TY - JOUR
T1 - D2 receptors regulate dopamine transporter function via an extracellular signal-regulated kinases 1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism
AU - Bolan, Elizabeth A.
AU - Kivell, Bronwyn
AU - Jaligam, Vanaja
AU - Oz, Murat
AU - Jayanthi, Lankupalle D.
AU - Han, Yang
AU - Sen, Namita
AU - Urizar, Eneki
AU - Gomes, Ivone
AU - Devi, Lakshmi A.
AU - Ramamoorthy, Sammanda
AU - Javitch, Jonathan A.
AU - Zapata, Agustin
AU - Shippenberg, Toni S.
PY - 2007/5
Y1 - 2007/5
N2 - The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D 2 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 D 2/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 D2SR autoreceptors may regulate DAT in the central nervous system.
AB - The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D 2 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 D 2/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 D2SR autoreceptors may regulate DAT in the central nervous system.
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U2 - 10.1124/mol.106.027763
DO - 10.1124/mol.106.027763
M3 - Article
C2 - 17267664
AN - SCOPUS:34247545124
SN - 0026-895X
VL - 71
SP - 1222
EP - 1232
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 5
ER -