Abstract
The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D2 dopamine receptors (D2R) has been reported. The high affinity of DA and other DAT substrates for the D2R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP+) with live cell imaging techniques to identify the role of two D2R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D2R regulation of DAT. Addition of the D2/D3 receptor agonist quinpirole (0.1–10 μM) to human embryonic kidney cells coexpressing human DAT and D2 receptor (short splice variant, D2SR) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP+ accumulation. The D2/D3 agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP+ accumulation. D2SR activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP+ accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D2R stimulation. These experiments demonstrate that D2SR stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D2R. Together, these results suggest a novel mechanism by which D2SRautoreceptors may regulate DAT in the central nervous system.
Footnotes
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J.J., A.Z., and T.S.S. contributed equally to this work.
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This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Drug Abuse, and National Institutes of Health grants MH57324, MH54137, and DA11495 (to J.A.J.) and DA08863 and DA0019521 (to L.A.D.), and National Institute on Drug Abuse and National Institutes of Health grants P50DA015369 and MH062612 (to S.R.).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.027763.
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ABBREVIATIONS: DA, dopamine; D2R, D2 dopamine receptor; DAT, dopamine transporter; D2SR, D2 receptor short splice variant; WT, wild type; BRET, bioluminescence resonance energy transfer; ERK1/2, extracellular signal-regulated kinases 1 and 2; PI3K, phosphoinositide 3 kinase; MEK, mitogen-activated protein kinase kinase; PKC, protein kinase C; D2LR, D2 receptor long splice variant; N2a, Neuro-2a cell; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; YFP, yellow fluorescent protein; KRH, Krebs-Ringer-HEPES; ANOVA, analysis of variance; SNK, Student Newman-Keuls test; HA, hemagglutinin; FACS, fluorescence-activated cell sorting; HEK, human embryonic kidney; κ-Luc, luciferase-tagged κ-opioid receptor; HA 2EL hDAT, hemagglutinin epitope-tagged human dopamine transporter; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PD128907, (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride; ASP+, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide; B135, 3-(2-pyridyl)-5-(3-allyloxy-5-carboxyphenyl)-1,2,4-oxadiazole.
- Received June 8, 2006.
- Accepted January 31, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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