科研之友
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摘要
The cDNA for the human D-2S dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The original D-2S gene and an elongated D-2S gene with an N-terminal fusion to the first 24 amino acids of the STE2 gene from S. cerevisiae were introduced into the episomal yeast expression vector YEp51 under the control of the GAL10 promoter. Expression studies performed in a wild-type strain and in two protease-deficient strains of S. cerevisiae revealed that the receptor was functionally expressed with respect to its ligand-binding properties. The K-D values for the binding of the dopamine antagonist [H-3]spiperone were calculated to be 1.6 nM for the D-2S receptor alone and 1.9 nM for the STE2-D-2S chimaera. Both membrane proteins could be further characterized by ligand-displacement studies using certain dopamine agonists and antagonists. D-2S dopamine-receptor-specific polyclonal antibodies were used to monitor the heterologous expression of the receptor. Western-blot analysis of membranes prepared from transformed yeast cells producing either the receptor protein alone or the receptor fusion protein revealed apparent molecular masses of 40 kDa (D-2S receptor alone) and 42 kDa (STE2/D-2S receptor fusion protein). It could be shown that, in comparison to the expression in a wild-type S. cerevisiae strain, the amount of receptor degradation was drastically reduced in the protease-deficient strains. The localizations of the heterologously produced dopamine receptor and of the chimaera in the recombinant yeast were studied by immunogold electron microscopy and were found to be restricted mainly to the vacuole of the cells.
- 出版日期1994-12-1
