Phosphorylation of dopamine transporter serine 7 modulates cocaine analog binding.

Moritz AE, Foster JD, Gorentla BK, Mazei-Robison MS, Yang JW, Sitte HH, Blakely RD, Vaughan RA
J Biol Chem. 2013 288 (1): 20-32

PMID: 23161550 · PMCID: PMC3537014 · DOI:10.1074/jbc.M112.407874

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (-)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.

MeSH Terms (20)

Animals Binding Sites Chromatography, Thin Layer Cocaine Corpus Striatum Dopamine Plasma Membrane Transport Proteins Dopamine Uptake Inhibitors HEK293 Cells Humans Kinetics Male Mass Spectrometry Mutagenesis, Site-Directed Mutation Phosphorylation Protein Binding Protein Conformation Rats Rats, Sprague-Dawley Serine

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