Işbilir Ali, Duan Sahbaz Burcin, Tuncgenc Gunes, Bünemann Moritz, Lohse Martin J, Birgül-Iyison Necla
Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Bogazici University, Istanbul 34342, Turkey.
Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin 13125, Germany.
ACS Omega. 2020 Dec 8;5(50):32183-32194. doi: 10.1021/acsomega.0c03382. eCollection 2020 Dec 22.
G protein-coupled receptors (GPCRs) play a pivotal role in regulating key physiological events in all animal species. Recent advances in collective analysis of genes and proteins revealed numerous potential neuropeptides and GPCRs from insect species, allowing for the characterization of peptide-receptor pairs. In this work, we used fluorescence resonance energy transfer (FRET)-based genetically encoded biosensors in intact mammalian cells to study the pharmacological features of the cognate GPCR of the type-C allatostatin (AST-C) peptide from the stick insect, . Analysis of multiple downstream pathways revealed that AST-C can activate the human Gi protein, and not Gs or Gq, through AST-C receptor (AlstRC). Activated AlstRC recruits β-arrestin2 independent of the Gi protein but stimulates ERK phosphorylation in a Gi protein-dependent manner. Identification of Gαi-, arrestin-, and GRK-like transcripts from revealed high evolutionary conservation at the G protein level, while β-arrestins and GRKs displayed less conservation. In conclusion, our study provides experimental and homology-based evidence on the functionality of vertebrate G proteins and downstream signaling biosensors to characterize early signaling steps of an insect GPCR. These results may serve as a scaffold for developing assays to characterize pharmacological and structural aspects of other insect GPCRs and can be used in deorphanization and pesticide studies.
G蛋白偶联受体（GPCRs）在调节所有动物物种的关键生理事件中起着关键作用。基因和蛋白质的集体分析的最新进展揭示了来自昆虫物种的众多潜在神经肽和GPCRs，从而能够对肽-受体对进行表征。在这项工作中，我们在完整的哺乳动物细胞中使用基于荧光共振能量转移（FRET）的基因编码生物传感器来研究来自竹节虫的C型咽侧体抑制素（AST-C）肽的同源GPCR的药理学特征。对多个下游途径的分析表明，AST-C可以通过AST-C受体（AlstRC）激活人类Gi蛋白，而不是Gs或Gq。激活的AlstRC独立于Gi蛋白招募β-抑制蛋白2，但以Gi蛋白依赖的方式刺激ERK磷酸化。从[具体物种]中鉴定出Gαi、抑制蛋白和GRK样转录本，表明在G蛋白水平上具有高度的进化保守性，而β-抑制蛋白和GRK的保守性较低。总之，我们的研究提供了关于脊椎动物G蛋白和下游信号生物传感器功能的实验和基于同源性的证据，以表征昆虫GPCR的早期信号步骤。这些结果可作为开发测定方法的支架，以表征其他昆虫GPCR的药理学和结构方面，并可用于孤儿受体鉴定和农药研究。
