Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for duanyu1531-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in duanyu1531-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls duanyu1531-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for duanyu1531-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that duanyu1531 activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate duanyu1531-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate DAT internalization and reveal unexpected region-specific differences in duanyu1531-stimulated DAT trafficking in bona fide DAergic terminals.

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