Abstract
The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant 32P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport.
Footnotes
This work was supported by the National Institutes of Health National Institute of Mental Health [Grant MH62612] (to S.R); the National Institutes of Health National Institute of General Medical Sciences [Grant GM081054] (to L.D.J.), and in part by the Intramural Research Program of the National Institutes of Health National Institute on Drug Abuse (to. T.S.S).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- 5-HT
- serotonin
- SERT
- serotonin transporter
- PKC
- protein kinase C
- CaMK
- calcium/calmodulin-dependent protein kinase
- MAPK
- mitogen-activated protein kinase
- PKG
- cGMP-dependent protein kinase
- GPCR
- G protein-coupled receptor
- LY294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
- PP2
- 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine
- PP3
- 4-amino-7-phenylpyrazol[3,4-d]pyrimidine
- ECL
- enhanced chemiluminescence
- PD98059
- 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
- PD169316
- 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole
- U0126
- 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene
- SB203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole
- SP600125
- anthra[1,9-cd]pyrazol-6(2H)-one
- siRNA
- small interfering RNA
- HTR
- human placental trophoblast cells
- KRH
- Krebs-Ringer-HEPES
- h
- human
- DAT
- dopamine transporter
- NET
- norepinephrine transporter
- TauT
- taurine transporter
- eGFP
- enhanced green fluorescent protein
- NE
- norepinephrine
- DA
- dopamine
- RIPA
- radioimmunoprecipitation assay
- PAGE
- polyacrylamide gel electrophoresis
- β-PMA
- phorbol 12-myristate 13-acetate
- OV
- orthovanadate
- PI3K
- phosphatidylinositol 3-kinase
- ERK
- extracellular signal-regulated kinase
- WT
- wild type
- ANOVA
- analysis of variance
- GBR12909
- vanoxerine
- sulfo-NHS-SS-biotin
- sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate.
- Received April 22, 2011.
- Accepted October 12, 2011.
- U.S. Government work not protected by U.S. copyright
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