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Two full-length cDNA encoding putative dopamine D2-like receptors were cloned from the brain of female European eel. The deduced protein sequences, termed D2A- and D2B-R, exhibit closer phylogenetic relationships to vertebrate D2 receptors compared with D3 and D4 or D1 receptors. The two protein sequences share 100% identity within the transmembrane domains containing the highly conserved amino acids involved in dopamine binding. Accordingly, an apparent single population of sites on eel brain membranes bound [(3)H]spiperone, a D2-R-specific antagonist, with a K(d) of 0.2 +/- 0.04 nM. However, D2A- and D2B-R significantly differ within the amino terminus and the third intracellular loop. As analyzed by quantitative PCR and in situ hybridization, both receptor transcripts were found, with different relative abundance, in the majority of brain areas and in the pituitary, whereas in the retina, olfactory epithelium, spinal cord, and adipose tissue, only D2A-R gene was expressed. Because sex steroid hormones recently have been shown to regulate eel brain dopamine systems, we analyzed the effect of steroids on the amount of D2-R transcripts by quantitative PCR and in situ hybridization. In eels treated with testosterone, the gene expression of the D2B-R, but not D2A-R, was increased in a region-dependent manner. The effect of testosterone on D2B-R transcript levels was mimicked by dihydrotestosterone, a nonaromatizable androgen, whereas estradiol had no stimulatory action, evidencing an androgen receptor-dependent mechanism. Although functionality of the two receptors awaits determination of D2-R proteins, we hypothesize that differences in the tissue expression pattern and hormonal regulation of eel D2A- and D2B-R gene expression could represent selective forces that have contributed to the conservation of the duplicated D2-R.

Two Distinct Dopamine D2 Receptor Genes in the European Eel: Molecular Characterization, Tissue-Specific Transcription, and Regulation by Sex Steroids