Molecular and Cellular Endocrinology 236 (2005) 17–30 Cloning, characterization and expression of the D 2 dopamine receptor from the tilapia pituitary Berta Levavi-Sivan ∗ , Joseph Aizen, Ayelet Avitan Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel Received 29 November 2004; received in revised form 16 March 2005; accepted 18 March 2005 Abstract 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 D 2 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-D 2 -R with all vertebrate D 2 -like receptor sequences cloned to date, and it was therefore designated taDA-D 2 -R. taDA-D 2 -R was transiently expressed in COS-7 cells together with the reporter construct CRE-luciferase. Addition of the specific D 2 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-D 2 -R for bromocriptine was higher than for quinpirole. As established for mammalian D2-like receptors, stimulation of the taDA-D 2 -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-D 2 -R. Pharmacological and genetic manipulation of the taDA-D 2 -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. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Dopamine receptor; Estradiol; cAMP; Adenylate cyclase; Steroid feedback; Tilapia; Mullet; Catfish; D2; Pituitary 1. Introduction Dopamine is a ubiquitous neurotransmitter found in both the central and peripheral nervous systems in many species. It is involved in a wide variety of cerebral and peripheral functions, such as control of movement, learning, behavior and regulation of the hypothalamo-pituitary axis. Disorders such as Parkinson’s disease, schizophrenia, Tourette’s syn- drome and pituitary tumors are related to dopaminergic path- ways. The transduction of dopaminergic signals is mediated by several subtypes of G-protein-coupled receptors (GPCRs). These receptors are divided into two major subclasses: the ∗ Corresponding author. Tel.: +972 8 9489988; fax: +972 8 9465763. E-mail address: sivan@agri.huji.ac.il (B. Levavi-Sivan). D1-like (D 1 and D 5 ) and D2-like (D 2 ,D 3 , and D 4 ) receptors, and are further characterized by their ability to stimulate or inhibit adenylate cyclase (AC) activity, respectively (Civelli et al., 1991). In mammals, dopamine released from the hypothalamic tuberinfundibular neurons serves as a physiological inhibitor of prolactin secretion, mediated through D 2 dopamine re- ceptors (DA-Rs) residing on the pituitary lactotroph mem- branes (Ben-Jonathan, 1985). In fish, dopamine also in- hibits prolactin release through D 2 -like receptors (James and Wigham, 1984) but stimulates growth hormone (GH) release through D 1 -like receptors (Melamed et al., 1995; Wong et al., 1993). Dopamine also inhibits the release of -melanocyte-stimulating hormone (-MSH) (Olivereau et al., 1987) through D2-like receptors in goldfish and tilapia (Lamers et al., 1991; Omeljaniuk et al., 1989) but has been 0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.03.010