Odorant response assays for a heterologously expressed olfactory receptor.
Odorant responsiveness of a mouse olfactory receptor, mOR-EG, was investigated in various heterologous cells using a variety of detection methods. Odorant-induced Ca(2+) response was observed in HEK293 cells that coexpressed mOR-EG and the promiscuous G protein, G alpha 15. Without G alpha 15, a robust increase in cAMP level was observed upon odorant-stimulation in various mammalian cells. A luciferase reporter gene assay using zif268 promoter was adopted to amplify the cAMP signals. In Xenopus laevis oocytes, odorant-stimulated currents were recorded when mOR-EG cRNA was co-injected with either G alpha 15 or cAMP-dependent channel. These results suggest that odorant responsiveness can be monitored via a signaling pathway mediated by endogenous G alphas or transfected G alpha 15 in heterologous cell systems. Various functional assays for a heterologously expressed olfactory receptor reported in this study, are potentially useful for high-throughput ligand screening and functional analyses of hundreds of olfactory receptors.
Katada
Sayako
S
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 277-8562, Chiba, Japan
Nakagawa
Takao
T
Kataoka
Hiroshi
H
Touhara
Kazushige
K
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D004553:Electric Conductivity; D005054:Eugenol / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D017930:Genes, Reporter; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000235:genetics; D051379:Mice; D009812:Odorants; D009865:Oocytes / Q000378:metabolism; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D051381:Rats; D018035:Receptors, Odorant / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020218:Response Elements; D015290:Second Messenger Systems; D014162:Transfection; D014982:Xenopus laevis
D018035:Receptors, Odorant; D005054:Eugenol; D000242:Cyclic AMP; D008156:Luciferases; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium