Cloning and expression of a zebrafish 5-HT2C receptor gene

Abstract

The 5-HT2C receptor is one of 14 different serotonin (5-HT) receptors that control neural function and behavior. Here, we present the entire sequence of a zebrafish 5-HT2C receptor cDNA including the 3′ untranslated region and the previously unknown 5′ untranslated region. The cloned 5-HT2C receptor gene is located on chromosome 7, is approximately 202kbp long, and contains six exons. The coding region of the gene is 1557bp long and flanked by a 504bp 5′ UTR and a 1474bp 3′ UTR. The deduced protein sequence of 518 amino acids aligns with orthologs of other vertebrates and is 54% identical to the human and mouse 5-HT2C receptor protein sequences. The region of the cDNA that encodes the 2nd cytoplasmic loop of the protein shows a 66% identity with vertebrate orthologs and clearly identifies the gene as a 5-HT2C receptor gene. Coupling sites for beta-arrestin and calmodulin are conserved in zebrafish. In-situ hybridization shows that the receptor is expressed in the brain and spinal cord including areas such as the olfactory bulb, the dorsal thalamus, the posterior tuberculum, the hypothalamus and the medulla oblongata. Reverse Transcriptase-PCR experiments indicate that the receptor gene can also be active in other tissues such as skin, ovaries, and axial muscle of adult zebrafish. Expression of the 5-HT2C receptor during ontogeny was found as early as 2.5hpf. Five edited adenines in the region of the human, rat and mouse mRNA that encodes the 2nd cytoplasmic loop are conserved in the zebrafish transcript. However, RNA editing was not detected in the zebrafish. The results characterize the zebrafish 5-HT2C receptor gene and gene expression pattern for the first time. The similarities to mammalian 5-HT2C receptor genes suggest the use of zebrafish for the study of 5-HT2C receptor function in behavior, development and drug discovery.