Petrol Note in Riesling - 1,1,6-Trimethyl-1,2-dihydronaphthalene (TDN) Selectively Activates Human Odorant Receptor OR8H1.

Grapevine (Vitis vinifera) is one of the most important perennial fruit plants. The variety Riesling stands out by developing a characteristic petrol-like odor note during aging, elicited by the aroma compound 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN). The UV-dependent TDN contents differ largely among Rieslings grown in the northern versus the southern hemisphere. Highest TDN concentrations were found in Australian Rieslings, where TDN is a scoring ingredient. In contrast, in Rieslings from Europe, for example, TDN may be a tending cause of rejection. A human receptor for TDN has been unknown. Here, we report on the identification of OR8H1 as a TDN-selective odorant receptor, out of a library of 766 odorant receptor variants. OR8H1 is selectively tuned to six carbon ring structures, identified by screening a collection of 180 key food odorants, using a HEK-293 cell-based cAMP luminescence assay equipped with the GloSensor technology.

Heterodimerization of Chemoreceptors TAS1R3 and mGlu2 in Human Blood Leukocytes.

The expression of canonical chemosensory receptors of the tongue, such as the heteromeric sweet taste (TAS1R2/TAS1R3) and umami taste (TAS1R1/TAS1R3) receptors, has been demonstrated in many extra-oral cells and tissues. Gene expression studies have revealed transcripts for all TAS1 and metabotropic glutamate (mGlu) receptors in different types of immune cells, where they are involved, for example, in the chemotaxis of human neutrophils and the protection of T cells from activation-induced cell death. Like other class-C G protein-coupling receptors (GPCRs), TAS1Rs and mGlu receptors form heteromers within their families. Since mGlu receptors and TAS1R1/TAS1R3 share the same ligand, monosodium glutamate (MSG), we hypothesized their hitherto unknown heteromerization across receptor families in leukocytes. Here we show, by means of immunocytochemistry and co-IP/Western analysis, that across class-C GPCR families, mGlu2 and TAS1R3 co-localize and heterodimerize in blood leukocytes. Expressing the recombinant receptors in HEK-293 cells, we validated their heterodimerization by bioluminescence resonance energy transfer. We demonstrate MSG-induced, mGlu2/TAS1R3 heteromer-dependent gain-of-function and pertussis toxin-sensitive signaling in luminescence assays. Notably, we show that mGlu2/TAS1R3 is necessary and sufficient for MSG-induced facilitation of N-formyl-methionyl-leucyl-phenylalanine-stimulated IL-8 secretion in neutrophils, using receptor-specific antagonists. In summary, our results demonstrate mGlu2/TAS1R3 heterodimerization in leukocytes, suggesting cellular function-tailored chemoreceptor combinations to modulate cellular immune responses.

The multi-faceted food odorant 4-methylphenol selectively activates evolutionary conserved receptor OR9Q2.

4-Methylphenol is a food-related odor-active volatile with a high recognition factor, due to its horse stable-like, fecal odor quality. Its ambivalent hedonic impact as key aroma compound, malodor, and semiochemical has spurred the search for its cognate, chemosensory odorant receptors across species. A human odorant receptor for the highly characteristic 4-methylphenol has been elusive. Here, we identified and characterized human receptor OR9Q2 to be tuned to purified 4-methylphenol, but not to its contaminant isomer 3-methylphenol. This highly selective function of OR9Q2 complements an exclusive phenol detection gap in the ancient, most broadly tuned human odorant receptor OR2W1. Moreover, a 4-methylphenol function is evolutionary conserved in phylogenetically related OR9Q2 orthologs from chimpanzee, mouse, and cow. Notably, the cow receptor outperformed human OR9Q2 10-fold in signal strength, consonant with previous reports of 4-methylphenol as a bovine pheromone. Our results suggest OR9Q2 as best sensor for the key food odorant, malodor, and semiochemical 4-methylphenol.

Sweetener System Intervention Shifted Neutrophils from Homeostasis to Priming.

BACKGROUND: Non-nutritive sweeteners (NNS) are part of personalized nutrition strategies supporting healthy glycemic control. In contrast, the consumption of non-nutritive sweeteners has been related to person-specific and microbiome-dependent glycemic impairments. Reports on the effects of NNS on our highly individual cellular immune system are sparse. The recent identification of taste receptor expression in a variety of immune cells, however, suggested their immune-modulatory relevance. METHODS: We studied the influence of a beverage-typical NNS system on the transcriptional profiling of sweetener-cognate taste receptors, selected cytokines and their receptors, and on Ca2+ signaling in isolated blood neutrophils. We determined plasma concentrations of saccharin, acesulfame-K, and cyclamate by HPLC-MS/MS, upon ingestion of a soft drink-typical sweetener surrogate. In an open-labeled, randomized intervention study, we determined pre- versus post-intervention transcript levels by RT-qPCR of sweetener-cognate taste receptors and immune factors. RESULTS: Here we show that the consumption of a food-typical sweetener system modulated the gene expression of cognate taste receptors and induced the transcriptional regulation signatures of early homeostasis- and late receptor/signaling- and inflammation-related genes in blood neutrophils, shifting their transcriptional profile from homeostasis to priming. Notably, sweeteners at postprandial plasma concentrations facilitated fMLF (N-formyl-Met-Leu-Phe)-induced Ca2+ signaling. CONCLUSIONS: Our results support the notion of sweeteners priming neutrophils to higher alertness towards their adequate stimuli.

Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1.

With approximately 400 encoding genes in humans, odorant receptors (ORs) are the largest subfamily of class A G protein-coupled receptors (GPCRs). Despite its high relevance and representation, the odorant-GPCRome is structurally poorly characterized: no experimental structures are available, and the low sequence identity of ORs to experimentally solved GPCRs is a significant challenge for their modeling. Moreover, the receptive range of most ORs is unknown. The odorant receptor OR5K1 was recently and comprehensively characterized in terms of cognate agonists. Here, we report two additional agonists and functional data of the most potent compound on two mutants, L1043.32 and L2556.51. Experimental data was used to guide the investigation of the binding modes of OR5K1 ligands into the orthosteric binding site using structural information from AI-driven modeling, as recently released in the AlphaFold Protein Structure Database, and from homology modeling. Induced-fit docking simulations were used to sample the binding site conformational space for ensemble docking. Mutagenesis data guided side chain residue sampling and model selection. We obtained models that could better rationalize the different activity of active (agonist) versus inactive molecules with respect to starting models and also capture differences in activity related to minor structural differences. Therefore, we provide a model refinement protocol that can be applied to model the orthosteric binding site of ORs as well as that of GPCRs with low sequence identity to available templates.

[6]-Gingerol Facilitates CXCL8 Secretion and ROS Production in Primary Human Neutrophils by Targeting the TRPV1 Channel.

SCOPE: Clarifying the function of sensory active TRP (transient receptor potential) channels in non-sensory tissue is of growing interest, especially with regard to food ingredients in nutritionally relevant concentrations. The study hypothesizes the TRPV1 agonist [6]-gingerol to facilitate cellular immune responses of primary human neutrophils, after treatment with 50 nM, a concentration that can be reached in the circulation after habitual dietary intake. METHODS AND RESULTS: qRT-PCR analyses reveal a high abundancy of TRP channel RNA expression in the types of primary leukocytes investigated, namely neutrophils, monocytes, NK cells, T cells, and B cells. Incubation of neutrophils with 50 nM of the known TRPV1 ligand [6]-gingerol led to increased surface expression of CD11b, CD66b, and the fMLF receptor FPR1, as shown by flow cytometry. Upon subsequent stimulation with fMLF, the neutrophils display an about 30% (p < 0.05) increase in CXCL8 secretion as well as in ROS production. Pharmacological inhibition of TRPV1 by trans-tert-butylcyclohexanol abolishes the [6]-gingerol induced effects. CONCLUSIONS: The TRPV1 channel is functionally expressed in human neutrophils. Activation of the channel with [6]-gingerol as a food-derived ligand in nutritionally relevant concentrations leads to an enhanced responsiveness in the cells towards activating stimuli, thereby facilitating a canonical cellular immune response in human neutrophils.

Metabolites of Key Flavor Compound 2,3,5-Trimethylpyrazine in Human Urine.

Pyrazines are among the most important compound class conveying the odor impressions "roasty", "nutty", and "earthy". They are formed by the Maillard reaction and occur ubiquitously in heated foods. The excretion of metabolites of the key flavor odorant 2,3,5-trimethylpyrazine, abundant in the volatile fraction of roasted coffee, was investigated. Based on literature suggestions, putative phase 1 and phase 2 metabolites were synthesized, characterized by nuclear magnetic resonance and mass spectroscopy data and used as standards for targeted, quantitative analysis of coffee drinkers' urine using stable-isotope-dilution-ultrahigh-performance liquid chromatography tandem mass spectroscopy (SIDA-UHPLC-MS/MS). The analysis of spot urine samples from a coffee intervention study revealed 3,6-dimethylpyrazine-2-carboxylic acid, 3,5-dimethylpyrazine-2-carboxylic acid, and 5,6-dimethylpyrazine-2-carboxylic acid were quantitatively dominating metabolites. Only negligible traces of pyrazinemethanols (3,6-dimethyl-2-pyrazinemethanol and 3,5,6-trimethylpyrazine-2-ol), glucuronides ((3,6-dimethylpyrazine-2-yl-)methyl-O-ß-D-glucuronide and (3,5-dimethylpyrazine-2-yl-)methyl-O-ß-D-glucuronide), and sulfates ((3,6-dimethylpyrazine-2-yl-)methyl-sulfate and (3,5-dimethylpyrazine-2-yl-)methyl-sulfate) were detected.

The human odorant receptor OR10A6 is tuned to the pheromone of the commensal fruit fly Drosophila melanogaster.

All living things speak chemistry. The challenge is to reveal the vocabulary, the odorants that enable communication across phylogenies and to translate them to physiological, behavioral, and ecological function. Olfactory receptors (ORs) interface animals with airborne odorants. Expression in heterologous cells makes it possible to interrogate single ORs and to identify cognate ligands. The cosmopolitan, anthropophilic strain of the vinegar fly Drosophila melanogaster depends on human resources and housing for survival. Curiously, humans sense the pheromone (Z)-4-undecenal (Z4-11Al) released by single fly females. A screening of all human ORs shows that the most highly expressed OR10A6 is tuned to Z4-11Al. Females of an ancestral African fly strain release a blend of Z4-11Al and Z4-9Al that produces a different aroma, which is how we distinguish these fly strains by nose. That flies and humans sense Z4-11Al via dedicated ORs shows how convergent evolution shapes communication channels between vertebrate and invertebrate animals.

The key food odorant receptive range of broadly tuned receptor OR2W1.

Mammals perceive a multitude of odorants by their chemical sense of olfaction, a high-dimensional stimulus-detection system, with hundreds of narrowly or broadly tuned receptors, enabling pattern recognition by the brain. Cognate receptor-agonist information, however, is sparse, and the role of broadly tuned odorant receptors for encoding odor quality remains elusive. Here, we screened IL-6-HaloTag®-OR2W1 and haplotypes against 187 out of 230 defined key food odorants using the GloSensor™ system in HEK-293 cells, yielding 48 new agonists. Altogether, key food odorants represent about two-thirds of now 153 reported agonists of OR2W1, the highest number of agonists known for a mammalian odorant receptor. In summary, we characterized OR2W1 as a human odorant receptor, with a chemically diverse but exclusive receptive range, complementary to chemical subgroups covered by evolutionary younger, highly selective receptors. Our data suggest OR2W1 to be suited for participating in the detection of many foodborne odorants.

Key Food Furanones Furaneol and Sotolone Specifically Activate Distinct Odorant Receptors.

Furanones formed during the Maillard reaction often are natural aroma-determining compounds found in numerous foods. Prominent economically relevant representatives are the structural homologues Furaneol and sotolone, which are important natural flavoring compounds because of their distinct caramel- and seasoning-like odor qualities. These, however, cannot be predicted by the odorants' molecular shape, rather their receptors' activation parameters help to decipher the encoding of odor quality. Here, the distinct odor qualities of Furaneol and sotolone suggested an activation of at least two out of our ca. 400 different odorant receptor types, which are the molecular biosensors of our chemical sense of olfaction. While an odorant receptor has been identified for sotolone, a receptor specific for Furaneol has been elusive. Using a bidirectional screening approach employing 616 receptor variants and 187 key food odorants in a HEK-293 cell-based luminescence assay, we newly identified OR5M3 as a receptor specifically activated by Furaneol and homofuraneol.

An evolutionary conserved olfactory receptor for foodborne and semiochemical alkylpyrazines.

Molecular recognition is a fundamental principle in biological systems. The olfactory detection of both food and predators via ecological relevant odorant cues are abilities of eminent evolutionary significance for many species. Pyrazines are such volatile cues, some of which act as both human-centered key food odorants (KFOs) and semiochemicals. A pyrazine-selective odorant receptor has been elusive. Here we screened 2,3,5-trimethylpyrazine, a KFO and semiochemical, and 2,5-dihydro-2,4,5-trimethylthiazoline, an innate fear-associated non-KFO, against 616 human odorant receptor variants, in a cell-based luminescence assay. OR5K1 emerged as sole responding receptor. Tested against a comprehensive collection of 178 KFOs, we newly identified 18 pyrazines and (2R/2S)-4-methoxy-2,5-dimethylfuran-3(2H)-one as agonists. Notably, OR5K1 orthologs in mouse and domesticated species displayed a human-like, potency-ranked activation pattern of pyrazines, suggesting a domestication-led co-evolution of OR5K1 and its orthologs. In summary, OR5K1 is a specialized olfactory receptor across mammals for the detection of pyrazine-based key food odors and semiochemicals.

Conserved C-terminal motifs in odorant receptors instruct their cell surface expression and cAMP signaling.

The highly individual plasma membrane expression and cAMP signaling of odorant receptors have hampered their ligand assignment and functional characterization in test cell systems. Chaperones have been identified to support the cell surface expression of only a portion of odorant receptors, with mechanisms remaining unclear. The presence of amino acid motifs that might be responsible for odorant receptors' individual intracellular retention or cell surface expression, and thus, for cAMP signaling, is under debate: so far, no such protein motifs have been suggested. Here, we demonstrate the existence of highly conserved C-terminal amino acid motifs, which discriminate at least between class-I and class-II odorant receptors, with their numbers of motifs increasing during evolution, by comparing C-terminal protein sequences from 4808 receptors across eight species. Truncation experiments and mutation analysis of C-terminal motifs, largely overlapping with helix 8, revealed single amino acids and their combinations to have differential impact on the cell surface expression and on stimulus-dependent cAMP signaling of odorant receptors in NxG 108CC15 cells. Our results demonstrate class-specific and individual C-terminal motif equipment of odorant receptors, which instruct their functional expression in a test cell system, and in situ may regulate their individual cell surface expression and intracellular cAMP signaling.

A Phenotyping Platform to Characterize Healthy Individuals Across Four Stages of Life - The Enable Study.

Introduction: Nutritional habits and requirements are changing over the lifespan, but the dynamics of nutritional issues and the diet-health relationship in the major stages of the human life cycle are not sufficiently understood. A human phenotyping research platform for nutrition studies was established to recruit and phenotype selected population groups across different stages of life. The project is the backbone of the highly interdisciplinary enable competence cluster of nutrition research aiming to identify dietary determinants of a healthy life throughout the lifespan and to develop healthier and tasty convenience foods with high consumer acceptance. Methods: The phenotyping program included anthropometry, body composition analysis, assessment of energy metabolism, health and functional status, multisensory perception, metabolic phenotyping, lifestyle, sociodemography, chronobiology, and assessment of dietary intake including food preferences and aversions. Results: In total, 503 healthy volunteers at four defined phases of life including 3-5-year old children (n = 44), young adults aged 18-25 years (n = 94), adults aged 40-65 years ("middle agers," n = 205), and older adults aged 75-85 years (n = 160) were recruited and comprehensively phenotyped. Plasma, serum, buffy coat, urine, feces and saliva samples were collected and stored at -80°C. Significant differences in anthropometric and metabolic parameters between the four groups were found. A major finding was the decrease in fat-free mass and the concomitant increase in % body fat in both sexes across the adult lifespan. Conclusions: The dataset will provide novel information on differences in diet-related parameters over the lifespan and is available for targeted analyses. We expect that this novel platform approach will have implications for the development of innovative food products tailored to promote healthy eating throughout life. Trial registration: DRKS, DRKS00009797. Registered on 20 January 2016, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&_ID=DRKS00009797.

Copper-mediated thiol potentiation and mutagenesis-guided modeling suggest a highly conserved copper-binding motif in human OR2M3.

Sulfur-containing compounds within a physiological relevant, natural odor space, such as the key food odorants, typically constitute the group of volatiles with the lowest odor thresholds. The observation that certain metals, such as copper, potentiate the smell of sulfur-containing, metal-coordinating odorants led to the hypothesis that their cognate receptors are metalloproteins. However, experimental evidence is sparse-so far, only one human odorant receptor, OR2T11, and a few mouse receptors, have been reported to be activated by sulfur-containing odorants in a copper-dependent way, while the activation of other receptors by sulfur-containing odorants did not depend on the presence of metals. Here we identified an evolutionary conserved putative copper interaction motif CC/CSSH, comprising two copper-binding sites in TMH5 and TMH6, together with the binding pocket for 3-mercapto-2-methylpentan-1-ol in the narrowly tuned human receptor OR2M3. To characterize the copper-binding motif, we combined homology modeling, docking studies, site-directed mutagenesis, and functional expression of recombinant ORs in a cell-based, real-time luminescence assay. Ligand activation of OR2M3 was potentiated in the presence of copper. This effect of copper was mimicked by ionic and colloidal silver. In two broadly tuned receptors, OR1A1 and OR2W1, which did not reveal a putative copper interaction motif, activation by their most potent, sulfur-containing key food odorants did not depend on the presence of copper. Our results suggest a highly conserved putative copper-binding motif to be necessary for a copper-modulated and thiol-specific function of members from three subfamilies of family 2 ORs.

Beyond the Flavour: The Potential Druggability of Chemosensory G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) belong to the largest class of drug targets. Approximately half of the members of the human GPCR superfamily are chemosensory receptors, including odorant receptors (ORs), trace amine-associated receptors (TAARs), bitter taste receptors (TAS2Rs), sweet and umami taste receptors (TAS1Rs). Interestingly, these chemosensory GPCRs (csGPCRs) are expressed in several tissues of the body where they are supposed to play a role in biological functions other than chemosensation. Despite their abundance and physiological/pathological relevance, the druggability of csGPCRs has been suggested but not fully characterized. Here, we aim to explore the potential of targeting csGPCRs to treat diseases by reviewing the current knowledge of csGPCRs expressed throughout the body and by analysing the chemical space and the drug-likeness of flavour molecules.

Food sources and biomolecular targets of tyramine.

Tyramine is a biogenic trace amine that is generated via the decarboxylation of the amino acid tyrosine. At pico- to nanomolar concentrations, it can influence a multitude of physiological mechanisms, exhibiting neuromodulatory properties as well as cardiovascular and immunological effects. In humans, the diet is the primary source of physiologically relevant tyramine concentrations, which are influenced by a large number of intrinsic and extrinsic factors. Among these factors are the availability of tyrosine in food, the presence of tyramine-producing bacteria, the environmental pH, and the salt content of food. The process of fermentation provides a particularly good source of tyramine in human nutrition. Here, the potential impact of dietary tyramine on human health was assessed by compiling quantitative data on the tyramine content in a variety of foods and then conducting a brief review of the literature on the physiological, cellular, and systemic effects of tyramine. Together, the data sets presented here may allow both the assessment of tyramine concentrations in food and the extrapolation of these concentrations to gauge the physiological and systemic effects in the context of human nutrition.

Current Status and Future Perspectives in Flavor Research: Highlights of the 11th Wartburg Symposium on Flavor Chemistry & Biology.

The 11th Wartburg Symposium on Flavor Chemistry & Biology, held at the hotel "Auf der Wartburg" in Eisenach, Germany, from June 21 to 24 in 2016, offered a venue for global exchange on cutting-edge research in chemistry and biology of odor and taste. The focus areas were (1) functional flavor genomics and biotechnology, (2) flavor generation and precursors, (3) new approaches and precursors, (4) new approaches and technologies, (5) new molecules and structure/activity relationships, (6) food-borne bioactives and chemosensory health prevention, and (7) chemosensory reception, processing, and perception. Selected from more than 250 applicants, 160 distinguished scientists and rising stars from academia and industry from 24 countries participated in this multidisciplinary event. This special issue comprises a selection of 33 papers from oral presentations and poster contributions and is prefaced by this symposium introduction to carve out essential achievements in odor and taste chemistry and to share future research perspectives.

Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1.

Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(-)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(-)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure-function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(-)-carvone-specific anosmia in humans.

IL-6-HaloTag® enables live-cell plasma membrane staining, flow cytometry, functional expression, and de-orphaning of recombinant odorant receptors.

The assignment of cognate odorant/agonist pairs is a prerequisite for an understanding of odorant coding at the receptor level. However, the identification of new ligands for odorant receptors (ORs) in cell-based assays has been challenging, due to their individual and rather sub-optimal plasma membrane expression, as compared with other G protein-coupled receptors. Accessory proteins, such as the chaperone RTP1S, or Ric8b, have improved the surface expression of at least a portion of ORs. Typically, recombinant ORs carry N-terminal tags, which proved helpful for their functional membrane expression. The most common tag is the 'Rho-tag', representing an N-terminal part of rhodopsin, but also 'Lucy-' or 'Flag-tag' extensions have been described. Here, we used a bi-functional N-terminal tag, called 'interleukin 6 (IL-6)-HaloTag®', with IL-6 facilitating functional cell surface expression of recombinant ORs, and the HaloTag® protein, serving as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by live-cell flow cytometry. Our experiments revealed on average an about four-fold increased surface expression, a four-fold higher signaling amplitude, and a significantly higher potency of odorant-induced cAMP signaling of six different human IL-6-HaloTag®-ORs across five different receptor families in NxG 108CC15 cells, as compared to their Rho-tag-HaloTag® constructs. We observed similar results in HEK-293 cells. Moreover, screening an IL-6-HaloTag®-odorant receptor library with allyl phenyl acetate, revealed both known receptors as best responders for this compound. In summary, the IL-6-HaloTag® represents a promising tool for the de-orphaning of ORs.

A bi-functional IL-6-HaloTag® as a tool to measure the cell-surface expression of recombinant odorant receptors and to facilitate their activity quantification.

The functional cell surface expression of recombinant odorant receptors typically has been investigated by expressing N-terminally extended, "tagged" receptors in test cell systems, using antibody-based immunocytochemistry or flow cytometry, and by measuring odorant/receptor-induced cAMP signaling, mostly by an odorant/receptor-induced and cAMP signaling-dependent transcriptional activation of a luciferase-based luminescence assay. In the present protocol, we explain a method to measure the cell-surface expression and signaling of recombinant odorant receptors carrying a bi-functional, N-terminal 'IL-6-HaloTag®'. IL-6, being a secreted cytokine, facilitates functional cell surface expression of recombinant HaloTag®-odorant receptors, and the HaloTag® protein serves as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by antibody-independent, chemical live-cell staining and flow cytometry. Here, we describe how to measure the cell surface expression of recombinant IL-6-HaloTag®-odorant receptors in HEK-293 cells or NxG 108CC15 cells, by live-cell staining and flow cytometry, and how to measure an odorant-induced activation of these receptors by the fast, real-time, luminescence-based GloSensor® cAMP assay.