The olfactory and gustatory systems are the principal chemosensory systems in many organisms and detect a diverse range of environmental cues important for survival including food, toxins, predators and prey1–3. Olfactory receptors (ORs) and taste receptors are primar-ily expressed in olfactory and gustatory sensory cells and are activated by specific groups of odorant and tastant ligands4,5, and the nasal olfactory and oral gustatory sys-tems transduce chemical information from odorants and tastants via electrical signals to the brain.

ORs and taste receptors are also expressed in non- olfactory and non- gustatory tissues, and this expression has often been described as ectopic. However, numer-ous reports have described widespread expression of ORs and taste receptors in various tissues6–8, suggest-ing that the extra- nasal and extra- oral expression of these receptors is normal, and their characterization as ectopically expressed appears to be inaccurate. This widespread expression of ORs and taste recep-tors throughout various tissues suggests that extra- nasal ORs and extra- oral taste receptors have distinct biological functions. Indeed, ORs have been reported to be involved in a variety of processes, including the regulation of sperm chemotaxis9, wound healing10,11, hair growth12, muscle regeneration13, cancer cell inhi-bition14–18 and adiposity19, whereas taste receptors are involved in controlling gut appetite hormone release20,21, innate immunity22 and adipogenesis23.

ORs and some taste receptors, including sweet, umami, bitter and potentially fat taste receptors, are clas-sified as G protein- coupled receptors (GPCRs). GPCRs are major drug targets in the pharmaceutical industry, with approximately 40% of approved drugs on the mar-ket targeting GPCR proteins24. Given their variety of biological functions, ectopic ORs and taste receptors

represent attractive potential novel therapeutic targets for a broad array of indications.

For example, activation of OR2AT4 in keratinocytes with a synthetic terpenoid sandalwood odorant called Sandalore induces cell proliferation and migration, thereby contributing to wound healing10. OR2AT4 is also expressed in human hair follicles, and its activation sub-stantially increases the active growth phase of the hair follicle by inducing insulin- like growth factor 1 (IGF1) production12. Thus, Sandalore may have potential ther-apeutic applications in the promotion of wound healing and the stimulation of hair growth.

In addition, as OR expression is upregulated in sev-eral cancers14–18,25,26, the potential application of ORs in cancer diagnostics and therapeutics is also beginning to emerge14–18,25,26. In particular, OR51E2 is a potential marker for prostate cancer17,27–33, and OR2B6, OR2C3 and OR10H1 could serve as biomarkers for breast carcinoma34, melanoma35 and urinary tract cancers26, respectively.

Ectopic ORs and taste receptors may also represent potential therapeutic targets in the treatment of obesity. Indeed, azelaic acid, an agonist of mouse OLFR544, has been shown to induce adipose lipolysis and hepatic fatty acid oxidation in mice19 and cultured human cells (S- J. Lee, unpublished observations). Although no human orthologue of OLFR544 has been defined by phylogeny, studies are underway to further assess the potential application of azelaic acid as a treatment for obesity and subcutaneous lipid reduction. Furthermore, bitter tastants (for example, quinine and denatonium ben-zoate) act on TAS2Rs expressed on adipocytes and on enteroendocrine cells in the gut to reduce appetite and body weight, with increased plasma levels of glucagon- like peptide 1 (GLP1) in rodents36,37. Notably, free fatty

Olfactory receptors(ORs). A type of membrane receptor expressed in the cilia of the olfactory sensory neurons, where they bind to odorants to give the sensation of smell. ORs are also expressed in diverse extra- nasal tissues and are involved in diverse biological functions.

Therapeutic potential of ectopic olfactory and taste receptorsSung- Joon Lee 1,4*, Inge Depoortere2,4 and Hanns Hatt3,4

Abstract | Olfactory and taste receptors are expressed primarily in the nasal olfactory epithelium and gustatory taste bud cells, where they transmit real- time sensory signals to the brain. However, they are also expressed in multiple extra- nasal and extra- oral tissues, being implicated in diverse biological processes including sperm chemotaxis, muscle regeneration, bronchoconstriction and bronchodilatation, inflammation, appetite regulation and energy metabolism. Elucidation of the physiological roles of these ectopic receptors is revealing potential therapeutic and diagnostic applications in conditions including wounds, hair loss, asthma, obesity and cancers. This Review outlines current understanding of the diverse functions of ectopic olfactory and taste receptors and assesses their potential to be therapeutically exploited.

1Department of Biotechnology, Laboratory of Ectopic Olfactory and Taste Receptors, School of Life Sciences and Biotechnology for BK21 PLUS, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea.2Translational Research Center for Gastrointestinal Disorders, Gut Peptide Research Lab, Catholic University of Leuven, Leuven, Belgium.3Faculty of Biology & Biotechnology, Department of Cell Physiology, Ruhr- University Bochum, Bochum, Germany.4These authors contributed equally: Sung- Joon Lee, Inge Depoortere, Hanns Hatt

*e- mail: junelee@korea.ac.kr

https://doi.org/10.1038/s41573-018-0002-3

REVIEWS

Nature reviews | Drug Discovery

acid receptor 4 (FFAR4), an omega-3 fatty acid receptor that has been implicated in the ability to taste fats38,39, has shown promise as a potential therapeutic target for obesity in randomized clinical trials40.

Taste receptors may also be important in asthma, as bitter tastants have been shown to act on TAS2Rs in lung epithelial cells and smooth muscle cells to increase the secretion of antibacterial peptides and to induce bronchodilatation41–43.

Although substantial advances in the understand-ing of the biological roles of ORs and taste receptors have been made, their potential as therapeutic tar-gets is only beginning to be investigated. This Review summarizes the current understanding of the key bio-logical functions of ectopic ORs and taste receptors and highlights their emerging potential therapeutic and diagnostic applications in a range of diseases. Limitations and challenges in the research of ectopic ORs and taste receptors, as well as future directions, are discussed.

Nasal olfactory systemOlfaction research began at the molecular level after Buck and Axel made the landmark discovery of a large multigene family of ORs in rats in 1991 (REF.44). The human and mouse genomes encode approximately 370 and 1,000 functional OR genes, respectively, making the ORs the largest gene family45–53. While different studies have yielded small discrepancies in the exact number of ORs owing to slight differences in the filtering criteria and bioinformatics search methods, it is clear that these receptors constitute 5–10% of the mammalian genome, including pseudogenes46,54–56.

ORs have several unique characteristics. Approximately 60% of human OR genes are non- functional pseu-dogenes, and an individual may have either an intact or a pseudogene version of a particular OR50,57–60. A sub-set of OR proteins require specific chaperones such as receptor transporting proteins 1 and 2 or the heat shock protein HSC70 (also known as HSPA8) for plasma membrane targeting61–64. Each OR recognizes multiple odorants, and each odorant can be detected by different ORs; thus, a single odorant has a specific OR activation pattern, which is translated into odour perception4,65,66. An increase in the concentration of an odorant can also change its receptor code so that it binds to a broader spectrum of ORs67. This may explain the altered percep-tion of odours at different concentrations. OR expres-sion is monogenic and monoallelic and, thus, a single olfactory sensory neuron expresses only one OR of the repertoire, according to the so- called ‘one neuron, one receptor’ hypothesis68–74. The monoallelic expression of OR genes is derived from either the paternal or mater-nal allele in different olfactory sensory neurons within an individual.

ORs belong to the class A (rhodopsin- like) GPCRs. The 3D structure of OR proteins has been deduced by homology modelling techniques with amino acid sequence analysis compared with known GPCR struc-tures75–82. Ligand binding is thought to take place in a pocket formed by the transmembrane helices. Odorant recognition is mainly based on the molecular shape

and matching electrostatics of the ligand structure and not on the vibration of chemicals, as has been shown by dynamic homology modelling80,83,84. With a combi-nation of in silico structure prediction, docking analy-sis, homology modelling and mutagenesis studies, the selective binding of odorants has been demonstrated for several ORs80–82,85–87.

ORs have a unique molecular receptive system. Once an odour molecule is bound to an OR in the cilia of an olfactory sensory neuron where all protein components for signalling pathways are present88,89, a signal transduction cascade transforms and amplifies the chemical information into an electrical signal88,90. According to the canonical model of olfactory signal transduction, ORs generally interact with a specific stimulatory G protein, Gαolf (REF.90), which initiates the signal transduction pathway. More specifically, when the odorant binds, ligand activation of the OR stimu-lates Gαolf by binding GTP, and this stimulates adenylyl cyclase III (ACIII; also known as ADCY3). This, in turn, increases the cytoplasmic cAMP level, which causes external sodium (Na+) and calcium (Ca2+) influx by opening nonspecific cation- selective cyclic nucleotide- gated (CNG) channels89,91. Subsequent depolarization of the plasma membrane is amplified at least in part by the Ca2+-activated chloride (Cl−) channel TMEM16B (also known as ANO2)92–94, further depolarizing the cilia by promoting Cl− efflux. The membrane depolarization triggers an action potential, which emits a frequency depending upon the intensity and duration of the olfactory message along the axons of olfactory sensory neurons towards the olfactory bulb, where the olfactory message is the first relayed for integration in the brain (reviewed previously1). Interestingly, the expression of canonical olfactory signalling molecules can also be demonstrated in diverse extra- nasal tissues. However, in some non- olfactory tissues, completely different signal-ling components are involved, as discussed throughout the manuscript.

Oral taste systemTaste receptors were first characterized in 2000 (REFs95,96), and six types of taste receptor and channel for sweet, umami, bitter, sour, salty and fat tastes have been iden-tified97. Sour and salty tastes are sensed by ion channels (for example, the epithelial sodium channel (ENaC))97, whereas other tastes are detected by membrane recep-tors, mainly by GPCR proteins. As this Review aims to focus on olfactory and taste GPCRs, detailed discussions of sour and salty taste detection are beyond the scope of this manuscript.

Sweet and umami receptors are heteromers of the GPCR taste receptor family 1 (TAS1Rs): TAS1R2–TAS1R3 for sweet and TAS1R1–TAS1R3 for umami. Umami receptors are broadly tuned to detect several amino acids but have a higher sensitivity for glutamate in humans98. In rodents, there is evidence for another umami receptor system mediated by mGluR1 and mGluR4 in addition to TAS1R1–TAS1R3 (REF.99). Other amino acid sensors (calcium- sensing receptor (CaSR), GPCR family C, group 6, member A (GPRC6A) and lysophosphatidic acid receptor 5 (LPA5R; also known

Taste receptorsA type of receptor that binds to tastants dissolved in saliva to facilitate the sensation of taste. Taste receptors are widely expressed in diverse tissues and play multiple biological roles.

Sperm chemotaxisA form of sperm guidance in which spermatozoa follow a concentration gradient of a chemoattractant secreted from the oocyte and thereby reach the oocyte.

AdipogenesisThe process of cell differentiation by which pre-adipocytes become adipocytes.

KeratinocytesCells that are a major component of the epidermis, the outer layer of the skin, which synthesizes keratin. Keratinocytes are involved in the intricate mechanisms of initiation, maintenance and completion of wound healing.

OrthologueA homologous gene that is related to those in different organisms by descent from the DNA of a common ancestor.

Pseudogenessequences of DNA resembling a gene but usually containing mutations that alter or abolish gene function; pseudogenes thus cannot produce a normal protein.

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as GPR92)) are expressed in taste cells, but their role in taste perception has not been investigated100. For bit-ter taste detection, humans and mice express approx-imately 25 and 35 putative receptors, respectively, in the genome101. TAS2Rs have a wide range of activation thresholds and tuning. Some can detect several bitter compounds, whereas others are narrowly tuned and detect one or only a few bitter compounds.

It is still a matter of debate whether fat can be con-sidered as the sixth taste in gustation because of its tex-tural and olfactory cues. However, mounting evidence suggests that several fatty acid receptors can be involved in fat taste sensation38,39,102. Scavenger receptor CD36 and the free fatty acid GPCRs FFAR1 and FFAR4 are expressed in the taste buds, and studies in knockout mice have demonstrated the involvement of these recep-tors in orosensory fat perception38,39,102. In fact, there are four types of FFAR depending on the chain length of the ligand fatty acids: FFAR2 and FFAR3 for short- chain fatty acids (SCFAs) (less than 6 carbons), FFAR1 for

medium- chain (6–12 carbons) to long- chain fatty acids (greater than 12 carbons) and FFAR4 for unsaturated long- chain fatty acids (for example, omega-3 fatty acids). To date, only FFAR1 and FFAR4 have been implicated in taste perception, and further studies are required to determine any potential role of FFAR2 and FFAR3 in fat taste sensation (BOx 1). FFAR1 and FFAR4 have been intensively studied as potential drug targets for several metabolic diseases23,103–105.

Despite their diversity, TAS1Rs and TAS2Rs have a common intracellular signalling pathway and cou-ple to several heterotrimeric G proteins such as Gα gustducin and transducin, Gβ3, Gγ13, Gα14 and Gi. Upon activation by a tastant, the βγ- subunit is dissociated from the α- subunit and activates the phospholipase Cβ2 (PLCβ2)–inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) pathway to induce the release of Ca2+ into the cyto-plasm97. Gα subunits may activate phosphodiesterase, leading to a breakdown of cAMP and inhibiting pro-tein kinase A (PKA) activation, which otherwise inhib-its the PLCβ2–Ins(1,4,5)P3 pathway106. The increase in Ca2+ opens transient receptor potential (TRP) channel M5 (TRPM5), resulting in Na+ influx and the activa-tion of other voltage- gated Na+ channels, which will induce the secretion of ATP through an atypical, non- vesicular mechanism that involves a channel composed of a heteromer of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (REFs107,108). ATP activates sensory nerves that communicate to the brain regions involved in taste perception. Each taste receptor cell is specifically tuned to detect one taste quality, which in turn relays information to the brain via single nerve fibres tuned for the same taste quality (labelled line model)109,110. Because taste receptor cells are constantly renewed, connections between existing ganglion pro-cesses and newly born taste receptor cells must be continuously re- established.

Ectopic olfactory receptorsORs are widely expressed throughout the body in a diverse range of tissues. It should be noted that most evidence for ectopic expression of ORs is at the level of mRNA and not protein owing to the lack of receptor- specific antibodies. These extra- nasally expressed ORs have different effects on cell biological functions owing to their versatility in activating different molec-ular and cellular signalling mechanisms. The pathways activated depend on the cell types and the signalling components involved.

Tissue expressionAfter the first report of ectopic expression of ORs in tes-tis by Parmentier et al.111, which was later confirmed by others9,112–114, OR transcripts were found in various tissues, including the adipose tissue19,115,116, airway117–119, heart120, kidney121–124, liver19,125–127, lung118,128,129, prostate16,17,27–33,130,131 and skin10,11,25 (TABlE 1). ORs are also expressed in the auto-nomic nervous system132, leukaemia cells18,133, brain134–139, colon14,140,141, salivary glands142, gut143–145, cardiac and skele-tal muscle13,120,146, pancreas147,148, placenta149, retina150,151 and tongue152,153. In addition, it has been suggested that ORs may be expressed during mammalian embryogenesis154

Box 1 | Therapeutic potential of FFAr2 and FFAr3

Free fatty acid receptor 2 (FFar2) and FFar3 are classified as free fatty acid receptors for short- chain fatty acids (sCFas) (less than six carbons), most notably, acetate, propionate and butyrate, which are primarily produced by colonic fermentation of dietary fibres. FFar2 is a promiscuous receptor that couples to Gαi/0 and Gαq/11, whereas FFar3 induces only Gαi/0-mediated signalling293. a recent study demonstrated the expression of FFar2 but not FFar3 in taste bud tissue from human fungiform papillae294. However, preference and taste nerve responses to sCFas in wild- type and knockout FFar2 and FFar3 mice are necessary to determine the role of these receptors in the detection of fat by taste receptor cells. Both receptors are also expressed in extra- oral tissue and show overlapping expression patterns in enteroendocrine cells and pancreatic β- cells295,296. FFar2, but not FFar3, is predominantly expressed in immune cells such as neutrophils, monocytes, eosinophils and regulatory t (treg) cells293,297–300, but dendritic cells appear to express both FFar2 and FFar3 (REF.299). While FFAR2 is expressed in both human and mouse white adipose tissue293,297, FFar3 expression in mouse adipocytes is controversial301. in contrast to the predominant expression of FFar2 in adipocytes and immune cells, FFar3, but not FFar2, is found on sympathetic ganglia and enteric neurons295,302,303. On the basis of some of these expression patterns and consequent functional studies, FFar2 and FFar3 have been considered as potential drug targets for the treatment of metabolic and inflammatory disorders.

Metabolic disordersLoss of FFar2 and FFar3 in pancreatic β- cells increases insulin secretion and improves glucose tolerance in high- fat diet- fed obese mice, suggesting a possible role for FFar2 and FFar3 antagonists in the treatment of diabetes296. On adipocytes, circulating sCFas activate FFar2 to suppress insulin- mediated fat accumulation while promoting the catabolism of lipids and glucose in liver and muscle304.

Long- term administration of prebiotic fibres (for example, fructans) consistently decreased weight gain in mice fed a high- fat diet, but effects on glucose intolerance have been contradictory305,306. in overweight adults, daily supplementation (24 weeks) with an inulin- propionate ester to deliver propionate to the colon reduced energy intake, ameliorated long- term weight gain and improved β- cell function with increased insulin secretion (NCt00750438)307,308. a placebo- controlled, randomized trial showed that daily supplementation of oligofructose- enriched inulin to overweight or obese children affected the intestinal microbiota and reduced their body weight z- scores, percent body and trunk fat, and interleukin-6 (iL-6) levels (NCt02125955)309.

inflammatory disorderssCFas regulate colonic treg cell homeostasis and protect against colitis in an FFar2-dependent manner in mice299. Nevertheless, studies using knockout mice failed to consistently determine whether FFar2 is protective or exacerbates the severity of inflammatory disease300,310. a clinical trial with the FFar2 antagonist GLPG0674 did not improve clinical symptoms in patients with inflammatory bowel disease after 4 weeks of treatment, although it reduced neutrophil activation and influx311.

Gustducin and transducinspecific types of G protein expressed in the gustatory system and extra- oral tissues that interact with G protein- coupled receptors that act as sweet, umami and bitter taste receptors. Gustducin and transducin are structurally and functionally similar and interact with taste receptors to generate diverse signal transduction pathways.

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Table 1 | overview of key ectopic olfactory receptor expression patterns and their functions

organ or tissue

cell type olfactory receptors

Liganda Function species Detection techniques

refs

Adipocyte White and brown adipose tissue cells

OLFR544 Azelaic acid Induction of lipolysis (white adipose tissue) and induction of thermogenesis (brown adipose tissue)

Mouse Microarray , RT-PCR or qPCR

19

Airway Human airway smooth muscle cells

OLFR78/OR51E2

SCFAs Mitigates airway obstruction

Mouse and human

RNA- seq, RT- PCR , ICC, immunoblot for OR51E2; immunoblot for OLFR78

117

OR1D2 and OR2AG1

• OR1D2: bourgeonal (antagonist undecanal)

• OR2AG1: amyl butyrate

• OR1D2: induction of cell contraction and IL-8 release

• OR2AG1: inhibition of histamine- induced contraction

Human RT- PCR , qPCR , immunoblot, ICC or IHC

118

Lung cancer tissue cells

OR51E1 MCFAs Novel diagnosis target for lung carcinoids

Human qPCR or IHC 129

Heart Cardiomyocytes and human myocardial tissue cells (ex vivo)

OR51E1 MCFAs Negative chronotropic and inotropic effects and reduction of contraction force of explanted heart

Human RNA- seq, RT- PCR , immunoblot or IHC

120

Kidney Kidney tissue cells and proximal tubule HK-2 cells

OR51E1 and OR11H7

Isovaleric acid and 4-methylvaleric acid

Increase of Ca2+ transients and cAMP levels

Human RT- PCR , qPCR , immunoblot, ICC or IHC

121

Renal juxtaglomerular apparatus cells

OLFR78 SCFAs Induction of renin secretion and blood pressure control

Mouse RT- PCR or IHC 122

Liver Huh7 cells OR1A2 (–)-Citronellal Inhibition of cancer proliferation (MAPK pathway and cAMP-dependent Ca2+ influx)

Human RT- PCR or ICC 125

HepG2 cells OR1A1 (–)-Carvone Reduction of lipid accumulation

Human RT- PCR , immunoblot or ICC

127

OR10J5 α- Cedrene Reduction of lipid accumulation

Human RT- PCR , ICC or immunoblot

126

Hapa1c1c cells and mouse liver cells

OLFR544 Azelaic acid Induction of fatty acid oxidation

Mouse Microarray , RT- PCR or qPCR

19

Lung Pulmonary neuroendocrine cells and human tracheobronchial epithelial cells

OR2F1, OR2W1, etc.

Bourgeonal, bergamot oil, citronellal, nonanal and hexadecanal

Decrease in serotonin secretion

Human Microarray , ISH, ICC, IHC or immunoblot

128

A549 lung cancer cells

OR2J3 Helional Inhibition of cell proliferation, apoptosis and migration

Human RT- PCR , ICC, IHC or immunoblot

15

Prostate Prostate cancer tissue cells and cell lines

OR51E2 β- Ionone (antagonist α- ionone)

Inhibition of cancer cell proliferation and cancer progression

Human Microarray , RT-PCR , qPCR , IHC or immunoblot

17,30,31,130,161

OR51E1 Nonanoic acid Inhibition of cell proliferation and inhibition of AR-mediated cellular senescence

Human RNA- seq, RT- PCR , ICC or IHC

16

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and neurogenesis136,155. Transcriptome analyses have shown that all cells and tissues investigated thus far express at least a few ORs6,7,156. Recent sequencing studies demonstrated that at least some ORs, such as OR2W3 and OR51E1, were expressed in all the tissues analysed, whereas others were exclusively expressed in only one tissue, such as OR4N4 in the testis, which is in line with other tran-scriptome analy ses7. These results suggest that ORs may have a unique functionality in the expressed tissues.

Signalling pathwaysInterestingly, ectopic ORs are involved in various signal-ling pathways in extra- nasal tissues that require unique and diverse signalling components. For example, Ca2+ signalling pathways are activated in a tissue- specific manner by ectopic ORs in different tissues. The canoni-cal OR signalling model involves heterotetrameric CNG channels, particularly CNGA2, and also CNGA4 and CNGB1; however, these channels were not detected in most non- nasal tissues7. CNGA1, which can form exog-enous functional homomeric channels and is activated by cGMP, is the most prominent channel in peripheral cells and tissues157 and may function as the CNG chan-nel used by ectopic ORs10,125. The native cone photo-receptor channel CNGA3 is expressed and functional in spermatozoa158. This channel, together with the cation channel of sperm (CatSper)159, may be responsible for OR- mediated chemotaxis in spermatozoa by controlling Ca2+ levels.

Other studies have suggested that OR- dependent intracellular Ca2+ elevation may be mediated by the PLC–Ins(1,4,5)P3–sarcoplasmic reticulum Ca2+-ATPase pathway in colorectal cancer cells14. In addition, calcium channel inhibitor studies have suggested the involvement of TRP channels, voltage- gated L- type Ca2+ channels or Ca2+ release- activated channels14,18,160. In melano-cytes, the OR51E2-triggered Ca2+ signal is generated by

both intracellular stores and extracellular Ca2+ influx25. Interestingly, a decrease in intracellular Ca2+, mediated by the cGMP- dependent protein kinase G (PKG) was observed when OR2J3 was activated by helional in non-small-cell lung cancer15.

Moreover, in prostate cancer cells, OR51E2 is acti-vated by β- ionone, and is involved in distinct processes, including sarcoma tyrosine kinase (SRC)-dependent induction of TRPV- mediated Ca2+ influx160, and the induction of various downstream signalling mole-cules, including p38 mitogen- activated protein kinase (MAPK) and JUN N- terminal kinase (JNK)–stress- activated protein kinase (SAPK)17,161. In advanced castration- resistant prostate cancer cells, β- ionone alternatively decreased the phosphorylation of riboso-mal protein S6 kinase (p70 S6K; also known as S6K1))31. In addition, at lower concentrations of β- ionone, a path-way involving Gβγ and downstream phosphoinositide 3-kinase (PI3K)–AKT signalling may be induced; this mediates different physiological processes in vitro and in vivo30,130.

These findings demonstrate that ectopic ORs medi-ate intracellular signal transduction through a variety of mechanisms. Given the diverse signalling pathways reg-ulated by ectopic ORs, these receptors have distinctive functions and may represent potential novel therapeutic targets for several disorders.

Functions and therapeutic potentialIt has been suggested that odorant phytochemicals in fruits and vegetables are sensory cues for health and nutritional value162 and that diverse odorant phyto-chemicals are bioactive substances with pharmacological activities162. The physiological and pathophysiologi-cal effects of odorants may be due to the activation of ectopic ORs in extra- nasal tissues. Key physiological functions and ORs that represent the most promising

Negative chronotropic and inotropic effectsNegative inotropic effects reduce cardiac contractility, while negative chronotropic effects lower the rate of heartbeat.

TranscriptomeA collection of all the mRNA expressed in a biological sample such as a cell or tissue.

organ or tissue

cell type olfactory receptors

Liganda Function species Detection techniques

refs

Skin Primary and cultured keratinocytes

OR2AT4 Sandalore (antagonists oxyphenylon and Phenirat)

Activation of keratinocyte proliferation

Human Microarray , RT-PCR or ICC

10

OR2A4/OR2A7 and OR51B5

• OR2A4/OR2A7: cyclohexyl salicylate

• OR51B5: isononyl alcohol

Cytokinesis and cell proliferation, migration and regeneration

Human RNA- seq, RT- PCR , ICC and IHC

11

Primary melanocytes

OR51E2 β- Ionone (antagonist α- ionone)

Inhibition of melanocyte proliferation and melanogenesis

Human RT- PCR , immunoblot or ICC

25

Testis Spermatozoa OR1D2 Bourgeonal (antagonist undecanal)

Sperm chemotaxis Human RT- PCR , ICC or immunoblot

9,166,167

OR7A5 and/or OR4D1

• OR7A5: Myrac, PI-21788 and vernaldehyde

• OR4D1: PI-23472 and PI-23474

Sperm swimming speed Human RT- PCR or qPCR 112

OLFR16 Lyral Sperm chemotaxis Mouse ISH or RT- PCR 114

AR , androgen receptor ; ICC, immunocytochemistry ; IHC, immunohistochemistry ; IL-8, interleukin-8; ISH, in situ hybridization; MAPK , mitogen- activated protein kinase; MCFA , medium- chain fatty acid; qPCR , quantitative PCR; RNA- seq, RNA- sequencing; RT- PCR , reverse transcription PCR; SCFA , short- chain fatty acid. aLigands are agonists unless indicated otherwise.

Table 1 (cont.) | overview of key ectopic olfactory receptor expression patterns and their functions

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therapeutic targets are discussed below and summarized in TABlE 2. It should be noted that ORs have been impli-cated in several other processes, including mamma-lian embryogenesis154 and neurogenesis136,155, but their discussion is beyond the scope of this manuscript.

Chemotaxis in sperm. Transcriptome analyses have deciphered all testicular and spermatozoal human OR expression patterns and demonstrated that the testis is the most OR- transcript-rich of all tissues, apart from the nose7.

To investigate endogenous ligands of ORs expressed in spermatozoa, odorants in vaginal secretions and fol-licular fluid were investigated by gas chromatography–olfactometry. The results revealed approximately 20 different odorants, including activators of OR1D2 (REF.163). Ca2+ levels in human sperm are elevated by the natu-rally occurring novel odorants 5α- androst-16-en-3-one and 4-hydroxy-2,5-dimethyl-3(2 H)-furanone, which bind to OR4D1 and OR7A5, respectively113,163. In sper-matozoa, Gαolf and nine subtypes of Gα proteins are expressed, which can stimulate ACIII activity; however, no downstream targets of ACIII have been identified.

Activation of OR1D2 (localized in the sperm mid-piece) with bourgeonal increased intracellular Ca2+ and may thus be responsible for the changes in chemotaxis behaviour and the swimming speed of spermatozoa, which were abrogated by undecanal, a specific antag-onist of OR1D2 (REF.9). Interestingly, a reduced sensitiv-ity to the odour of bourgeonal may be correlated with idiopathic infertility in humans164,165.

The activation of OR1D2 also initiates the trans-location of cytosolic β- arrestin 2 to the nucleus, which may stimulate the expression of genes required for ferti-lization or early embryogenesis166. Tracking and sperm flagellar analyses showed that Ca2+ increase caused by OR1D2 activation may lead to modulation of flagellar configuration, resulting in chemotaxis.

The motility of spermatozoa might also be influenced by the activation of OR4D1 and OR7A5 by the odorants Myrac and PI-23472, respectively112. The activation of both ORs might induce sperm capacitation and elicit a specific Ca2+ increase with unique spatiotemporal kinet-ics, correlated with stimulus- specific stereotyped behav-ioural responses that may play important functional roles in different stages of chemical communication in sperm and egg cells. In mouse sperm cells, OLFR16 functions as a chemosensing receptor. Experiments on transgenic mice expressing high levels of OLFR16 and Lyral, a cognate ligand of OLFR16, revealed that Lyral- induced Ca2+ increases were mediated by OLFR16 and may cause sperm chemotaxis114.

Together, these findings suggest that modulation of specific ORs may have applications in the modulation of human fertility9,167,168 (FiG. 1).

Kidney function and blood pressure regulation. Several ORs are significantly expressed in the kidney. For example, a recent study demonstrated the expression of OR51E1 and OR11H7 in the human proximal tubule cell line HK-2 (REF.121). Activation of the two ORs by the ligands isovaleric acid and 4-methylvaleric acid led

to intracellular increases in Ca2+ via the cAMP- mediated pathway121.

In addition, OLFR78 (the mouse homologue of OR51E2) has been shown to be expressed in the renal arterioles of the juxtaglomerular apparatus122,123, which secrete the hormone renin into the bloodstream. Renin hydrolyses angiotensinogen to angiotensin I, which is further cleaved by angiotensin- converting enzyme (ACE) to angiotensin II, a potent vasoconstrictor.

The endogenous ligands that bind OLFR78 are SCFAs, including acetate and propionate. These SCFAs are primarily produced by the gut microbiota following fermentation of dietary fibres and are absorbed into the bloodstream, where they play important roles in differ-ent physiological processes, including inflammation and metabolism169.

Studies involving mice lacking OLFR78 or FFAR3 (another SCFA receptor) revealed that these SCFA receptors exert substantial modulatory effects on blood pressure in response to signals generated via gut micro-organisms through the stimulation of renin secretion and vasorelaxation, respectively122. The authors hypoth-esized that the opposing responses of these receptors may protect against changes in blood pressure as a con-sequence of normal physiological variations in SCFA concentration. This study expands the range of physio-logical processes that are modulated by SCFA receptors to include blood pressure regulation and adds an OR to the SCFA receptor family.

Hypoxia sensing. Animals have an intrinsic homeostatic response to changes in oxygen availability, and ORs have been implicated in the regulation of the acute response to hypoxia.

OLFR78 is expressed in several oxygen- sensitive tissues such as heart, lung and glomus cells of the carotid body7,122,170. The carotid body is a chemosensory organ at the carotid artery bifurcation that monitors blood oxy-gen and stimulates breathing within seconds when oxygen declines, thus functioning as a hypoxia sensor171. Chang et al.171 suggested that OLFR78 is activated by lactate, a signature metabolite present during hypoxia, to induce hyperventilation under hypoxic conditions. In OLFR78-deficient mice, hypoxia- induced carotid body activity was reported to be diminished, although glomus cells are structurally intact and normal171. However, a recent study was unable to replicate these findings and reported that Olfr78−/− mice had a normal ventilatory response to hypoxia and that the physiologi-cal responses of single glomus cells to hypoxia and lactate were indistinguishable between wild- type and Olfr78−/− mice317. These discrepant findings may be related to the high sensitivity of the behavioural response to hypoxia to differences in assay conditions and genetic background.Although lactate has been reported as a partial agonist of OLFR78, it has not been confirmed to be a ligand for the human homologue, OR51E2 (REF.117). Moreover, the reported effective concentration for half- maximum response (EC50) of lactate for OLFR78 has varied between reports117,171,172. Therefore, there is still no clear consensus as to whether lactate is a functional ligand for OLFR78 under physiological conditions.

HyperventilationAbnormal fast and deep breathing; the result of either an emotional status or a physiological condition such as asthma.

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Table 2 | olfactory and taste receptors representing promising therapeutic targets

receptor Ligand Target disease or condition

Biological evidence Application or route of administration

clinical development refs

OR2AT4 Brahmanol and Sandalore

Wounds Increased keratinocyte proliferation, migration, regeneration and wound healing (humans)

Cream, ointment or oil

On the market in Spain 10,312,313

Hair loss Increased hair growth and prolonged anagen phase in the hair follicles (humans)

Shampoo or lotion Preliminary studies have been performed and clinical trials are planned in Italy

12

OR10H1 Santalol and Sandranol

Bladder cancer Decreased tumour cell proliferation

Urethral application Not tested in clinical trials but is being using to treat patients in German clinics

26,314

OR51B6 Troenan Colon cancer Reduced cell proliferation and migration (humans)

Suppository capsules

Not tested in clinical trials but is being using to treat patients in German clinics

14

OLFR544a Azelaic acid Obesity and subcutaneous fat reduction

Reduced subcutaneous fat by adipose lipolysis and hepatic fat oxidation (mice and human adipocytes)

Potential oral drug or supplemental intervention (obesity) or patch or injectables (subcutaneous fat reduction)

Preclinical studies in progress for obesity (azelaic acid has been approved as an anti- inflammatory topical agent for the treatment of rosacea and acne, but the target protein is unknown)

19

FFAR4 Omega-3 PUFAs Metabolic disorders Reduction in body weight, macrophage- induced tissue inflammation, improved glucose tolerance, insulin sensitivity , brown and beige adipocyte differentiation and thermogenic activation (humans)

Oral drug or supplemental intervention

Phase II: NCT03406897; phase III: NC03361501; phase IV: NCT01733147 and NCT03120299

200–203,278

TAS2R38 and TAS2R14

• TAS2R38: acyl homoserine lactone quorum- sensing molecules secreted by Gram- negative bacteria, phenyl-thiocarbamide, propylthiouracil, etc.

• TAS2R14: flavones

Upper respiratory infections

Improvement of ciliary beat frequency and mucus clearance and direct antibacterial activity (humans)

Potential topical nasal application

In vitro and ex vivo studies only

43,213

TAS2R47 and TAS1R2–TAS1R3

• TAS2R47: denatonium benzoate and absinthin

• TAS1R2–TAS1R3: glucose and d- amino acids

Upper respiratory infections

Secretion of bitter- induced antimicrobial peptides that are inhibited by glucose (humans)

Potential topical nasal application

Ex vivo studies only 42

Mouse TAS2R108 or human TAS2R1

Isohumulone (KDT501) or emetine

Metabolic disorders Stimulation of GLP1 secretion, improvement of glucose tolerance, weight loss, normalization of plasma lipids, suppression of inflammatory markers in mice, reduction of inflammation and post- meal triglyceride levels in insulin- resistant humans who are prediabetic

Oral drug Open- label phase II trial: NCT02444910

275

FFAR4, free fatty acid receptor 4; GLP1, glucagon- like peptide 1; PUFA , polyunsaturated fatty acid; TAS1R , taste receptor family 1. aMouse receptor ; studies are underway to identify the target of azelaic acid in humans.

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Muscle regeneration. Several ORs are expressed in mus-cle, particularly during myogenesis and muscle regen-eration. OLFR16 plays a critical role in muscle tissue regeneration13. The signalling pathways of OLFR16 in muscle are unclear; however, activation of OLFR16 by

the ligand Lyral increases intracellular cAMP levels and results in myocyte migration, myofibre branching and myogenesis, which are all associated with muscle regeneration13. OLFR16 in muscle tissue regulates mus-cle cell migration and adhesion173. These effects were

Undecanal Bourgeonal

Sperm chemotaxis and chemokinesis

Cytokines

OR1D2

OLFR16

CNGA3 orCatSperchannel

a Spermatozoa

Lyral

cAMP

Sandalore Oxyphenylon

Wound healing

OR2AT4

CNGA1or CNGB1

channel

c Skin (keratinocytes)

cAMP

ERK1/ERK2 andp38 MAPK

Pannexin

(–)-Carvone

↓ Hepatic steatosis

OR1A1 OLFR544 OLFR544

d Hepatocyte e White adipose tissue

Azelaic acid

cAMP

PKA/CREB/HES1

PPARγ

↓ Fatty acidsynthesis

↑ Fatty acidoxidation

cAMP

PKA/CREB

PPARα

↓ Adiposity

cAMP

PKA

HSL

Triglyceride Free fattyacid

Free fatty acid

ATP

Propionate

Blood pressure regulation

Renin secretion

↑ Proliferationand migration

OLFR78

FFAR3(VSMC)

b Renal arteriole

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+

Fig. 1 | Major signalling pathways and functions regulated by ectopic olfactory receptors. a | OR1D2 and OLFR16 activation mediates sperm chemotaxis and chemokinesis. b | In the kidney , activation of OLFR78 in the renal afferent artery induces renin secretion and regulates blood pressure with the combined activity of free fatty acid receptor 3 (FFAR3), an alternative receptor for short- chain fatty acids. c | Activation of OR2AT4 in keratinocytes stimulates cell proliferation and migration to aid wound healing. d | Activation of OR1A1 ameliorates hepatic steatosis. e | Activation of OLFR544 by azelaic acid reduces hepatic steatosis and stimulates adipose lipolysis to decrease adiposity. CatSper, cation channel of sperm; CNG, cyclic nucleotide- gated; CREB, cAMP- response element- binding protein; ERK , extracellular- signal-regulated kinase; HES1, hairy and enhancer split 1; HSL , hormone- sensitive lipase; MAPK , mitogen- activated protein kinase; PKA , protein kinase A ; PPAR , peroxisome proliferator- activated receptor ; VSMC, vascular smooth muscle cell.

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negated in muscle cells with Olfr16 gene knockdown. Although the endogenous ligand remains uncharacter-ized, the treatment of myocytes with crushed muscle extracts results in a dramatic increase in myocyte migra-tion in a cAMP- dependent manner, which is abrogated in Olfr16 small interfering RNA- treated cells173. These findings suggest that muscle cells secrete a soluble ligand for OLFR16 during muscle regeneration and that ligand- dependent activation of OLFR16 is required for proper skeletal muscle regeneration.

In addition, OLFR16 regulates cell–cell adhesion and myotube formation. In vivo myogenesis and the loss of OLFR16 result in increased myofibre branch-ing, which is commonly associated with muscular dys-trophy; this involvement was further confirmed in a mouse model of muscular dystrophy (mdx)173. OLFR16 overexpression in mdx mice reduced myofibre branch-ing after muscle regeneration in non- dystrophic mouse muscle and decreased the severity of myofibre branch-ing in mdx mouse muscle. Muscles from mdx mice overexpressing OLFR16 showed significantly less dam-age to eccentric contractions than control mdx mus-cle173. These results indicate that OLFR16 is necessary for proper regeneration of muscle tissue, suggesting a novel function for ORs in tissue repair. Alternatively, in human skeletal myoblasts, activation of OR2H2 with its ligand aldehyde 13-13 might lead to a reduction of myoblast fusion146.

Hepatic lipid metabolism. Several ectopic ORs have been implicated in the regulation of hepatic lipid metabolism. For example, OR1A1 is expressed in the plasma membrane of HepG2 cultured human hepatocytes. When activated by the aromatic ligand (–)-carvone, a major compound in spearmint essential oil, the PKA signalling pathway is stimulated without affecting intracellular Ca2+ levels127 (FiG. 1); this ulti-mately decreases intracellular triglyceride concentra-tions. In addition, OR1A1 and its mouse homologue, OLFR43, have been implicated in GLP1 secretion in enteroendocrine cells. Injection of geraniol, a ligand of OLFR43, in diabetic mice improves their glucose and insulin tolerance with increased GLP1, provid-ing another mechanism for reducing hepatic lipid accumulation in diabetes174.

Furthermore, OLFR16 and OR10J5 receptors are involved in the regulation of hepatic lipid metabolism through the cAMP–PKA pathway in cultured cells126. OR10J5 is activated by Lyral as well as α- cedrene, and the stimulation of cells with α- cedrene reduces hepatic lipid concentrations.

Together, these studies suggest that hepatocyte- expressed ORs may represent potential therapeutic targets for hepatic steatosis and nonalcoholic fatty liver disease. Although endogenous ligands for OR1A1, OR10J5 and OLFR16 have not been identified, these ORs can be activated by exogenous aromatic ligands such as Lyral and (–)-carvone, which may be inhaled or derived from the diet. As the regulation of lipid metabolism in hepatocytes is critical, it is possible that many regulatory pathways finely tune metabolism in a redundant manner.

Bronchoconstriction. Chronic inflammatory lung dis-eases such as asthma, chronic obstructive pulmonary disease and allergies occur owing to airway smooth muscle contraction, increased smooth muscle mass and mucous plugs175. ORs expressed in the airway play a role in regulating bronchoconstriction. The expression of several ORs and of the canonical olfactory machin-ery, ACIII and Gαolf , was confirmed by RNA- sequencing (RNA- seq) analysis and quantitative PCR in human air-way smooth muscle cells117. Ainsberg et al.117 confirmed the expression of four ORs (OR51E2, OR1J1, OR2A1 and OR6A2) in human airway smooth muscle cells, with OR51E2 representing the most highly enriched OR transcript in lung- resident cells, and further investigated the functions of OLFR78 and OR51E2 in airway smooth muscle stiffness and relaxation. Cytoskeletal remodel-ling was measured by beads tethered to the cytoskeleton through cell surface integrin receptors117. Experiments assessing intracellular cytoskeletal remodelling by meas-uring spontaneous nanoscale bead motion, demon-strated that SCFAs significantly reduced cytoskeletal remodelling in both mouse and human airway smooth muscle cells, and these effects were negated in cells with OLFR78 and/or OR51E2 knockdown. Proliferation of airway smooth muscle cells was reduced by SCFAs, which may explain the reduced cytoskeletal remodelling, at least in part. SCFAs also mitigated the increased mass of asthmatic airway smooth muscle cells117.

These findings suggest that OR51E2 is a functional receptor in airway smooth muscle, and its ligand could ameliorate the increased airway smooth muscle mass associated with asthma pathology117,176.

Kalbe118 et al. showed that OR2AG1 and OR1D2 are also expressed in human airway smooth muscle cells. Application of OR2AG1 and OR1D2 agonists triggered transient Ca2+ increases in human airway smooth mus-cle cells via a cAMP- dependent signal transduction cascade. Interestingly, activation of OR2AG1 using amyl butyrate might inhibit the histamine- induced contrac-tion of human airway smooth muscle cells, resulting in muscle relaxation. By contrast, stimulation of OR1D2 using bourgeonal increased cell contractility and elicited the secretion of interleukin-8 (IL-8) and granulocyte–macrophage colony- stimulating factor. These effects were inhibited by the OR1D2-specific antagonist unde-canal. These findings support further investigation into the effects of OR modulation (for example, OR51E2 agonists, OR2AG1 agonists or OR1D2 antagonists) in models of early- stage chronic inflammatory lung dis-eases such as asthma, chronic obstructive pulmonary disease and allergies.

Obesity and subcutaneous fat reduction. Genetic varia-tion of ORs has been linked with obesity177–181. For exam-ple, copy number variations in OR7D4 and other OR7 genes on chromosome 19q13 are reportedly linked to eating behaviours and reduced levels of adiposity178,181. In addition, a frequently duplicated region on chro-mosome 11q11 and three OR genes (OR4P4, OR4S2 and OR4C6) have all been associated with obesity177. A genetic association between variations in the OR14C36 gene at the 1q44 locus and extreme obesity has

ChemokinesisAn increased nondirectional activity of cells owing to the presence of chemical substances; this is opposed to the oriented movement of chemotaxis.

MyogenesisThe formation of muscle fibres from primordial muscle cells known as myoblasts.

RNA- sequencing(RNA- seq). A powerful technique used to profile the transcriptome of biological samples to examine the presence and quality of mRNA.

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also been reported180. These associations between OR gene variants and obesity may be related to an altered sense of smell and altered eating behaviour; however, it is intriguing to speculate that these ORs may have crit-ical functions in extra- nasal tissues, where they influ-ence adiposity. Further investigations should examine whether ligands binding to OR variants can affect food choices and eating behaviours and whether ectopic ORs found in extra- nasal tissues have critical roles in regulating adiposity.

Transcriptome profiling has indicated that many OR genes are significantly expressed in adipose tis-sue7,19,115,116, although the expressed OR genes differ depending on the study design, the type of fat tissue used and the analyses employed. Lee and colleagues19 reported that liver and adipose tissue in mice fed either normal chow or a high- fat diet showed high- level expression of an OR, OLFR544, which plays a role in regulating energy and lipid metabolism (FiG. 1). When the OLFR544 ligand azelaic acid was administered orally to wild- type mice fed a high- fat diet for 6 weeks or obese (ob/ob) mice, the body weight and adiposity (especially subcutaneous fat) of the mice decreased without affecting their food intake. These azelaic acid- induced metabolic alterations are associated with increased hepatic fatty acid oxidation and the stimulation of brown adipose tissue thermo-genesis. Although the presence of a human homologue of mouse OLFR544 currently remains unclear on the basis of sequence analysis, azelaic acid was found to regulate lipid metabolism in human primary adipocytes and cul-tured hepatocytes, inducing adipose lipolysis and hepatic fatty acid oxidation (S- J. Lee, unpublished observations). Thus, it is possible that azelaic acid may have therapeutic applications in the treatment of obesity.

Wound healing, hair growth and skin hypopigmenta-tion. The skin is the outer barrier of the human body and is constantly exposed to a variety of external chemicals. Several ORs have been identified in dis-tinct skin- specific cell types of the epidermis, including keratinocytes and melanocytes10,11,25. Basal keratinocytes, the main cell type responsible for natural wound heal-ing, express high levels of OR2AT4 (REF.10). Activation of OR2AT4 by Sandalore strongly increases Ca2+ via a cAMP- dependent pathway and the phosphorylation of protein kinases, promoting human keratinocyte prolif-eration and migration (FiG. 1). Additionally, activation of OR2AT4 might induce pannexin- mediated cell–cell communication via ATP between keratinocytes and trigeminal neurons in a co- culture system182. The effects of Sandalore on keratinocyte proliferation and migra-tion improved cutaneous wound healing in a human ex vivo system10. The antagonist oxyphenylon inhibited this process.

Keratinocytes express at least two other ORs, OR2A4/OR2A7 and OR51B5, which participate in keratinocyte cytokinesis11. The activation of the receptor OR2A4/OR2A7 by the specific agonist cyclohexyl salicylate may increase cell proliferation and IL-1 production, whereas isononyl alcohol- dependent activation of OR51B5 may increase the migration and regeneration of keratinocytes and the secretion of cytokines, such as IL-6 (REF.11). These

findings suggest that several ectopic ORs in the skin may be involved in the regulation of keratinocyte func-tion and may have potential applications in shortening wound- healing time.

OR2AT4 is also expressed in human scalp hair follicles12. Stimulation of OR2AT4 in organ- cultured hair follicles with Sandalore or Brahmanol, another mild woody- like odour, significantly retarded sponta-neous catagen (involuting or repressing phase of hair growth) development of the hair follicle ex vivo and significantly decreased apoptosis of hair matrix kerati-nocytes. OR2AT4 stimulation also prolonged the anagen (active growth phase) of the hair follicle by increasing IGF1 production12. Thus, OR2AT4-mediated signal-ling is required for both maintaining human scalp hair follicles in anagen phase and suppressing keratino-cyte apoptosis in the hair matrix. Silencing OR2AT4 by coadministering the specific OR2AT4 antagonist Phenirat significantly reversed the Sandalore- induced intrafollicular upregulation of IGF1 in organ- cultured human hair follicles12. A preliminary pilot clinical trial or hair pull test confirmed the ex vivo studies: application of a Sandalore- containing lotion for 12 weeks signifi-cantly lowered hair loss by about 20%, as compared with the control group (H. Hatt, unpublished observation). These results suggest that OR- dependent chemore-ceptors may represent novel targets for the regulation of hair growth.

Cutaneous melanogenesis occurs when skin is exposed to the Sun and provides protection against the harmful effects of ultraviolet radiation; however, pathological hyperpigmentation can also occur. The activation of OR51E2 by β- ionone leads to improved melanogenesis and dendritogenesis, as well as reduced cell proliferation in primary cultures of human mel-anocytes25 (FiG. 2); effects which were compromised by cells with OR51E2 knockdown or antagonist treatment. These results were associated with decreases in cell pro-liferation and an increased capacity for differentiation among melanocytes; melanin content also increased significantly. It is possible that β- ionone, which is often found in beauty care products, may play a role in the hyperpigmentation induced by cosmetics. In addition, the activation of OR2A4/OR2A7 by cyclohexyl salic-ylate elevated intracellular cAMP and Ca2+ levels and might induce p38 MAPK and reduce p42 MAPK (also known as MAPK1) and/or p44 MAPK (also known as MAPK3) phosphorylation in melanocytes, in conjunc-tion with increased melanin biosynthesis and inhibition of melanocyte growth183. Together, these finding suggest that some ORs may represent potential targets for the treatment of pigmentation disorders.

Heart rate. Next- generation sequencing analysis deter-mined the expression pattern of more than ten differ-ent ORs in adult and fetal human heart muscle cells7,120, with OR51E1 representing the most highly expressed OR in both cardiac development stages120. Medium- chain fatty acids (MCFAs), consisting of 6–12 carbon atoms, were found to be potent agonists for OR51E1, with the C9-nonanoic acid showing the highest affinity, while 2-ethylhexanoic acid functioned as a competitive

MelanogenesisThe production of melanin from the amino acid tyrosine via complex metabolic pathways; the process is carried out by melanocytes located in the bottom layer of the skin epidermis.

Next- generation sequencingA high- speed nucleic acid sequencing technique, typically characterized by being highly scalable, allowing the entire genome to be sequenced at once.

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Helional

Cancer inhibition

↓Proliferation and/or migration↑Apoptosis

OR2J3

c Lung carcinoma cells

TRPA1

PI3K

Nonanoic acid

Cancer inhibition

OR51E1

a Prostate cancer cells b Melanoma/melanocytes

SRC

p38 MAPK

CREB

ER

MEK1/MEK2

ERK1/ERK2

RSK1/RSK2/RSK3

Troenan

Cancer inhibition

↑Apoptosis

OR51B4

d Colon cancer cells

Store-operatedcalcium entryby ORAI channels

PLC Ins(1,4,5)P3

ER

Sandalore

Cancer inhibition

OR2AT4

e Leukaemia cells

Oxyphenylon

cAMPPKA

DAG

PKCθ AKT

RAF1

p38 MAPK

Caspase 9/caspase 3

CAMKII

p42 MAPK/p44 MAPK

Caspase 3

mTOR/BAD/actin

Ca2+

Ca2+

Ca2+Ca2+

Ca2+

p38 MAPK

↑Apoptosis

↓Proliferation

Ca2+

Ca2+

Ca2+

L-type Ca2+

channel

• PSA secretion• Actin cytoskeleton

β-Ionone

Cancer promotion

OR51E2 OR51E2

19-OHAD

α-Ionone

AKT

↑ Invasivenessand metastasis

↓Proliferationand migration

↓Proliferationand migration↑Apoptosis Melanogenesis

Trans-differentiation (19-OHAD)

STAT3

AR

p70 S6KPI3Kγ

PYK2p38 MAPK

SAPK/JNK

SRC

Cancer inhibition Melanin synthesis

cAMP

PKA

MITF

CREBERK1/ERK2 andp38 MAPK

TRPV6

Ca2+

Ca2+TRPM

ER

AKT

Fig. 2 | Activation of ectopic olfactory receptors regulates cancer cell growth. Activation of olfactory receptors (ORs) OR51E1 (prostate; part a), OR51E2 (melanoma; part b), OR2J3 (lung; part c), OR51B4 (colon; (part d) and OR2AT4 (leukaemia; part e) can inhibit cancer in the cells shown through the regulation of different signalling pathways, as indicated. Stimulation of OR51E2 inhibits cell proliferation but also induces invasiveness, metastasis and trans-differentiation of prostate cells. Receptor ligands are shown in green. Dash ed lines indicate that these steps are postulated and were not demonstrated with experimental data. 19-OHAD, 19-hydroxyandrostenedione; AR , androgen receptor ; CAMKII, Ca2+/calmodulin- dependent kinase II; CREB, cAMP- response element- binding protein; DAG, diacylglycerol;

ER , endoplasmic reticulum; ERK , extracellular- signal-regulated kinase; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; JNK , JUN N- terminal kinase; MAPK , mitogen- activated protein kinase; MEK , mitogen- activated protein kinase kinase; MITF, microphthalmia- associated transcription factor ; ORAI, calcium release- activated calcium; p70 S6K , ribosomal protein S6 kinase; PI3K , phosphoinositide 3-kinase; PKA , protein kinase A ; PKCθ, protein kinase Cθ; PLC, phospholipase C; PSA , prostate- specific antigen; PYK2, protein tyrosine kinase 2; RSK , ribosomal S6 kinase; SAPK , stress- activated protein kinase; SRC, sarcoma tyrosine kinase; STAT3, signal transducer and activator of transcription protein 3; TRPA1, transient receptor potential ion channel subfamily A , member 1; TRPM, TRP subfamily M; TRPV6, TRP subfamily V, member 6.

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antagonist. Studies have suggested that MCFAs can be used to identify potential therapeutic targets for various metabolic and inflammatory disorders, including obe-sity, type 2 diabetes, cardiovascular and intestinal dis-eases and atherosclerosis184. OR51E1-activating MCFAs are available from diets or from lipolysis products from adipose tissues. Interestingly, some MCFAs including nonanoic acid have been detected at receptor- activating concentrations in the plasma and in epicardial adipose tissue, particularly in patients with diabetes mellitus185. The activation of OR51E1 by MCFAs has been shown to induce negative inotropic effects in human explanted heart preparations (cardiac trabeculae and slice prepa-rations of explanted ventricles) and leads to negative chronotropic effects in human stem cell- derived cardio-myocytes120. These findings suggest that OR51E1 may be involved in the regulation of heart rate.

Cancer. OR expression is upregulated in several cancer tissues compared with levels in normal tissues14,17,26,34,35,186 — for example, OR51E2, OR2B6, OR2C3 and OR10H1 are upregulated in prostate cancer cells17,27–33, breast carcinoma cells34, melanoma cells35 and urinary cancer cells26, respectively.

The expression of OR51E2 has been studied in par-ticular detail (FiG. 2). The expression of OR51E2 and the closely related OR51E1 is strongly upregulated in pros-tate carcinomas compared with healthy prostate cells, suggesting their potential use in the diagnosis of prostate tumours and as therapeutic targets16,17,27–33.

Overexpression of OR51E2 in phosphatase and ten-sin homologue (PTEN)-deficient mice accelerates pros-tate cancer development and progression187. Intriguingly — consistent with the long- known anti- proliferative effect of the isoprenoid β- ionone on carcinoma cells188–191 — this OR51E2 agonist decreased proliferation but increased the invasiveness of human prostate cancer cells17,30,31. Meanwhile, stimulation of OR51E2 with the endogenous agonist 19-hydroxyandrostenedione facilitated cellular transformation, resulting in neu-roendocrine trans- differentiation, indicating that the activation of OR51E2 in prostate cancer may contribute to the development of non- proliferating foci131. Similarly, the activation of OR51E2 in primary cultures of mela-noma cells derived from metastatic growth and vertical growth phases appears to inhibit cell proliferation and migration and might induce apoptosis192 (FiG. 2).

OR51E2 has also been identified as a prostate tumour antigen with implications for cancer immuno-therapy193. Novel HLA- A2-restricted OR51E2-derived peptides, which frequently induced peptide- specific T cell responses and killed LNCaP prostate cancer cells, have been developed, with the potential to be used as diagno-stic markers and immune targets for the development of anticancer vaccines193.

OR51E1 has also been reported to influence prostate cancer growth, and stimulation of cells with the ligand nonanoic acid significantly reduced proliferation and induced cellular senescence in LNCaP cells16 (FiG. 2). Interestingly, activation of OR51E1 may interfere with the androgen receptor (AR), as the expression levels of AR target genes decrease in response to nonanoic acid16.

Studies have demonstrated the overexpression and functional role of ORs in many other cancers. For exam-ple, OR2J3 is expressed in human lung carcinoma tis-sues and in the non- small-cell lung cancer cell line A549 (REF.15), and its activation with the OR2J3 agonist helional triggered the release of intracellular Ca2+ and induced extracellular- signal-regulated kinase 1 (ERK1) and/or ERK2 phosphorylation via PI3K signalling pathways, resulting in the induction of apoptosis and the inhibition of cell proliferation and migration15 (FiG. 2).

OR1A2, the paralogous receptor to OR1A1, is expressed in hepatic cancer cells125. The (–)-citronellal- activated olfactory receptor OR1A2 evokes a cAMP- dependent cytosolic Ca2+ increase in the hepatocellular carcinoma Huh7 cell line, in which the olfactory signal-ling components ACIII, Gαolf and the CNG channel sub-unit CNGA1 are expressed125. Moreover, (–)-citronellal mediates the phosphorylation of p38 MAPK and pro-motes a reduction in cell proliferation125. These findings are consistent with various studies that have described the anti- carcinogenic properties of terpenes194,195.

In addition, activation of the recently identified and deorphaned OR51B4 affects the physiology of colorec-tal cancer cells and native human colon cancer tissues14 (FiG. 2). The agonist Troenan inhibits cell proliferation and migration and actin filament formation, and it stimulates apoptosis and the release of serotonin14. These inhibitory effects on cell growth may be medi-ated via a PLC- dependent signalling pathway, which might include a Ca2+ influx through calcium release from calcium release- activated calcium (ORAI) chan-nels, increased p38 MAPK phosphorylation to induce apoptosis and decreased AKT and mTOR kinase phos-phorylation for cancer inhibition. These results reveal OR51B4 as a potential novel target for the treatment of colorectal cancer. As colon cancer is accessible from the lumen, the rectal or oral application of Troenan is conceivable.

Finally, OR expression and function have also been implicated in acute and chronic myelogenous leukaemia (FiG. 2). Sandalore- dependent activation of OR2AT4 and a cAMP- dependent pathway leads to reduced prolifer-ation and increased apoptosis in the acute and chronic myelogenous leukaemia cell line K562, by arresting the cell cycle at the G0–1 and G2–M phases18. Furthermore, in the presence of an agonist, growth inhibition is accompanied by an increased number of haemoglobin- carrying erythroid cells that originate from immature myeloblasts. The inhibition of myeloid leukaemia cell proliferation can also be mediated by another OR, the isononyl alcohol- activated OR51B5 (REF.133), which is the most highly expressed OR in K562 cells. These find-ings highlight novel potential targets for the treatment of acute and chronic myeloid leukaemias.

It is striking that although approximately 40% of pharmacological tools target GPCRs24,196, relatively few of these are used to treat cancer197. ORs are easily the largest group within the mammalian GPCR gene super-family. As many ORs are dysregulated in malignant tissues, they are promising targets for treating and diag-nosing cancer, although further studies are necessary to further delineate the role of ectopic ORs in cancer.

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Ectopic taste receptorsTissue expressionHofer et al. showed that the gustatory G protein α-gustducin is expressed in brush cells of the stomach and duode-num in rats, introducing the concept that taste signalling also occurs in extra- oral tissues198. Following the dis-covery of a large family of gustducin- linked bitter taste receptors in taste receptor cells95, the expression of bitter TAS2Rs was identified in mouse enteroendocrine cells199. Subsequent studies showed ectopic expression of sweet, bitter and free fatty acid taste receptors in the gut but also in various other tissues, including adipose tissue36,200–205, airways41–43,206–213, blood vessels214, bone205,214, brain215,216, breast217, heart218–220, immune cells201,202,221–224, kidney225, pancreas226–229, skin230,231, testis232–234, thyroid235, urethra236 and urinary bladder237 (FiGs 3,4; TABlE 3).

Signalling pathwaysThe signalling pathways in extra- oral tissues have been studied mainly for TAS2Rs. Cell- autonomous regulation of TAS2Rs has been demonstrated in the airway epithelia and smooth muscle cells. In human sinonasal motile cilia, a bitter- induced Ca2+ release regulates the ciliary beat fre-quency via nitric oxide and PKG, which phosphorylates ciliary proteins43. In smooth muscle cells, bitter tastants induce localized Ca2+ release, which activates large con-ductance Ca2+-activated K+ channels to induce hyperpol-arization and consequent muscle relaxation41. Although several other mechanisms have been proposed, including a critical role for the βγ- subunits of gustducin, the relaxant properties of TAS2R agonists are confirmed in all studies206.

TAS2Rs play a paracrine role in mouse solitary che-mosensory cells in the nasal cavity and in gut entero-endocrine cells, in which a bitter- induced Ca2+ release causes acetylcholine or cholecystokinin (CCK) release, respectively238,239. Acetylcholine activates trigeminal afferent nerves containing nociceptors to evoke neuro-genic inflammation, whereas CCK upregulates the efflux transporter ATP- binding cassette B1 in enterocytes to pump bitter toxins out of the cells239.

The endocrine mechanism of TAS2Rs in the gut epithe lium involves gustducin and activation of several elements of the canonical taste signalling pathway to induce the release of satiety hormones37,240. A similar gustducin-mediated pathway is involved after the acti-vation of sweet taste receptors by glucose to induce the release of the incretin hormone GLP1 but not the hunger hormone ghrelin241,242. The signalling pathway activated by FFAR1 and FFAR4 involves Gαq in GLP1-containing cells and Gαi in ghrelin-containing cells. Downstream of FFAR1 and FFAR4, the Ca2+ signalling pathway involves TRP channels243,244.

Functions and therapeutic potentialEctopic taste receptors are involved in a variety of pro-cesses, including diabetes and metabolic disease23,245, asthma22 and inflammatory diseases224. Their functions in the gastrointestinal tract in appetite regulation have been investigated intensively20,21,246. Those functions and receptors that likely represent the most promising targets for therapeutic application are discussed below and sum-marized in TABlE 2.

Appetite regulation in the gastrointestinal tract. The gut senses meal composition and volume. Taste receptors have been described in almost all enteroendocrine cells in the gut and regulate the release of satiety hormones in response to a meal (reviewed previously8,21). Both in vitro studies (cell lines, mucosal segments and isolated crypts) and in vivo studies of taste receptor- deficient mice have been used to demonstrate a role for taste receptors in gut hormone release (FiG. 3a).

In the stomach, the products of protein and lipid breakdown are sensed by fatty acid receptors (FFAR4), the umami receptor (TAS1R1–TAS1R3) and other amino acid sensors (CaSR and GPRC6A) on P/D1 cells, which regulate the release of the hunger hormone ghre-lin247–250 (FiG. 3a). Dietary protein hydrolysates further interact with amino acid taste sensors (CaSR, GRPC6A and LPA5R) on G cells and S cells to stimulate the release of gastrin and somatostatin, which are two important regulators of acid and pepsinogen secretion251,252. Dietary toxins are sensed by bitter TAS2Rs on enteroendocrine cells and smooth muscle cells to prevent further inges-tion of food but are also present on parietal cells to regulate acid secretion253–255.

After stomach emptying, food arrives in the small intestine, which is the major site of digestion and absorp-tion. Fatty acids (via FFAR1 and FFAR4) and amino acids (via CaSR and TAS1R1–TAS1R3) are sensed by taste receptors on I cells, which secrete CKK, a satiety hormone that delays gastric emptying and triggers the release of digestive enzymes from the pancreas and bile from the gallbladder256–260. Carbohydrate sensing mainly occurs in the L cells and K cells, which secrete the incre-tin hormones GLP1 and gastric inhibitory polypeptide (GIP), respectively.

Bitter taste receptors on enteroendocrine cells are an important target to reduce appetite. Indeed, intra-gastric administration of denatonium benzoate or quinine reduced hunger scores in healthy female volunteers by suppressing the release of orexigenic gut hormones and gastric motility261,262. Furthermore, intragastric quinine decreased prospective and hedonic eating by alter-ing brain activity in homeostatic and hedonic brain regions, which covaried with decreases in circulating levels of orexigenic gut hormones in healthy females263. In obese mice, daily gavage of denatonium benzoate or quinine over a 4-week period decreased weight gain in an α- gustducin-dependent manner36.

The therapeutic benefits of bitter plant extracts for treating obesity and diabetes are recognized by tradi-tional medicine practitioners across the world and, in some cases, are supported by scientific evidence264,265. For example, some bitter- tasting herbal medicines (for example, berberine, Hoodia gordonii and bitter melon extract) affect the release of appetite- suppressing hor-mones (for example, GLP1 and CCK) via TAS2Rs266–268 and have exhibited weight- reducing or antidiabetic effects in animal models268–270. However, clinical studies on the effects of bitter melon extracts in patients with obesity and diabetes lacked an appropriate study design and were inconclusive271. The bitter herbal tea yerba maté delayed gastric emptying and promoted weight loss in patients with obesity who were treated for 45 days272.

Gut hormonesA group of hormones (for example, ghrelin, glucagon- like peptide 1 (GlP1) and cholecystokinin (CCK)) secreted by enteroendocrine cells in the gastrointestinal tract that regulate several biological functions involved in the regulation of food intake, digestion, motility and so on.

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Enteroendocrine cell

White adipocyte

Beige adipocyte

Pre-adipocyte

a Gut mucosa

b Adipose tissue

Pre-adipocyte

Amino acids• TAS1R1–TAS1R3

Bitter• 25 TAS2Rs

Sweet• TAS1R2–TAS1R3

TAS1R2–TAS1R3

Gustducin/transducin

Gustducin

cAMP

Sucralose

Saccharin

Omega-3 PUFAs/FFAR4 agonists

Gs

Ca2+

Bloodvesssel

Vagusnerve

• Food intake• Gastric emptying

↑ Insulinsensitivity

Browning

↓ Inflammation

FFAR4

FFAR4

FFAR4

TAS2R,denatonium

benzoateand quinine

Brown adipocyte

?

FFAR4

FFAR4

FFAR4

White adiposetissue macrophage

FGF21

Gustducin

• Ghrelin• CCK• GLP-1• PYY• GIP

Fat• FFAR1/ FFAR4

Fig. 3 | Pathways activated by ectopic taste receptors in the gut and adipose tissue. a | Tastants (for example, sweet, amino acids, bitter and fat) activate specific chemosensors on enteroendocrine cells in the gut that regulate the release of gut hormones involved in appetite signalling. b | Bitter and sweet tastants inhibit the differentiation of pre-adipocytes to mature white adipocytes. Omega-3 fatty acids or free fatty acid receptor 4 (FFAR4) agonists promote adipogenesis in white and brown pre- adipocytes via FFAR4 activation. In white adipocytes, they increase insulin sensitivity and reduce inflammation by activating macrophages. FFAR4 activation promotes the browning of white fat and regulates thermogenic activation in beige and brown adipocytes to increase energy expenditure and to ameliorate insulin resistance, in part by releasing fibroblast growth factor 21 (FGF21). CCK , cholecystokinin; GIP, gastric inhibitory polypeptide; GLP1, glucagon- like peptide 1; PUFA , polyunsaturated fatty acid; PYY, peptide YY; TAS1R , taste receptor family 1.

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In animal models of obesity, it decreased lipid accu-mulation and improved diabetes phenotypes273,274. Of note, given the complex and non- selective nature of bitter herbs, a direct link to the activation of TAS2Rs and the precise mechanisms mediating their health benefits still require further investigation. Recently, Kok et al.275 identified a pure derivative of a hops isohumulone, KDT501, that signals through TAS2R108 (also known as TAS2R4) to improve multiple features of metabolic disease in diet- obese mice. In humans with insulin resist-ance and prediabetes, KDT501 reduced plasma trigly-ceride levels276 (NCT02444910). Additional randomized trials of a single bitter compound are needed.

Adipogenesis. The taste- associated G protein α- gustducin is expressed in adipose tissue, and high- fat diet- induced α- gustducin–/– mice show an obesity- resistant phenotype owing to increased brown adipose tissue thermo genesis36 (FiG. 3b). The specific taste receptor involved was not identified, but a similar obesity- resistant phenotype has been described in sweet taste receptor- deficient mice205. Furthermore, both α- gustducin and bitter taste recep-tors (TAS2R108 and TAS2R135) are expressed in white adipose tissue and mouse 3T3-F442A pre- adipocytes. In addition, a direct effect on adipocyte metabolism has been suggested, as bitter agonists inhibited 3T3-F442A differentiation into mature adipocytes, as shown by a decrease in lipid accumulation36.

FFAR4 is expressed in white adipose tissue, and high- fat diet- fed FFAR4–/– mice have an obese pheno-type with increased fatty liver and hepatic lipogenesis but decreased adipocyte differentiation and lipogenesis; this indicates that FFAR4 contributes to the process of adipogenesis203. A recent study showed that FFAR4 acti-vation promotes the browning of white fat and regulates thermogenesis in beige and brown mouse adipocytes, increasing energy expenditure and ameliorating insulin resistance in part by releasing fibroblast growth factor 21 (FGF21)200 (FiG. 3b). Given that human adipocytes can acquire a beige phenotype after incubation with eicosa-pentaenoic acids277, further studies are needed to inves-tigate whether FFAR4 activation also induces brown and beige adipocyte differentiation and thermogenic activa-tion in humans. In addition, FFAR4 is highly expressed in adipose macrophages, and the administration of omega-3 fatty acids, which are ligands of FFAR4, to high- fat diet- fed obese mice reduced inflammation and increased insulin sensitivity through FFAR4 (REFs201,202).

Randomized clinical trials have shown that omega-3 polyunsaturated fatty acids reduce waist circumference and triglyceride levels but not body weight. A sex- specific effect was observed on insulin resistance40,278,279. The identification of a mutation in the FFAR4 gene, which resulted in attenuated signalling associated with an increased risk of obesity in European populations, further supports a role for FFAR4 in obesity203.

Finally, FFAR1 is highly expressed in pancreatic β- cells, but divergent effects have been reported on insulin secretion in mice228,229. In contrast to FFAR4, the development of FFAR1 agonists has experienced a seri-ous setback owing to the failure of a phase III trial with fasiglifam that induced liver injury280.

Immunity, inflammation and asthma. Bitter taste recep-tors are expressed in several cell types within the lungs, including epithelial, chemosensory, smooth muscle and immune cells, and play an important role in the innate immune response and bronchodilatation22. In the upper airways, TAS2R38 is expressed on motile cilia of human sinonasal ciliated epithelial cells, and upon activation, Ca2+-dependent nitric oxide production increases ciliary beat frequency and mucus clearance43 (FiG. 4). Further dif-fusion of nitric oxide in the airway surface liquid elicits direct antibacterial activity. The effect was mimicked by acyl homoserine lactones, which are quorum- sensing mol-ecules produced by Gram- negative bacteria43,281. Human motile ciliated cells from the trachea and bronchi also express TAS2Rs, which increase ciliary beating207.

Solitary chemosensory cells (SCCs) in human primary sinonasal cultures express TAS2Rs that, upon stimula-tion with the bitter agonist denatonium benzoate, induce the secretion of antimicrobial peptides to kill bacteria42. Sweet taste receptors in SCCs, activated by sugars in the airway lumen or sweet- tasting d- amino acids (d- Leu and d- Phe) produced by non- pathogenic Staphylococcus spp., blocked bitter- induced antimicrobial peptide secretion and inhibited biofilm formation of Pseudomonas aerug-inosa42,212 (FiG. 4). Antimicrobial peptide secretion does not occur in human bronchial tissue and is thus specific for human upper airways. High glucose concentrations in the airway lining of patients with diabetes mellitus or chronic rhinosinusitis can promote airway infections by inhibiting bitter- induced antimicrobial production. Topical application of sweet taste receptor antagonists (for example, lactisole) may therefore be a useful thera-peutic strategy to generate an antimicrobial response to bitter bacterial molecules and may represent a promising alternative to antibiotics. In mouse SCCs, activation of TAS2R38 results in a local inflammatory response and breath holding238,282. However, it is unknown whether a similar mechanism exists in human SCCs.

Finally, human airway smooth muscle cells also express TAS2Rs, which upon stimulation with bitter agonists induce airway smooth muscle relaxation41. In a mouse model of allergic airway inflammation and bronchial hyperresponsiveness, the TAS2R agonist quinine, administered as an aerosol, reduced bron-choconstriction and exceeded the therapeutic efficacy of β2-adrenergic agonists41. However, as quinine has multiple mechanisms of action, it is possible that these beneficial effects may not be mediated by TAS2Rs, and similar experiments using more specific bitter agonists should be performed. In mouse models challenged with allergens, (chloro) quinine, administered as an aerosol or intra- nasally, reduced hyperresponsiveness, allergen- induced airway inflammation, tissue remodelling and mucus accumulation283. Finally, bitter agonists inhibited the lipopolysaccharide- induced release of inflammatory mediators from the blood leukocytes of patients with asthma expressing TAS2Rs222 and suppressed histamine and prostaglandin D2 release from human primary mast cells223. These studies indicate that, in contrast to β2-adrenergic agonists, TAS2R agonists exert multi-ple effects that interfere with some of the key features of asthma.

Beige adipocyteUnder certain circumstances (such as prolonged cold exposure), a ‘beiging’ process occurs in white adipocytes resulting in the formation of beige adipocytes, which function similarly to brown adipocytes and produce heat by activation of oxidative metabolism.

Innate immune responseThe component of the immune system that is nonspecific, as distinguished from the adaptive immune response. it provides the first line of defence against pathogens.

BronchodilatationA widening of the lumen of the bronchi, allowing increased airflow to and from the lungs.

Rhinosinusitisinflammation of the sinuses resulting in clinical symptoms of thick nasal mucus, a plugged nose and pain in the face.

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Future directions and challengesORs and taste receptors are widely expressed in extra- nasal and extra- oral tissues, being highly expressed in several cancer cells and metabolically active tis-sues including gut, liver, muscle and adipose tissues (TABlEs 1,3). These ectopically expressed ORs and taste receptors exert diverse physiological effects depending on the tissue and cell type in which they are expressed and the signal transduction pathways they trigger (FiGs 1–4). These ORs and taste receptors show sub-stantial diversity in the activation of signalling path-ways in different tissues. As the understanding of the functionalities of these receptors increases, their poten-tial application in the diagnosis and treatment of sev-eral clinical disorders including cancer, obesity and subcutaneous fat reduction, wound healing, hair loss, metabolic disorders and upper respiratory infections is emerging (TABlE 2).

Regarding taste receptors, the TAS2R family repre-sents a promising future target for the treatment of both asthma and obesity22,206. Other promising novel targets within the taste receptor family are members of the FFAR family which influence inflammatory processes and are involved in the regulation of energy metabolism. Indeed, FFAR4 may influence metabolic disorders284.

Regarding ORs, cancer represents one of the most promising therapeutic areas for further development.

However, target candidates should fulfil the following criteria: good accessibility, high specificity and sensitiv-ity, and high selectivity for a specific tissue. There are only a few cancer types that express ORs that at least partially fulfil these conditions, such as colorectal can-cer (OR7C1)14, bladder cancer (OR10H1)26, melanoma (OR51E2)192 and leukaemia (OR51B5 and OR2AT4)18,133. In addition, some cancer tissues express OR mRNAs at very high levels, and these OR transcripts could be used as biomarkers for cancer diagnostics. Exemplary ORs include OR51E2 for prostate cancer17,27–33, OR51E1 for small intestine carcinoma186, OR2B6 for breast carci-noma34, OR2J3 for non- small-cell lung cancer15, OR7C1 for colorectal cancer14 and OR10H1 for bladder cancer26. Recent studies have shown that OR transcripts (OR51E2 and OR10H1) can be measured by PCR in urine; thus, these samples can potentially be used for liquid biopsies for cancer diagnosis26,29.

Other exciting directions involving the olfactory receptor family include the potential of activating OLFR544 in the treatment of obesity and subcutane-ous fat reduction19 and activating OR2AT4 to promote wound healing10 and hair growth12.

However, despite these promising findings, there are several challenges and limitations facing research on ORs and taste receptors. Deorphanization of ORs is still a major challenge. Only 10–20% of mammalian

Mucin

Bacterial killing

Goblet cellCiliated epithelial cell Epithelial cell Solitary chemosensory cell

Antimicrobialpeptides

Nitric oxide

TAS2R

Gapjunction

TAS1R2–TAS1R3

Pseudomonas aeruginosa Staphylococcus spp.

Denatoniumbenzoate

Phenylthio-carbamide

PLCβ2PKG

D-Leu andD-Phe Glucose

P

TAS2R38

NH

RO

O

O

Ca2+Ca2+

Acyl homoserine lactones

Tuft ofmicrovilli

Fig. 4 | Antibacterial effects of ectopic taste receptors in the lung. Bitter compounds induce the release of nitric oxide to stimulate ciliary beating and mucociliary clearance and induce direct antibacterial effects in human ciliated epithelial cells. In human solitary chemosensory cells, bitter compounds initiate, via the release of Ca2+, the release of antimicrobial peptides to induce bacterial killing. This effect is inhibited by sweet taste receptors activated by glucose and sweet-tasting d- amino acids. PKG, protein kinase G; PLCβ2, phospholipase Cβ2; TAS1R , taste receptor family 1; TAS2R , taste receptor family 2.

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Table 3 | overview of key ectopic taste receptor expression patterns and functions

organ or tissue

cell type Taste receptor

Ligand Function species Detection technique

refs

Adipose tissue White adipose tissue cells

TAS2R Bitter agonists Inhibition of adipocyte differentiation

Mouse RT- PCR 36

FFAR4 Omega-3 PUFAs Regulation of adipogenesis, promoting browning of white fat

Mouse and human

RT- PCR or qPCR 200,203

Brown adipose tissue cells

FFAR4 Omega-3 PUFAs Increase of thermogenic activity

Mouse RNA- seq, RT- PCR , qPCR or immunoblot

200

Macrophages FFAR4 Omega-3 PUFAs Anti- inflammatory effects

Mouse RT- PCR or qPCR 201,202

Airways (sinonasal cavity)

Ciliated epithelial cells

TAS2R38 and TAS2R14

• TAS2R38: PTC and acyl homoserine lactone

• TAS2R14: flavone

Increase of ciliary beating and mucociliary clearance and bactericidal effects

Mouse and human

ICC or IHC 43,213,281

Solitary chemosensory cells

TAS2Rs Denatonium benzoate

Stimulation of antimicrobial peptides

Human ICC 42

TAS1R2–TAS1R3

Glucose, artificial sweeteners and d- amino acids (d- Leu and d- Phe)

Inhibition of bitter- induced antimicrobial peptides

Human ICC 42,212

Airways (trachea and bronchi)

Ciliated epithelial cells

TAS2Rs Bitter agonists Ciliary beating Human Microarray , RT- PCR or ICC

207

Chemosensory cells

TAS2R Bitter agonists and acyl homoserine lactone

Trigeminal nerve activation and drops in respiratory rate

Mouse RT- PCR 210,211

Smooth muscle cells

TAS2Rs Bitter agonists Bronchodilatation Rodent and human

qPCR or ICC 41,206

Gastrointestinal tract

Enteroendocrine cells

TAS2Rs Bitter agonists, berberine, Hoodia gordonii and wild bitter gourd

Ghrelin, CCK and GLP1 release

Mouse and human

RT- PCR or immunoblot

253,266–268

FFAR1 and FFAR4

• FFAR1: medium- chain and long- chain free fatty acids

• FFAR4: omega-3 PUFAs

Ghrelin, somatostatin, gastrin, CCK , GLP1 and GIP release

Mouse and human

RT- PCR , ISH for FFAR1, qPCR or IHC for both receptors

248,249,258–260

TAS1R1–TAS1R3

Amino acids and oligopeptides

Ghrelin, CCK and GLP1 release

Mouse and human

qPCR , IHC for both receptors or immunoblot for TAS1R1

241,247,250,256

TAS1R2–TAS1R3

Glucose (antagonist lactisole)

GLP1 release Mouse and human

qPCR or IHC 241,315,316

Parietal cells TAS2R Bitter agonists Stimulation of gastric acid secretion

Human qPCR or IHC 254

Pancreas β- Cells TAS1R2–TAS1R3

Sweet agonists Potentiation of glucose- induced insulin release by fructose and artificial sweeteners

Mouse and human

qPCR 227

FFAR1 Medium- chain and long- chain fatty acids

Contradictory findings: potentiation of glucose- induced insulin secretion but also impaired glucose homeostasis

Rodent and human

ISH or qPCR 228,229

δ- Cells FFAR4 Omega-3 PUFAs Inhibition of somatostatin to regulate insulin secretion

Mouse and human

ISH 226

CCK , cholecystokinin; FFAR , free fatty acid receptor ; GIP, gastric inhibitory polypeptide; GLP1, glucagon- like peptide 1; ICC, immunocytochemistry ; IHC, immunohistochemistry ; ISH, in situ hybridization; PTC, phenylthiocarbamide; PUFA , polyunsaturated fatty acid; qPCR , quantitative PCR; RNA- seq, RNA-sequencing; RT-PCR , reverse transcription PCR; TAS1R , taste receptor family 1; TAS2R , taste receptor family 2.

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ORs have been deorphanized1. Notably, although 25 functional TAS2R genes have been identified in humans, several receptors are yet to be deorphanized101.

In addition, although ectopic functions for several OR and taste receptors have been identified, most stud-ies have been performed in cell lines and mouse models. Confirmation of the role of these receptors in humans is therefore vital. While some orthologues have been clearly identified, for example, human OR51E2 and mouse OLFR78, it is often that a human orthologue of a mouse receptor cannot be identified by sequence analy-sis. In addition, the identification of endogenous ligands is challenging, and a human orthologue may not always share ligands with a mouse receptor. Another challenge in the translation of findings in models to humans is whether, even with dietary supplementation, the levels of tastants or odorants are enough to sustain chronic ago-nism of the receptor or whether a potent agonist and/or antagonist with high levels of bioavailability can be iden-tified for clinical application. Notably, it has been shown that aroma and flavour compounds can be delivered to the circulation via inhalation, percutaneous application and food intake. The concentration of particular com-ponents of essential oils, such as the monoterpenes lin-alool and linalyl acetate, can reach concentrations in the micromolar range285.

Achieving a high level of functional expression of receptors in a heterologous system can also be diffi-cult. Co- expression of several chaperones increases the membrane targeting of ORs and taste receptors; how-ever, the molecular basis for the specificity and sensi-tivity of these chaperones to specific receptors is poorly understood. Until now, no full- length 3D structure of ORs, TAS1Rs and TAS2Rs has been elucidated. Thus, key information on the molecular mechanisms of ligand binding, which triggers signal transduction pathways, is incomplete, and the development of potent and specific agonists and antagonists is a time- intensive and labour- intensive process. However, there has been substantial progress in understanding the structure of these recep-tors. For example, the crystal structure of the extracel-lular ligand- binding domains of TAS1R2 and TAS1R3

has been recently reported286. Single- particle cryo- electron microscopy has been applied to determine GPCR structures287,288, which might also be used in the structure studies of ORs and taste receptors.

As in the case of other GPCRs, it is often difficult to develop a high- quality antibody to detect these recep-tors; this is especially true for ORs, which share high sequence homology (from 50% to 99%). Thus, most of the evidence for ectopic expression of ORs has been con-firmed at the level of mRNAs not proteins. Therefore, nucleic acid aptamers289,290 and protein scaffolds291 have also been suggested to detect proteins of interest. These newer options offer opportunities to rectify problems stemming from using poorly validated antibodies in research.

Finally, when contemplating targeting ectopic ORs and taste receptors, given their often multiple functions, the potential for off- target effects must be considered. Indeed, while some TAS2Rs are narrowly tuned and detect only a limited number of bitter compounds, other TAS2Rs are broadly tuned. As a result, when tar-geting TAS2Rs, many off- target effects may occur and are believed to play a role in the side effects of bitter- tasting drugs in clinical use292. In addition, some TAS2Rs are highly polymorphic (such as TAS2R38 (REF.43)), which may affect the sensitivity of patients to bitter tastants. These issues are also important in OR research.

OutlookMore than half of the approximately 370 ORs from the nasal epithelium have been shown to be extra- nasally expressed7, and many taste receptors are found in extra- oral tissues. Considering that only approximately 10% have been functionally characterized, the potential roles of unexamined ORs and taste receptors are obvi-ously broad. ORs and taste receptors should no longer be considered as pure odour or taste receptors but rather as general chemoreceptors involved in physio-logical and pathophysiological processes throughout the human body, representing a largely unexplored therapeutic field.

Published online xx xx xxxx

1. Silva Teixeira, C. S., Cerqueira, N. M. & Silva Ferreira, A. C. Unravelling the olfactory sense: from the gene to odor perception. Chem. Senses 41, 105–121 (2016). This is a comprehensive review of the role played by ORs and associated signalling pathways in sensory olfactory neurons.

2. Wackermannova, M., Pinc, L. & Jebavy, L. Olfactory sensitivity in mammalian species. Physiol. Res. 65, 369–390 (2016).

3. Breslin, P. A. S. An evolutionary perspective on food and human taste. Curr. Biol. 23, R409–R418 (2013).

4. Dalton, R. P. & Lomvardas, S. Chemosensory receptor specificity and regulation. Annu. Rev. Neurosci. 38, 331–349 (2015).

5. Ihara, S., Yoshikawa, K. & Touhara, K. Chemosensory signals and their receptors in the olfactory neural system. Neuroscience 254, 45–60 (2013).

6. Feldmesser, E. et al. Widespread ectopic expression of olfactory receptor genes. BMC Genomics 7, 121 (2006). This study systematically profiles OR gene expression in different tissues, which express specific, relatively small OR gene subsets at particularly high levels, and suggests that such small OR subsets may play functional roles in different tissues.

7. Flegel, C., Manteniotis, S., Osthold, S., Hatt, H. & Gisselmann, G. Expression profile of ectopic olfactory receptors determined by deep sequencing. PLOS ONE 8, e55368 (2013). This study uses next- generation sequencing to perform comprehensive RNA- seq experiments investigating the ectopic OR profiles of 16 human tissues. All tissues examined express at least some ORs, and several ORs such as OR2W3 and OR51E1 are widely distributed. OR4N4 is shown to be specifically expressed in the testis.

8. Steensels, S. & Depoortere, I. Chemoreceptors in the gut. Annu. Rev. Physiol. 80, 117–141 (2017). This is a comprehensive review of the role played by taste receptors in enteroendocrine cells of the gut.

9. Spehr, M. et al. Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science 299, 2054–2058 (2003). This is the first study to prove the functionality of an OR located outside the nose. In this study, OR1D2 is reported to be highly expressed in human spermatozoa and is activated by the odorant bourgeonal, eliciting positive chemotactic and chemokinetic swimming behaviour by spermatozoa.

10. Busse, D. et al. A synthetic sandalwood odorant induces wound- healing processes in human keratinocytes via the olfactory receptor OR2AT4. J. Invest. Dermatol. 134, 2823–2832 (2014). This study is the first to investigate the role of an OR in skin keratinocytes. OR2AT4 activation by Sandalore is shown to induce wound healing by stimulating keratinocyte proliferation via activation of ERK1, ERK2 and p30 in ex vivo human skin. Novel wound- healing therapeutics were developed on the basis of these findings.

11. Tsai, T. et al. Two olfactory receptors- OR2A4/7 and OR51B5-differentially affect epidermal proliferation and differentiation. Exp. Dermatol. 26, 58–65 (2017).

12. Cheret, J. et al. Hair follicles engage in chemosensation: olfactory receptor OR2AT4 regulates human hair growth. Nat. Commun. 9, 3624 (2018). This study demonstrates the functionality of OR2AT4 in human hair growth. OR2AT4 is expressed in the human hair follicles, and activation by Sandalore prolongs the active growth phase of hair by inducing the expression of IGF1. Results from the preliminary test on humans support the findings from ex vivo culture of human follicles.

Nucleic acid aptamersOligonucleotides (single- stranded DNAs or RNAs) that bind to a specific target molecule with high affinity and specificity. Aptamers can target proteins that remain inaccessible to antibodies and could be functional in a wider range of conditions.

Protein scaffoldssmall proteins designed to bind to a specific target molecule with high affinity and specificity. These scaffolds can target proteins that remain inaccessible to antibodies and could be functional in a wider range of conditions.

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13. Griffin, C. A., Kafadar, K. A. & Pavlath, G. K. MOR23 promotes muscle regeneration and regulates cell adhesion and migration. Dev. Cell 17, 649–661 (2009). This study investigates the role played by the mouse OR OLFR16 (MOR23) in skeletal muscle. In this study, the receptor stimulates skeletal muscle regeneration by inducing myocyte migration and adhesion, thus contributing to tissue repair.

14. Weber, L. et al. Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis. PLOS ONE 12, e0172491 (2017).

15. Kalbe, B. et al. Helional- induced activation of human olfactory receptor 2J3 promotes apoptosis and inhibits proliferation in a non- small-cell lung cancer cell line. Eur. J. Cell Biol. 96, 34–46 (2017).

16. Massberg, D. et al. The activation of OR51E1 causes growth suppression of human prostate cancer cells. Oncotarget 7, 48231–48249 (2016).

17. Neuhaus, E. M. et al. Activation of an olfactory receptor inhibits proliferation of prostate cancer cells. J. Biol. Chem. 284, 16218–16225 (2009).

18. Manteniotis, S. et al. Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia. Cell Death Discov. 2, 15070 (2016). This study is the first to describe an OR- mediated pathway in human myelogenous leukaemia cells; the pathway is shown to regulate cell proliferation, apoptosis and differentiation after stimulation of OR2AT4 with an odorant.

19. Wu, C. et al. Olfactory receptor 544 reduces adiposity by steering fuel preference toward fats. J. Clin. Invest. 127, 4118–4123 (2017). This study is the first to investigate the role played by ectopic ORs in obesity and energy metabolism in vivo. In this study, OLFR544 activation by azelaic acid reduces adiposity via induction of fatty acid oxidation, thermogenesis and lipolysis in the liver, brown adipose tissue and white adipose tissue, respectively.

20. Calvo, S. S. & Egan, J. M. The endocrinology of taste receptors. Nat. Rev. Endocrinol. 11, 213–227 (2015).

21. Depoortere, I. Taste receptors of the gut: emerging roles in health and disease. Gut 63, 179–190 (2014).

22. Devillier, P., Naline, E. & Grassin- Delyle, S. The pharmacology of bitter taste receptors and their role in human airways. Pharmacol. Ther. 155, 11–21 (2015).

23. Suckow, A. T. & Briscoe, C. P. Key questions for translation of FFA receptors: from pharmacology to medicines. Handb. Exp. Pharmacol. 236, 101–131 (2017).

24. Hutchings, C. J., Koglin, M., Olson, W. C. & Marshall, F. H. Opportunities for therapeutic antibodies directed at G- protein-coupled receptors. Nat. Rev. Drug Discov. 16, 787–810 (2017).

25. Gelis, L. et al. Functional characterization of the odorant receptor 51E2 in human melanocytes. J. Biol. Chem. 291, 17772–17786 (2016). This study examines the role played by OR51E2 in melanogenesis. In this study, activation of the receptor by β- ionone is shown to reduce melanogenesis and increase the levels of cAMP and intracellular calcium, and cell proliferation is inhibited by receptor activation.

26. Weber, L. et al. Characterization of the olfactory receptor OR10H1 in human urinary bladder cancer. Front. Physiol. 9, 456 (2018).

27. Xu, L. L. et al. PSGR, a novel prostate- specific gene with homology to a G protein- coupled receptor, is overexpressed in prostate cancer. Cancer Res. 60, 6568–6572 (2000).

28. Xu, L. L. et al. Quantitative expression profile of PSGR in prostate cancer. Prostate Cancer Prostat. Dis. 9, 56–61 (2006).

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AcknowledgementsThe authors thank C. Wu and Y.-J. Kim for assistance in man-uscript preparations. This work was supported by a grant from the National Research Foundation (NRF) of Korea (NRF-2018R1A4A1022589 and 2016R1A2A2A05005483), funded by the South Korean government (MSIP), and was supported by a grant from the University of Leuven (Methusalem grant) and Research Foundation Flanders (G073615N).

Competing interestsThe authors declare no competing interests.

Publisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

RElatEd liNksClinicalTrials.gov: https://clinicaltrials.gov/

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