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Subpopulations of Dopamine Transporters Show Enrichment in Phosphorylation or Palmitoylation
Author(s) -
Storandt Michael H,
Hovde Moriah J,
Stanislowski Daniel J,
Vaughan Roxanne A,
Foster James D
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb189
Subject(s) - palmitoylation , phosphorylation , dopamine transporter , dopaminergic , dopamine plasma membrane transport proteins , gap 43 protein , microbiology and biotechnology , transmembrane protein , transmembrane domain , transporter , chemistry , dopamine , biology , biochemistry , neuroscience , receptor , cysteine , enzyme , gene , immunohistochemistry , immunology
The dopamine transporter (DAT) is a key regulator of duration and intensity of dopaminergic signaling through its uptake of dopamine from the extraneuronal space, and DAT dysregulation has been hypothesized to play a role in dopaminergic diseases such as autism, attention deficit hyperactivity disorder, and drug addiction. DAT consists of 12 transmembrane spanning domains that form the substrate permeation pathway, and cytoplasmic N‐ and C‐termini that contain sites for regulatory post‐translational modifications. Phosphorylation of the N‐terminus occurs on Ser7 and Thr53, which leads to decreased transport V max , and S‐palmitoylation occurs on Cys580 at the membrane‐cytoplasm interface of the most C‐terminal transmembrane helix, which leads to increased transport V max . We previously showed that palmitoylation and phosphorylation occur in a reciprocal manner, with high phosphorylation promoting reduced palmitoylation and visa versa, consistent with their opposing functional outcomes, but it has not been established if these modifications can coexist on a single molecule of DAT or if they are mutually exclusive. To address this issue, we isolated palmitoylated DAT by acyl‐biotin exchange (ABE) and neutravidin affinity chromatography, followed by phospho‐specific immunodetection of Thr53 phosphorylation. Our results indicate that only a small percentage of total DATs are simultaneously phosphorylated and palmitoylated, indicating that the modifications are predominantly mutually exclusive. We also found that S7A DATs display increased palmitoylation, consistent with previous findings, but also show a trend towards increased Thr53 phosphorylation, indicating complex interplay between all three of these modifications. These results support the presence of communication between subregions of the N‐terminus as well as between N‐ and C‐termini, and highlight the mechanistic integration of these post‐translational modifications in the establishment of dopamine reuptake capacity. Support or Funding Information National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)