Elsevier

Neuropharmacology

Volume 49, Issue 6, November 2005, Pages 759-768
Neuropharmacology

Differential effects of dopamine and psychoactive drugs on dopamine transporter phosphorylation and regulation

https://doi.org/10.1016/j.neuropharm.2005.08.011Get rights and content

Abstract

The dopamine transporter (DAT) is a phosphoprotein whose activity and phosphorylation state are acutely regulated by both protein kinase C (PKC) and substrate transport. DAT is a major site of action for psychostimulant and therapeutic drugs that either block transport or are transported substrates, but the effects of such drugs on DAT phosphorylation and regulation are not well understood. To examine these issues we subjected rDAT LLC-PK1 cells to acute in vitro pretreatments with the endogenous, psychostimulant, and therapeutic compounds dopamine (DA), (āˆ’)-cocaine, 2Ī²-carbomethoxy-3Ī²-(4-fluorophenyl)tropane (Ī²-CFT), GBR 12909, mazindol, and methylphenidate (MPH), in the presence or absence of the PKC activator phorbol 12 myristate 13 acetate (PMA), followed by analysis of DAT metabolic phosphorylation and transport activity. Basal phosphorylation of DAT was not affected by any of the uptake blockers tested, and PMA-stimulated phosphorylation was not affected by cocaine, Ī²-CFT, mazindol or MPH, but was strongly suppressed by GBR 12909. Pretreatment of cells with cocaine or MPH had no effect on subsequent DA transport activity or the extent of PMA-induced transport down-regulation, whereas GBR 12909 inhibited PMA-induced DAT internalization. These findings indicate that these DAT phosphorylation and down-regulation properties are unaffected by some classes of uptake blocking drugs, but that differential regulatory effects may be exerted by GBR compounds. Pretreatment of cells with DA had no obvious effect on basal or PMA-stimulated DAT phosphorylation but led to cocaine-blockable transport down-regulation. DA-induced down-regulation was blocked by the PKC inhibitor bisindoylmaleimide I and was not additive with down-regulation induced by PMA, consistent with PKC serving as a common step and point of integration for these DA and PMA induced processes. The results of this study provide information on the potential for endogenous and psychoactive compounds to modulate or be modulated by DAT phosphorylation-mediated regulatory mechanisms that may contribute to drug behavioral or therapeutic properties.

Introduction

The dopamine transporter (DAT) is a neuronal phosphoprotein that removes the neurotransmitter dopamine (DA) from the synaptic space and regulates its availability to pre- and post-synaptic DA receptors. The activity of DAT and its ability to clear DA is acutely controlled by protein kinase C (PKC) activators such as phorbol 12 myristate 13 acetate (PMA), which induces transport down-regulation and increases the level of DAT phosphorylation (Zahniser and Doolen, 2001; Vaughan, 2004). Although in vivo signals leading to these processes have not yet been identified, regulation of DAT activity by PKC is presumed to function as a neuronal mechanism for fine-tuning DA levels under diverse physiological conditions.

Dysregulation of DA levels potentially caused by inappropriate DAT function is associated with many neurological disorders, including depression, attention deficit hyperactivity disorder (ADHD), and psychostimulant abuse (Miller et al., 1999). Many compounds used to treat these disorders or that lead to drug addiction exert their effects by acting on DAT and increasing synaptic DA levels. Some of these compounds including cocaine, 2Ī²-carbomethoxy-3Ī²-(4-fluorophenyl)tropane (Ī²-CFT), GBR 12909, mazindol, and methylphenidate (MPH) are uptake blockers that bind to DAT and inhibit substrate transport, while others such as amphetamine (AMPH) and methamphetamine (METH) are structurally similar to DA and are transported by DAT. Substrates can induce DA efflux through transport reversal (Eshleman et al., 1994; Sulzer et al., 1995), and once inside the cell may induce cytotoxicity and neuronal death, postulated to be a mechanism leading to Parkinson's disease and related dopaminergic pathologies (Storch et al., 2004).

PKC-induced down-regulation of DA transport is associated with DAT endocytosis, which moves the protein from the plasma membrane into intracellular vesicles (Daniels and Amara, 1999; Melikian and Buckley, 1999). Although DAT undergoes PKC-stimulated phosphorylation concomitantly with these processes, down-regulation and endocytosis occur in phosphorylation-deficient DAT constructs (Granas et al., 2003; Cervinski et al., 2005), implicating phosphoproteins associated with DAT as additional PKC targets. DAT endocytosis and down-regulation are also induced by DA, AMPH, and METH (Chi and Reith, 2003; Saunders et al., 2000; Sandoval et al., 2001; Cervinski et al., 2005). Various aspects of these processes have been demonstrated to require substrate transport and/or PKC activity, suggesting the presence of a transport-sensing regulatory feedback process that acts through PKC. Substrate efflux is also regulated by a mechanism that requires PKC (Kantor and Gnegy, 1998; Cowell et al., 2000; Johnson et al., 2005) and N-terminal DAT serines (Khoshbouei et al., 2004), and DAT undergoes PKC-dependent phosphorylation stimulated by in vivo and in vitro AMPH and METH (Cervinski et al., 2005). Many properties and functions of DAT are thus regulated by PKC, but their relationships to each other and the underlying molecular mechanisms are not understood.

In contrast to down-regulation effects of substrates, cocaine has been reported to suppress PMA-induced DAT phosphorylation (Cowell et al., 2000), and to either increase (Daws et al., 2002) or have no effect (Sandoval et al., 2001; Chi and Reith, 2003; Cervinski et al., 2005) on DAT surface expression and activity. These studies suggest the potential for the cocaine-bound form of DAT and/or the absence of transport activity to serve as biochemical signals that may block or counteract substrate- and PKC-activated DAT functions.

In most of these studies drugs were examined primarily in isolation for their effects on DAT regulation, and less has been done to examine drugs effects in combination with PKC activators to investigate potential interactions of the processes. The goal of this study was to assess DA and DAT blockers for effects on PKC-dependent and independent properties and to extend observations to therapeutically used uptake-blocking drugs outside the cocaine class.

Section snippets

Cell culture

LLC-PK1 cells stably expressing rDAT (Gu et al., 1994) were maintained in Alpha Minimum Essential Medium supplemented with 10% heat inactivated fetal bovine serum, 2Ā mM l-glutamine, and 1Ɨ penicillin/streptomycin/G418 in an incubation chamber gassed with 5% CO2/95% O2 at 37Ā Ā°C. Cells were grown to 70% confluence in 6- or 12-well plates for use.

Dopamine uptake

For [3H]DA uptake assays rDAT LLC-PK1 cells were washed twice with 1Ā ml of Krebs-Ringer HEPES (KRH) buffer (25Ā mM HEPES, 125Ā mM NaCl, 4.8Ā mM KCl, 1.2Ā mM KH2PO4,

Effects of uptake blockers on DAT phosphorylation

rDAT LLC-PK1 cells were metabolically labeled with 32PO4 and treated with vehicle or 1Ā Ī¼M PMA in the presence or absence of 10Ā Ī¼M (āˆ’)-cocaine, followed by immunoprecipitation, SDS-PAGE, and autoradiography of DAT (Fig. 1, left). In control cells DAT displayed a basal level of constitutive phosphorylation (100 Ā± 3%) that was stimulated approximately two-fold by PMA (215 Ā± 9% of basal, p < 0.001 relative to basal), as previously reported (Huff et al., 1997; Cervinski et al., 2005). In four

Discussion

This study provides an initial characterization of the acute effects of multiple DA uptake blockers on basal and PKC-stimulated DAT phosphorylation and down-regulation. To our knowledge this is the first such study to examine Ī²-CFT, mazindol, or MPH with respect to most of these properties. Our results show that in rDAT LLC-PK1 cells the levels of basal and PMA stimulated DAT phosphorylation were not affected by (āˆ’)-cocaine, Ī²-CFT, mazindol, or MPH. The cocaine results differ from the findings

Acknowledgments

This work was supported by grant RO1 DA13147 to R.A.V. from the National Institute on Drug Abuse. We thank Mark Cervinski and Dr James Foster for advice with aspects of this study.

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