Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells, expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G protein-coupled Receptor), an endogenously overexpressed OR, by β-ionone, its odorant agonist, resulted in the same phenotypic change. We also showed the involvement of a PI3 kinase γ dependent signaling pathway in this promotion of tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist β-ionone significantly enhanced metastasis emergence and spreading.

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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.

We describe how mammalian olfactory receptors (ORs) could be used as sensing elements of highly specific and sensitive bioelectronic noses. An OR and an appropriate G(alpha) protein were co-expressed in Saccharomyces cerevisiae cells from which membrane nanosomes were prepared, and immobilized on a sensor chip. By Surface Plasmon Resonance, we were able to quantitatively evaluate OR stimulation by an odorant, and G protein activation. We demonstrate that ORs in nanosomes discriminate between odorant ligands and unrelated odorants, as in whole cells. This assay also provides the possibility for quantitative assessment of the coupling efficiency of the OR with different G(alpha) subunits, without the interference of the cellular transduction pathway. Our findings will be useful to develop a new generation of electronic noses for detection and discrimination of volatile compounds, particularly amenable to micro- and nano-sensor formats.

Mechanical properties of nano-sized vesicles made up of natural membranes are crucial to the development of stable, biocompatible nanocontainers with enhanced functional, recognition and sensing capabilities. Here we measure and compare the mechanical properties of plasma and inner membrane nanovesicles ∼80 nm in diameter obtained from disrupted yeast Saccharomyces cerevisiae cells. We provide evidence of a highly deformable behaviour for these vesicles, able to support repeated wall-to-wall compressions without irreversible deformations, accompanied by a noticeably high Young's modulus (∼300 MPa) compared to that obtained for reconstituted artificial liposomes of similar size and approaching that of some virus particles. Surprisingly enough, the results are approximately similar for plasma and inner membrane nanovesicles, in spite of their different lipid compositions, especially on what concerns the ergosterol content. These results point towards an important structural role of membrane proteins in the mechanical response of natural membrane vesicles and open the perspective to their potential use as robust nanocontainers for bioapplications.

G-protein-coupled receptors (GPCRs) constitute the largest but the most divergent class of cell surface proteins. Although they are thought to share a common 3D-structure composed of seven transmembrane helical domains, they can be activated by extracellular signals as diverse as light, peptides, proteins, lipids, organic odorants, taste molecules, nucleotides or nucleosides. They are involved in an extraordinarily large number of physiological functions and are therefore potential drug targets for many human diseases. During the last decade various GPCRs have been successfully expressed in S. cerevisiae. Yeast is an attractive expression system because it offers the genetic engineering tools typical of a microorganism while possessing an eukaryotic type of secretory pathway and post-translational machinery. This host is particularly attractive for in-vivo manipulation of these receptors due to the high homology between the yeast pheromone signaling pathway and that of mammalian GPCRs. When expressed in yeast, mammalian GPCRs have been shown to couple functionally to either the endogenous yeast Galpha (Gpa1), or co-expressed mammalian Galpha subunits (wild-type or chimeric), and are characterized by a similar pharmacology in response to agonists or antagonists as in native cells. Heterologous expression of wild type or mutant GPCRs in S. cerevisiae allows a rapid assessment of their ability to detect and transduce extracellular stimulations, through the use of a reporter system. Furthermore, this approach is amenable to high-throughput screening of new drugs, which would provide a determinant advantage in the field of therapeutic research, and also for investigation of the still unknown ligands of orphan receptors. This review will focus on the latest developments of yeast-based technology to screen for potential GPCR agonists/antagonists.

The molecular mechanisms underlying odorant detection have been investigated using the chip based SPR technique by focusing on the dynamic interactions between transmembrane Olfactory Receptor OR1740, odorant ligands and soluble Odorant-Binding Protein (OBP-1F). The OR1740 present in the lipid bilayer of nanosomes derived from transformed yeasts specifically bound OBP-1F. The receptor preferential odorant ligand helional released bound OBP-1F from the OR-OBP complex, while unrelated odorants failed to do so. OBP-1F modified the functional OR1740 dose-response to helional, from a bell-shaped to a saturation curve, thus preserving OR activity at high ligand concentration. This unravels an active role for OBPs in olfaction, in addition to passive transport or a scavenger role. This sensorchip technology was applied to assessing native OBP-1F in a biological sample: rat olfactory mucus also displayed significant binding to OR1740 nanosomes, and the addition of helional yielded the dissociation of mucus OBP from the receptor.

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