title: Tyrosine phosphorylation of the human serotonin transporter: a role in the transporter stability and function.
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 (32)P-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.
authors: Department of Neurosciences, Division of Neuroscience Research, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors: Balasubramaniam
authors: B
authors: Annamalai
authors: Padmanabhan
authors: P
authors: Mannangatti
authors: Obulakshmi
authors: O
authors: Arapulisamy
authors: Toni S
authors: TS
authors: Shippenberg
authors: Lankupalle D
authors: LD
authors: Jayanthi
authors: Sammanda
authors: S
authors: Ramamoorthy
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D010766:Phosphorylation / Q000502:physiology; D055550:Protein Stability; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism* / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D050486:Serotonin Plasma Membrane Transport Proteins; D014443:Tyrosine