Department of Biology, University of Toronto Mississauga, Mississauga, Ontario, Canada ; Center for Functional and Comparative Insect Genomics, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2013 Nov 29;8(11):e82466. doi: 10.1371/journal.pone.0082466. eCollection 2013.
Several families of diuretic hormones exist in insects, one of which is the calcitonin-like diuretic hormone (CT/DH) family. CT/DH mediates its effects by binding to family B G-protein coupled receptors (GPCRs). Here we isolate and functionally characterize two R. prolixusCT/DH receptor paralogs (Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2) using a novel heterologous assay utilizing a modified human embryonic kidney 293 (HEK293) cell line. Rhopr-CT/DH-R1 is orthologous to the previously characterized D. melanogasterCT/DH receptor (CG17415) while Rhopr-CT/DH-R2 is orthologous to the D. melanogaster receptor (CG4395), an orphan receptor whose ligand was unknown until now. We determine the cDNA sequences of three splice variants encoding Rhopr-CT/DH-R1 (Rhopr-CT/DH-R1-A, Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C) and two splice variants encoding Rhopr-CT/DH-R2 (Rhopr-CT/DH-R2-A and Rhopr-CT/DH-R2-B). Rhopr-CT/DH-R1-A and Rhopr-CT/DH-R2-A encode truncated receptors that lack six and seven of the characteristic seven transmembrane domains, respectively. Rhopr-CT/DH-R1-B and Rhopr-CT/DH-R1-C, which only differ by 2 amino acids in their C-terminal domain, can both be activated by Rhopr-CT/DH at equal sensitivities (EC50 = 200-300 nM). Interestingly, Rhopr-CT/DH-R2-B is much more sensitive to Rhopr-CT/DH (EC50 = 15 nM) compared to Rhopr-CT/DH-R1-B/C and also yields a much greater response (amplitude) in our heterologous assay. This is the first study to reveal that insects possess at least two CT/DH receptors, which may be functionally different. Quantitative PCR demonstrates that Rhopr-CT/DH-R1 and Rhopr-CT/DH-R2 have distinct expression patterns, with both receptors expressed centrally and peripherally. Moreover, the expression analysis also identified novel target tissues for this neuropeptide, including testes, ovaries and prothoracic glands, suggesting a possible role for Rhopr-CT/DH in reproductive physiology and development.
存在几种利尿激素家族的昆虫，其中之一是降钙素样利尿激素（CT/DH）家族。CT/DH 通过与家族 B G 蛋白偶联受体（GPCR）结合来发挥其作用。在这里，我们使用一种新型的异源测定法，利用修饰的人胚肾 293（HEK293）细胞系，分离并功能表征了两种 R. prolixusCT/DH 受体同源物（Rhopr-CT/DH-R1 和 Rhopr-CT/DH-R2）。Rhopr-CT/DH-R1 与先前表征的 D. melanogasterCT/DH 受体（CG17415）同源，而 Rhopr-CT/DH-R2 与 D. melanogaster 受体（CG4395）同源，后者是一种孤儿受体，直到现在才知道其配体。我们确定了编码 Rhopr-CT/DH-R1 的三种剪接变体（Rhopr-CT/DH-R1-A、Rhopr-CT/DH-R1-B 和 Rhopr-CT/DH-R1-C）和编码 Rhopr-CT/DH-R2 的两种剪接变体（Rhopr-CT/DH-R2-A 和 Rhopr-CT/DH-R2-B）的 cDNA 序列。Rhopr-CT/DH-R1-A 和 Rhopr-CT/DH-R2-A 编码缺少六个和七个特征性七跨膜结构域的截断受体。Rhopr-CT/DH-R1-B 和 Rhopr-CT/DH-R1-C 仅在其 C 末端结构域中相差 2 个氨基酸，都可以被 Rhopr-CT/DH 以相同的敏感性激活（EC50 = 200-300 nM）。有趣的是，与 Rhopr-CT/DH-R1-B/C 相比，Rhopr-CT/DH-R2-B 对 Rhopr-CT/DH 更为敏感（EC50 = 15 nM），并且在我们的异源测定中也产生了更大的反应（幅度）。这是第一项表明昆虫至少具有两种 CT/DH 受体的研究，这两种受体可能具有不同的功能。定量 PCR 表明，Rhopr-CT/DH-R1 和 Rhopr-CT/DH-R2 具有不同的表达模式，两种受体在中枢和外周均有表达。此外，表达分析还鉴定了这种神经肽的新靶组织，包括睾丸、卵巢和前胸腺，这表明 Rhopr-CT/DH 在生殖生理学和发育中可能具有重要作用。
