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

Abstract

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 (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated 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 PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC 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 PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Dynamic control of the dopamine transporter in neurotransmission and homeostasis

“…However, attempts to validate PKC-mediated degradation for endogenous DAT have proven challenging as multiple groups report PMA treatment fails to induce DAT internalization in mid-brain neuronal cultures 77 , 80 , 81 . Although PKC-induced loss of surface DAT can be robustly reproduced using a biotinylation assay of ex-vivo striatal and mid-brain slices, the total level of DAT is not significantly reduced compared to heterologous systems 27 , 30 , 49 . Conflicting results may be due to differences in PMA treatment times and/or use of heterologous model systems overexpressing DAT.…”

“…Clathrin-mediated endocytosis (CME) was the first mechanism identified for DAT regulation and is best characterized by PKC activation 44 , 45 , which robustly drives the loss of surface DAT in heterologous systems and striatal tissues 46 . New data suggests PKC activation may disrupt DAT interactions with Rit2, and with cytosolic nonreceptor tyrosine kinase (TNK2, formerly called activated p21CDC42 kinase; ACK1), that normally stabilize DAT in the plasma membrane 30 , 47 . AIM-100 may also inhibit TNK2 and lower surface DAT 47 , 48 .…”

“…The C-terminus contains domains for interactions with other proteins including α-synuclein 25 , parkin 26 , flotillin 27 and protein kinase C (PKC) 28 . Recently, RAS-like GTPase, Rit2 was shown to complex with DAT and regulate PKC-stimulated DAT trafficking 29 , 30 . Genetic variability in the Rit2 locus has been robustly associated with PD and several other dopaminergic disorders 29 , 31 , 32 .…”

GeneCup: mine PubMed for gene relationships using custom ontology and deep learning

“…For example, RIT2 is associated with smoking initiation 1 and autism 23 . Recent publications indicated that RIT2 is involved in dopamine transporter trafficking 24 and plays a sex-specific role in acute cocaine response 25 . SYT4 is expressed in the hippocampus and entorhinal cortex 26 and regulates synaptic growth 27,28 .…”

Direct dopamine terminal regulation by local striatal microcircuitry

“…All of the above effectors—ERK, PKC, PP1, PP2A, CaMKII, Akt, and phosphoinositide 3‐kinase (PI3K)—are directly linked to activity‐dependent signaling, providing a potential mechanism by which changes in terminal excitability can regulate clearance on a rapid timescale. Indeed, changes in membrane potential alter DAT membrane expression and dopamine clearance via rapid DAT trafficking on a second timescale, through interactions Rit2 (Fagan, Kearney, & Sweeney, 2020; Richardson et al., 2016; Sweeney et al., 2020). Additionally, increasing dopamine neuron activity increased phosphorylated‐ERK, DAT phosphorylation at threonine53 (known to be phosphorylated by ERK), and leads to functional increases in DAT‐mediated dopamine clearance rates (Calipari, Juarez, et al, 2017).…”