Heterologous functional expression system for odorant receptors.
Heterologous functional expression system for odorant receptors (ORs) is essential for investigating the structure-activity relationship (SAR) of various ligands. Different systems that coexpressed ORs with different G-protein alpha subunits (Galpha) demonstrated inconsistent effects on weak agonists and antagonists, but retained original relative sensitivities to potent agonists. In order to maintain the binding specificity of Galpha to ORs, we constructed a chimeric Galpha(15_olf), which contained the Galpha(15) sequence with the conserved C-terminal region of Galpha(olf). The Ca(2+) responses of the HEK293 cells that coexpressed OR-S6 with Galpha(15_olf) were more robust and reproducible compared to those of cells that coexpressed OR-S6 with Galpha(15). Furthermore, Galpha(15) sometimes induced unstable Ca(2+) responses that limited the accuracy of quantitative comparison of peak responses. Our results showed that a heterologous expression system that coexpressed ORs with Galpha(15_olf) and receptor transporting proteins was suitable for SAR analysis of various ligands.
Hamana
Hiroshi
H
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan.
Shou-xin
Li
L
Breuils
Laure
L
Hirono
Junzo
J
Sato
Takaaki
T
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000235:genetics; D002461:Cell Line, Transformed; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D006801:Humans; D011485:Protein Binding; D018035:Receptors, Odorant / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods
D044385:GTP-Binding Protein alpha Subunits; D018035:Receptors, Odorant; D002118:Calcium