FMRFamide G protein-coupled receptors (GPCR) in the cuttlefish Sepiella japonica: Identification, characterization and expression profile

Abstract

FMRFamide is a ubiquitous neuromodulator in the animal kingdom. Once FMRFamide or similar neuropeptides bind to their G protein-coupled receptors (GPCR), a series of signal transduction events are triggered, thereby mediating various physiological effects. FMRFamide had been reported to be involved in the regulation of sexual maturation in Sepiella japonica. In this research, the full-length cDNA of FMRFamide G protein-coupled receptor of S. japonica (SjFaGPCR) was cloned. The sequence is 1396 bp long and encodes a protein consisting of 418 amino acid residues, lacking a signal peptide at the N-terminal region. The 3D structure of SjFaGPCR was predicted using Todarodes pacificus rhodopsin as a template, and the result indicated the presence of seven transmembrane regions. Multiple sequence alignments and phylogenetic trees indicated that SjFaGPCR is conserved among invertebrates, and shares highly similar sequence characteristics with other cephalopods. In situ hybridization (ISH) results revealed that significant signals of SjFaGPCR were detected in the central medulla and the granular layer cells of the optic lobe, and were also observed in the supraesophageal and subesophageal masses of the brain. Meanwhile, quantitative real-time PCR (qRT-PCR) results showed that a higher expression level of SjFaGPCR mRNA was detected in the brain and optic lobe of female cuttlefish at stage III and stage VI, and also in the brain (stage V) and optic lobe (stages IV and V) of male cuttlefish than that in other tissues. The co-localization results demonstrated that fluorescence signals of SjFMRFamide and SjFaGPCR were overlapped in HEK293 cells, suggesting a possible interaction between the SjFMRFamide and SjFaGPCR. These findings provide molecular support for further exploring the roles of FMRFamide and FaGPCR in the reproductive regulation of S. japonica.