HETEROLOGOUS EXPRESSION OF THE HUMAN D-2S DOPAMINE-RECEPTOR IN PROTEASE-DEFICIENT SACCHAROMYCES-CEREVISIAE STRAINS

The cDNA for the human D-2S dopamine receptor has been functionally ex pressed in the unicellular yeast Saccharomyces cerevisiae. The origina l D-2S gene and an elongated D-2S gene with an N-terminal fusion to th e first 24 amino acids of the STE2 gene from S. cerevisiae were introd uced into the episomal yeast expression vector YEp51 under the control of the GAL10 promoter. Expression studies performed in a wild-type st rain and in two protease-deficient strains of S. cerevisiae revealed t hat the receptor was functionally expressed with respect to its ligand -binding properties. The K-D values for the binding of the dopamine an tagonist [H-3]spiperone were calculated to be 1.6 nM for the D-2S rece ptor alone and 1.9 nM for the STE2-D-2S chimaera. Both membrane protei ns could be further characterized by ligand-displacement studies using certain dopamine agonists and antagonists. D-2S dopamine-receptor-spe cific polyclonal antibodies were used to monitor the heterologous expr ession of the receptor. Western-blot analysis of membranes prepared fr om 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 pro tein). It could be shown that, in comparison to the expression in a wi ld-type S. cerevisiae strain, the amount of receptor degradation was d rastically reduced in the protease-deficient strains. The localization s of the heterologously produced dopamine receptor and of the chimaera in the recombinant yeast were studied by immunogold electron microsco py and were found to be restricted mainly to the vacuole of the cells.