School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
State Key Laboratory for Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, 999078, China.
Pharmacol Res. 2020 Nov;161:105117. doi: 10.1016/j.phrs.2020.105117. Epub 2020 Aug 5.
Formyl peptide receptor 2 (FPR2) is a Class A G protein-coupled receptor (GPCR) that interacts with multiple ligands and transduces both proinflammatory and anti-inflammatory signals. These ligands include weak agonists and modulators that are produced during inflammation. The present study investigates how prolonged exposure to FPR2 modulators influence receptor signaling.
Fluorescent biosensors of FPR2 were constructed based on single-molecule fluorescent resonance energy transfer (FRET) and used for measurement of ligand-induced receptor conformational changes. These changes were combined with FPR2-mediated signaling events and used as parameters for the conformational states of FPR2. Ternary complex models were developed to interpret ligand concentration-dependent changes in FPR2 conformational states.
Incubation with Ac, an anti-inflammatory ligand of FPR2, decreased FRET intensity at picomolar concentrations. In comparison, WKYMVm (W-pep) and Aβ, both proinflammatory agonists of FPR2, increased FRET intensity. Preincubation with Ac at 10 pM diminished W-pep-induced Ca flux but potentiated W-pep-stimulated β-arrestin2 membrane translocation and p38 MAPK phosphorylation. The opposite effects were observed with 10 pM of Aβ. Neither Ac nor Aβ competed for W-pep binding at the picomolar concentrations.
The results support the presence of two allosteric binding sites on FPR2, each for Ac and Aβ, with high and low affinities. Sequential binding of the two allosteric ligands at increasing concentrations induce different conformational changes in FPR2, providing a novel mechanism by which biased allosteric modulators alter receptor conformations and generate pro- and anti-inflammatory signals.
甲酰肽受体 2（FPR2）是一种 A 类 G 蛋白偶联受体（GPCR），可与多种配体相互作用，并转导促炎和抗炎信号。这些配体包括在炎症过程中产生的弱激动剂和调节剂。本研究旨在探讨 FPR2 调节剂的长期暴露如何影响受体信号。
构建了基于单分子荧光共振能量转移（FRET）的 FPR2 荧光生物传感器，用于测量配体诱导的受体构象变化。这些变化与 FPR2 介导的信号事件相结合，用作 FPR2 构象状态的参数。开发了三元复合物模型来解释 FPR2 构象状态随配体浓度依赖性的变化。
在皮摩尔浓度下孵育 FPR2 的抗炎配体 Ac 会降低 FRET 强度。相比之下，FPR2 的促炎激动剂 WKYMVm（W-肽）和 Aβ均增加了 FRET 强度。用 10 pM 的 Ac 预孵育会减弱 W-肽诱导的 Ca 流，但会增强 W-肽刺激的β-arrestin2 膜转位和 p38 MAPK 磷酸化。用 10 pM 的 Aβ观察到相反的效果。在皮摩尔浓度下，Ac 和 Aβ均不与 W-肽竞争结合。
研究结果支持 FPR2 上存在两个变构结合位点，每个位点与 Ac 和 Aβ结合，亲和力高低不同。两种变构配体在增加浓度时的顺序结合会导致 FPR2 发生不同的构象变化，为偏态变构调节剂改变受体构象并产生促炎和抗炎信号提供了一种新的机制。
