Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Mol Brain. 2009 Aug 5;2:26. doi: 10.1186/1756-6606-2-26.
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.
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, PKGIalpha specifically associates with hSERT.
Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIalpha that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIalpha 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.
钠离子/氯离子依赖性血清素（5-羟色胺，5-HT）转运体（SERT）是神经元 5-HT 信号传递的关键要素，负责在释放后有效消除 5-HT。SERT 不仅是外源性成瘾和治疗药物的靶点，还可以被内源性、受体相关的信号通路调节。我们已经表明，神经元 A3 腺苷受体的激活导致体外 5-HT 转运的增强和体内 SERT 介导的 5-HT 清除率的增加。A3 腺苷受体对 SERT 的刺激源于 cGMP 的升高，随后激活 cGMP 依赖性蛋白激酶（PKG）和 p38 有丝分裂原激活蛋白激酶。已知 PKG 激活剂，如 8-Br-cGMP，可导致转运体磷酸化，尽管这种修饰如何支持 SERT 调节尚不清楚。
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。我们使用先前已显示支持 8-Br-cGMP 刺激 SERT 表面转运的永生化大鼠 5-羟色胺能中缝神经元（RN46A），证明了 PKGI 的表达，以及较低程度的 PKGII 的表达。使用透化或非透化条件对染色谱进行定量分析表明，SERT 与 PKGI 在 RN46A 细胞体的细胞内和细胞表面区域共定位，并在神经突过程中表现出更局限的、细胞内的共定位模式。在相同的细胞中，SERT 与 PKGII 在细胞内或质膜中均无共定位。与膜通透激酶抑制剂 DT-2 阻断 8-Br-cGMP 刺激 SERT 的能力一致，我们发现 DT-2 处理消除了通过液相色谱分离的 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 处理转染 SERT 的 HeLa 细胞，消除了 8-Br-cGMP 诱导的 SERT 活性调节。共免疫沉淀研究表明，在转运体/激酶共转染细胞中，PKGIalpha 特异性与 hSERT 结合。
我们的发现提供了 SERT 和 PKGIalpha 之间物理和区室化关联的证据，支持 8-Br-cGMP 诱导的 SERT 的快速调节。我们讨论了一种模型，其中 SERT 相关的 PKGIalpha 依次支持包含细胞内转运体的囊泡的动员，导致表面表达增强，以及产生催化调节性 SERT 磷酸化，导致 5-HT 清除能力的最大增强。
