Expression and intracellular localisation of odorant receptors in mammalian cell lines using Semliki Forest virus vectors

Odorant receptors are members of the G protein-coupled receptor superfamily. They are expressed on the surface of cilia of olfactory neurons, where they bind ligand (odorant). Studies of the molecular mechanisms of olfaction are complicated by the extremely large number of receptor genes, and difficulties in pairing a particular mammalian receptor to a specific odorant ligand in vivo. Here we report expression and localisation studies of two rat odorant receptor genes (17 and OR5), and C. elegans odr-10, using the Semliki Forest virus (SFV) system. All receptors were epitope-tagged at the N- or C-terminus in order to facilitate their detection in infected cells, and determine the localisation and membrane-orientation of recombinant proteins. The immortalised mouse olfactory neuronal cell line OLF 442, rat cortical and striatal primary neuron cultures, and the baby hamster kidney (BHK) cells, were infected and tested. Immunofluorescence and confocal microscopy studies performed on permeabilised, non-permeabilised and native cells revealed that in BHK cells the rat receptors 17 and OR5 were not targeted to the plasma membrane and remained in the endoplasmic reticulum. In contrast, in the mouse olfactory cell line OLF 442 both rat receptors were correctly inserted into the plasma membrane. Similar results were obtained using primary neurons, indicating that like mature neurons, the immortalised OLF 442 cells are capable of providing for correct odorant receptor processing and targeting.     

Cloning, functional expression and characterization of a human olfactory receptor.

The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.     

Functional analysis of olfactory receptors expressed in a HEK-293 cell system by using cameleons

Cameleon is a genetically engineered Ca2+ sensing molecule consisting of two variants of the green fluorescent protein (GFP), calmodulin and calmodulin-binding protein, M13. HEK-293 cells stably expressing three types of cameleons, yellow cameleon-2, cameleon-3er, and cameleon-2nu, were constructed, and the expression and localization of these cameleons were confirmed by fluorescent imaging. Among the cameleons, the yellow cameleon-2 was selected for analyzing the change in Ca2+ induced by the olfactory receptor-mediated signal transduction, because it is localized in the cytosol and binds to cytosolic Ca2+ ions. Cells stably expressing yellow cameleon-2 were transfected with each of the test olfactory receptor genes, odr-l0 and 17, and the expression of the olfactory receptor genes were examined using immunocytochemical methods and RT-PCR. Stimulating each olfactory receptor with its specific odorant caused an increase in the intracellular Ca2+ level, which was measured using yellow cameleon-2. These results demonstrate that yellow cameleon-2 can be conveniently used to examine the function of the olfactory receptors expressed in heterologous cells.     

Piezoelectric olfactory biosensor: ligand specificity and dose-dependence of an olfactory receptor expressed in a heterologous cell system

An olfactory receptor protein of rats, I7, was expressed on the surface of human embryonic kidney (HEK)-293 cells. For targeting and detecting the protein, rho-tag import sequence was fused with the I7 protein. The olfactory receptor was expressed on the plasma membrane of HEK-293 cells, and stable cell lines regulated by an inducer were obtained. The expression on the cell surface was confirmed by immunocytochemical and Western blotting methods, and the binding of specific odorant molecules to the olfactory receptor was measured using quartz crystal microbalance (QCM). The results for QCM coated with cells containing the olfactory receptor showed that the expressed protein I7 strongly interacted with octyl aldehyde (octanal), which is an odorant specific to the I7 protein. Several other odorants were tested, and the results showed that I7 interacted differently with them. The QCM response to the serial concentrations of octyl aldehyde showed that the response is dose dependent. All these results indicate that the I7 receptor protein expressed on the surface of the heterologous cell system is sensitive to the specific odorant and can be used for the quantitative measurement of the odorant.     

Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds

Molecular properties of odorant compounds essential for activation of the human olfactory receptor hOR17-40 were investigated using a collection of 23 variants of its cognate ligand helional. Coupling receptor activation to an optically detectable intracellular Ca(2+) ion flux allowed dose-dependent screening of different odorant molecules in human embryonic kidney (HEK)293 cells. We found an extended collection of activating ligands and provide first evidence for hOR17-40-specific antagonists. The C-terminal fusion of enhanced green fluorescent protein to the hOR17-40 retained full receptor function and permitted the selection of cells with defined receptor expression levels, which was an essential step for optimizing our screening protocol. Interestingly, cells with a low EGFP fluorescence intensity exhibited efficient hOR17-40 cell surface targeting and odorant-evoked signal transduction; in contrast, highly fluorescent cells displayed mainly incorrectly targeted, intracellular receptors. Fluorescence-activated cell sorting was used to separate hOR17-40-expressing cells on the basis of their endogenous EGFP fluorescence intensity, thereby increasing the fraction of odorant-responsive cells to up to 80% of the total cell number.     

Enhancement of odorant detection sensitivity by the expression of odorant-binding protein

Odorant-binding proteins are low molecular weight, soluble proteins that are secreted by glands of the nasal cavity. Their function is known to be the transport of hydrophobic odorants. This feature is important to artificial olfactory biosensors, which operate in the aqueous phase. In this study, one of rat odorant-binding proteins, OBP3, was inserted into a mammalian expression vector pcDNA3, expressed, and secreted from human embryonic kidney-293 (HEK-293) cells. The his(6) tag and signal peptide of the prelysozyme (plys) were fused with OBP3 for the detection and secretion of the proteins, respectively. The secretion level of OBP3 was maximal at 3h of incubation time. The secreted OBP3 increased the solubility of a hydrophobic odorant, octanal, which is the specific odorant of rat olfactory receptor I7. The secreted OBP3 also bound to olfactory receptor I7. These interactions consequently increased the cellular signal intensity stimulated by the odorant in the cells expressing olfactory receptor I7. Our findings indicate that odorant-binding protein can be effectively used to increase the sensitivity of olfactory receptor-based biosensors.     

Dual signal transduction mediated by a single type of olfactory receptor expressed in a heterologous system

Controversy exists over the relationship between the cAMP and IP3 pathways in vertebrate olfactory signal transduction, as this process is known to occur by either of the two pathways. Recent studies have shown that a single olfactory neuron responds to both cAMP- and IP3-producing odorants, suggesting the existence of an olfactory receptor protein that can recognize both ligands. In this study we found that the rat olfactory receptor I7, stably expressed in HEK-293 cells, triggers the cAMP pathway upon stimulation by a specific odorant (octanal) at concentrations lower than 10(-4) M; however, the receptor triggers both pathways at higher concentrations. This indicates that a single olfactory receptor, stimulated by a single pathway-inducing odorant, can evoke both pathways at high odorant concentrations. Using this heterologous system, both the dose-dependent response and receptor I7 specificity were analyzed. The dose-dependent Ca2+ response curve, which also includes the release of Ca2+ ions from internal stores at high odorant concentrations, was not monotonous, but had a local maximum and minimum with 10(-10) and 10(-7) M octanal, respectively, and reached a plateau at 10(-2) M octanal. The specificity of the I7 receptor was lower when exposed to higher concentrations of odorants.     

A specific heat shock protein enhances the expression of mammalian olfactory receptor proteins

Multiple trials failed to express significant amounts of olfactory receptors in heterologous cells as they are typically retained in the endoplasmic reticulum (ER). Evidence is accumulating that cell-type-specific accessory proteins regulate the folding of olfactory receptors, their exit from the ER, and the trafficking to the plasma membrane of the olfactory cilia where the receptors gain access to odorants. We found Hsc70t, a testis-enriched variant of the Hsp70 family of heat shock proteins which is specifically expressed in post-meiotic germ cells, in the olfactory epithelium of mouse and human. Cotransfected HEK293 cells with Hsc70t and different green fluorescent protein-tagged odorant receptors (ORs) from mouse and man showed a significantly enhanced OR expression. Hsc70t expression also changed the amount of cells functionally expressing olfactory receptors at the cell surface as the number of cells responding to odorants in Ca2+-imaging experiments significantly increased. Our results show that Hsc70t helps expression of ORs in heterologous cell systems and helped the characterization of an "orphan" human olfactory receptor.     

Specificity of odorant-binding proteins: a factor influencing the sensitivity of olfactory receptor-based biosensors

Odorant-binding proteins (OBPs) primarily function in the transport of hydrophobic odorants. In this study, OBPs originating from rat and pig were cloned into a mammalian expression vector, pcDNA3, and expressed in HEK-293 cells, and their specificity for odorants and olfactory receptors was examined. Results suggest that OBPs have a high affinity for the olfactory receptors when both the OBP and receptor originate from the same species. The rat OBPs were bound not only to the rat olfactory receptor I7 but also to the odorant specific to I7. The solubility of the odorant was increased by both OBP2 and OBP3, which originate from rat, but with different efficiencies. These results demonstrate that OBPs specifically interact with odorants as well as olfactory receptors, and these interactions can influence the sensitivity of olfactory receptor-based biosensors.     

Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.     

Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.     

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

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

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(alpha) 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(alpha15) 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.     

Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

Functional analysis of a subset of canine olfactory receptor genes

In this paper, we explored the level complexity of the combinatorial olfactory code that allows mammals with a repertoire of about thousand putatively active olfactory receptors encoded in their genomes to recognize and identify a much larger repertoire of odorant molecules. To that end, we cloned 38 canine OR genes belonging to the same OR gene family and transiently expressed them in a subclone of embryonic human kidney cells (HEK293) permanently expressing the G(olf) subunit. Using a Ca(2+) imaging approach, we established for example that as many as 26 out of the 38 cloned OR elicited a Ca(2+) response when exposed to octanal, whereas 10 responded to nonanal, other aldehydes providing intermediate responses. Altogether, these results demonstrated that the combinatorial code is quite complex in support to the highly developed sense of olfaction demonstrated by dogs.     

A new concept of olfactory biosensor based on interdigitated microelectrodes and immobilized yeasts expressing the human receptor OR17-40

This work shows the feasibility of an olfactory biosensor based on the immobilization of Saccharomyces cerevisiae yeast cells genetically modified to express the human olfactory receptor OR17-40 onto interdigitated microconductometric electrodes. This olfactory biosensor has been applied to the detection of its specific odorant (helional) with a high sensitivity (threshold 10⁻¹⁴ M). In contrast, no significant response was observed using a non-specific odorant (heptanal), which suggests a good selectivity. Thus, this work may represent a first step towards a new kind of bioelectronic noses based on whole yeast cells and allowing a real time monitoring of olfactory receptor activation. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet, France, 14–19 October, 2006.     

RTP family members induce functional expression of mammalian odorant receptors

Transport of G protein-coupled receptors (GPCRs) to the cell surface membrane is critical in order for the receptors to recognize their ligands. However, mammalian GPCR odorant receptors (ORs), when heterologously expressed in cells, are poorly expressed on the cell surface. Here we show that the transmembrane proteins RTP1 and RTP2 promote functional cell surface expression of ORs expressed in HEK293T cells. Genes encoding these proteins are expressed specifically in olfactory neurons. These proteins are associated with OR proteins and enhance the OR responses to odorants. Similar although weaker effects were seen with a third protein, REEP1. These findings suggest that RTP1 and RTP2 in particular play significant roles in the translocation of ORs to the plasma membrane as well as in the functioning of ORs. We have used this approach to identify active odorant ligands for ORs, providing a platform for screening the chemical selectivity of the large OR family.     

Molecular and cellular basis of human olfaction

The human olfactory systems recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein in the ciliary membrane of olfactory neurons. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed, and characterized some of the human olfactory receptors from chromosome 17. Our results show that a receptor protein is capable of recognizing the particular chemical substructure of an odor molecule and, therefore, is able to respond only to odorants that have a defined molecular structure. These findings represent the beginning of the molecular understanding of odorant recognition in humans. In the future, this knowledge could be used for the design of synthetic ideal receptors for specific odors (biosensors), or the perfect odor molecule for a given receptor.     

The N-terminal replacement of an olfactory receptor for the development of a yeast-based biomimetic odor sensor

For the development of a biomimetic odor-sensing system, we investigated the effects of replacing the N-terminus of an olfactory receptor (OR) on its functional expression in the budding yeast, Saccharomyces cerevisiae. Using the mouse olfactory receptor OR226 (mOR226), three types of chimeric ORs were constructed by replacing N-terminal regions of mOR226 with the corresponding regions of the rat I7 receptor, which is known to be functionally expressed in yeast. The replacement of the N-terminal region of mOR226 dramatically affected the expression and localization of the receptor and improved the sensing ability of the yeast cells for the odorant. Furthermore, the replacement of the endogenous yeast G-protein α subunit (Gpa1) by the OR-specific G(olf) drastically elevated the odorant-sensing ability of the yeast cells and caused the cells to display a dose-dependent responsiveness to the odorant. Because of the suitability of yeast cells for screening large-scale libraries, the strategy presented here would be useful for the establishment of advanced biomimetic odor-sensing systems.     

Deorphanizing vertebrate olfactory receptors: recent advances in odorant-response assays

Olfactory receptors (ORs) comprise the largest multigene G protein-coupled receptor families in organisms from fish to primates, and play a critical role in recognizing thousands of odorant molecules. Recent achievement of functional OR expression in heterologous cells led to identification of ligands for some ORs, revealing a combinatorial receptor coding scheme in the olfactory sensory system. Using the functional assay, the odorant-binding site in ORs has been elucidated, showing that a binding pocket constructed by transmembrane helices provides the molecular basis for agonist and antagonist specificity. To retrospectively identify ORs that recognize a particular odorant of interest, two functional cloning strategies have been developed: one is a strategy wherein OR genes are amplified from single olfactory neurons that show odorant responsiveness in Ca(2+) imaging, and another is an approach based on glomerular activity by combining in vivo bulbar Ca(2+) imaging and retrograde dye labeling of innervating olfactory neurons. The conventional ligand-screening approach and the functional cloning strategies in an odorant-directed manner have allowed us to match ORs to the cognate odorants both in vitro and in vivo.