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首页> 外文期刊>Molecular brain >cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake
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cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

机译：cGMP依赖性蛋白激酶Iα与抗抑郁药敏感的5-羟色胺转运蛋白相关，并指示5-羟色胺摄取的快速调节

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Background The Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear. Results In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIα specifically associates with hSERT. Conclusion Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.
机译：背景依赖于Na + / Cl的血清素（5-羟色胺，5-HT）转运蛋白（SERT）是神经元5-HT信号传导中的关键元素，负责释放后有效消除5-HT。 SERTs不仅是外源性成瘾剂和治疗剂的靶标，而且还可以通过内源性受体连接的信号传导途径进行调节。我们已经表明神经元A3腺苷受体激活导致增强的突触前5-HT体外转运和体内SERT介导的5-HT清除率增加。 A3腺苷受体对SERT的刺激源自cGMP的升高以及cGMP依赖性蛋白激酶（PKG）和p38丝裂原活化蛋白激酶的激活。众所周知，PKG激活剂（例如8-Br-cGMP）会导致转运蛋白磷酸化，尽管这种修饰如何支持SERT调控尚不清楚。结果在本报告中，我们探讨了PKG激活剂快速刺激SERT活性的激酶同工型特异性。使用以前显示支持SERT表面贩运的8-Br-cGMP刺激的永生化大鼠血清素能神经元（RN46A），我们记录了PKGI的表达，而在较低程度上，PKGII的表达。使用透化或非透化条件对染色图谱进行定量分析显示，SERT与PKGI在RN46A细胞体的细胞内和细胞表面域中共定位，并在神经过程中表现出更局限的细胞内共定位模式。在同一细胞中，SERT证明在细胞内或表面膜中缺乏与PKGII的共定位。为了与膜渗透激酶抑制剂DT-2阻断SERT的8-Br-cGMP刺激的能力保持一致，我们发现DT-2处理消除了液相色谱解析的PKGI免疫反应性提取物中的cGMP依赖性激酶活性。同样，用靶向内源性PKGI的小干扰RNA处理SERT转染的HeLa细胞，可消除8-Br-cGMP诱导的SERT活性调节。免疫共沉淀研究表明，在转运蛋白/激酶共转染的细胞中，PKGIα与hSERT特异性结合。结论我们的发现提供了SERT与PKGIα之间物理和分区关联的证据，该关联支持快速的8-Br-cGMP诱导的SERT调节。我们讨论了一个模型，其中与SERT相关的PKGIα依次支持细胞内含转运蛋白的囊泡的动员，从而导致表面表达增强，以及催化调节性SERT磷酸化的产生，从而导致5-HT清除能力的最大增强。

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