PHARMACOLOGICAL AND BIOCHEMICAL-CHARACTERIZATION OF THE MOUSE 5HT(5A)SEROTONIN RECEPTOR HETEROLOGOUSLY PRODUCED IN THE YEAST SACCHAROMYCES-CEREVISIAE

The cDNA for the mouse 5HT(5A) receptor has been functionally expresse d in the unicellular yeast Saccharomyces cerevisiae. The NH2-terminal end of the receptor gene was fused to the Bacillus macerans (1-3, 1-4) -beta-lucanase signal sequence to ensure proper membrane insertion and to the c-myc epitope to permit immunological detection of the heterol ogously expressed protein. In the resulting episomal yeast expression plasmid pCNNmm5HT5A the modified 5HT(5A) gene is under the transcripti onal control of the endopeptidase B gene promoter (PRB1). After transf ormation of the vector into the protease deficient S. cerevisiae strai n cI3-ABYS-86, recombinant clones were examined for the presence of fu nctional receptor by radioligand binding using [H-3]LSD. Whole cells a s well as crude membrane preparations of recombinant clones showed sat urable binding of the receptor with a K-D of approximate to 2.2 nM. Th e pharmacological properties for the heterologous expressed receptor, estimated by ligand-displacement studies using certain serotonin agoni sts and antagonists, were comparable to those reported for the recepto r expressed in mammalian systems. Western blot analysis of membranes p repared from a recombinant clone using the monoclonal antibody 9E10, d irected against the c-myc epitope of the modified receptor, revealed a n apparent molecular mass of about 43 kDa for the receptor expressed i n S. cerevisiae. Glycosylation of the receptor was analysed by EndoH d igestion. A heat shock of recombinant yeast significantly increased th e number of specific binding sites per cell and also improved the affi nity of the receptor. Immunogold electron microscopy was used to study the localization of the heterologously expressed protein within the y east cells.