Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity
Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity
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DOI：
10.1073/pnas.0610964104
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发表时间：
2007-05-08
影响因子：
11.1
通讯作者：
Marin, P.
中科院分区：
文献类型：
--
作者：
Chanrion, B.;la Cour, C. Mannoury;Marin, P.
神经递质转运蛋白的时空调控涉及与其胞内结构域相互作用的蛋白质。使用蛋白质组学的方法，我们确定了几个蛋白质与C端的5-羟色胺转运蛋白（SERT）。这些包括神经元型一氧化氮合酶（nNOS），PSD-95/光盘大/ZO-1（PDZ）结构域的蛋白质招募的非典型PDZ结合基序的SERT。HEK 293细胞中nNOS与SERT的共表达降低了SERT细胞表面定位和5-羟色胺（5-HT）摄取。这些影响是不存在的细胞转染SERT突变的PDZ基序，以防止与nNOS的物理关联，5-HT摄取不受激活或抑制nNOS酶活性。nNOS缺陷型和野生型小鼠肌肉注射5-HT进入脑突触体的摄取增加。用SERT C末端的膜渗透肽基模拟物注射，其破坏SERT和nNOS之间的相互作用，表明nNOS降低体内SERT活性。此外，用模拟肽处理培养的中脑神经元同样增加5-HT摄取。相反，表明5-HT摄取刺激nNOS活性，NO的产生增加与nNOS和SERT共转染的细胞暴露于5-HT。这种作用被5-HT摄取抑制剂消除，并且在表达SERT的细胞中不存在PDZ基序突变。总之，nNOS和SERT之间的物理关联为它们的相互功能调节提供了分子底物。除了显示nNOS控制SERT的细胞表面定位外，这些发现还为底物携带转运蛋白调节细胞信号传导（NO产生）提供了证据。
The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/ Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface localization and 5-hydroxytryptamine (5-HT) uptake. These effects were absent in cells transfected with SERT mutated in its PDZ motif to prevent physical association with nNOS, and 5-HT uptake was unaffected by activation or inhibition of nNOS enzymatic activity. 5-HT uptake into brain synaptosomes was increased in both nNOS-deficient and wild-type mice im. injected with a membrane-permeant peptidyl mimetic of SERT C terminus, which disrupted interaction between SERT and nNOS, suggesting that nNOS reduces SERT activity in vivo. Furthermore, treating cultured mesencephalic neurons with the mimetic peptide similarly increased 5-HT uptake. Reciprocally, indicating that 5-HT uptake stimulates nNOS activity, NO production was enhanced on exposure of cells cotransfected with nNOS and SERT to 5-HT. This effect was abolished by 5-HT uptake inhibitors and absent in cells expressing SERT mutated in its PDZ motif. In conclusion, physical association between nNOS and SERT provides a molecular substrate for their reciprocal functional modulation. In addition to showing that nNOS controls cell surface localization of SERT, these findings provide evidence for regulation of cellular signaling (NO production) by a substrate-carrying transporter.