Cloning and expression of a fish α₂‐adrenoceptor

Abstract

Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an α2‐adrenoceptor pharmacology. We now report the cloning of the α2‐F, a fish skin α2‐receptor from the cuckoo wrasse (Labrus ossifagus). Degenerate oligonucleotides corresponding to conserved regions of the human α2‐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 α2‐adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 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 α2‐adrenoceptors and thus appeared to encode a fish α2‐adrenoceptor. In the 5′‐untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the α2‐F might be regulated by cyclic AMP, calcium and/or steroids. The α2‐F was expressed in COS‐7 cells and radioligand binding studies were performed with [3H]‐rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 ± 0.1 nm and a Bmax of 5.7 ± 1.0 pmol mg−1 of protein. Competition curves for the displacement of specific [3H]‐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. These results show that α2‐F has characteristics of both the human α2‐C10 and α2‐C4 and that it might represent an ancestral α2‐adrenoceptor subtype.

Comparative anatomy of α2 and β adrenoceptors in the adult and developing brain of the marine teleost the red porgy (Pagrus pagrus, Sparidae): [3H]clonidine and [3H]dihydroalprenolol quantitative autoradiography and receptor subtypes immunohistochemistry

“…it might represent an ancestral ␣ 2 AR subtype (Svensson et al, 1993), highly correlated with a frog ␣ 2 AR (Herman et al, 1996). Moreover, recent cloning and mapping of zebrafish ␣ 2 AR indicates conservation of ␣ 2 AR subtypes across species (Ruuskanen et al, 2004).…”

“…All antibodies used have been raised to recognize C-terminus receptor domains. Even though studies on mammalian adrenergic receptors indicate a certain degree of diversity for those domains (Kobilka, 1992), molecular studies on fish skin ␣ 2 AR have shown that the greatest identity of fish and mammalian ␣ 2 AR is in the membrane-spanning and intracellular domains, which are thought to be evolutionarily conserved (Svensson et al, 1993). In addition, as in earlier studies (Aoki et al, 1987(Aoki et al, , 1994, the lack of labeling following preadsorption of the primary antibodies with the peptides used to raise those antibodies in mammals suggests that the antibodies recognized a protein fraction common for ␣ 2 , ␤ 1 , and ␤ 2 AR in mammals and teleost brain.…”

“…Adrenergic receptors of both the ␣ 2 and the ␤ type have been identified in the peripheral nervous system and skin of several teleosts (Holmgren and Nilsson, 1982;Svensson et al, 1993;Katayama et al, 1999). These studies report some differences in the potencies of certain compounds but also point out similarities between vertebrate AR.…”

Functional autoradiography and gene expression analysis applied to the characterization of the α2-adrenergic system in the chicken brain

“…Although chicken genes codifying for a 2 -adrenoceptors have not yet been sequenced, the comparison of primary and secondary structures of a 2 -adrenoceptors cloned for different species of mammals and fish shows a high correlation coefficient (Svensson et al, 1993;Ruuskanen et al, 2005). Now, in an attempt to better a 2 -Adrenoceptors Adenosine receptor Tissue section RT-PCR…”

Neuronal and glial localization of α2A‐adrenoceptors in the adult zebrafish (Danio rerio) brain

“…ARs of ␣ 2 and ␤ types have been identified in the nervous system and skin of some teleost species (Holmgren and Nilsson, 1982;Svensson et al, 1993;Katayama et al, 1999; Zikopoulos and Dermon, 2005), including zebrafish (Ruuskanen et al, 2004(Ruuskanen et al, , 2005a. The ␣ 2A -ARs are located both postsynaptically and presynaptically, the latter limiting the release of NA and other neurotransmitters, such as serotonin and glutamate.…”