Cloning, characterization and expression of the D₂ dopamine receptor from the tilapia pituitary

A full-length cDNA encoding a dopamine receptor (DA-R) was obtained from the pituitary of tilapia (ta). This cDNA encodes a protein of 469 amino acids that exhibits the typical arrangement of GPCR. The taDA-R shows high similarity to the DA-Rs of mullet and fugu, and over 70% similarity to Xenopus, mouse and turkey D₂ DA-Rs. Northern blot analysis revealed transcript for a single transcript in the pituitary, of approximately 3 kb. In a Southern analysis, the tilapia probe recognized specific bands in the genomic DNA of both mullet and catfish, suggesting high similarity between the corresponding genes. Phylogenetic analysis clearly aligned the taDA-D₂-R with all vertebrate D₂-like receptor sequences cloned to date, and it was therefore designated taDA-D₂-R. taDA-D₂-R was transiently expressed in COS-7 cells together with the reporter construct CRE-luciferase. Addition of the specific D₂ dopamine agonists quinpirole or bromocriptine, in the presence of forskolin, led to a dose-dependent decrease in forskolin-induced cAMP levels. Both agonists yielded the same maximal inhibition (around 40%). However, the potency of taDA-D₂-R for bromocriptine was higher than for quinpirole. As established for mammalian D2-like receptors, stimulation of the taDA-D₂-R with quinpirole triggers pertussis-toxin-sensitive Gi/o-mediated, but not Gs-mediated signaling. In contrast to mammals, PCR analysis gave no evidence of alternative splicing in taDA-D₂-R. Pharmacological and genetic manipulation of the taDA-D₂-R should enable us to better define its physiological role and to further explore the usefulness of fish as a model system for understanding dopaminergic function in higher organisms.