Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization
Regulated Norepinephrine Transporter Interaction with the Neurokinin-1 Receptor Establishes Transporter Subcellular Localization
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DOI：
10.1074/jbc.m113.472878
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发表时间：
2013-10-04
影响因子：
4.8
通讯作者：
Jayanthi, Lankupalle D.
中科院分区：
文献类型：
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作者：
Arapulisamy, Obulakshmi;Mannangatti, Padmanabhan;Jayanthi, Lankupalle D.
神经激肽-1受体（NK 1 R）通过蛋白激酶C（PKC）介导人去甲肾上腺素（NE）转运体（hNET）的下调。然而，NK 1 R对NET的天然调控以及NK 1 R靶向NET的机制尚不清楚。使用表达天然NET和NK 1 R的大鼠脑突触体以及共表达WT-hNET或NK 1 R/PKC抗性hNET-T258 A、S259 A双突变体（NET-DM）和hNK 1 R的人胎盘滋养层（HTR）细胞研究NK 1 R激活对天然NET调节和NET/NK 1 R相互作用的影响。选择性NK 1 R激动剂GR 73632和P物质（SP）抑制NE转运，并降低NET和NK 1 R的质膜表达。用NK 1 R拮抗剂EMEND（阿瑞匹坦）预处理可防止这些NK 1 R介导的效应。免疫沉淀实验表明NET与NK 1 R形成稳定的复合物。在HTR细胞中，结合生物素化和免疫沉淀研究显示NETNK 1 R复合物的质膜定位。受体活化导致NETNK 1 R复合物的内化。脂筏和免疫沉淀分析揭示了NETNK 1 R复合物的存在下，只在非筏膜组分基础/未刺激的条件下。然而，NK 1 R活化导致NETNK 1 R复合物易位到富含筏的膜组分。重要的是，发现PKC与SP治疗后筏定位的NET相关。与WT-NET相似，PKC抗性NET-DM仅在非筏组分中与NK 1 R相关。然而，SP处理未能将NET-DMNK 1 R复合物从非筏级分移位到筏级分。总的来说，这些结果表明，NK 1 R与NET形成物理复合物，并且NETNK 1 R复合物的受体介导的Thr（258）+ Ser（259）基序依赖性易位进入富含筏的微结构域促进了NET/NK 1 R与PKC的相互作用，以协调空间限制的NET调节。
Neurokinin-1 receptor (NK1R) mediates down-regulation of human norepinephrine (NE) transporter (hNET) via protein kinase C (PKC). However, native NET regulation by NK1R and the mechanism by which NK1R targets NET among other potential effectors are unknown. Effect of NK1R activation on native NET regulation and NET/NK1R interaction were studied using rat brain synaptosomes expressing native NET and NK1R as well as human placental trophoblast (HTR) cells coexpressing WT-hNET or NK1R/PKC-resistant hNET-T258A,S259A double mutant (NET-DM) and hNK1R. The selective NK1R agonist, GR73632, and Substance-P (SP) inhibited NE transport and reduced plasma membrane expression of NET and NK1R. Pretreatment with the NK1R antagonist, EMEND (aprepitant) prevented these NK1R-mediated effects. Immunoprecipitation experiments showed that NET forms stable complexes with NK1R. In HTR cells, combined biotinylation and immunoprecipitation studies revealed plasma membrane localization of NETNK1R complexes. Receptor activation resulted in the internalization of NETNK1R complexes. Lipid raft and immunoprecipitation analyses revealed the presence of NETNK1R complexes exclusively in non-raft membrane fractions under basal/unstimulated conditions. However, NK1R activation led to translocation of NETNK1R complexes to raft-rich membrane fractions. Importantly, PKC was found in association with raft-localized NET following SP treatment. Similar to WT-NET, PKC-resistant NET-DM was found in association with NK1R exclusively in non-raft fractions. However, SP treatment failed to translocate NET-DMNK1R complexes from non-raft fractions to raft fractions. Collectively, these results suggest that NK1R forms physical complexes with NET and that the receptor-mediated Thr(258) + Ser(259) motif-dependent translocation of NETNK1R complexes into raft-rich microdomains facilitates NET/NK1R interaction with PKC to coordinate spatially restricted NET regulation.