The human beta 2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties.
We have developed a rapid and efficient expression system to study the human beta 2 adrenergic receptor (hu beta 2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the hu beta 2AR gene, modified by replacement of the 5' untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (alpha-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu beta 2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu beta 2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.
EniChem SpA, Istituto Guido Donegani, Monterotondo/Rome, Italy.
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Tocchini Valentini
D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D005656:Fungal Proteins / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007118:Immunoassay; D008024:Ligands; D008969:Molecular Sequence Data; D010957:Plasmids; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics
D004274:DNA, Recombinant; D005656:Fungal Proteins; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C489241:STE12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors