Cloning and expression of a fish α2-adrenoceptor

S. P S Svensson, T. J. Bailey, D. J. Pepperl, N. Grundstrom, S. Ala-Uotila, M. Scheinin, J. O G Karlsson, John W Regan

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

1. 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). 2. 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. 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 ~2kb 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 encoded a fish α2-adrenoceptor. 4. 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. 5. 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 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 [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. 7. 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.

Original languageEnglish (US)
Pages (from-to)54-60
Number of pages7
JournalBritish Journal of Pharmacology
Volume110
Issue number1
StatePublished - 1993

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Adrenergic Receptors
Organism Cloning
Fishes
Yohimbine
Skin
Medetomidine
Melanophores
Polymerase Chain Reaction
Proteins
Genomic Library
Prazosin
Phentolamine
5' Untranslated Regions
COS Cells
Clonidine
Oligonucleotides
Base Pairing
Cyclic AMP
Open Reading Frames
Amino Acid Sequence

Keywords

  • Adrenoceptor
  • G-protein coupled receptor
  • Labrus ossifagus
  • Melanophore
  • Yohimbine

ASJC Scopus subject areas

  • Pharmacology

Cite this

Svensson, S. P. S., Bailey, T. J., Pepperl, D. J., Grundstrom, N., Ala-Uotila, S., Scheinin, M., ... Regan, J. W. (1993). Cloning and expression of a fish α2-adrenoceptor. British Journal of Pharmacology, 110(1), 54-60.

Cloning and expression of a fish α2-adrenoceptor. / Svensson, S. P S; Bailey, T. J.; Pepperl, D. J.; Grundstrom, N.; Ala-Uotila, S.; Scheinin, M.; Karlsson, J. O G; Regan, John W.

In: British Journal of Pharmacology, Vol. 110, No. 1, 1993, p. 54-60.

Research output: Contribution to journalArticle

Svensson, SPS, Bailey, TJ, Pepperl, DJ, Grundstrom, N, Ala-Uotila, S, Scheinin, M, Karlsson, JOG & Regan, JW 1993, 'Cloning and expression of a fish α2-adrenoceptor', British Journal of Pharmacology, vol. 110, no. 1, pp. 54-60.
Svensson SPS, Bailey TJ, Pepperl DJ, Grundstrom N, Ala-Uotila S, Scheinin M et al. Cloning and expression of a fish α2-adrenoceptor. British Journal of Pharmacology. 1993;110(1):54-60.
Svensson, S. P S ; Bailey, T. J. ; Pepperl, D. J. ; Grundstrom, N. ; Ala-Uotila, S. ; Scheinin, M. ; Karlsson, J. O G ; Regan, John W. / Cloning and expression of a fish α2-adrenoceptor. In: British Journal of Pharmacology. 1993 ; Vol. 110, No. 1. pp. 54-60.
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AU - Svensson, S. P S

AU - Bailey, T. J.

AU - Pepperl, D. J.

AU - Grundstrom, N.

AU - Ala-Uotila, S.

AU - Scheinin, M.

AU - Karlsson, J. O G

AU - Regan, John W

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N2 - 1. 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). 2. 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. 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 ~2kb 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 encoded a fish α2-adrenoceptor. 4. 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. 5. 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 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 [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. 7. 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.

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