The effects of phosphorylation on the functional regulation of an expressed recombinant human dopamine transporter

Vrindavanam, Nandagopal; Arnaúd, Philippe; Ma, Jian; Altman-Hamamdzic, Sanja; Parratto, N.P.; Sallee, Floyd R.

doi:10.1016/0304-3940(96)13034-2

Cited by 16 publications

(11 citation statements)

References 12 publications

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“…Whether PMAmediated internalization is the result of the direct phosphoryl- ation of the hDAT remains unclear. The primary amino acid sequence of the hDAT contains multiple consensus sites for PKC phosphorylation, and increased phosphorylation of the hDAT has been shown in response to PMA stimulation (10,11,33). However, mutation of the canonical PKC phosphorylation sites within the GABA and glycine transporters, which are also regulated by PKC activation, has no effect on PMA-mediated inhibition of transporter activity (30,34).…”

Section: Discussionmentioning

confidence: 99%

Regulated Trafficking of the Human Dopamine Transporter

Daniels¹,

Amara²

1999

Journal of Biological Chemistry

301

126

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The dopamine transporter plays an essential role in the modulation of dopaminergic neurotransmission by mediating the reuptake of dopamine into presynaptic neurons. In cells expressing the dopamine transporter, activation of protein kinase C by phorbol esters results in a significant reduction in dopamine uptake. This phorbol ester-mediated inhibition of dopamine transport is associated with a decrease in V max , although the apparent affinity of the transporter for dopamine remains unchanged. Using a green fluorescent proteintagged dopamine transporter stably expressed in Madin-Darby canine kidney cells, we show in live cells that the decrease in transporter activity is caused by the rapid internalization of carriers from the plasma membrane. This redistribution of the transporter is specific to phorbol ester activation and is unaffected by the presence of either substrates or inhibitors of the carrier. Upon the addition of phorbol esters, transporters at the cell surface are rapidly endocytosed through a clathrinmediated and dynamin-dependent mechanism into early endosomes, where they colocalize with transferrin. The internalized carrier is targeted to the endosomal/lysosomal pathway and is completely degraded within 2 h of protein kinase C activation. Phorbol estermediated alterations in the trafficking of the dopamine transporter may serve as a mechanism for controlling extracellular dopamine levels in the central nervous system. The dopamine transporter (DAT)1 mediates the reuptake of dopamine into presynaptic neurons. This process of reaccumulation effectively reduces extracellular dopamine concentrations and limits activation of both presynaptic and postsynaptic dopamine receptors. The DAT is the primary target of psychostimulant drugs, which block the reuptake of released dopamine, resulting in an increase in synaptic dopamine levels (reviewed in Ref. 1). Functional loss of dopamine transport either through pharmacological inhibition (2) or genetic knockout (3) results in profound physical, physiological, and behavioral changes. Given the essential role played by the DAT in the modulation of dopaminergic neurotransmission, the regulation of transporter activity is of considerable interest.Cloning of the DAT and elucidation of its amino acid sequence revealed the presence of several consensus sites for protein kinase C (PKC) phosphorylation (3-5). Inhibition of DAT activity in response to activation of PKC by phorbol 12-myristate 13-acetate (PMA) has been observed in striatal synaptosomes and in several systems using cloned transporters expressed in either mammalian cells or Xenopus oocytes (6 -11). Increased phosphorylation of the DAT in response to phorbol esters has been demonstrated in both endogenous and exogenous expression systems; however, no direct link between DAT phosphorylation and changes in transporter activity has been established (10, 11). The inhibition of DAT activity is the result of a decrease in V max , with little or no change in the apparent affinity of the transporter for substrate...

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Section: Discussionmentioning

confidence: 99%

Regulated Trafficking of the Human Dopamine Transporter

Daniels¹,

Amara²

1999

Journal of Biological Chemistry

301

126

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“…The most well documented example of neurotransmitter transporter phosphorylation is DAT, which has now been characterized as a phosphoprotein both in neuronal tissue and heterologous expression systems (16,38). The phosphorylation of DATs in LLC-PK 1 cells displays activator, inhibitor, and kinetic characteristics that parallel those shown here for neuronal DATs, indicating that heterologous expression systems provide valid models for examining DAT phosphorylation and that results from studies such as deletion or site-directed mutagenesis using transfected DATs will be relevant to the neuronal form of the protein.…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase C-mediated Phosphorylation and Functional Regulation of Dopamine Transporters in Striatal Synaptosomes

Vaughan¹,

Huff²,

Uhl³

et al. 1997

Journal of Biological Chemistry

231

206

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Dopamine transporters (DATs) are members of a family of Na ؉ -and Cl؊ -dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [ 32 P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 M PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, (؊)-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4␣-phorbol 12,13-didecanoate at 10 M was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in V max with no significant effect on the K m for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by phosphorylation of DAT and present a potential mechanism for local neuronal control of synaptic neurotransmitter levels and consequent downstream neural activity. Dopamine transporters (DATs)1 are integral membrane neuronal proteins that function to terminate dopaminergic neurotransmission by the rapid reuptake of synaptic dopamine into presynaptic neurons. As the primary mechanism for the clearance of synaptic dopamine, DAT is the main determinant that regulates the intensity and duration of dopaminergic neurotransmission (1). DAT is implicated in the etiology of psychostimulant drug abuse, as binding of cocaine and amphetamine to the protein inhibit dopamine transport (2), and the resulting elevation of synaptic dopamine levels is believed to underlie the reinforcing properties of these drugs (3, 4). DAT is also a dopaminergic-specific mode of entry for the neurotoxins 6-hydroxydopamine and 1-methyl-4-phenylpyridinium (5, 6), implicating it in mechanisms of neurotoxicity that serve as the best current models for Parkinsonian neurodegeneration.DAT is a membe...

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“…The hDAT and the rDAT contain 47 and 44 Ser's, respectively, in their primary sequences (Giros et al, 1992;Shimada et al, 1991) and Western blot analysis indicates that the DAT is phosphorylated on at least one of these Ser's. It is likely that the phosphorylation of the DAT is substantially affected by ligand occupancy (Vrindavanam et al, 1996) and that phosphorylation-mediated DAT trafficking is affected by phosphorylation of mediator proteins (Chang et al, 2001). (Foster et al, 2002;Surratt et al, 1993).…”

Section: Serinementioning

confidence: 99%

“…However, the DAT does not appear to be directly phosphorylated at its Thr consensus sites and phosphorylation may thus occur at nonconsensus sites . It is also likely that the phosphorylation of the DAT is substantially affected by ligand occupancy (Vrindavanam et al, 1996) and that phosphorylation-mediated DAT trafficking is affected by phosphorylation of mediator proteins (Chang et al, 2001).…”

Section: Threoninementioning

confidence: 99%

A comprehensive atlas of the topography of functional groups of the dopamine transporter

Volz

Schenk

2005

Synapse

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The neuronal dopamine transporter (DAT) is a transmembrane transporter that clears DA from the synaptic cleft. Knowledge of DAT functional group topography is a prerequisite for understanding the molecular basis of transporter function, the actions of psychostimulant drugs, and mechanisms of dopaminergic neurodegeneration. Information concerning the molecular interactions of drugs of abuse (such as cocaine, amphetamine, and methamphetamine) with the DAT at the functional group level may also aid in the development of compounds useful as therapeutic agents for the treatment of drug abuse. This review will provide a cumulative and comprehensive focus on the amino acid functional group topography of the rat and human DATs, as revealed by protein chemical modification and the techniques of site-directed mutagenesis. The results from these studies, represented mostly by site-directed mutagenesis, can be classified into several main categories: modifications without substantial affects on substrate transport, DAT membrane expression, or cocaine analog binding; those modifications which alter both substrate transport and cocaine analog binding; and those that affect DAT membrane expression. Finally, some modifications can selectively affect either substrate transport or cocaine analog binding. Taken together, these literature results show that domains for substrates and cocaine analogs are formed by interactions with multiple and sometimes distinct DAT functional groups.

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The effects of phosphorylation on the functional regulation of an expressed recombinant human dopamine transporter

Cited by 16 publications

References 12 publications

Regulated Trafficking of the Human Dopamine Transporter

Regulated Trafficking of the Human Dopamine Transporter

Protein Kinase C-mediated Phosphorylation and Functional Regulation of Dopamine Transporters in Striatal Synaptosomes

A comprehensive atlas of the topography of functional groups of the dopamine transporter

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