D₂ receptors regulate dopamine transporter function via an extracellular signal-regulated kinases 1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism

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, D_2SR) 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 D_2SR 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 D_2SR autoreceptors may regulate DAT in the central nervous system.