Cloning and expression of a fish α 2 ‐adrenoceptor

1 Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an α 2 ‐adrenoceptor pharmacology. We now report the cloning of the α 2 ‐F, a fish skin α 2 ‐receptor from the cuckoo wrasse ( Labrus ossifagus ). 2 Degenerate oligonucleotides corresponding to conserved regions of the human α 2 ‐adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human α 2 ‐adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3 A clone (pTB17BS) consisting of ∼5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and ∼2 kb of 5′‐untranslated sequence. The deduced amino acid sequence of this protein showed 47–57% identity with the human α 2 ‐adrenoceptors and thus appeared to encode a fish α 2 ‐adrenoceptor. 4 In the 5′‐untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the α 2 ‐F might be regulated by cyclic AMP, calcium and/or steroids. 5 The α 2 ‐F was expressed in COS‐7 cells and radioligand binding studies were performed with [ 3 H]‐rauwolscine. The binding was of high affinity and it was saturable with a K D of 0.8 ± 0.1 n m and a B max of 5.7 ± 1.0 pmol mg −1 of protein. 6 Competition curves for the displacement of specific [ 3 H]‐rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine> p ‐aminoclonidine > B‐HT 920 > (−)‐noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin. 7 These results show that α 2 ‐F has characteristics of both the human α 2 ‐C10 and α 2 ‐C4 and that it might represent an ancestral α 2 ‐adrenoceptor subtype.