Department of Plant Medicals, Andong National University, Andong, Korea.
Faculty of Agriculture, Department of Plant Protection, KahramanMaras Situ Imam University, KahramanMaras, Turkey.
Arch Insect Biochem Physiol. 2024 Nov;117(3):e70005. doi: 10.1002/arch.70005.
Prostaglandin E (PGE) is an eicosanoid that mediates a range of physiological actions in vertebrates and invertebrates, including reproduction and immunity. The PGE receptor was identified and functionally assessed in two lepidopteran insects, Manduca sexta and Spodoptera exigua. However, its binding affinity to the receptor has not been reported. The PGE receptor is a G-protein coupled receptor (GPCR) although its corresponding G-protein is not identified. PGE binding assays were performed with membrane preparations from hemocytes of M. sexta larvae. We recorded an optimal binding in 4 h reactions conducted at pH 7.5 with 12 nM tritium-labeled PGE. We found that hemocytes express a single population of PGE binding sites with a high affinity (Kd = 35 pmol/mg protein), which are specific and saturable. The outcomes of experiments on the influence of purine nucleotides suggested these are functional GPCRs. A bioinformatics analysis led to a proposed trimeric G-protein in the S. exigua transcriptome, in which the Gα subunit is classified into five different types: Gα(o), Gα(q), Gα(s), Gα(12), and Gα(f). After confirming expressions of these five types in S. exigua, individual RNA interference (RNAi) treatments were applied to the larvae using gene-specific double-stranded RNAs. RNAi treatments specific to Gα(s) or Gα(12) gene expression significantly suppressed the cellular immune responses although the RNAi treatments specific to other three Gα components did not. While PGE treatments led to elevated hemocyte cAMP or Ca levels, the RNAi treatments specific to Gα(s) or Gα(12) genes led to significantly reduced second messenger levels under PGE although the RNAi treatments specific to the other three Gα components did not. These results showed that the PGE receptor has high PGE affinity in the nanomolar range and binds G-proteins containing a Gα(s) or Gα(12) trimeric component in S. exigua and M. sexta, and likely, all lepidopteran insects.
前列腺素 E (PGE) 是一种类二十烷酸，在脊椎动物和无脊椎动物中介导多种生理作用，包括繁殖和免疫。PGE 受体在两种鳞翅目昆虫，烟草天蛾和斜纹夜蛾中被鉴定并进行了功能评估。然而，其与受体的结合亲和力尚未报道。PGE 受体是一种 G 蛋白偶联受体（GPCR），尽管其对应的 G 蛋白尚未确定。PGE 结合测定是用来自烟草天蛾幼虫血细胞的膜制剂进行的。我们记录到在 pH 7.5 下进行的 4 小时反应中，最佳结合在 12 nM 氚标记的 PGE 时发生。我们发现血细胞表达具有高亲和力（Kd = 35 pmol/mg 蛋白）的单一 PGE 结合位点群体，该群体是特异性和饱和的。嘌呤核苷酸影响实验的结果表明这些是功能性 GPCR。生物信息学分析导致在 S. exigua 转录组中提出了一种三聚体 G 蛋白，其中 Gα 亚基分为五种不同类型：Gα(o)、Gα(q)、Gα(s)、Gα(12)和 Gα(f)。在确认 S. exigua 中表达了这五种类型后，使用基因特异性双链 RNA 对幼虫进行了单独的 RNA 干扰 (RNAi) 处理。Gα(s)或 Gα(12)基因表达的 RNAi 处理显著抑制了细胞免疫反应，尽管针对其他三种 Gα 成分的 RNAi 处理没有。虽然 PGE 处理导致血细胞 cAMP 或 Ca 水平升高，但针对 Gα(s)或 Gα(12)基因的 RNAi 处理导致 PGE 下第二信使水平显著降低，尽管针对其他三种 Gα 成分的 RNAi 处理没有。这些结果表明，PGE 受体在纳摩尔范围内具有高 PGE 亲和力，并与包含 Gα(s)或 Gα(12)三聚体成分的 G 蛋白结合在 S. exigua 和 M. sexta 中，并且可能在所有鳞翅目昆虫中都是如此。
