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.
Levavi-Sivan
Berta
B
Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel. sivan@agri.huji.ac.il
Aizen
Joseph
J
Avitan
Ayelet
A
D000818:Animals; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000032:analysis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010902:Pituitary Gland / Q000737:chemistry*; D017448:Receptors, Dopamine D2 / Q000819:agonists / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D017210:Tilapia / Q000235:genetics*; D014018:Tissue Distribution
D017448:Receptors, Dopamine D2; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go