The olfactory receptors (ORs) are a large group of proteins belonging to subfamily I of G protein coupled receptors (GPCRs) that bind odorant ligands. These receptors are predicted to contain seven transmembrane helices that change their relative orientation upon odorant stimulation, resulting in the conformational change of the receptor and productive interaction of its intracellular loops with G olf , the a subunit of the heterotrimeric G protein [1][2][3]. Several lines of evidence suggest that the mechanism of OR activation by an odorant is central to understanding odorant perception and coding. Each OR recognizes multiple odorants and most odorants are recognized by several ORs [4][5][6][7]. One OR can discriminate between odorants with different functional groups, molecular size or shape and can even be sensitive to odorant concentration [8][9][10]. In addition, receptor perception of an odorant can be enhanced or antagonized by the presence of another odorant [8,11,12]. Despite the importance of OR pharmacology to olfactory detection and The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G a subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G olf compared to the G a15 subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.
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