Abstract
Dopamine (DA) transporters (DATs) are regulated by trafficking and modulatory processes that probably rely on stable and transient interactions with neighboring proteins and lipids. Using proximity-dependent biotin identification (BioID), we found novel potential partners for DAT, including several membrane proteins, such as the transmembrane chaperone 4F2hc, the proteolipid M6a and a potential membrane receptor for progesterone (PGRMC2). We also detected two cytoplasmic proteins: a component of the Cullin1-dependent ubiquitination machinery termed F-box/LRR-repeat protein 2 (FBXL2), and the enzyme inositol 5-phosphatase 2 (SHIP2). Immunoprecipitation (IP) and immunofluorescence studies confirmed either a physical association or a close spatial proximity between these proteins and DAT. M6a, SHIP2 and the Cullin1 system were shown to increase DAT activity in coexpression experiments, suggesting a functional role for their association. Deeper analysis revealed that M6a, which is enriched in neuronal protrusions (filopodia or dendritic spines), colocalized with DAT in these structures. In addition, the product of SHIP2 enzymatic activity (phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]) was tightly associated with DAT, as shown by co-IP and by colocalization of mCherry-DAT with a specific biosensor for this phospholipid. PI(3,4)P2 strongly stimulated transport activity in electrophysiological recordings, and conversely, inhibition of SHIP2 reduced DA uptake in several experimental systems including striatal synaptosomes and the dopaminergic cell line SH-SY5Y. In summary, here we report several potential new partners for DAT and a novel regulatory lipid, which may represent new pharmacological targets for DAT, a pivotal protein in dopaminergic function of the brain.
Highlights
The dopamine (DA) transporter (DAT) is a neuronal protein that controls DA flux in brain, and is, a major regulator of dopaminergic functions including control of movement, cognition, mood or reward
We have identified three membrane proteins and two cytoplasmic proteins as potential partners of DAT: 4F2hc, M6a and PGRMC2, F-box/LRR-repeat protein 2 (FBXL2) and SHIP2
M6a and SHIP2, potentiated DA transport upon coexpression with DAT, as did the ubiquitinating system based in the Cullin-1 scaffold, which might be recruited to the transporter through FBXL2 or other F-box-related adaptors
Summary
The dopamine (DA) transporter (DAT) is a neuronal protein that controls DA flux in brain, and is, a major regulator of dopaminergic functions including control of movement, cognition, mood or reward. Therapeutic or abuse drugs affect DAT activity either by inhibition (in the case of Ritalin or cocaine) or by promoting heteroexchange and the consequent release of DA into the synaptic cleft (in the case of amphetamines). Both mechanisms result in potentiation of the activity of diverse DA receptors during drug therapy or abuse [3, 4]. A member of the SLC6 family of neurotransporters, DAT co-transports one molecule of DA with two sodium ions and one chloride. Recent advances in structural biology have revealed the 3-D structure of DAT, improving our understanding of
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