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PMID:19357284

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Citation

Egaña, LA, Cuevas, RA, Baust, TB, Parra, LA, Leak, RK, Hochendoner, S, Peña, K, Quiroz, M, Hong, WC, Dorostkar, MM, Janz, R, Sitte, HH and Torres, GE (2009) Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3. J. Neurosci. 29:4592-604

Abstract

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.

Links

PubMed PMC2846176 Online version:10.1523/JNEUROSCI.4559-08.2009

Keywords

Animals; Dopamine Plasma Membrane Transport Proteins/genetics; Dopamine Plasma Membrane Transport Proteins/metabolism; Dopamine Plasma Membrane Transport Proteins/physiology; Humans; Male; Membrane Proteins/genetics; Membrane Proteins/metabolism; Membrane Proteins/physiology; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins/genetics; Nerve Tissue Proteins/metabolism; Nerve Tissue Proteins/physiology; PC12 Cells; Rats; Synaptic Vesicles/genetics; Synaptic Vesicles/metabolism; Synaptic Vesicles/physiology; Transfection

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

RAT:D4ABK1

involved_in

GO:0001504: neurotransmitter uptake

ECO:0006063: over expression analysis evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:VMAT2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q8R191

F

Seeded From UniProt

complete

MOUSE:SNG3

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q8BRU6

F

Seeded From UniProt

complete

MOUSE:SNG3

involved_in

GO:0032411: positive regulation of transporter activity

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

MOUSE:SNG3

located_in

GO:0008021: synaptic vesicle

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

MOUSE:SNG3

enables

GO:0047485: protein N-terminus binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q61327

F

Seeded From UniProt

complete

MOUSE:SC6A3

enables

GO:0047485: protein N-terminus binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q8R191

F

Seeded From UniProt

complete


See also

References

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