Dopamine transporter is downregulated and its association with chaperone protein Hsc70 is enhanced by activation of dopamine D₃ receptor

Synaptic dopamine (DA) concentrations are largely determined by the activities of presynaptic D₂ and D₃ autoreceptors (D₂R and D₃R) and DA transporter (DAT). Furthermore, the activity of DAT is regulated by phosphorylation events and protein interactions that affect its surface expression. Because DA autoreceptors and DAT coordinately maintain synaptic DA homeostasis, we hypothesized that D₃R might crosstalk with DAT to fine-tune synaptic DA concentrations. To test this hypothesis, we established [³H]DA uptake and DAT surface expression assays in hD₃/rDAT-double-transfected HEK-293 cells or limbic forebrain synaptosomal preparations. Ropinirole, a preferential D₃R agonist, reduced [³H]DA uptake in HEK-hD₃/rDAT cells in a dose-dependent manner, an effect which could be blocked by the D₂R/D₃R antagonist, raclopride. Furthermore, ropinirole also reduced DAT surface expression in limbic forebrain synaptosomes, and this effect could be blocked by raclopride or the internalization inhibitor, concanavalin A. To identify potential mediators of this apparent D₃R-DAT crosstalk, DAT-associated proteins were co-immunoprecipitated from limbic forebrain synaptosomes after D₃R activation and identified by MALDI-TOF. From this analysis, the Hsc70 chaperone was identified as a DAT-associated protein. Interestingly, ropinirole induced the association of Hsc70/Hsp70 with DAT, and the Hsc70/Hsp70 inhibitor, apoptozole, prevented the ropinirole-induced reduction of DAT surface expression. Together, these results suggest that D₃R negatively regulates DAT activity by promoting the association of DAT and Hsc70/Hsp70.