Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor
Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor
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DOI：
10.1074/jbc.m114.612614
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发表时间：
2014-12-05
影响因子：
4.8
通讯作者：
Mouledous, Lionel
中科院分区：
文献类型：
--
作者：
Bray, Lauriane;Froment, Carine;Mouledous, Lionel
背景：G蛋白偶联受体的功能受磷酸化调节。结果如下：质谱被用来映射的NPFF 2神经肽FF受体的磷酸化位点，和定点诱变允许识别其在受体调节中的作用。结论：脱敏和内化所涉及的位点并不完全重叠。重要性：神经肽FF 2（NPFF 2）受体属于G蛋白偶联受体的视紫红质家族，并介导几种相关的RFamide神经肽的作用。该系统的主要药理学兴趣之一在于其调节内源性阿片系统的能力，使其成为减少慢性阿片类药物使用的负面影响的潜在靶点。细胞内残基的磷酸化是研究最广泛的调节G蛋白偶联受体活性的翻译后修饰。然而，到目前为止，还没有有关NPFF 2受体磷酸化的信息。在这项研究中，我们结合质谱分析和定点突变分析的第一次NPFF 2受体的磷酸化模式和受体信号，脱敏和贩运在SH-SY 5 Y模型细胞系中的各种磷酸化位点的作用。我们确定了可能是GRK依赖性的主要磷酸化簇，其负责急性脱敏，在受体C末端的（TNST 415）-T-412，以及涉及脱敏（（TS 373）-T-372）和内化（Ser（395））的其他位点。因此，我们证明了磷酸化在调节NPFF 2受体活性和贩运中发挥的关键作用。我们的数据还提供了额外的证据支持脱敏和内化是依赖于不同磷酸化模式的部分独立过程的概念。
Background: G protein-coupled receptor functions are regulated by phosphorylation. Results: Mass spectrometry was used to map the phosphorylation sites of the NPFF2 neuropeptide FF receptor, and site-directed mutagenesis permitted the identification of their role in receptor regulation. Conclusion: Sites involved in desensitization and internalization do not fully overlap. Significance: This is the first mapping of NPFF2 receptor phosphorylation.The neuropeptide FF2 (NPFF2) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF2 receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF2 receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, (TNST415)-T-412 at the end of the C terminus of the receptor, and additional sites involved in desensitization ((TS373)-T-372) and internalization (Ser(395)). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF2 receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.