Department of Neurobiology, Brudnick Neuropsychiatric Research Institute; Melikian Lab; Graduate School of Biomedical Sciences
Amino Acids, Peptides, and Proteins | Biochemistry | Enzymes and Coenzymes | Neuroscience and Neurobiology | Nucleic Acids, Nucleotides, and Nucleosides
Following its evoked release, DA signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DAT. DAT surface availability is dynamically regulated by endocytic trafficking, and direct 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.
GTPase, dopamine transporter, endocytosis, membrane trafficking, protein kinase C (PKC), ras-like without CAAX 2, short hairpin RNA (shRNA), striatum
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© 2020 The Author(s). Publisher's paper in press version posted as allowed by publisher's author rights policy at https://www.asbmb.org/journals-news/editorial-policies.
DOI of Published Version
This research was originally published in: Fagan RR, Kearney PJ, Sweeney CG, Luethi D, Schoot Uiterkamp FE, Schicker K, Alejandro BS, O'Connor LC, Sitte HH, Melikian HE. Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact. J Biol Chem. 2020 Mar 4:jbc.RA120.012628. doi: 10.1074/jbc.RA120.012628. Epub ahead of print. PMID: 32132171. Link to article on publisher's site
The Journal of biological chemistry
Fagan RR, Kearney P, Sweeney CG, Luethi D, Schoot Uiterkamp FE, Schicker K, Alejandro BS, O'Connor LC, Sitte HH, Melikian H. (2020). Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact. Open Access Articles. https://doi.org/10.1074/jbc.RA120.012628. Retrieved from https://escholarship.umassmed.edu/oapubs/4187