Psychoactive substrates stimulate dopamine transporter phosphorylation and down-regulation by cocaine-sensitive and protein kinase C-dependent mechanisms.
Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine (METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by (32)PO(4) metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK(1) cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (-)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine- and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.
Cervinski
Mark A
MA
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA.
Foster
James D
JD
Vaughan
Roxanne A
RA
D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D001552:Benzazepines / Q000494:pharmacology; D003042:Cocaine / Q000494:pharmacology*; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D015259:Dopamine Agents / Q000494:pharmacology*; D065127:Dopamine D2 Receptor Antagonists; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D008694:Methamphetamine / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017447:Receptors, Dopamine D1 / Q000037:antagonists & inhibitors; D012694:Serine / Q000378:metabolism; D013469:Sulpiride / Q000494:pharmacology
D001552:Benzazepines; D015259:Dopamine Agents; D065127:Dopamine D2 Receptor Antagonists; D050483:Dopamine Plasma Membrane Transport Proteins; D007211:Indoles; D008301:Maleimides; D017447:Receptors, Dopamine D1; D008694:Methamphetamine; D012694:Serine; D013469:Sulpiride; D000661:Amphetamine; D011493:Protein Kinase C; D003042:Cocaine; C088060:bisindolylmaleimide; D004298:Dopamine