Heterologous expression of the human D2S dopamine receptor in protease-deficient Saccharomyces cerevisiae strains.
The cDNA for the human D2S dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The original D2S gene and an elongated D2S 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 KD values for the binding of the dopamine antagonist [3H]spiperone were calculated to be 1.6 nM for the D2S receptor alone and 1.9 nM for the STE2-D2S chimaera. Both membrane proteins could be further characterized by ligand-displacement studies using certain dopamine agonists and antagonists. D2S 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 (D2S receptor alone) and 42 kDa (STE2/D2S 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.
Sander
P
P
Max-Planck-Institut für Biophysik, Abt. Molekulare Membranbiologie, Frankfurt/M., Germany.
Grünewald
S
S
Bach
M
M
Haase
W
W
Reiländer
H
H
Michel
H
H
D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D003593:Cytoplasm / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000648:ultrastructure; D013134:Spiperone / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D013134:Spiperone; D010450:Endopeptidases