CCHamides are newly identified insect neuropeptides, which are widely occurring in most insects. However, our knowledge about their signaling characteristics and physiological roles is still limited. Here, we cloned two full-length cDNAs encoding putative CCHamide receptors, Bombyx neuropeptide GPCR A14 (BNGR-A14) and -A15 (BNGR-A15), from the brain of B. mori larvae. Characterization of signaling indicated that Bombyx CCHamide-1 and CCHamide-2 are specific endogenous ligands for BNGR-A15 and BNGR-A14, respectively. Further functional assays combined with specific inhibitors demonstrated that upon activation by CCHamide-2, BNGR-A14 elicited significant increases in CRE-driven luciferase activity, intracellular Ca²⁺ mobilization and ERK1/2 phosphorylation in a G_q inhibitor-sensitive manner, while BNGR-A15 was activated by CCHamide-1, thus leading to intracellular accumulation of cAMP, Ca²⁺ mobilization, and ERK1/2 phosphorylation in a G_s and G_q inhibitor-sensitive manner. Based on these findings, we designated the receptors BNGR-A15 and -A14 as Bommo-CCHaR-1 and -2, respectively. In addition, our results showed that CCHamides are considered to require intrachain disulfide bonds to activate their respective receptor in the physiological concentration range. Moreover, quantitative RT-PCR analysis revealed that CCHamide-1 is more likely to serve as a brain-gut peptide to regulate feeding behavior and growth through BNGR-A15, whereas the CCHamide-2 signaling system might play an important role in the control of multiple physiological processes. Our findings provide in-depth information on CCHamide-1 and -2-mediated signaling, facilitating further elucidation of their endocrinological roles in the regulation of fundamental physiological processes.

CCHamides 是新发现的昆虫神经肽，广泛存在于大多数昆虫中。然而，我们对它们的信号特征和生理作用的了解仍然有限。在这里，我们从家蚕幼虫的大脑中克隆了两个编码假定 CCHamide 受体的全长 cDNA，即家蚕神经肽 GPCR A14 (BNGR-A14) 和 -A15 (BNGR-A15) 。信号表征表明Bombyx CCHamide-1 和 CCHamide-2 分别是 BNGR-A15 和 BNGR-A14 的特定内源性配体。结合特定抑制剂的进一步功能分析表明，在被 CCHamide-2 激活后，BNGR-A14 引起 CRE 驱动的荧光素酶活性、细胞内 Ca ²⁺显着增加以 G _q抑制剂敏感的方式动员和 ERK1/2 磷酸化，而 BNGR-A15 被 CCHamide-1 激活，从而导致 cAMP、Ca ²⁺动员和 ERK1/2 磷酸化在 G _s和 G 的细胞内积累_q抑制剂敏感方式。基于这些发现，我们将受体 BNGR-A15 和 -A14 命名为Bommo-CCHaR-1 和 -2，分别。此外，我们的结果表明，CCHamides 被认为需要链内二硫键才能在生理浓度范围内激活它们各自的受体。此外，定量 RT-PCR 分析表明，CCHamide-1 更有可能作为脑肠肽通过 BNGR-A15 调节摄食行为和生长，而 CCHamide-2 信号系统可能在控制进食行为和生长方面发挥重要作用。多种生理过程。我们的研究结果提供了有关 CCHamide-1 和 -2 介导的信号传导的深入信息，有助于进一步阐明它们在调节基本生理过程中的内分泌作用。