Cloning and functional characterization of PACAP-specific receptors in zebrafish

Two PACAP receptors were isolated from total zebrafish cDNA library prepared from 6-day old fish by a homology-based cloning strategy. The two zebrafish PACAP receptors have the same topology as the one found in other members of this class of seven membrane-spanning G-protein-coupled receptors. Each of the two zebrafish PACAP receptors shares about 70% sequence identity at the amino acid level with the human PACAP-type 1 receptor, and about 50% amino acid identity with PACAP/VIP R-1 and PACAP/VIP R-2 receptors. One of these zebrafish receptors contains the hop2 configuration found in the human and rat PACAP-type 1 receptors. On the basis of these structural characteristics the zebrafish PACAP receptors were classified as PACAP-type 1 and PACAP-type 2 receptors. In competitive binding experiments zebrafish PACAP-type 1 and PACAP-type 2 receptors showed similar binding specificity for zebrafish and human PACAP-38 and PACAP-27. Furthermore, the specificity of PACAP-type 1 and PACAP-type 2 receptors for zebrafish and human PACAPs is about 1,000-fold higher than for human VIP. These results demonstrate that zebrafish PACAP-type 1 receptor is a structural and pharmacological homolog of the mammalian PACAP-type 1 receptor. Additional pharmacological characterization is needed in order to classify the zebrafish PACAP-type 2 receptor.