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

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-PK(1) cells to acute in vitro pretreatments with the endogenous, psychostimulant, and therapeutic compounds dopamine (DA), (-)-cocaine, 2 beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (beta-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, beta-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.