Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

Abstract

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.

Relationship between Homo-oligomerization of a Mammalian Olfactory Receptor and Its Activation State Demonstrated by Bioluminescence Resonance Energy Transfer

“…Some ORs, including human OR1740, were reported not to heterodimerize with a non-olfactory GPCR, the ␤ 2 -adrenergic receptor (15). Our previous studies (6,16,17), in line with others (18,19), showed a bell-shaped OR dose-response curve upon odorant stimulation. Several assumptions have been raised to elucidate the decreased OR response at high ligand concentrations (20 -22).…”

“…For this purpose, we heterologously expressed the human OR1740 receptor under optimized experimental conditions in the yeast Saccharomyces cerevisiae (17,23) and investigated its homo-oligomerization status. Besides a biochemical approach, we used bioluminescence resonance energy transfer (BRET), a well developed biophysical technology employed largely in the past decade to explore GPCR oligomerization to monitor not only OR homodimerization but also the odorant ligand-mediated conformational changes in the receptors.…”

Nanobiosensors Based on Individual Olfactory Receptors

“…In the framework of this project, an OR and an appropriate G α protein were co-expressed in yeast cells [2] from which membrane nanosomes, with size homogenized to around 50 nm by sample sonication, were prepared.…”

Bioelectronic Noses Based on Olfactory Receptors

“…Such systems are easily adaptable for a high throughput format (Minic et al, 2005a). Upon stimulation of the olfactory receptor by its odorant ligands in yeasts engineered to co-express either rat I7 or human OR 17-40 receptor and the mammalian Gα subunit, an activation of a MAP kinase signaling pathway takes place, which induces the synthesis of a functional reporter luciferase (Minic et al, 2005b). In that way bioluminescence responses are detected upon odorant binding to the receptor.…”