Supplementary Material for: Functional characterization of gonadotropin-releasing hormone and corazonin signaling systems in Pacific abalone: toward reclassification of invertebrate neuropeptides

Introduction: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. Methods: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. Results: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. Conclusion: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

引言：依托受体脱孤儿化（receptor deorphanization）技术，学界对两侧对称动物促性腺激素释放激素（gonadotropin-releasing hormone, GnRH）相关神经肽的演化起源与对应命名体系的认知已产生巨大变革。然而，冠轮动物门（Lophotrochozoa）内GnRH与心激肽（corazonin, CRZ）信号系统的重新分类仍不明确。
方法：本研究针对软体动物盘鲍（Haliotis discus hannai, 简称Hdh）中的GnRH与CRZ受体开展表征研究，采用的实验手段包括系统发育分析、基因表达分析、生物发光报告基因实验、蛋白质印迹（western blotting）、肽段氨基酸取代实验、体内神经肽注射实验及RNA干扰（RNA interference, RNAi）检测。
结果：本研究共鉴定出2种Hdh CRZ样受体（Hdh-CRZR-A与Hdh-CRZR-B）及3种Hdh GnRH样受体（Hdh-GnRHR1-A、Hdh-GnRHR1-B与Hdh-GnRHR2）。系统发育分析结果显示，Hdh-CRZR-A与Hdh-CRZR-B归属于CRZ型受体家族，而Hdh-GnRHR1-A/-B及Hdh-GnRHR2则聚类于GnRH/脂动激素（adipokinetic hormone, AKH）/CRZ相关肽型受体家族。Hdh-CRZR-A/-B与Hdh-GnRHR1-A分别可被Hdh CRZ（pQNYHFSNGWHA-NH2）与Hdh GnRH（pQISFSPNWGT-NH2）激活。Hdh-CRZR-A/-B可同时偶联Gαq与Gαs信号通路，而Hdh-GnRHR1-A仅能与Gαq信号通路耦合。针对取代肽段[I2S3]Hdh CRZ与[N2Y3H4]Hdh GnRH的分析及虚拟对接（in silico docking）模型结果显示，肽段的N端氨基酸是决定Hdh-CRZR与Hdh-GnRHR配体选择性的关键因素。Hdh CRZ与Hdh GnRH肽段的2种前体转录本及其受体主要在神经节中表达，且在饥饿盘鲍体内的表达水平显著上调。向盘鲍体内注射Hdh CRZ肽段可降低其摄食量，而敲低Hdh-CRZR的表达则可提升盘鲍的摄食量。此外，Hdh CRZ可诱导成熟卵母细胞发生生发泡破裂（germinal vesicle breakdown）。
结论：对Hdh CRZR、Hdh GnRHR及其同源配体肽段的表征研究，为两侧对称动物中GnRH相关信号系统的演化路径提供了全新的研究视角。