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Titolo:
THE DOPAMINE TRANSPORTER CARBOXYL-TERMINAL TAIL - TRUNCATIONS SUBSTITUTION MUTANTS SELECTIVELY CONFER HIGH-AFFINITY DOPAMINE UPTAKE WHILE ATTENUATING RECOGNITION OF THE LIGAND-BINDING DOMAIN/
Autore:
LEE FJS; PRISTUPA ZB; CILIAX BJ; LEVEY AI; NIZNIK HB;
Indirizzi:
CLARKE INST PSYCHIAT,MOL NEUROBIOL LAB,250 COLL ST TORONTO ON M5T 1R8CANADA CLARKE INST PSYCHIAT,MOL NEUROBIOL LAB TORONTO ON M5T 1R8 CANADA UNIV TORONTO,DEPT PSYCHIAT TORONTO ON M5S 1A8 CANADA UNIV TORONTO,DEPT PHARMACOL TORONTO ON M5S 1A8 CANADA EMORY UNIV,DEPT NEUROL ATLANTA GA 30322
Titolo Testata:
The Journal of biological chemistry
fascicolo: 34, volume: 271, anno: 1996,
pagine: 20885 - 20894
SICI:
0021-9258(1996)271:34<20885:TDTCT->2.0.ZU;2-K
Fonte:
ISI
Lingua:
ENG
Soggetto:
AMINOBUTYRIC-ACID TRANSPORTER; GLUT4 GLUCOSE-TRANSPORTER; BRAIN GABA TRANSPORTER; H-3 WIN 35,428; SEROTONIN TRANSPORTER; RAT-BRAIN; NOREPINEPHRINE TRANSPORTERS; SACCHAROMYCES-CEREVISIAE; RADIATION INACTIVATION; COCAINE RECEPTORS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
85
Recensione:
Indirizzi per estratti:
Citazione:
F.J.S. Lee et al., "THE DOPAMINE TRANSPORTER CARBOXYL-TERMINAL TAIL - TRUNCATIONS SUBSTITUTION MUTANTS SELECTIVELY CONFER HIGH-AFFINITY DOPAMINE UPTAKE WHILE ATTENUATING RECOGNITION OF THE LIGAND-BINDING DOMAIN/", The Journal of biological chemistry, 271(34), 1996, pp. 20885-20894

Abstract

In order to delineate structural motifs regulating substrate affinityand recognition for the human dopamine transporter (DAT), we assessed[H-3]dopamine uptake kinetics and [H-3]CFT binding characteristics ofCOS-7 cells transiently expressing mutant DATs in which the COOH terminus was truncated or substituted, Complete truncation of the carboxyltail from Ser(582) allowed for the expression of biphasic [H-3]dopamine uptake kinetics displaying both a low capacity (V-max similar to-0.4 pmol/10(5) cells/min) high affinity (K-m similar to 300 nM) component and one exhibiting low affinity (K-m similar to 15 mu M) and high capacity (V-max similar to 5 pmol/10(5) cells/min) with a concomitant 40% decrease in overall apparent V-max 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 ofbiphasic dopamine uptake kinetics is likely conferred by removal of DAT-specific sequence motifs distal to Pro(597). 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 mu M), did not affect the kinetics of [H-3]dopamine transport. The estimated affinity of dopamine (K-i similar to 180 nM) for all truncated/substituted DAT mutants was 10-fold lower than that of WT DAT (similar to 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 [H-3]CFT binding interactions with estimated K-i 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 activitiesassociated with both transporters. Instead, complete restoration of uniphasic low affinity [H-3]dopamine uptake kinetics (K-m similar to 2000 nM) and high affinity substrate and inhibitor [H-3]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 ex pression 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.

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Documento generato il 04/04/20 alle ore 02:33:02