Identification, molecular structure and expression of two cloned serotonin receptors from the pond snail, Helisoma trivolvis

Abstract

SUMMARY Helisoma trivolvis has served as a model system to study the functions of serotonin (5-HT) from cellular, developmental, physiological and behavioural perspectives. To further explore the serotonin system at the molecular level, and to provide experimental knockout tools for future studies, in this study we identified serotonin receptor genes from the H. trivolvis genome, and characterized the molecular structure and expression profile of the serotonin receptor gene products. Degenerate oligonucleotide primers, based on conserved regions of the Lymnaea stagnalis 5-HT1Lym receptor, were used to amplify G protein-coupled biogenic amine receptor sequences from H. trivolvisgenomic cDNA, resulting in the cloning of two putative serotonin receptors. The deduced gene products both appear to be G protein-coupled serotonin receptors, with well-conserved structure in the functional domains and high variability in the vestibule entrance of the receptor protein. Phylogenetic analysis placed these receptors in the 5-HT1 and 5-HT7families of serotonin receptors. They are thus named the 5-HT1Heland 5-HT7Hel receptors, respectively. In situhybridization and immunofluorescence studies revealed that these genes and gene products are expressed most heavily in the ciliated pedal and mantle epithelia of H. trivolvis embryos. In adults, widespread expression occurred in all ganglia and connectives of the central nervous system. Expression of both receptor proteins was localized exclusively to neurites when examined in situ. In contrast, when isolated neurons were grown in culture, 5-HT1Hel and 5-HT7Hel immunoreactivity were located primarily in the cell body. This is the first study to reveal a 5-HT7 receptor in a molluscan species.