Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine.
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.
Li
Yan
Y
Department of Psychiatry, New York University School of Medicine, New York, New York 10016, USA.
Cheng
Shu-Yuan
SY
Chen
Nianhang
N
Reith
Maarten E A
ME
D000661:Amphetamine / Q000737:chemistry* / Q000494:pharmacology; D002460:Cell Line; D004298:Dopamine / Q000737:chemistry* / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008959:Models, Neurological; D009154:Mutation / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D013499:Surface Properties
D050483:Dopamine Plasma Membrane Transport Proteins; C489427:MYC protein, human; D046911:Macromolecular Substances; D016271:Proto-Oncogene Proteins c-myc; D000661:Amphetamine; D004298:Dopamine