Regulation of the dopamine transporter - Addiction Research ...

Schmitt & Reith DAT Regulation
diarylethene moiety (with excitation and emission
spectra of 488 nm and 609 nm, respectively 99 ).
Hence, unlike other potential surrogate DAT substrates
(e.g., [ 3 H]MPP + and [ 3 H]amphetamine), it
is possible to measure the cellular uptake and accumulation
of ASP + in real time by using fluorescent
microscopy. In EM4 cells coexpressing hDAT and
D2R transcripts, receptor activation with quinpirole
increases the rate of ASP + uptake compared
with vehicle treatment—this effect was abolished
in cells treated with PTX, corroborating the necessity
of Gi/Go protein–coupled signaling for functional
DAT upregulation. 100 Moreover, inhibition of
MEK (which, in turn, prevents ERK1/2 activation)
blocked the effects of quinpirole, whereas PI3K inhibition
had no effect. Treatment with quinpirole
also increases surface expressed DAT (with a concomitant
decrease in intracellular-localized DAT),
as measured by biotinylation. Similarly, quinpirole
also increases plasmalemmal DAT expression and
ASP + uptake in EM4 cells coexpressing hDAT and
D3 receptors. 101 Like the DAT upregulation mediated
by D2 receptor activation, this effect is PTX
sensitive; however, both the MAPK and PI3K pathways
are required for the quinpirole-induced increase
in DAT function. Interestingly, much like the
observations of Johnson et al. 31 for amphetaminergic
substrates, D3 activation results in a rapid increase
in surface DAT expression that eventually
gives way to significantly reduced plasmalemmal
transporter expression upon prolonged D3 agonist
exposure. 101 As a tritiated alternative to fluorescent
ASP + , it also appears possible to use tracer amounts
of [ 3 H]tyramine for monitoring DAT function in
experiments disentangling transporter and receptor
phenomena, because tyramine has been shown to
display rather low potency (micromolar) in activating
D2 dopamine receptors. 100 It will be important
to show that the tritiated ligand does not appreciably
bind to dopamine receptors; caution is required
because the thorough study of Zapata et al. 101
shows considerable binding of [ 3 H]dopamine to D3
dopamine receptors that can be displaced by 0.1 �M
spiroperidol in cells that do not express hDAT compared
with cells subjected to the same conditions
with hDAT on board.
One unexpected finding in this series of receptormediated
ASP + -uptake experiments was the
observation of positive resonance energy transfer
between fluorescent-tagged hDAT and a biolumi-
nescent D2R–luciferase construct, suggesting that
the DAT and D2R proteins are in proximity (