Bray Lauriane, Froment Carine, Pardo Pierre, Candotto Cédric, Burlet-Schiltz Odile, Zajac Jean-Marie, Mollereau Catherine, Moulédous Lionel
From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France.
From the Institut de Pharmacologie et Biologie Structurale, UMR5089 CNRS, Université de Toulouse, 31077 Toulouse, France
J Biol Chem. 2014 Dec 5;289(49):33754-66. doi: 10.1074/jbc.M114.612614. Epub 2014 Oct 17.
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, (412)TNST(415) at the end of the C terminus of the receptor, and additional sites involved in desensitization ((372)TS(373)) 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.
神经肽FF2（NPFF2）受体属于G蛋白偶联受体的视紫红质家族，介导几种相关RFamide神经肽的作用。该系统的一个主要药理学研究兴趣在于其调节内源性阿片系统的能力，使其成为减轻慢性阿片类药物使用负面影响的潜在靶点。细胞内残基的磷酸化是调节G蛋白偶联受体活性的研究最为广泛的翻译后修饰。然而，迄今为止，尚无关于NPFF2受体磷酸化的信息。在本研究中，我们结合质谱分析和定点诱变，首次分析了NPFF2受体的磷酸化模式以及各种磷酸化位点在SH-SY5Y模型细胞系中受体信号传导、脱敏和转运中的作用。我们确定了负责急性脱敏的主要的、可能依赖于G蛋白偶联受体激酶（GRK）的磷酸化簇，即受体C末端的（412）TNST（415），以及参与脱敏（（372）TS（373））和内化（Ser（395））的其他位点。因此，我们证明了磷酸化在调节NPFF2受体活性和转运中所起的关键作用。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这一概念。
