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 of the alpha2-F, a fish skin alpha2-receptor from the cuckoo wrasse (Labrus ossifagus). 2 Degenerate oligonucleotides corresponding to conserved regions of the human alpha2-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 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 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 approximately 2 kb of 5'-untranslated sequence.The deduced amino acid sequence of this protein showed 47-57% identity with the human alpha2-adrenoceptors and thus appeared to encode a fish alpha2-adrenoceptor. 4 In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the alpha2-F might be regulated by cyclic AMP, calcium and/or steroids. 5 The alpha2-F was expressed in COS-7 cells and radioligand binding studies were performed with [H-3]-rauwolscine. The binding was of high 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 the displacement of specific [H-3]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine > p-aminoclonidine > B-HT920 > (-)-noradrenaline; for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin. 7 These results show that alpha2-F has characteristics of both the human alpha2-CIO and alpha2-C4 and that it might represent an ancestral alpha2-adrenoceptor subtype.