G protein-coupled receptors (GPCRs) are membrane-bound receptors that are considered prime candidates for the development of novel insect pest management strategies. However, the molecular signaling properties of insect GPCRs remain poorly understood. In fact, most studies on insect GPCR signaling are limited to analysis of fluctuations in the secondary messenger molecules calcium (Ca2+) and/or cyclic adenosine monophosphate (cAMP). In the current study, we characterized a corticotropin-releasing factor-related diuretic hormone (CRF-DH) receptor of the desert locust, Schistocerca gregaria. This Schgr-CRF-DHR is mainly expressed in the nervous system and in brain-associated endocrine organs. The neuropeptide Schgr-CRF-DH induced Ca2+-dependent aequorin-based bioluminescent responses in CHO cells co-expressing this receptor with the promiscuous Gα16 protein. Furthermore, when co-expressed with the cAMP-dependent bioluminescence resonance energy transfer (BRET)-based CAMYEL biosensor in HEK293T cells, this receptor elicited dose-dependent agonist-induced responses with an EC50 in the nanomolar range (4.02 nM). In addition, we tested if vertebrate BRET-based G protein biosensors, can also be used to detect direct Gα protein subunit activation by an insect GPCR. Therefore, we analyzed ten different human BRET-based G protein biosensors, representing members of all four Gα protein subfamilies; Gαs, Gαi/o, Gαq/11 and Gα12/13. Our data demonstrate that stimulation of Schgr-CRF-DHR by Schgr-CRF-DH can dose-dependently activate Gαi/o and Gαs biosensors, while no significant effects were observed with the Gαq/11 and Gα12/13 biosensors. Our study paves the way for future biosensor-based studies to analyze the signaling properties of insect GPCRs in both fundamental science and applied research contexts.

中文翻译：

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可以使用基于BRET的生物传感器来表征昆虫GPCR的G蛋白介导的信号通路，即Gistaria Cregaria CRF相关的利尿激素受体吗？

G蛋白偶联受体（GPCR）是膜结合受体，被认为是开发新型害虫管理策略的主要候选对象。但是，昆虫GPCR的分子信号传导特性仍然知之甚少。实际上，大多数有关昆虫GPCR信号传导的研究仅限于分析次级信使分子钙（Ca2 +）和/或环状单磷酸腺苷（cAMP）的波动。在当前的研究中，我们表征了沙漠蝗Schistocerca gregaria的促肾上腺皮质激素释放因子相关的利尿激素（CRF-DH）受体。这种Schgr-CRF-DHR主要在神经系统和与大脑相关的内分泌器官中表达。神经肽Schgr-CRF-DH在共表达该受体与混杂Gα16蛋白的CHO细胞中诱导Ca2 +依赖性水母发光蛋白基生物发光反应。此外，当与基于cAMP的生物发光共振能量转移（BRET）的CAMYEL生物传感器在HEK293T细胞中共表达时，该受体以纳摩尔浓度（4.02 nM）的EC50引发剂量依赖性激动剂诱导的应答。此外，我们测试了基于脊椎动物BRET的G蛋白生物传感器是否也可以用于通过昆虫GPCR检测直接Gα蛋白亚基的激活。因此，我们分析了十种不同的基于人BRET的G蛋白生物传感器，它们代表了所有四个Gα蛋白亚家族的成员。Gαs，Gαi/ o，Gαq/ 11和Gα12/ 13。我们的数据表明，Schgr-CRF-DH对Schgr-CRF-DHR的刺激可以剂量依赖性地激活Gαi/ o和Gαs生物传感器，而Gαq/ 11和Gα12/ 13生物传感器未观察到明显的影响。我们的研究为将来基于生物传感器的研究在基础科学和应用研究背景下分析昆虫GPCR的信号传导铺平了道路。