Structural Determinants of the Dopamine Transporter Regulation Mediated by G Proteins

Dopamine clearance in the brain is
controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which
drives reuptake of extracellular dopamine into presynaptic neurons.
Studies have revealed that the βγ subunits of heterotrimeric
G proteins modulate DAT function through a physical association with
the C-terminal region of the transporter. Regulation of neurotransmitter
transporters by Gβγ subunits is unprecedented in the literature;
therefore, it is interesting to investigate the structural details
of this particular protein–protein interaction. Here, we refined
the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal
domains; subsequently, we used the full-length dDAT structure to generate
a comparative model of human DAT (hDAT). Both proteins were assembled
with Gβ1γ2 subunits employing protein–protein docking,
and subsequent molecular dynamics simulations were run to identify
the specific interactions governing the formation of the hDAT:Gβγ
and dDAT:Gβγ complexes. A [L/F]­R­[Q/E]­R sequence motif
containing the residues R588 in hDAT and R587 in dDAT was found as
key to bind the Gβγ subunits through electrostatic interactions
with a cluster of negatively charged residues located at the top face
of the Gβ subunit. Alterations of DAT function have been associated
with multiple devastating neuropathological conditions; therefore,
this work represents a step toward better understanding DAT regulation
by signaling proteins, allowing us to predict therapeutic target regions.