- Email: [email protected]
Functional cloning and reconstitution of vertebrate odorant receptors
Life Sciences 68 (2001) 2199–2206
Functional cloning and reconstitution of vertebrate odorant receptors Kazushige Touhara* Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Abstract The olfactory systems of vertebrates have a remarkable capacity to recognize and discriminate thousands of different odorant molecules. The initial step in the process of odorant perception is the recognition of volatile odorant molecules by a group of roughly one thousand G protein-coupled odorant receptors that are expressed on the surface of olfactory neuronal cilia. The aims of this study were to obtain functional evidence that these putative odorant receptors recognize and respond to specific odorant molecules, and to elucidate the mechanisms of odorant discrimination in vertebrate olfaction at a receptor level. In order to identify odorant receptors that specifically recognize a particular odorant of interest, we developed a functional cloning strategy in an odorant-directed manner by combining Ca21-recording and single cell RT-PCR techniques. We then adopted an adenovirus-mediated expression system or a chimeric receptor approach to reconstitute the functionally cloned receptors for further biochemical analyses. We herein describe how we obtained experimental evidence for a combinatory mechanism of odorant recognition by examining the diversity of odorant receptors that recognize a particular odorant of interest, and by determining ligand specificity and structure-function relationships for individual odorant receptors. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Odorant; Olfactory; Cloning; Receptor
Introduction The discovery of a huge multigene superfamily of G protein-coupled receptors expressed in olfactory neurons provided supportive evidence for a receptor-mediated secondary messenger pathway in olfaction [1] (Figure 1A). This superfamily is widely diversified with low homology especially in the transmembrane domains that are thought to be the regions that bind odorant molecules. The relationship between structural diversity and ligand specificity has been unclear up to this point because of the lack of functional evidence that these puta* Corresponding author. Tel.: 181-3-5841-5135; fax: 181-3-5841-8039. E-mail address: [email protected] (K. Touhara) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 0 6 -2
2200
K. Touhara / Life Sciences 68 (2001) 2199–2206
Fig. 1. A. Structure of an olfactory receptor neuron, and mechanism of olfactory signal transduction. Odorants are detected on the surface of the olfactory neuronal cilia in the olfactory epithelium. An odorant molecule is recognized by a seven-transmembrane receptor, followed by activation of G protein (Golf) and adenylyl cyclase (ACIII). The resulting cAMP activates a cyclic nucleotide-gated channel (CNG channel) that elicits a Ca21/Na1influx, leading to membrane depolarization. B. A scheme for the functional cloning and reconstitution of an odorant receptor. The olfactory receptor neuron (Cell B), which responds to a specific odorant (Odor 1), is identified by Ca21-imaging, followed by single cell RT-PCR analysis to isolate an odorant receptor expressed in the responsive neuron. The functionally cloned receptor is reconstituted in various expression systems that allow for detection of the response to Odor 1 by Ca21-imaging. The screening of other ligands reveals a structurally similar odorant (Odor 1’) that also elicits receptor-mediated signaling, leading to Ca21-influx.
tive odorant receptors bind specific odorant molecules. Although many attempts have been focused on providing proof that a given receptor functions as an odorant perceptive molecule, the experimental data has been inconclusive due to the difficulties in expressing these receptors for functional analyses. The functional expression and ligand identification of the rat orphan olfactory receptor I7, were achieved using an adenovirus-mediated expression strategy in the olfactory epithelium [2], providing the first evidence that the olfactory G
K. Touhara / Life Sciences 68 (2001) 2199–2206
2201
protein-coupled receptor superfamily mediates responses to odorant molecules in the olfactory neurons. The purpose of our studies when we started nearly five years ago, was to obtain experimental evidence that a putative odorant receptor binds to and recognizes odorant molecules. Further, we sought to examine the molecular receptive range of a variety of odorant receptors. The functional expression of the receptor in heterologous expression systems, however, turned out to be rather difficult. Since we also wanted to determine if multiple receptors exist for a specific odorant and subsequently to characterize structural aspects of such receptors, we designed a novel functional cloning strategy to identify the olfactory receptor gene(s) from individual olfactory neurons that show responses to defined odorants (Figure 1B). Materials and methods Isolation and Ca21-imaging of olfactory receptor neurons Olfactory receptor neurons were isolated on a cover glass from the olfactory epithelium of 3–4 weeks old BALB/c CrSlc mice as described previously [3]. The fura-2 based Ca21-imaging of the neurons was performed with an ARGUS50 Ca21-imaging processing system (Hamamatsu Photonics). Odorant solutions were applied to cells for 5–10 sec at an interstimulus interval long enough to wash the chamber. Single cell RT-PCR Crude RNA was purified from each odorant-responsive cell using GlassMAX spin cartridges (GIBCO) as previously described [3]. After treatment with DNase, the samples were subjected to a reverse transcriptase reaction, followed by amplification of olfactory receptor cDNA using degenerate primers designed from conserved amino acid sequences in the odorant receptor superfamily. Functional expression of odorant receptors A total of 2.5z5.0 ml recombinant adenovirus solution at a titer of 109 pfu/ml was injected into the nostril of a 3–4-week-old C57bl/6NCrj mouse (Charles River) to express the receptor protein of interest and GFP bicistronically. The infected olfactory neurons were isolated from the epithelium 2z3 days post-infection, and the recordings of odorant responses were performed by Ca21-imaging as described above. Results and discussion Functional cloning of odorant receptors from single olfactory neurons Based on the observation that a single olfactory neuron expresses only one of roughly 1000 receptor genes [4, 5], we reasoned that the combination of an odorant response assay with single cell RT-PCR analysis would lead to the isolation of odorant receptors expressed by single olfactory neurons and would allow us to correlate the functions of the receptors with the observed physiological responses. To monitor responses of olfactory neurons to var-
2202
K. Touhara / Life Sciences 68 (2001) 2199–2206
ious odorants, we adopted a fura-2 based Ca21-imaging technique to measure increases in intracellular Ca21-levels in response to odorant stimuli. Odorant-evoked elevations in cAMP are thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca21-influx, leading to membrane depolarization (Figure 1A). The Ca21imaging method was suitable for simultaneous recording of odorant responses in several cells, which greatly facilitated the screening of odorant responsive cells, compared to the typical single cell-based electrophysiological approach. Sequential applications of various odorants by injection into a continuous stream of wash buffer flowing over the cells, allowed for the identification of odorant responsive cells from which the expressed odorant receptor cDNA was amplified by single cell RT-PCR using primers designed from conserved amino acid sequences in the olfactory receptor family [3]. We have so far isolated a total of 16 odorant receptor genes from cells that responded to various odorants, including six receptor genes for eugenol, four receptor genes for cresol, one for carvone, one for ethylvanillin, one for pyridine, and one receptor gene from a cell that responded to both carvone and ethylvanillin (Inaki et al., manuscript in preparation). Although reconstitution and functional analyses of these receptors are necessary to determine the ligand specificity of these receptors, the results clearly suggest that a particular odorant can be recognized by more than one receptor. The multiple receptors, which recognized the same odorant (i.e. eugenol or cresol in our studies), were widely diversified according to phylogenic analyses based on the primary amino acid sequences (Inaki et al., manuscript in preparation). The receptors that recognize a spicy smell, eugenol, are less diverse in the phylogenic tree than the ones which recognize the more simple molecule, cresol, possibly because the recognition of a more complex molecule requires a binding site that is relatively more conserved among the receptors. One receptor for carvone is closely related to the receptor for limonene [6], a molecule that is structurally similar to carvone, while one of the other carvone receptors [6] shows proximity to one of the receptors for cresol, a molecule that has minimum structural similarity with carvone. These analyses suggest that multiple receptors for a certain odorant recognize different epitopes on the target odorant molecule, and that the phylogenic analyses of odorant receptors do not provide clear information for predicting candidate ligands or identifying residues involved in ligand binding. In one study, it was found that many of the receptors, which recognize structurally similar odorants, are more closely related to each other than to most other previously isolated receptors [7]. The level of homology reported, however, is still insufficient for identifying putative receptor genes for particular odorants from existing genome data bases. Functional expression and ligand-specificity of odorant receptors The combination of Ca21-imaging and single cell RT-PCR techniques enabled us to isolate specific receptor genes that encode a receptor for a particular odorant. In order to ascertain the reliability of this funtional cloning approach and to be able to proceed with further biochemical characterization of the cloned receptors, functional expression and reconstitution of the receptors were required (Figure 1B). To overcome the difficulties in expressing odorant receptors in typical expression systems, including mammalian cell lines, we decided to target the olfactory neuron itself as an expression system for the reconstitution of odorant receptors using recombinant adenoviruses as gene transfer mediators.
K. Touhara / Life Sciences 68 (2001) 2199–2206
2203
A receptor isolated from a cell that responded to lyral, which turned out to be identical to the previously characterized mouse receptor MOR23, was expressed via recombinant adenovirus in olfactory neurons, and the response to lyral was successfully demonstrated for reconstituted MOR23 [3]. The reconstitution of MOR23 proved that the lyral response, which was originally observed in the single olfactory neuron, was indeed derived from the MOR23-lyral interaction and that the type of odorant receptors expressed by single neurons correlated with the physiological response of the cell to the specific odorant. Although the adenovirus-mediated expression system was also successfully utilized for ligand-screening of the rat orphan receptor I7 [2], we observed that not all of the functionally cloned receptors were successfully reconstituted by this adenovirus approach (Touhara, unpublished observation). We then utilized other expression systems that had been reported during the course of our studies. It should be noted that attempts to express the odorant receptors in various primary culture preparations or cell lines derived from olfactory neurons have thus far been unsuccessful (Kajiya et al., unpublished observation). The chimeric receptor approach using N-terminal sequences of rhodopsin [6], however, was successfully applied to some of our receptors, although again not all of them were functionally expressed (Kajiya et al., manuscript in preparation). An important caveat from these studies is that there has been no approach applicable to all odorant receptors, and that receptors successfully expressed in the adenovirus system were not necessarily expressed using the chimeric receptor approach for unknown reasons. In any case, proper post-translational modification of an odorant receptor and its appropriate translocation to the plasma membrane are required for functional expression. Our reconstitution studies not only supported the reliability of our single cell RT-PCR approach, but provided information on ligand specificity and the structure-function relationships of odorant receptors. For example, a screening of other ligands for MOR23 revealed that hydroxycitronellol and its dimethyl acetal evoked Ca21-increase, suggesting that the tertiary alcohol group common to lyral, hydroxycitronellol, and its dimethyl acetal is essential for recognition by MOR23, and that the cyclohexenecarbaldehyde moiety in lyral is not critical although the binding pocket prefers a relatively polar group in that position [3]. It is interesting to note that lyral and hydroxycitronellol have different aromas, indicating that an odorant receptor does not necessarily recognize odorants with the same aroma but responds to odorants that are similar in structure (Figure 1B). A combinatory mechanism of odorant recognition How is the enormous diversity of aromas recognized by the odorant receptors? It has been speculated that more than hundred thousand different odorants are discriminated by a combinatory mechanism involving the recognition of single odorant compounds by multiple receptor molecules, and the diverse molecular receptive range of each odorant receptor (Figure 2). This combinatory mechanism was proposed based on the observation that a single olfactory neuron seemed to express only one of z1000 receptor genes [4, 5], and that the olfactory receptor neurons expressing a given odorant receptor converged onto defined glomeruli in the olfactory bulb [8, 9]. Thus, given the number of odorant receptor genes, an almost infinite number of odorants could be discriminated by a combinatory receptor code for each odorant
2204
K. Touhara / Life Sciences 68 (2001) 2199–2206
Fig. 2. A model for the formation of activation pattern in olfactory bulb by a combinatorial mechanism of odorantreceptor interaction. A. One odorant is recognized by multiple receptors and one receptor recognizes a wide range of odorants. The structural epitopes in odorant molecules determine the type of receptors that recognize the particular odorant. B. The olfactory receptor neurons expressing a given odorant receptor converge onto defined glomeruli in the olfactory bulb. C. Activation of multiple receptors for a particular odorant elicits a specific spatial activation pattern in the olfactory bulb that defines the aroma of an odorant molecule.
K. Touhara / Life Sciences 68 (2001) 2199–2206
2205
that provides a specific pattern of neuronal activation both in the olfactory epithelium and bulb. Our two-step approach (Figure 1B), the receptor screening for a given odorant(s) from single neurons and the functional expression of the clone receptor, supports the hypothesis of a combinatory mechanism of odorant recognition. A single olfactory receptor does not recognize odorants with the same aroma but recognizes similarities in structure of the odorant molecules as is the case for other G protein coupled receptors. The creation of a certain aroma or ‘odor’ is established by the activation of multiple receptors that lead to the formation of specific activity patterns in the olfactory bulb where the tuning events occur [10] (Figure 2). In this aspect, although the prediction or designing of agonists or antagonists for a particular olfactory receptor is possible using standard pharmacological techniques, the antagonist to a specific olfactory receptor would not alter the recognition of an aroma, unless all receptors that bind the particular odorant are antagonized. Thus, knocking-out an odorant receptor gene would not cause a significant change in overall perception of the ligand odorant. Living things utilize olfaction as a highly complex molecular recognition system to discriminate among thousands of structurally diverse odorant molecules. The recent progress in understanding the molecular mechanism of olfaction at a receptor level have begun to shed new light on how living things detect and discriminate odors. Amazingly, the multigene olfactory receptor family in mammals consists of more than 1% of the total genome [11]. This unique molecular recognition system has a long evolutionary history and is present in organisms as diverse as nematodes to mammals. Information derived from olfactory research in various living things will undoubtedly have far-reaching effects, impacting various fields including anatomy, physiology, behavioral biology, neurobiology, pharmacology, and evolution.
Acknowledgments I thank Drs. Tatsuya Haga, Ushio Kikkawa, and Hiroshi Kataoka for support, Koichiro Inaki and Kentaro Kajiya for experimental help, Drs. Takaaki Sato and Hitoshi Sakano for collaboration, and Jennifer Ito for English correction. This work has been supported by grants from the Ministry of Education, Science, and Culture, and the Ministry of International Trade and Industry. The author is a recipient of a grant from NOVARTIS Foundation for the Promotion of Science.
References 1. Buck L, Axel R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 1991; 65: 175–87 2. Zhao H, Lidija I, Otaki JM, Hashimoto M, Mikoshiba K, Firestein S. Functional expression of a mammalian odorant receptor. Science 1998; 279: 237–42. 3. Touhara K, Sengoku S, Inaki K, Tsuboi A, Hirono J, Sato T, Sakano H, Haga T. Functional identification and reconstitution of an odorant receptor in single olfactory neurons. Proc. Natl. Acad. Sci. USA; 96: 4040–5. 4. Ressler KJ, Sullivan SL, Buck LB. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 1993; 73: 597–609
2206
K. Touhara / Life Sciences 68 (2001) 2199–2206
5. Vassar R, Ngai J, Axel R. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 1993; 74: 309–318. 6. Krautwurst D, Yan KW, Reed RR. Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 1998; 95: 917–26. 7. Malnic B, Hirono J, Sato T, Buck LB. Combinatorial receptor codes for odors. Cell 1999; 96: 713–23. 8. Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB, Axel R. Topographic organization of sensory projections to the olfactory bulb. Cell 1994; 79: 981–91. 9. Ressler KJ, Sullivan SL, Buck LB. Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 1994; 79: 1245–55. 10. Mori K, Nagao H, Yoshihara Y. The olfactory bulb: coding and processing of odor molecule information. Science 1999; 286: 711– 11. Mombaerts P. Seven-transmembrane proteins as odorant and chemosensory receptors. Science 1999; 286: 707–11.
Functional cloning and reconstitution of vertebrate odorant receptors Kazushige Touhara* Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
Abstract The olfactory systems of vertebrates have a remarkable capacity to recognize and discriminate thousands of different odorant molecules. The initial step in the process of odorant perception is the recognition of volatile odorant molecules by a group of roughly one thousand G protein-coupled odorant receptors that are expressed on the surface of olfactory neuronal cilia. The aims of this study were to obtain functional evidence that these putative odorant receptors recognize and respond to specific odorant molecules, and to elucidate the mechanisms of odorant discrimination in vertebrate olfaction at a receptor level. In order to identify odorant receptors that specifically recognize a particular odorant of interest, we developed a functional cloning strategy in an odorant-directed manner by combining Ca21-recording and single cell RT-PCR techniques. We then adopted an adenovirus-mediated expression system or a chimeric receptor approach to reconstitute the functionally cloned receptors for further biochemical analyses. We herein describe how we obtained experimental evidence for a combinatory mechanism of odorant recognition by examining the diversity of odorant receptors that recognize a particular odorant of interest, and by determining ligand specificity and structure-function relationships for individual odorant receptors. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Odorant; Olfactory; Cloning; Receptor
Introduction The discovery of a huge multigene superfamily of G protein-coupled receptors expressed in olfactory neurons provided supportive evidence for a receptor-mediated secondary messenger pathway in olfaction [1] (Figure 1A). This superfamily is widely diversified with low homology especially in the transmembrane domains that are thought to be the regions that bind odorant molecules. The relationship between structural diversity and ligand specificity has been unclear up to this point because of the lack of functional evidence that these puta* Corresponding author. Tel.: 181-3-5841-5135; fax: 181-3-5841-8039. E-mail address: [email protected] (K. Touhara) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 0 6 -2
2200
K. Touhara / Life Sciences 68 (2001) 2199–2206
Fig. 1. A. Structure of an olfactory receptor neuron, and mechanism of olfactory signal transduction. Odorants are detected on the surface of the olfactory neuronal cilia in the olfactory epithelium. An odorant molecule is recognized by a seven-transmembrane receptor, followed by activation of G protein (Golf) and adenylyl cyclase (ACIII). The resulting cAMP activates a cyclic nucleotide-gated channel (CNG channel) that elicits a Ca21/Na1influx, leading to membrane depolarization. B. A scheme for the functional cloning and reconstitution of an odorant receptor. The olfactory receptor neuron (Cell B), which responds to a specific odorant (Odor 1), is identified by Ca21-imaging, followed by single cell RT-PCR analysis to isolate an odorant receptor expressed in the responsive neuron. The functionally cloned receptor is reconstituted in various expression systems that allow for detection of the response to Odor 1 by Ca21-imaging. The screening of other ligands reveals a structurally similar odorant (Odor 1’) that also elicits receptor-mediated signaling, leading to Ca21-influx.
tive odorant receptors bind specific odorant molecules. Although many attempts have been focused on providing proof that a given receptor functions as an odorant perceptive molecule, the experimental data has been inconclusive due to the difficulties in expressing these receptors for functional analyses. The functional expression and ligand identification of the rat orphan olfactory receptor I7, were achieved using an adenovirus-mediated expression strategy in the olfactory epithelium [2], providing the first evidence that the olfactory G
K. Touhara / Life Sciences 68 (2001) 2199–2206
2201
protein-coupled receptor superfamily mediates responses to odorant molecules in the olfactory neurons. The purpose of our studies when we started nearly five years ago, was to obtain experimental evidence that a putative odorant receptor binds to and recognizes odorant molecules. Further, we sought to examine the molecular receptive range of a variety of odorant receptors. The functional expression of the receptor in heterologous expression systems, however, turned out to be rather difficult. Since we also wanted to determine if multiple receptors exist for a specific odorant and subsequently to characterize structural aspects of such receptors, we designed a novel functional cloning strategy to identify the olfactory receptor gene(s) from individual olfactory neurons that show responses to defined odorants (Figure 1B). Materials and methods Isolation and Ca21-imaging of olfactory receptor neurons Olfactory receptor neurons were isolated on a cover glass from the olfactory epithelium of 3–4 weeks old BALB/c CrSlc mice as described previously [3]. The fura-2 based Ca21-imaging of the neurons was performed with an ARGUS50 Ca21-imaging processing system (Hamamatsu Photonics). Odorant solutions were applied to cells for 5–10 sec at an interstimulus interval long enough to wash the chamber. Single cell RT-PCR Crude RNA was purified from each odorant-responsive cell using GlassMAX spin cartridges (GIBCO) as previously described [3]. After treatment with DNase, the samples were subjected to a reverse transcriptase reaction, followed by amplification of olfactory receptor cDNA using degenerate primers designed from conserved amino acid sequences in the odorant receptor superfamily. Functional expression of odorant receptors A total of 2.5z5.0 ml recombinant adenovirus solution at a titer of 109 pfu/ml was injected into the nostril of a 3–4-week-old C57bl/6NCrj mouse (Charles River) to express the receptor protein of interest and GFP bicistronically. The infected olfactory neurons were isolated from the epithelium 2z3 days post-infection, and the recordings of odorant responses were performed by Ca21-imaging as described above. Results and discussion Functional cloning of odorant receptors from single olfactory neurons Based on the observation that a single olfactory neuron expresses only one of roughly 1000 receptor genes [4, 5], we reasoned that the combination of an odorant response assay with single cell RT-PCR analysis would lead to the isolation of odorant receptors expressed by single olfactory neurons and would allow us to correlate the functions of the receptors with the observed physiological responses. To monitor responses of olfactory neurons to var-
2202
K. Touhara / Life Sciences 68 (2001) 2199–2206
ious odorants, we adopted a fura-2 based Ca21-imaging technique to measure increases in intracellular Ca21-levels in response to odorant stimuli. Odorant-evoked elevations in cAMP are thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca21-influx, leading to membrane depolarization (Figure 1A). The Ca21imaging method was suitable for simultaneous recording of odorant responses in several cells, which greatly facilitated the screening of odorant responsive cells, compared to the typical single cell-based electrophysiological approach. Sequential applications of various odorants by injection into a continuous stream of wash buffer flowing over the cells, allowed for the identification of odorant responsive cells from which the expressed odorant receptor cDNA was amplified by single cell RT-PCR using primers designed from conserved amino acid sequences in the olfactory receptor family [3]. We have so far isolated a total of 16 odorant receptor genes from cells that responded to various odorants, including six receptor genes for eugenol, four receptor genes for cresol, one for carvone, one for ethylvanillin, one for pyridine, and one receptor gene from a cell that responded to both carvone and ethylvanillin (Inaki et al., manuscript in preparation). Although reconstitution and functional analyses of these receptors are necessary to determine the ligand specificity of these receptors, the results clearly suggest that a particular odorant can be recognized by more than one receptor. The multiple receptors, which recognized the same odorant (i.e. eugenol or cresol in our studies), were widely diversified according to phylogenic analyses based on the primary amino acid sequences (Inaki et al., manuscript in preparation). The receptors that recognize a spicy smell, eugenol, are less diverse in the phylogenic tree than the ones which recognize the more simple molecule, cresol, possibly because the recognition of a more complex molecule requires a binding site that is relatively more conserved among the receptors. One receptor for carvone is closely related to the receptor for limonene [6], a molecule that is structurally similar to carvone, while one of the other carvone receptors [6] shows proximity to one of the receptors for cresol, a molecule that has minimum structural similarity with carvone. These analyses suggest that multiple receptors for a certain odorant recognize different epitopes on the target odorant molecule, and that the phylogenic analyses of odorant receptors do not provide clear information for predicting candidate ligands or identifying residues involved in ligand binding. In one study, it was found that many of the receptors, which recognize structurally similar odorants, are more closely related to each other than to most other previously isolated receptors [7]. The level of homology reported, however, is still insufficient for identifying putative receptor genes for particular odorants from existing genome data bases. Functional expression and ligand-specificity of odorant receptors The combination of Ca21-imaging and single cell RT-PCR techniques enabled us to isolate specific receptor genes that encode a receptor for a particular odorant. In order to ascertain the reliability of this funtional cloning approach and to be able to proceed with further biochemical characterization of the cloned receptors, functional expression and reconstitution of the receptors were required (Figure 1B). To overcome the difficulties in expressing odorant receptors in typical expression systems, including mammalian cell lines, we decided to target the olfactory neuron itself as an expression system for the reconstitution of odorant receptors using recombinant adenoviruses as gene transfer mediators.
K. Touhara / Life Sciences 68 (2001) 2199–2206
2203
A receptor isolated from a cell that responded to lyral, which turned out to be identical to the previously characterized mouse receptor MOR23, was expressed via recombinant adenovirus in olfactory neurons, and the response to lyral was successfully demonstrated for reconstituted MOR23 [3]. The reconstitution of MOR23 proved that the lyral response, which was originally observed in the single olfactory neuron, was indeed derived from the MOR23-lyral interaction and that the type of odorant receptors expressed by single neurons correlated with the physiological response of the cell to the specific odorant. Although the adenovirus-mediated expression system was also successfully utilized for ligand-screening of the rat orphan receptor I7 [2], we observed that not all of the functionally cloned receptors were successfully reconstituted by this adenovirus approach (Touhara, unpublished observation). We then utilized other expression systems that had been reported during the course of our studies. It should be noted that attempts to express the odorant receptors in various primary culture preparations or cell lines derived from olfactory neurons have thus far been unsuccessful (Kajiya et al., unpublished observation). The chimeric receptor approach using N-terminal sequences of rhodopsin [6], however, was successfully applied to some of our receptors, although again not all of them were functionally expressed (Kajiya et al., manuscript in preparation). An important caveat from these studies is that there has been no approach applicable to all odorant receptors, and that receptors successfully expressed in the adenovirus system were not necessarily expressed using the chimeric receptor approach for unknown reasons. In any case, proper post-translational modification of an odorant receptor and its appropriate translocation to the plasma membrane are required for functional expression. Our reconstitution studies not only supported the reliability of our single cell RT-PCR approach, but provided information on ligand specificity and the structure-function relationships of odorant receptors. For example, a screening of other ligands for MOR23 revealed that hydroxycitronellol and its dimethyl acetal evoked Ca21-increase, suggesting that the tertiary alcohol group common to lyral, hydroxycitronellol, and its dimethyl acetal is essential for recognition by MOR23, and that the cyclohexenecarbaldehyde moiety in lyral is not critical although the binding pocket prefers a relatively polar group in that position [3]. It is interesting to note that lyral and hydroxycitronellol have different aromas, indicating that an odorant receptor does not necessarily recognize odorants with the same aroma but responds to odorants that are similar in structure (Figure 1B). A combinatory mechanism of odorant recognition How is the enormous diversity of aromas recognized by the odorant receptors? It has been speculated that more than hundred thousand different odorants are discriminated by a combinatory mechanism involving the recognition of single odorant compounds by multiple receptor molecules, and the diverse molecular receptive range of each odorant receptor (Figure 2). This combinatory mechanism was proposed based on the observation that a single olfactory neuron seemed to express only one of z1000 receptor genes [4, 5], and that the olfactory receptor neurons expressing a given odorant receptor converged onto defined glomeruli in the olfactory bulb [8, 9]. Thus, given the number of odorant receptor genes, an almost infinite number of odorants could be discriminated by a combinatory receptor code for each odorant
2204
K. Touhara / Life Sciences 68 (2001) 2199–2206
Fig. 2. A model for the formation of activation pattern in olfactory bulb by a combinatorial mechanism of odorantreceptor interaction. A. One odorant is recognized by multiple receptors and one receptor recognizes a wide range of odorants. The structural epitopes in odorant molecules determine the type of receptors that recognize the particular odorant. B. The olfactory receptor neurons expressing a given odorant receptor converge onto defined glomeruli in the olfactory bulb. C. Activation of multiple receptors for a particular odorant elicits a specific spatial activation pattern in the olfactory bulb that defines the aroma of an odorant molecule.
K. Touhara / Life Sciences 68 (2001) 2199–2206
2205
that provides a specific pattern of neuronal activation both in the olfactory epithelium and bulb. Our two-step approach (Figure 1B), the receptor screening for a given odorant(s) from single neurons and the functional expression of the clone receptor, supports the hypothesis of a combinatory mechanism of odorant recognition. A single olfactory receptor does not recognize odorants with the same aroma but recognizes similarities in structure of the odorant molecules as is the case for other G protein coupled receptors. The creation of a certain aroma or ‘odor’ is established by the activation of multiple receptors that lead to the formation of specific activity patterns in the olfactory bulb where the tuning events occur [10] (Figure 2). In this aspect, although the prediction or designing of agonists or antagonists for a particular olfactory receptor is possible using standard pharmacological techniques, the antagonist to a specific olfactory receptor would not alter the recognition of an aroma, unless all receptors that bind the particular odorant are antagonized. Thus, knocking-out an odorant receptor gene would not cause a significant change in overall perception of the ligand odorant. Living things utilize olfaction as a highly complex molecular recognition system to discriminate among thousands of structurally diverse odorant molecules. The recent progress in understanding the molecular mechanism of olfaction at a receptor level have begun to shed new light on how living things detect and discriminate odors. Amazingly, the multigene olfactory receptor family in mammals consists of more than 1% of the total genome [11]. This unique molecular recognition system has a long evolutionary history and is present in organisms as diverse as nematodes to mammals. Information derived from olfactory research in various living things will undoubtedly have far-reaching effects, impacting various fields including anatomy, physiology, behavioral biology, neurobiology, pharmacology, and evolution.
Acknowledgments I thank Drs. Tatsuya Haga, Ushio Kikkawa, and Hiroshi Kataoka for support, Koichiro Inaki and Kentaro Kajiya for experimental help, Drs. Takaaki Sato and Hitoshi Sakano for collaboration, and Jennifer Ito for English correction. This work has been supported by grants from the Ministry of Education, Science, and Culture, and the Ministry of International Trade and Industry. The author is a recipient of a grant from NOVARTIS Foundation for the Promotion of Science.
References 1. Buck L, Axel R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 1991; 65: 175–87 2. Zhao H, Lidija I, Otaki JM, Hashimoto M, Mikoshiba K, Firestein S. Functional expression of a mammalian odorant receptor. Science 1998; 279: 237–42. 3. Touhara K, Sengoku S, Inaki K, Tsuboi A, Hirono J, Sato T, Sakano H, Haga T. Functional identification and reconstitution of an odorant receptor in single olfactory neurons. Proc. Natl. Acad. Sci. USA; 96: 4040–5. 4. Ressler KJ, Sullivan SL, Buck LB. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 1993; 73: 597–609
2206
K. Touhara / Life Sciences 68 (2001) 2199–2206
5. Vassar R, Ngai J, Axel R. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 1993; 74: 309–318. 6. Krautwurst D, Yan KW, Reed RR. Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell 1998; 95: 917–26. 7. Malnic B, Hirono J, Sato T, Buck LB. Combinatorial receptor codes for odors. Cell 1999; 96: 713–23. 8. Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB, Axel R. Topographic organization of sensory projections to the olfactory bulb. Cell 1994; 79: 981–91. 9. Ressler KJ, Sullivan SL, Buck LB. Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 1994; 79: 1245–55. 10. Mori K, Nagao H, Yoshihara Y. The olfactory bulb: coding and processing of odor molecule information. Science 1999; 286: 711– 11. Mombaerts P. Seven-transmembrane proteins as odorant and chemosensory receptors. Science 1999; 286: 707–11.