CONSTITUTIVE EXPRESSION OF THE HUMAN D-2S-DOPAMINE RECEPTOR IN THE UNICELLULAR YEAST SACCHAROMYCES-CEREVISIAE

The cDNA for the human D-2S-dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. Two expression plasmids pRS421D2 (original D-2S-gene coding region) and pRS421D2S (the first 24 aa of the yeast STE2-gene are fused to the N-terminus of the D-2S-gene) were constructed and transformed into the protease deficient S. cerevisiae strain cI3-ABYS-86. Northern blot analysis of total RNA from transformed yeast clones revealed that for both constructs the D-2S-gene was constitutively transcribed from the plasmids PMA1 promoter. Membranes prepared from recombinant S. cerevisiae exhibited saturable binding with the antagonist [H-3]methylspiperone. Competition studies revealed pharmacological properties for these sites which were comparable to those reported for the D-2-receptor heterologously expressed in mammalian cells. The expression of the receptor was monitored by Western blot analysis using an antiserum raised against a peptide from the third intracellular domain of the receptor protein and by ligand binding assay.