title: D2 receptors regulate dopamine transporter function via an extracellular signal-regulated kinases 1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism.
abstract: The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D(2) dopamine receptors (D(2)R) has been reported. The high affinity of DA and other DAT substrates for the D(2)R, 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 D(2)R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D(2)R regulation of DAT. Addition of the D(2)/D(3) receptor agonist quinpirole (0.1-10 muM) to human embryonic kidney cells coexpressing human DAT and D(2) receptor (short splice variant, D(2S)R) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP(+) accumulation. The D(2)/D(3) 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. D(2S)R 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 D(2)R stimulation. These experiments demonstrate that D(2S)R 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 D(2)R. Together, these results suggest a novel mechanism by which D(2S)R autoreceptors may regulate DAT in the central nervous system.
authors.affiliation: Integrative Neuroscience Section, National Institute on Drug Abuse Intramural Research Program/National Institutes of Health/Department of Health and Human Services, Baltimore, MD 21224, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Bolan
authors.forename: Bronwyn
authors.initials: B
authors.lastname: Kivell
authors.forename: Vanaja
authors.initials: V
authors.lastname: Jaligam
authors.forename: Murat
authors.initials: M
authors.lastname: Oz
authors.forename: Lankupalle D
authors.initials: LD
authors.lastname: Jayanthi
authors.forename: Yang
authors.initials: Y
authors.lastname: Han
authors.forename: Namita
authors.initials: N
authors.lastname: Sen
authors.forename: Eneki
authors.initials: E
authors.lastname: Urizar
authors.forename: Ivone
authors.initials: I
authors.lastname: Gomes
authors.forename: Lakshmi A
authors.initials: LA
authors.lastname: Devi
authors.forename: Sammanda
authors.initials: S
authors.lastname: Ramamoorthy
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: Agustin
authors.initials: A
authors.lastname: Zapata
authors.forename: Toni S
authors.initials: TS
authors.lastname: Shippenberg
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D004735:Energy Transfer / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011726:Pyridinium Compounds / Q000378:metabolism / Q000494:pharmacology; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000187:drug effects; D014439:Tyramine / Q000378:metabolism / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; D011726:Pyridinium Compounds; D017448:Receptors, Dopamine D2; C052247:4-(4-diethylaminostyryl)-N-methylpyridinium; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D014439:Tyramine