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.

突触多巴胺 (DA) 浓度很大程度上取决于突触前 D ₂和 D ₃自身受体（D ₂ R 和 D ₃ R）和 DA 转运蛋白 (DAT) 的活动。此外，DAT 的活性受磷酸化事件和影响其表面表达的蛋白质相互作用的调节。由于 DA 自身受体和 DAT 协同维持突触 DA 稳态，我们假设 D ₃ R 可能与 DAT 串扰以微调突触 DA 浓度。为了验证这一假设，我们在 hD _{3 中}建立了 [ ³ H]DA 摄取和 DAT 表面表达测定/rDAT-双转染的 HEK-293 细胞或边缘前脑突触体制剂。Ropinirole 是一种优先的 D ₃ R 激动剂，以剂量依赖性方式降低HEK-hD ₃ /rDAT 细胞中的[ ³ H]DA 摄取，这种作用可以被 D ₂ R/D ₃ R 拮抗剂 raclopride 阻断。此外，罗匹尼罗还降低了边缘前脑突触体中 DAT 表面的表达，这种作用可以被 raclopride 或内化抑制剂伴刀豆球蛋白 A 阻断。为了鉴定这种明显的 D ₃ R-DAT 串扰的潜在介质，DAT 相关蛋白是共同D ₃后从边缘前脑突触体免疫沉淀R 激活并由 MALDI-TOF 鉴定。从该分析中，Hsc70 分子伴侣被鉴定为 DAT 相关蛋白。有趣的是，罗匹尼罗诱导 Hsc70/Hsp70 与 DAT 的结合，而 Hsc70/Hsp70 抑制剂 apoptozole 阻止了罗匹尼罗诱导的 DAT 表面表达降低。总之，这些结果表明 D ₃ R 通过促进 DAT 和 Hsc70/Hsp70 的结合来负调节 DAT 活性。