The dopamine transporter carboxyl-terminal tail. Truncation/substitution mutants selectively confer high affinity dopamine uptake while attenuating recognition of the ligand binding domain.
In order to delineate structural motifs regulating substrate affinity and recognition for the human dopamine transporter (DAT), we assessed [3H]dopamine uptake kinetics and [3H]CFT binding characteristics of COS-7 cells transiently expressing mutant DATs in which the COOH terminus was truncated or substituted. Complete truncation of the carboxyl tail from Ser582 allowed for the expression of biphasic [3H]dopamine uptake kinetics displaying both a low capacity (Vmax approximately 0.4 pmol/10(5) cells/min) high affinity (Km approximately 300 nM) component and one exhibiting low affinity (Km approximately 15 microM] and high capacity (Vmax approximately 5 pmol/10(5)cells/min) with a concomitant 40% decrease in overall apparent Vmax relative to wild type (WT) DAT. Truncation of the last 22 amino acids or substitution of the DAT-COOH tail with sequences encoding the intracellular carboxyl-terminal of either dopamine D1 or D5 receptors produced results that were identical to those with the fully truncated DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely conferred by removal of DAT-specific sequence motifs distal to Pro597. The attenuation of WT transport activity, either by lowering levels of DAT expression or by pretreatment of cells with phorbol 12-myristate 13-acetate (1 microM), did not affect the kinetics of [3H]dopamine transport. The estimated affinity of dopamine (Ki approximately 180 nM) for all truncated/substituted DAT mutants was 10-fold lower than that of WT DAT (approximately 2000 nM) and appears selective for the endogenous substrate, since the estimated inhibitory constants for numerous putative substrates or uptake inhibitors were virtually identical to those obtained for WT DATs. In marked contrast, DAT truncation/substitution mutants displayed significantly reduced high affinity [3H]CFT binding interactions with estimated Ki values for dopamine and numerous other substrates and inhibitors tested from 10-100-fold lower than that observed for WT DAT. Moreover, co-expression of truncated and/or substituted DATs with WT transporter failed to reconstitute functional or pharmacological activities associated with both transporters. Instead, complete restoration of uniphasic low affinity [3H]dopamine uptake kinetics (Km approximately 2000 nM) and high affinity substrate and inhibitor [3H]CFT binding interactions attributable to WT DATs were evident. These data clearly suggest the functional independence and differential regulation of the dopamine translocation process from the characteristics exhibited by its ligand binding domain. The lack of functional phenotypic expression of mutant DAT activities in cells co-expressing WT transporter is consistent with the contention that native DATs may exist as multisubunit complexes, the formation and maintenance of which is dependent upon sequences encoded within the carboxyl tail.
Lee
F J
FJ
Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Pristupa
Z B
ZB
Ciliax
B J
BJ
Levey
A I
AI
Niznik
H B
HB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003042:Cocaine / Q000031:analogs & derivatives / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D017931:DNA Primers; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008024:Ligands; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C493215:Slc6a3 protein, mouse; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D003042:Cocaine; D004298:Dopamine