Stable expression of recombinant human α2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay

Anne Marjamäki, Sari Ala-Uotila, Kirsti Luomala, Merja Perälä, Christian Jansson, Markku Jalkanen, John W Regan, Mika Scheinin

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Cloning of the genes encoding distinct subtypes of human α2-adrenergic receptors (α2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human α2-AR subtypes α2-C4 and α2-C10 at densities of approx. 2·105 receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5′-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the α2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.

Original languageEnglish (US)
Pages (from-to)169-177
Number of pages9
JournalBBA - Molecular Cell Research
Volume1134
Issue number2
DOIs
StatePublished - Mar 16 1992

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Yohimbine
Ribonucleases
Adenylyl Cyclases
Adrenergic Receptors
Cell Line
Pharmacology
Transfection
Norepinephrine
Oxymetazoline
Adrenergic Agonists
Prazosin
Clonidine
Colforsin
G-Protein-Coupled Receptors
Guanosine Triphosphate
Genes
Organism Cloning
Suspensions
Breast Neoplasms
Ligands

Keywords

  • Adenylate cyclase activity
  • Cell line
  • RNAase protection assay
  • Stable transfection
  • [H]Rauwolscine binding
  • α-Adrenoceptor subtype

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Stable expression of recombinant human α2-adrenoceptor subtypes in two mammalian cell lines : characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay. / Marjamäki, Anne; Ala-Uotila, Sari; Luomala, Kirsti; Perälä, Merja; Jansson, Christian; Jalkanen, Markku; Regan, John W; Scheinin, Mika.

In: BBA - Molecular Cell Research, Vol. 1134, No. 2, 16.03.1992, p. 169-177.

Research output: Contribution to journalArticle

Marjamäki, Anne ; Ala-Uotila, Sari ; Luomala, Kirsti ; Perälä, Merja ; Jansson, Christian ; Jalkanen, Markku ; Regan, John W ; Scheinin, Mika. / Stable expression of recombinant human α2-adrenoceptor subtypes in two mammalian cell lines : characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay. In: BBA - Molecular Cell Research. 1992 ; Vol. 1134, No. 2. pp. 169-177.
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