Human Olfactory Receptors: Novel Cellular Functions Outside of the Nose.

Olfactory receptors (ORs) are not exclusively expressed in the olfactory sensory neurons; they are also observed outside of the olfactory system in all other human tissues tested to date, including the testis, lung, intestine, skin, heart, and blood. Within these tissues, certain ORs have been determined to be exclusively expressed in only one tissue, whereas other ORs are more widely distributed in many different tissues throughout the human body. For most of the ectopically expressed ORs, limited data are available for their functional roles. They have been shown to be involved in the modulation of cell-cell recognition, migration, proliferation, the apoptotic cycle, exocytosis, and pathfinding processes. Additionally, there is a growing body of evidence that they have the potential to serve as diagnostic and therapeutic tools, as ORs are highly expressed in different cancer tissues. Interestingly, in addition to the canonical signaling pathways activated by ORs in olfactory sensory neurons, alternative pathways have been demonstrated in nonolfactory tissues. In this review, the existing data concerning the expression, as well as the physiological and pathophysiological functions, of ORs outside of the nose are highlighted to provide insights into future lines of research.

Modulation of the combinatorial code of odorant receptor response patterns in odorant mixtures.

The perception of odors relies on combinatorial codes consisting of odorant receptor (OR) response patterns to encode odor identity. Modulation of these patterns by odorant interactions at ORs potentially explains several olfactory phenomena: mixture suppression, unpredictable sensory outcomes, and the perception of odorant mixtures as unique objects. We determined OR response patterns to 4 odorants and 3 binary mixtures in vivo in mice, identifying 30 responsive ORs. These patterns typically had a few strongly responsive ORs and a greater number of weakly responsive ORs. ORs responsive to an odorant were often unrelated sequences distributed across several OR subfamilies. Mixture responses predicted pharmacological interactions between odorants, which were tested in vitro by heterologous expression of ORs in cultured cells, providing independent evidence confirming odorant agonists for 13 ORs and identifying both suppressive and additive effects. This included 11 instances of antagonism of ORs by an odorant, 1 instance of additive responses to a binary mixture, 1 instance of suppression of a strong agonist by a weak agonist, and the discovery of an inverse agonist for an OR. Interactions between odorants at ORs are common even when the odorants are not known to interact perceptually in humans, and in some cases interactions at mouse ORs correlate with the ability of humans to perceive an odorant in a mixture.

Machine Learning Assisted Approach for Finding Novel High Activity Agonists of Human Ectopic Olfactory Receptors.

Olfactory receptors (ORs) constitute the largest superfamily of G protein-coupled receptors (GPCRs). ORs are involved in sensing odorants as well as in other ectopic roles in non-nasal tissues. Matching of an enormous number of the olfactory stimulation repertoire to its counterpart OR through machine learning (ML) will enable understanding of olfactory system, receptor characterization, and exploitation of their therapeutic potential. In the current study, we have selected two broadly tuned ectopic human OR proteins, OR1A1 and OR2W1, for expanding their known chemical space by using molecular descriptors. We present a scheme for selecting the optimal features required to train an ML-based model, based on which we selected the random forest (RF) as the best performer. High activity agonist prediction involved screening five databases comprising ~23 M compounds, using the trained RF classifier. To evaluate the effectiveness of the machine learning based virtual screening and check receptor binding site compatibility, we used docking of the top target ligands to carefully develop receptor model structures. Finally, experimental validation of selected compounds with significant docking scores through in vitro assays revealed two high activity novel agonists for OR1A1 and one for OR2W1.

Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors.

Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.

Position Review: Functional Selectivity in Mammalian Olfactory Receptors.

There is increasing appreciation that G-protein-coupled receptors (GPCRs) can initiate diverse cellular responses by activating multiple G proteins, arrestins, and other biochemical effectors. Structurally different ligands targeting the same receptor are thought to stabilize the receptor in multiple distinct active conformations such that specific subsets of signaling effectors are engaged at the exclusion of others, creating a bias toward a particular outcome, which has been referred to as ligand-induced selective signaling, biased agonism, ligand-directed signaling, and functional selectivity, among others. The potential involvement of functional selectivity in mammalian olfactory signal transduction has received little attention, notwithstanding the fact that mammalian olfactory receptors comprise the largest family of mammalian GPCRs. This position review considers the possibility that, although such complexity in G-protein function may have been lost in the specialization of olfactory receptors to serve as sensory receptors, the ability of olfactory receptor neurons (ORNs) to function as signal integrators and growing appreciation that this functionality is widespread in the receptor population suggest otherwise. We pose that functional selectivity driving 2 opponent inputs have the potential to generate an output that reflects the balance of ligand-dependent signaling, the direction of which could be either suppressive or synergistic and, as such, needs to be considered as a mechanistic basis for signal integration in mammalian ORNs.

Mammalian class I odorant receptors exhibit a conserved vestibular-binding pocket.

Odorant receptors represent the largest family of mammalian G protein-coupled receptors. Phylogenetically, they are split into two classes (I and II). By analyzing the entire subclass I odorant receptors sequences, we identified two class I-specific and highly conserved motifs. These are predicted to face each other at the extra-cellular portion of the transmembrane domain, forming a vestibular site at the entrance to the orthosteric-binding cavity. Molecular dynamics simulation combined with site-directed mutagenesis and in vitro functional assays confirm the functional role of this vestibular site in ligand-driven activation. Mutations at this part of the receptor differentially affect the receptor response to four agonists. Since this vestibular site is involved in ligand recognition, it could serve ligand design that targets specifically this sub-genome of mammalian odorant receptors.

Characterizing novel olfactory receptors expressed in the murine renal cortex.

The kidney uses specialized G protein-coupled receptors, including olfactory receptors (ORs), to act as sensors of molecules and metabolites. In the present study, we cloned and studied seven renal ORs, which we previously found to be expressed in the murine renal cortex. As most ORs are orphan receptors, our goal was to identify ligands for these ORs in the hope that this will guide future research into their functional roles. We identified novel ligands for two ORs: Olfr558 and Olfr90. For Olfr558, we confirmed activation by previously reported ligands and identified 16 additional carboxylic acids that activated this OR. The strongest activation of Olfr558 was produced by butyric, cyclobutanecarboxylic, isovaleric, 2-methylvaleric, 3-methylvaleric, 4-methylvaleric, and valeric acids. The primary in vivo source of both butyric and isovaleric acids is gut microbial metabolism. We also identified 14 novel ligands that activated Olfr90, the strongest of which were 2-methyl-4-propyl-1,3-oxathiane, 1-octen-3-ol, 2-octanol, and 3-octanol. Interestingly, 8 of these 14 ligands are of fungal origin. We also investigated the tissue distribution of these receptors and found that they are each found in a subset of "nonsensory" tissues. Finally, we examined the putative human orthologs of Olfr558 and Olfr90 and found that the human ortholog of Olfr558 (OR51E1) has a similar ligand profile, indicating that the role of this OR is likely evolutionarily conserved. In summary, we examined seven novel renal ORs and identified new ligands for Olfr558 and Olfr90, which imply that both of these receptors serve to detect metabolites produced by microorganisms.

Small-chain fatty acid activates astrocytic odorant receptor Olfr920.

Odorant receptors are the largest subfamily of G protein-coupled receptors and were recently suggested to play critical roles in nonolfactory tissues. However, the expression and function of odorant receptors in astrocytes, the most abundant cells in the brain, are not well known. We demonstrate that Olfr920 is highly expressed and propose that it functions as a short-chain fatty acid sensor in primary cortical astrocytes. The short-chain fatty acid isobutyric acid (IBA) was identified via a luciferase assay as an Olfr920 ligand. We show that IBA activates the Gs protein-adenylyl cyclase-cAMP pathway via Olfr920 in primary cortical astrocytes by using cAMP and knockdown analyses. In addition, IBA reduces lipopolysaccharide-induced glial fibrillary acidic protein expression in reactive astrocytes. These results suggest that astrocytic Olfr920 is a potential novel target for increased reactive astrocytes.

Effects of lactate ions on the cardiorespiratory system in rainbow trout (Oncorhynchus mykiss).

Lactate ions are involved in several physiological processes, including a direct stimulation of the carotid body, causing increased ventilation in mammals. A similar mechanism eliciting ventilatory stimulation in other vertebrate classes has been demonstrated, but it remains to be thoroughly investigated. Here, we investigated the effects of lactate ions on the cardiorespiratory system in swimming rainbow trout by manipulating the blood lactate concentration. Lactate elicited a vigorous, dose-dependent elevation of ventilation and bradycardia at physiologically relevant concentrations at constant pH. After this initial confirmation, we examined the chiral specificity of the response and found that only l-lactate induced these effects. By removal of the afferent inputs from the first gill arch, the response was greatly attenuated, and a comparison of the responses to injections up- and downstream of the gills collectively demonstrated that the lactate response was initiated by branchial cells. Injection of specific receptor antagonists revealed that a blockade of serotonergic receptors, which are involved in the hypoxic ventilatory response, significantly reduced the lactate response. Finally, we identified two putative lactate receptors based on sequence homology and found that both were expressed at substantially higher levels in the gills. We propose that lactate ions modulate ventilation by stimulating branchial oxygen-sensing cells, thus eliciting a cardiorespiratory response through receptors likely to have originated early in vertebrate evolution.

Optimization of Insect Odorant Receptor Trafficking and Functional Expression Via Transient Transfection in HEK293 Cells.

Insect odorant receptors (ORs) show a limited functional expression in various heterologous expression systems including insect and mammalian cells. This may be in part due to the absence of key components driving the release of these proteins from the endoplasmic reticulum and directing them to the plasma membrane. In order to mitigate this problem, we took advantage of small export signals within the human HCN1 and Rhodopsin that have been shown to promote protein release from the endoplasmic reticulum and the trafficking of post-Golgi vesicles, respectively. Moreover, we designed a new vector based on a bidirectional expression cassette to drive the functional expression of the insect odorant receptor coreceptor (Orco) and an odor-binding OR, simultaneously. We show that this new method can be used to reliably express insect ORs in HEK293 cells via transient transfection and that is highly suitable for downstream applications using automated and high-throughput imaging platforms.

Drosophila Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit.

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.

Odorant Receptor 7D4 Activation Dynamics.

Deciphering how an odorant activates an odorant receptor (OR) and how changes in specific OR residues affect its responsiveness are central to understanding our sense of smell. A joint approach combining site-directed mutagenesis and functional assays with computational modeling has been used to explore the signaling mechanics of OR7D4. In this OR, a genetic polymorphism affects our perception of androstenone. Molecular simulations totaling 0.12 ms predicted that, similarly to observations for other G-protein-coupled receptors with known experimental structures, an activation pathway connects the ligand and the G-protein binding site. The 3D model activation mechanism correlates with in vitro data and notably predicts that the OR7D4 WM variant is not activated. Upon activation, an OR-specific sequence motif is the convergence point of the mechanism. Our study suggests that robust homology modeling can serve as a powerful tool to capture OR dynamics related to smell perception.

Two olfactory receptors-OR2A4/7 and OR51B5-differentially affect epidermal proliferation and differentiation.

Olfactory receptors (ORs), which belong to the G-protein coupled receptor family, are expressed in various human tissues, including skin. Cells in non-olfactory tissues tend to express more than one individual OR gene, but function and interaction of two or more ORs in the same cell type has only been marginally analysed. Here, we revealed OR2A4/7 and OR51B5 as two new ORs in human skin cells and identified cyclohexyl salicylate and isononyl alcohol as agonists of these receptors. In cultured human keratinocytes, both odorants induce strong Ca2+ signals that are mediated by OR2A4/7 and OR51B5, as demonstrated by the receptor knockdown experiments. Activation of corresponding receptors induces a cAMP-dependent pathway. Localization studies and functional characterization of both receptors revealed several differences. OR2A4/7 is expressed in suprabasal keratinocytes and basal melanocytes of the epidermis and influences cytokinesis, cell proliferation, phosphorylation of AKT and Chk-2 and secretion of IL-1. In contrast, OR51B5 is exclusively expressed in suprabasal keratinocytes, supports cell migration and regeneration of keratinocyte monolayers, influences Hsp27, AMPK1 and p38MAPK phosphorylation and interestingly, IL-6 secretion. These findings underline that different ORs perform diverse functions in cutaneous cells, and thus offering an approach for the modulated treatment of skin diseases and wound repair.

The Broadly Tuned Odorant Receptor OR1A1 is Highly Selective for 3-Methyl-2,4-nonanedione, a Key Food Odorant in Aged Wines, Tea, and Other Foods.

Key food odorants are the most relevant determinants by which we detect, recognize, and hedonically evaluate the aroma of foods and beverages. Odorants are detected by our chemical sense of olfaction, comprising a set of approximately 400 different odorant receptor types. However, the specific receptor activity patterns representing the aroma percepts of foods or beverages, as well as the key food odorant agonist profiles of single-odorant receptors, are largely unknown. We aimed to establish comprehensive key food odorant agonist profiles of 2 unrelated, broadly tuned receptors, OR1A1 and OR2W1, that had been associated thus far with mostly non-key food odorants and shared some of these agonists. By screening both receptors against 190 key food odorants in a cell-based luminescence assay, we identified 14 and 18 new key food odorant agonists for OR1A1 and OR2W1, respectively, with 3-methyl-2,4-nonanedione emerging as the most potent agonist for OR1A1 by 3 orders of magnitude, with a submicromolar half maximal effective concentration. 3-Methyl-2,4-nonanedione has been associated with a prune note in oxidized wine and is an aroma determinant in tea and apricots. Further screening against the entire set of 391 human odorant receptors revealed that 30 or 300 µmol/L 3-methyl-2,4-nonanedione activated only 1 receptor, OR1A1, suggesting a unique role of OR1A1 for the most sensitive detection of this key food odorant in wine, tea, and other food matrices.

ORA1, a zebrafish olfactory receptor ancestral to all mammalian V1R genes, recognizes 4-hydroxyphenylacetic acid, a putative reproductive pheromone.

The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.

Monoterpene (-)-citronellal affects hepatocarcinoma cell signaling via an olfactory receptor.

Terpenes are the major constituents of essential oils in plants. In recent years, terpenes have become of clinical relevance due to their ability to suppress cancer development. Their effect on cellular proliferation has made them promising agents in the prevention or treatment of many types of cancer. In the present study, a subset of different monoterpenes was investigated for their molecular effects on the hepatocellular carcinoma cell line Huh7. Using fluorometric calcium imaging, acyclic monoterpene (-)-citronellal was found to induce transient Ca(2+) signals in Huh7 cells by activating a cAMP-dependent signaling pathway. Moreover, we detected the (-)-citronellal-activated human olfactory receptor OR1A2 at the mRNA and protein levels and demonstrated its potential involvement in (-)-citronellal-induced calcium signaling in Huh7 cells. Furthermore, activation of OR1A2 results in phosphorylation of p38 MAPK and reduced cell proliferation, indicating an effect on hepatocellular carcinoma progression. Here, we provide for the first time data on the molecular mechanism evoked by (-)-citronellal in human hepatocellular carcinoma cells. The identified olfactory receptor could serve as a potential therapeutic target for cancer diagnosis and treatment.

An inhibitor of Na(+)/Ca(2+) exchange blocks activation of insect olfactory receptors.

Earlier we showed that the Na(+)/Ca(2+) exchanger inhibitor, KB-R7943, potently blocks the odor-evoked activity of lobster olfactory receptor neurons. Here we extend that finding to recombinant mosquito olfactory receptors stably expressed in HEK cells. Using whole-cell and outside-out patch clamping and calcium imaging, we demonstrate that KB-R7943 blocks both the odorant-gated current and the odorant-evoked calcium signal from two different OR complexes from the malaria vector mosquito, Anopheles gambiae, AgOr48+AgOrco and AgOr65+AgOrco. Both heteromeric and homomeric (Orco alone) OR complexes were susceptible to KB-R7943 blockade when activated by VUAA1, an agonist that targets the Orco channel subunit, suggesting the Orco subunit may be the target of the drug's action. KB-R7943 represents a valuable tool to further investigate the functional properties of arthropod olfactory receptors and raises the interesting specter that activation of these ionotropic receptors is directly or indirectly linked to a Na(+)/Ca(2+) exchanger, thereby providing a template for drug design potentially allowing improved control of insect pests and disease vectors.

Discrimination between olfactory receptor agonists and non-agonists.

A joint approach combining free-energy calculations and calcium-imaging assays on the broadly tuned human 1G1 olfactory receptor is reported. The free energy of binding of ten odorants was computed by means of molecular-dynamics simulations. This state function allows separating the experimentally determined eight agonists from the two non-agonists. This study constitutes a proof-of-principle for the computational deorphanization of olfactory receptors.

Narrow SAR in odorant sensing Orco receptor agonists.

The systematic exploration of a series of triazole-based agonists of the cation channel insect odorant receptor is reported. The structure-activity relationships of independent sections of the molecules are examined. Very small changes to the compound structure were found to exert a large impact on compound activity. Optimal substitutions were combined using a 'mix-and-match' strategy to produce best-in-class compounds that are capable of potently agonizing odorant receptor activity and may form the basis for the identification of a new mode of insect behavior modification.

Functional agonism of insect odorant receptor ion channels.

In insects, odor cues are discriminated through a divergent family of odorant receptors (ORs). A functional OR complex consists of both a conventional odorant-binding OR and a nonconventional coreceptor (Orco) that is highly conserved across insect taxa. Recent reports have characterized insect ORs as ion channels, but the precise mechanism of signaling remains unclear. We report the identification and characterization of an Orco family agonist, VUAA1, using the Anopheles gambiae coreceptor (AgOrco) and other orthologues. These studies reveal that the Orco family can form functional ion channels in the absence of an odor-binding OR, and in addition, demonstrate a first-in-class agonist to further research in insect OR signaling. In light of the extraordinary conservation and widespread expression of the Orco family, VUAA1 represents a powerful new family of compounds that can be used to disrupt the destructive behaviors of nuisance insects, agricultural pests, and disease vectors alike.