Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.107 (DAT)
 1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The psychostimulants cocaine and amphetamine increase expression of the immediate early gene (IEG) c-fos indirectly, via D1 dopamine receptor activation. To determine whether dopamine transporter substrates and inhibitors can affect c-Fos expression directly, we investigated their effects on c-Fos protein and c-fos mRNA in HEK-293 (HEK) cells transfected with the human dopamine transporter (hDAT). In untransfected HEK cells, methylphenidate and cocaine produced a small but statistically significant increase in c-Fos, whereas dopamine and amphetamine did not. In hDAT cells, DAT substrates (dopamine, amphetamine) increased c-Fos immunoreactivity 6- and 3-fold (respectively). The DAT inhibitors cocaine, methylphenidate, and bupropion also increased c-Fos approximately 3-fold in hDAT cells. If coincubated with dopamine, the inhibitors attenuated dopamine-induced c-Fos in hDAT cells. The magnitude of c-fos mRNA induction by substrates and inhibitors paralleled induction of c-Fos protein immunoreactivity. The results indicate that substrates or inhibitors of the DAT can trigger induction of IEG expression in the absence of D1 dopamine receptor. For substrates, IEG induction is DAT-dependent, but for certain DAT inhibitors the cellular response can be elicited in the absence of the DAT in HEK cells. Oxidative stress may partly, but not fully, account for the DA-induced c-Fos induction as an inhibitor of oxidative stress Trolox C, attenuated DA-induced c-Fos induction. Protein kinase C (PKC) may also partially account for c-Fos induction as a specific inhibitor of PKC Bisindolylmaleimide I (BIS) attenuated DA-induced c-Fos by 50%. DAT substrate and inhibitor effects on IEGs, other fos-related antigens, and possible mechanisms that contribute to c-Fos induction warrant investigation in presynaptic neurons as a potential contribution to the long-term effects of psychostimulants.
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PMID:Dopamine transporter-dependent induction of C-Fos in HEK cells. 1211 14

Cocaine, methylphenidate and other drugs that block dopamine transport indirectly promote immediate early gene expression, via dopamine-mediated activation of D1 dopamine receptors. Increased expression of the immediate early gene (IEG) c-fos, initiates a cascade of intracellular events that may underlie neuroadaptive changes following repeated exposure to the drugs. We investigated whether substrates (dopamine, norepinephrine) of the human dopamine (hDAT) and norepinephrine (hNET) transporters can directly induce c-Fos protein in HEK-293 (HEK) cells transfected with the hDAT and hNET and whether PKC modulators affect this process. Dopamine and norepinephrine robustly induced c-Fos immunofluorescence in both hDAT and hNET cells, but not in untransfected HEK-293 cells, demonstrating that catecholamine-induced c-Fos induction was DAT- and NET-dependent. The PKC activator PMA induced c-Fos in hDAT, hNET and HEK cell lines indicating that PKC stimulated c-Fos independent of transporters. The PKC inhibitor bisindolylmaleimide I (BIS) significantly increased c-Fos expression in hDAT cells, but not in hNET or HEK-293 cells, suggesting that inhibition of DAT-mediated phosphorylation results in c-Fos induction. BIS pretreatment abolished norepinephrine-induced c-Fos expression hNET but not dopamine-induced c-Fos induction in hDAT cells. In conclusion, induction of c-Fos by dopamine and norepinephrine requires the presence of hDAT and hNET but the contributions of hDAT and hNET to c-Fos induction is distinguishable on the basis of differing responses to a PKC inhibitor. These findings present a cell system and methodology for investigating the potential contribution of monoamine transporters to pre-synaptic neuroadaptation.
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PMID:Dopamine and norepinephrine transporter-dependent c-Fos production in vitro: relevance to neuroadaptation. 1576 38

Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.
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PMID:The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking. 2195 39

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.
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PMID:Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton. 2419 73

Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40-50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.
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PMID:PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination. 2641 48

The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or "endocytic brake," controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.
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PMID:Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant. 2662 48