Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay
Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay
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使用基于 NanoBiT 的结合测定探索松弛素家族肽受体 3 和 4 与配体的静电相互作用
DOI：
10.1016/j.bbamem.2019.01.010
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发表时间：
2019
影响因子：
3.4
通讯作者：
Guo Zhan-Yun
中科院分区：
文献类型：
--
作者：
Wang Jia-Hui;Nie Wei-Han;Shao Xiao-Xia;Li Hao-Zheng;Hu Meng-Jun;Liu Ya-Li;Xu Zeng-Guang;Guo Zhan-Yun
松弛素家族肽通过激活四种G蛋白偶联受体（即松弛素家族肽受体1-4（RXFP 1 -4））来执行多种生物学功能。我们最近公开了同源RXFP 3和RXFP 4与基于激活互补的一些激动剂的静电相互作用。然而，这种基于活化测定的方法不能应用于不活化受体的拮抗剂。在此，我们提出了一种通用的方法，适用于激动剂和拮抗剂的基础上，我们新开发的NanoBiT为基础的结合试验。我们首先使用激动剂松弛素-3验证基于结合测定的方法，然后将其应用于嵌合拮抗剂R3（ΔB23-27）R/I5。激动剂和拮抗剂的3个带正电荷的B链Arg残基分别被带负电荷的Glu残基取代;同时，两种受体的必需WxxExxxD基序中带负电荷的Glu和Asp残基分别被带正电荷的Arg残基取代。基于突变配体对突变受体的结合互补作用，我们推断激动剂和拮抗剂与RXFP 3和RXFP 4可能的静电相互作用：它们的B链C端Arg残基与两种受体的WxxExxxD基序中深埋的Glu残基相互作用，并且它们的B链中心Arg残基中的一个或两个与两种受体的WxxExxxD基序中浅埋的Asp残基相互作用。本工作为RXFP 3和RXFP 4与激动剂和拮抗剂的相互作用机制提供了新的思路，也为研究某些质膜受体与其配体的相互作用提供了新的途径。
Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.