAT phosphorylation may also be responsible for signaling via ubiquitination. For example, Miranda et al. demonstrated that activation of PKC by treatment with �-PMA increases ubiquitination of the DAT and that the major proportion of ubiquitin-conjugated DAT was present as the monoubiquitinated species. 89 Using Förster resonance energy transfer, the authors also showed that fluorescent-tagged hDAT and ubiquitin are associated in late endosomes and multivesicular bodies destined for lysosomal degradation. Much like PKC-associated transporter phosphorylation, ubiquitination of the DAT depends upon residues residing in the N terminus of the transporter, because simultaneous mutation of three amino-terminal lysine residues (Lys-19, Lys-27, and Lys-35) inhibits PKC-mediated ubiquitination. 90 Ubiquitin conjugation to the DAT is catalyzed by the ubiquitin ligase Nedd4-2. 91 Although it is clear that ubiquitination is involved in the regulated endocytosis of the DAT, a relationship between substrate-induced transporter trafficking and ubiquitination has not yet been established. Presynaptic G protein-coupled receptors affecting DAT function Because DAT protein function and membrane distribution are acutely regulated via several different intracellular signaling cascades, it is not surprising that activation of various types of GPCRs expressed Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 325
DAT Regulation Schmitt & Reith by dopaminergic neurons can affect the DAT. Of the possible GPCR subtypes that can affect DAT function, dopamine D2-like autoreceptors are the most logical candidates, with their presynaptic localization and clearly defined role in regulatory inhibition of both tyrosine hydroxylase 92 and vesicular dopamine release. 93 However, although D2-like autoreceptors are the most thoroughly investigated, other GPCRs also appear to modulate dopaminergic neurotransmission by altering DAT function. In particular, activation of both �-opioid receptors and TAAR1 receptors (a member of a recently discovered family of receptors for endogenous trace amines) has been shown to elicit changes in DAT function. Dopamine D2 and D3 autoreceptors The D2-like family of dopamine receptors comprises D2,D3,andD4 receptor subtypes. In addition to their role as classical postsynaptic receptors, the D2 and D3 subtypes are expressed by dopaminergic neurons themselves, serving as presynaptic autoreceptors. Activation of D2-like autoreceptors attenuates dopaminergic neurotransmission via several parallel mechanisms: in PC12 cells, for example, treatment with the D2/D3 agonist quinpirole causes feedback inhibition of tyrosine hydroxylase—the rate-determining enzyme in dopamine synthesis— and decreases K + -evoked dopamine release. 93 In addition to reducing stimulated dopamine release, recent evidence suggests that D2 and D3 receptor activation also reduces extracellular dopamine concentration via acute upregulation of DAT function. This effect was first demonstrated in the early 1990s by using rotating disk electrode voltammetry to measure the rate of extracellular dopamine clearance. That is, Meiergerd et al. demonstrated 94 that the D2/D3 agonist quinpirole increases the V max of dopamine transport into rat striatal synaptosomes, an effect that is reversed by concomitant administration of the antagonist sulpiride. The authors also showed in vivo evidence of the inverse situation: a reductionintheV max of dopamine clearance in rats chronically treated with the potent D2 antagonist haloperidol. In support of this finding, localized intrastriatal application of the selective D2 antagonist raclopride has also been shown to decrease clearance of exogenous dopamine applied locally via a second micropipette. 95 Studies using selective D3 receptor ligands paint a similar picture: the selective D3 agonist PD128907 increases electrochemically measured dopamine clearance in rat nucleus accumbens slices, producing an increase in transport V max with no effect on the K m of dopamine after a 10min preincubation. 96 In contrast, the D3 receptor antagonist GR103691 decreased dopamine significantly below vehicle levels. In Xenopus oocytes coexpressing hDAT and D2R transcripts, activation of D2 receptors with apomorphine for as little as 5 min results in a 40% increase of whole-cell [ 3 H]CFT binding (Bmax) with no alteration in the Ki value for CFT, suggesting that activation of D2 receptors triggers rapid trafficking of intracellular DATs to the plasma membrane. 97 Upregulation of surface DAT expression by D2 receptor agonists requires Gi/Go protein coupling, because pretreatment with pertussis toxin (PTX) blocked the increase in [ 3 H]CFT binding. In our collaborative study with the group of Garris, in vivo voltammetry was used to monitor dopamine uptake and release in rat striatum and nucleus accumbens. 98 We found that D2R antagonism reduced uptake at all frequencies used to stimulate the medial forebrain bundle, whereas release was enhanced only at lower frequencies. Be that as it may, both processes—D2R regulation of dopamine uptake as well as release—serve as parallel feedback mechanisms aimed at reducing extracellular dopamine levels when high dopamine levels activate D2 receptors. Most investigations into the effects of D2-like receptor ligands on DAT function in vitro have used electrochemical methods to assess dopamine uptake in animal tissues, in lieu of more commonly used measurement of [ 3 H]dopamine uptake by heterologous cells. The reason for this methodological preference is purely technical: dopamine is a high-affinity agonist at D2 and D3 receptors, creating an obvious confound because the radioligand will activate the receptors as well. Although electrochemical detection has greater temporal resolution than is possible with cell models, 16 it does not allow direct observation of effects on DAT trafficking in concert with kinetic measures of DAT function. Recent studies have overcome this issue by using a novel synthetic DAT substrate that is easily detected yet exhibits negligible affinity for dopamine receptors. The substrate 4-(4-(dimethylamino)styryl)- N-methylpyridinium (ASP + )—a styryl analogue of the neurotoxic substrate MPP + —has the added benefit of possessing a fluorescent �-conjugated 326 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.
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