Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Biochim Biophys Acta Biomembr. 2019 Apr 1;1861(4):776-786. doi: 10.1016/j.bbamem.2019.01.010. Epub 2019 Jan 24.
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.
松弛素家族肽通过激活四个 G 蛋白偶联受体，即松弛素家族肽受体 1-4（RXFP1-4），发挥多种生物学功能。我们最近根据激活互补性揭示了同源 RXFP3 和 RXFP4 与一些激动剂的静电相互作用。然而，这种基于激活测定的方法不能应用于不激活受体的拮抗剂。在此，我们提出了一种基于我们新开发的 NanoBiT 结合测定的适用于激动剂和拮抗剂的通用方法。我们首先使用激动剂松弛素-3 验证了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 B 链三个正电荷 Arg 残基分别被负电荷 Glu 残基取代；同时，两个受体必需的 WxxExxxD 基序中的负电荷 Glu 和 Asp 残基分别被正电荷 Arg 残基取代。基于突变配体对突变受体的结合互补性，我们推断出激动剂和拮抗剂与 RXFP3 和 RXFP4 的可能静电相互作用：它们的 B 链 C 末端 Arg 残基与两个受体的 WxxExxxD 基序中深埋的 Glu 残基相互作用，其 B 链中心的一个或两个 Arg 残基与两个受体的 WxxExxxD 基序中浅埋的 Asp 残基相互作用。我们的工作为 RXFP3 和 RXFP4 与激动剂和拮抗剂的相互作用机制提供了新的见解，也为一些质膜受体与其配体的相互作用研究提供了一种新方法。
