Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of Biosciences and Applications, National Centre for Scientific Research "Demokritos", Athens, Greece.
Research Programme on Biomedical Informatics (GRIB) - Department of Experimental and Health Sciences, Hospital del Mar Medical Research Institute, Pompeu Fabra University, Barcelona, Spain.
FEBS J. 2020 Feb;287(4):736-748. doi: 10.1111/febs.15033. Epub 2019 Sep 9.
The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ-OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ-ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ-ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ-opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4-binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein.
δ-阿片受体（δ-OR）与 Gi/Go 蛋白偶联，调节神经系统中的各种反应。先前的研究表明，G 蛋白信号调节因子 4（RGS4）通过其 N 端结构域直接与 δ-OR 的 C 端区域相互作用，从而负调控阿片受体信号。在此，我们使用分子动力学模拟和体外下拉实验，将这种相互作用限定在 δ-OR 的 12 个 8 螺旋残基和 RGS4 的前 17 个 N 端残基（NT）。通过分子动力学模拟结合突变研究监测 RGS4 和 δ-OR 之间的复合物排列和稳定，我们确定形成了两个关键相互作用：一个是 δ-OR 第 8 螺旋的 Phe329 与 RGS4 的 NT 第 9 位 Pro 之间的相互作用，另一个是 δ-OR 的 Glu323 与 RGS4 的 Lys17 之间的盐桥。我们的观察结果首次为包括 δ-阿片受体、G 蛋白和 RGS 蛋白在内的三元复合物绘制了结构模型。此外，与 RGS4 结合区域的阿片受体之间具有高度的保守性，表明阿片受体与这种重要的调节蛋白之间存在保守的相互作用模式。
