Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function
Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function
复制标题
DOI：
10.1124/mol.111.073171
复制
发表时间：
2012-01-01
影响因子：
3.6
通讯作者：
Ramamoorthy, Sammanda
中科院分区：
文献类型：
--
作者：
Annamalai, Balasubramaniam;Mannangatti, Padmanabhan;Ramamoorthy, Sammanda
5-羟色胺转运体（5-HT transporter，SERT）通过清除释放到突触间隙的5-HT来调节肾上腺素能神经传递。丝氨酸和苏氨酸上的SERT磷酸化介导SERT调节。酪氨酸磷酸化是否调节SERT尚不清楚。在这里，我们测试的假设，酪氨酸磷酸化的SERT调节5-HT运输。为了支持这一点，在大鼠血小板中发现了耐碱性P-32标记的SERT，Src-酪氨酸激酶抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3，4，d]嘧啶（PP 2）降低了血小板SERT的功能和表达。在表达SERT的人胎盘滋养层细胞中，PP 2降低了转运蛋白的功能、表达和稳定性。虽然Src表达的siRNA沉默降低了SERT功能和表达，但Src的共表达导致了SERT功能和表达的PP 2敏感性增加。PP 2处理显著降低了SERT蛋白的稳定性。与WT-SERT相比，SERT酪氨酸突变体Y 47 F和Y142 F表现出降低5-HT转运，尽管它们的总表达和细胞表面表达水平较高。此外，Src共表达增加了Y 47 F和Y142 F SERT突变体的总表达和细胞表面表达，而不影响其5-HT转运能力。值得注意的是，Y 47 F和Y142 F突变体与WT-SERT相比表现出更高的蛋白质稳定性。然而，与WT-SERT相似，PP 2处理降低了Y 47 F和Y142 F突变体的稳定性。此外，与WT-SERT相比，Y 47 F和Y142 F突变体表现出较低的基础酪氨酸磷酸化，并没有进一步增强酪氨酸磷酸化响应Src共表达。这些结果提供了SERT酪氨酸磷酸化支持转运蛋白稳定性和5 HT转运的第一个证据。
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 P-32-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino5-(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.