Molecular cloning and characterization of a VPAC receptor in the inshore hagfish, Eptatretus burgeri

Abstract

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are pleiotropic polypeptides with diverse tissue distribution. They belong to the same VIP/PACAP/secretin peptide superfamily and have the ability to interact with the class II G protein-coupled receptors. Three receptor subtypes namely VPAC1, VPAC2, and PAC1 are responsible for interaction with VIP and PACAP, each differing in ligand-binding affinities and signal-transduction mechanisms. However, characterization of these ligand–receptor pairs has not yet been well studied in living primitive fish species such as lampreys and hagfish, which represent the earliest vertebrates to exist. In this study, a VPAC receptor (hfVPAC) sequence was identified in the inshore hagfish, Eptatretus burgeri. By real-time PCR, it was found predominately in the brain, while trace amounts were also detected in the muscles. This contrasts the diverse tissue distribution pattern of VIP/ PACAP receptors in other vertebrates, suggesting the first function of the ligand–receptor pair to be restricted to the brain and that functions elsewhere evolved only later. Functionally, both VIP and PACAP ligands from fish and mammals were able to stimulate cAMP responses in the hfVPAC-transfected cell line, further suggesting it's identity as a VPAC receptor. This is also verified by phylogenetic analyses showing the hfVPAC to be clustered together with other vertebrate VIP and PACAP receptors. As the hfVPAC does not belong exclusively to VPAC1, VPAC2, or PAC1 receptor subtype groups, this may suggest a more ancient evolutionary origin and a closer resemblance to the ancient VIP- and PACAP-receptor genes. Through the discovery of this receptor in the hagfish, we hope to provide clues to better understand the early events in the molecular evolution of VIP and PACAP receptors in vertebrates.