Expression of the human D2S dopamine receptor in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe: A comparative study

FEBS Letters

Michel, Hartmut¹  Grünewald, Sylvia¹  Reiländer, Helmut¹  Sander, Peter¹ 

[1] Max-Planck-Institut für Biophysik, Abt. Molekulare Membranbiologie, Heinrich-Hoffmann-Straße 7, 60528 Frankfurt/M, Germany

关键词: Saccharomyces cerevisiae;    Schizosaccharomyces pombe;    D2S dopamine receptor;    G-protein coupled receptor;    Membrane protein;    Heterologous expression;    AP;    alkaline phosphatase;    BCIP;    5-bromo-4-chloro-3-indolyl-phosphate p-toluidinium salt;    BSA;    bovine serum albumin;    DMF;    dimethylformamide;    NBT;    Nitroblue-tetrazoliumchloride;    mcs;    multiple cloning site;    PNGaseF;    peptide-N-glycosidase F;   

DOI  :  10.1016/0014-5793(94)00335-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were tested for heterologous expression of the human D_2S dopamine receptor. The cDNA coding for the dopamine receptor was cloned into high copy number plasmids with inducible promoters. After transformation into the yeasts recombinant clones were examined for the presence of functional receptor by radioligand binding using the antagonist [³H]spiperone. Subsequent Western blot analysis of positive recombinants with an antiserum raised against a peptide from the third intracellular domain of the receptor protein revealed the production of a protein with an apparent molecular mass of 40 kDa in both yeasts. Membranes harvested from recombinant yeast clones exhibited saturable binding of the dopaminergic antagonist [³H]spiperone with K _d values of 1.3 nM in S. cerevisiae and 0.25 nM in S. pombe. The rank order of potencies for several dopaminergic ligands to displace specific [³H]spiperone binding to membranes were the same in both yeasts, whereas the affinities for ligands differed significantly.

【 授权许可】

Unknown