Concomitant action of structural elements and receptor phosphorylation determines arrestin-3 interaction with the free fatty acid receptor FFA4.
Concomitant action of structural elements and receptor phosphorylation determines arrestin-3 interaction with the free fatty acid receptor FFA4.
复制标题
DOI：
10.1074/jbc.m114.568816
复制
发表时间：
2014-06-27
期刊：
影响因子：
--
通讯作者：
Tobin AB
中科院分区：
文献类型：
--
作者：
Butcher AJ;Hudson BD;Shimpukade B;Alvarez-Curto E;Prihandoko R;Ulven T;Milligan G;Tobin AB
背景：FFA 4是长链脂肪酸的受体，是糖尿病和炎症性疾病的可能靶点。结果：磷酸化位点和与arrestin-3相互作用的位点被定位在C-末端尾内。结论：arrestin-3与磷酸化和结构元件的相互作用是必需的。重要性：深入了解arrestin-3与G蛋白信号传导和偏向配体发展的影响可能会推动改进治疗方法的鉴定。除了作为营养物质，游离脂肪酸还通过激活G蛋白偶联受体家族作为信号分子。其中包括FFA 4，以前称为GPR 120，它对中链和长链脂肪酸有反应，包括促进健康的ω-3脂肪酸，这些脂肪酸与代谢和炎症反应的调节有关。在这里，我们显示，使用质谱，诱变，和磷酸特异性抗体，激动剂调节磷酸化的人FFA 4受体主要发生在5个残基（Thr 347，Thr 349，Ser 350，Ser 357，Ser 360）的C-末端尾部。这些残基的突变降低了配体介导的arrestin-3募集的功效和效力，并影响募集动力学。所有这五个残基的组合诱变不足以完全消除与arrestin-3的相互作用，但带负电荷的残基的进一步诱变揭示了在受体的C-末端尾内与arrestin-3相互作用的另外的结构组分。这些元件由位于磷酸化位点附近的酸性残基Glu 341、Asp 348和Asp 355组成。因此，受体磷酸化与FFA 4的C末端尾内的结构元件协同作用，以允许募集抑制蛋白-3。重要的是，这些抑制蛋白-3募集的机制独立于Gq/11偶联起作用，从而提供了开发利用这些差异偶联机制的显示刺激偏倚的配体的可能性。此外，这提供了一种设计偏向受体的策略，以探测生理相关的信号。
Background: FFA4 is a receptor for long chain fatty acids and possible target for diabetes and inflammatory diseases. Results: Sites of phosphorylation and interaction with arrestin-3 were mapped within the C-terminal tail. Conclusion: Both phosphorylation and structural elements are required for interaction with arrestin-3. Significance: Insight gained into arrestin-3 versus G protein signaling and implications for biased ligand development may drive identification of improved therapeutics. In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr347, Thr349, Ser350, Ser357, and Ser360) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu341, Asp348, and Asp355 located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling.