Functional expression of odorant receptors of the zebrafish Danio rerio and of the nematode C. elegans in HEK293 cells.
Odorant receptors of zebrafish and C elegans were functionally expressed in vertebrate kidney cells (HEK293) using the eucaryotic expression vector pSMyc. Receptor-encoding cDNA cloned into this vector was expressed as a fusion protein with the N-terminal membrane import sequence of the guinea-pig serotonin receptor followed by a myc tag. Immunocytochemical evidence indicates that this strategy directs a protein with the predicted immunoreactivity and approximate molecular weight to the plasma membrane. Fish food extract (TetraMin) evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for three fish odorant receptors and converted to stable cell lines. The effect of the extract was concentration dependent and limited to the fraction of the extract < 5 kDa. Pretreating the transfected cells with the PLC inhibitor U73122 reduced the odor-evoked signal. Fish food extract also evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for single fish odorant receptors. Diacetyl evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids encoding the cDNA of ODR10, an odorant receptor of C. elegans suggested in other work to be specific for diacetyl. These results strongly imply that odorant receptors can be functionally expressed in HEK293 cells using this novel expression protocol.
Wellerdieck
C
C
Lehrstuhl für Zellphysiologie, Ruhr-Universität Bochum, Germany.
Oles
M
M
Pott
L
L
Korsching
S
S
Gisselmann
G
G
Hatt
H
H
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D017173:Caenorhabditis elegans / Q000235:genetics*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000502:physiology; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008969:Molecular Sequence Data; D018035:Receptors, Odorant / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection / Q000235:genetics / Q000502:physiology; D015027:Zebrafish / Q000235:genetics*
D018076:DNA, Complementary; D008024:Ligands; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D002118:Calcium