Prolonged dopamine D₃ receptor stimulation promotes dopamine transporter ubiquitination and degradation through a...

The dopamine transporter (DAT) is a membrane glycoprotein in dopaminergic neurons, which modulates extracellular and intracellular dopamine levels. DAT is regulated by different presynaptic proteins, including dopamine D₂ (D₂R) and D₃ (D₃R) receptors. While D₂R signalling enhances DAT activity, some data suggest that D₃R has a biphasic effect. However, despite the extensive therapeutic use of D₂R/D₃R agonists in neuropsychiatric disorders, this phenomenon has been little studied. In order to shed light on this issue, DAT activity, expression and posttranslational modifications were studied in mice and DAT-D₃R-transfected HEK cells. Consistent with previous reports, acute treatment with D₂R/D₃R agonists promoted DAT recruitment to the plasma membrane and an increase in DA uptake. However, when the treatment was prolonged, DA uptake and total striatal DAT protein declined below basal levels. These effects were inhibited in mice by genetic and pharmacological inactivation of D₃R, but not D₂R, indicating that they are D₃R-dependent. No changes were detected in mesostriatal tyrosine hydroxylase (TH) protein expression and midbrain TH and DAT mRNAs, suggesting that the dopaminergic system is intact and DAT is posttranslationally regulated. The use of immunoprecipitation and cell surface biotinylation revealed that DAT is phosphorylated at serine residues, ubiquitinated and released into late endosomes through a PKCβ-dependent mechanism. In sum, the results indicate that long-term D₃R activation promotes DAT down-regulation, an effect that may underlie neuroprotective and antidepressant actions described for some D₂R/D₃R agonists.