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Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine.

ABSTRACT: Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d-amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co-immunoprecipitation and cross-linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild-type (WT) DAT with W84L mutant, effects of d-amphetamine on oligomerization (decrease) but not surface DAT are observed. Internalization of W84L monomers appears to be slow as inferred from the inability of d-amphetamine to reduce surface Myc upon co-expressing Flag-WT with Myc-W84L but not Myc-WT with Flag-W84L, and from the sluggish Myc-W84L endocytosis rate (both with or without d-amphetamine). Results obtained for D313N, D345N, or D436N mutants can all be accommodated by a model in which D-amphetamine is unable to dissociate mutant protomers from oligomers (tetramers or higher-order assemblies) that contain them; this interpretation is confirmed in experiments with both tag reversal in co-expression and antibody reversal in western blotting. Upon co-transfecting Myc- and Flag-tagged constructs, resulting tetramers can be calculated to be composed of different species (MycMycMycMyc, MycMycMycFlag, MycMycFlagFlag, MycFlagFlagFlag, and FlagFlagFlagFlag), but it is shown that outcomes predicted by models based on MycMycFlagFlag oligomers are not changed in a major way by the occurrence of the additional species.

SUBMITTER: Li Y

PROVIDER: S-EPMC2910181 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine.

Li Yan Y Cheng Shu-Yuan SY Chen Nianhang N Reith Maarten E A ME

Journal of neurochemistry 20100518 3

Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d-amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co-immunoprecipitation and cross-linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild-type (WT) ...[more]

PMID: 20492355

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Project description:Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22-normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a "reluctant" state to a "willing" state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.

Dataset Information

Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine.

Publications

Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine.

Similar Datasets

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