Vol 442|10 August 2006|doi:10.1038/nature05066 ARTICLES A second class of chemosensory receptors in the olfactory epithelium Stephen D. Liberles1 & Linda B. Buck1 The mammalian olfactory system detects chemicals sensed as odours as well as social cues that stimulate innate responses. Odorants are detected in the nasal olfactory epithelium by the odorant receptor family, whose ,1,000 members allow the discrimination of a myriad of odorants. Here we report the discovery of a second family of receptors in the mouse olfactory epithelium. Genes encoding these receptors, called ‘trace amine-associated receptors’ (TAARs), are present in human, mouse and fish. Like odorant receptors, individual mouse TAARs are expressed in unique subsets of neurons dispersed in the epithelium. Notably, at least three mouse TAARs recognize volatile amines found in urine: one detects a compound linked to stress, whereas the other two detect compounds enriched in male versus female urine—one of which is reportedly a pheromone. The evolutionary conservation of the TAAR family suggests a chemosensory function distinct from odorant receptors. Ligands identified for TAARs thus far suggest a function associated with the detection of social cues. The first step in odour perception is the detection of odorants by G which we then used in real-time quantitative PCR (qPCR) reactions protein-coupled odorant receptors on olfactory sensory neurons with primers matching GPCRs not previously implicated in odour, (OSNs) in the nasal olfactory epithelium1–3. In response to odorants, pheromone or taste detection19. cDNAs encoding individual GPCRs OSNs transmit signals to the brain, thereby generating odour were quantified using standard curves obtained from qPCR reactions perceptions2,4. Each OSN expresses a single functional odorant with titrations of mouse genomic DNA. receptor gene5, and OSNs with the same odorant receptor are To confirm whether the GPCRs identified by qPCR are actually randomly dispersed within one olfactory epithelial zone6,7. Consist- expressed by OSNs, we used RNA in situ hybridization. Initial studies ent with their ability to detect and discriminate diverse odorants, revealed two GPCR genes—Taar7d and Taar9—that are expressed in mammals have as many as 1,000 different odorant receptors that vary small subsets of OSNs. Digoxigenin-labelled antisense RNA probes in protein sequence8–10 and are used combinatorially to detect for each of these genes hybridized to messenger RNA in a small different odorants and encode their unique identities5. These features percentage of OSNs that were dispersed in certain olfactory epithelial of the odorant receptor family would seem to account easily for the regions (Fig. 1), an expression pattern similar to that of individual odorant recognition abilities of mammals. However, a small percen- odorant receptor genes6,7. Both of these genes encode members of the 11 20,21 tage of OSNs lack Gaolf , the G protein through which odorant trace amine-associated receptor (TAAR) family . On the basis of receptors signal12, suggesting that they might express another class of genome sequence data, this family has 15 members in mouse and 6 in chemosensory receptor. In addition, small peptides that bind major human, and is also found in fish22. TAARs are unrelated to odorant histocompatibility complex (MHC) proteins can stimulate some receptors, with their closest relatives being receptors for biogenic OSNs13, suggesting that those OSNs might express a class of receptor amines such as serotonin and dopamine. For example, mouse TAAR1 that detects peptides rather than small volatile odorants. Finally, is 33% identical to the mouse 5-hydroxytryptamine (serotonin) although many pheromones are detected in the vomeronasal organ— receptor 4, but only 16% identical to the most closely related an olfactory structure with receptors that differ from odorant mouse odorant receptor (OLFR461), and it lacks sequence motifs receptors3,14—responses to some mouse pheromones involve the characteristic of odorant receptors. olfactory epithelium15–17, raising the possibility that the olfactory To examine whether other TAARs are also expressed in the epithelium also contains a dedicated class of pheromone receptors. olfactory epithelium, we used primers specific for each mouse Taar gene in qPCR reactions with cDNAs from the olfactory epithelium A second family of receptors in the olfactory epithelium and other mouse tissues (Fig. 2). These experiments indicated that all To explore whether there might be other types of chemosensory mouse Taar genes, except Taar1, are expressed in the olfactory receptors in the olfactory epithelium, we initiated a search for epithelium. They further showed that the expression levels of additional G protein-coupled receptors (GPCRs) expressed by individual Taar genes in the olfactory epithelium resemble those of mouse OSNs. In preliminary studies, we found that tissue fixation odorant receptor genes (Fig. 2). with glutaraldehyde reveals endogenous b-galactosidase activity Although TAARs have been proposed to function as receptors for present in OSNs, but not in other olfactory epithelial cells (Sup- trace amines (for example, tyramine and octopamine) in the brain, plementary Fig. S1). To obtain an enriched population of OSNs, we we obtained no evidence for Taar gene expression in any tissue— treated dissociated olfactory epithelial cells with a fluorescent b- including the brain—apart from the olfactory epithelium, even galactosidase substrate—fluorescein di-(b-galactopyranoside)18— though we detected high-level expression of genes encoding several and then isolated labelled cells by fluorescence-activated cell sorting. biogenic amine receptors in the brain. It could be that TAARs are Using RNA from the sorted cells, we prepared complementary DNA, expressed in small subsets of brain neurons below the detection level 1Howard Hughes Medical Institute, Basic Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109, USA. 645 © 2006 Nature Publishing Group ARTICLES NATURE|Vol 442|10 August 2006 of these assays (,100 copies of mRNA per cell in 60,000 brain cells). or exclusively in the olfactory epithelium. However, the expression of The Taar1 gene is reportedly expressed in mouse and human brain23 some TAARs in small subsets of cells in other tissues cannot be and the Taar9 gene in human pituitary (and skeletal muscle)24, but excluded. qPCR reactions with 50-fold more mouse brain cDNA (detection Zebrafish reportedly has 57 intact taar genes22, only one of threshold, ,100 copies of mRNA per cell in 1,200 cells) also failed to which—taar9—is listed as such at the NCBI. Interestingly, UniGene reveal the expression of either Taar1 or Taar9 in mouse brain (data lists three ESTs for zebrafish taar9, all of which are from olfactory not shown). Consistent with these results, no expressed sequence tags rosettes—the location of the fish olfactory epithelium. Using the (ESTs) were listed for any mouse Taar gene in the UniGene database zebrafish Taar9 protein to search ESTs translated in silico by of the National Center for Biotechnology Information (NCBI; http:// TBLASTN, we found a large number of zebrafish ESTs encoding www.ncbi.nlm.nih.gov/) and, except for two ESTs from stomach related proteins, with 33 of the 37 highest matches being sequences (TAAR1) and eye (TAAR2), all ESTs listed for human TAAR genes from a zebrafish olfactory epithelium cDNA library (NIH_ZGC_14). came from sequence collections derived, in part, from genomic DNA. The olfactory epithelium ESTs included 24 different taar gene Together, these data suggest that TAARs may be expressed primarily sequences, suggesting that numerous different TAARs are expressed in the fish olfactory epithelium. TAAR expression patterns in the olfactory epithelium We next examined the expression of each mouse Taar gene in the olfactory epithelium by RNA in situ hybridization25. Similar to results obtained with odorant receptor probes6,7,everyTaar probe, except a Taar1 probe, labelled a small subset of OSNs that were scattered in the olfactory epithelium in a seemingly random fashion (Fig. 1). The labelled neurons were confined to certain olfactory epithelial zones, which varied among the Taar genes, another feature characteristic of odorant receptor gene expression6,7 (Fig. 1). Each Taar probe was specific for one Taar gene (see below) except the Taar7 and Taar8 probes, which are likely to hybridize with all of the highly related members of the Taar7 (Taar7a, b, d, e and f)orTaar8 (Taar8a, b and c) subfamilies, respectively. Expression of each Taar, except Taar1, was seen in both male and female mice by qPCR as well as RNA in situ hybridization. Quantification of OSNs labelled by individual Taar Figure 2 | Taar genes are selectively expressed in the olfactory epithelium. qPCR analysis was performed in triplicate (^s.d.) using primers specific for nine mouse Taar genes, two mouse odorant receptor genes (OR-M5 Figure 1 | Taar genes are expressed in subsets of olfactory sensory (Olfr139) and OR-EG (Olfr73)), and the mouse beta-Actin gene with cDNAs neurons. Digoxigenin-labelled antisense RNA probes for the mouse Taar prepared from different mouse tissues (A, heart; B, spleen; C, intestine; D, genes indicated were hybridized to coronal sections of mouse olfactory liver; E, brain; F, vomeronasal organ; G, olfactory epithelium; H, taste epithelium. Each Taar probe hybridized to mRNA in a small percentage of papillae; I, olfactory bulb; J, testis; K,