CLONING AND EXPRESSION OF A FISH ALPHA-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 alpha2-adrenoceptor pharmacology. We now report the cloning o f the alpha2-F, a fish skin alpha2-receptor from the cuckoo wrasse (La brus ossifagus). 2 Degenerate oligonucleotides corresponding to conser ved regions of the human alpha2-adrenoceptor subtypes were used in a p olymerase chain reaction (PCR) with cDNA prepared from mRNA isolated f rom the skin of the cuckoo wrasse. An 876 base pair (bp) product was o btained that was homologous with that of the human alpha2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3 A clone (pTB 17BS) consisting of approximately 5 kb of genomic DNA was o btained which contained the nucleotide sequence of the initial PCR pro duct. In addition, it contained an open reading frame that encoded a p rotein of 432 amino acids and approximately 2 kb of 5'-untranslated se quence.The deduced amino acid sequence of this protein showed 47-57% i dentity with the human alpha2-adrenoceptors and thus appeared to encod e a fish alpha2-adrenoceptor. 4 In the 5'-untranslated region of the g ene, nucleotide sequences were present suggesting that transcription o f the alpha2-F might be regulated by cyclic AMP, calcium and/or steroi ds. 5 The alpha2-F was expressed in COS-7 cells and radioligand bindin g studies were performed with [H-3]-rauwolscine. The binding was of hi gh affinity and it was saturable with a K(D) of 0.8 +/- 0.1 nm and a B (max) of 5.7 +/- 1.0 pmol mg-1 of protein. 6 Competition curves for th e displacement of specific [H-3]-rauwolscine binding showed the follow ing order of potency: for agonists, medetomidine > clonidine > p-amino clonidine > B-HT920 > (-)-noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin. 7 These results s how that alpha2-F has characteristics of both the human alpha2-CIO and alpha2-C4 and that it might represent an ancestral alpha2-adrenocepto r subtype.