Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase
Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase
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DOI：
10.1073/pnas.0501432102
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发表时间：
2005-08-09
影响因子：
11.1
通讯作者：
Blakely, RD
中科院分区：
文献类型：
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作者：
Prasad, HC;Zhu, CB;Blakely, RD
人血清素[5-羟色胺（5-HT）]转运体（hSERT、5HTT和SLC6A4）在释放后使5-HT失活，是情绪、焦虑和强迫症治疗干预的重要靶点。多个hSERT编码变体已被确定，尽管迄今为止尚未报道对这些变体进行全面的功能分析。我们转染了hSERT或10个hSERT编码变体，并通过翻译后的调控途径检测了总蛋白和表面蛋白的表达、拮抗剂的识别和转运蛋白的调节。两个变体Pro339Leu和lle425Val表现出显著的表面表达变化，支持5-HT转运能力的改变（V-max）。无论基础转运活性如何，所有SERT变体都表现出快速的、磷酯触发的下调能力。值得注意的是，5个变异（Thr4Ala、Gly56Ala、Glu215Lys、Lys605Asn和Pro612Ser）在急性蛋白激酶G (PKG)/p38丝裂原活化蛋白激酶（MAPK）激活后没有表现出5-HT摄取刺激的能力。Epstein-Barr病毒（EBV）转化淋巴细胞表达最常见的这些变体（Gly56AIa）表现出类似的PKG/p38 MAPK激活剂对5-HT摄取刺激的丧失。转染Gly56AIa变体的HeLa细胞显示基础磷酸化升高，并且与hSERT不同，在8-溴cGMP （8BrcGMP）处理后不能进一步磷酸化。这些研究揭示了与自然发生的人类SERT编码变异相关的细胞表型，并表明通过PKG/p38 mapk相关途径改变的转运体调节可能影响5-HT信号受损引起的疾病风险。
Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and lle425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity V-max). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56AIa) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56AIa variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.