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.