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Menini A, editor. The Neurobiology of Olfaction. Boca Raton (FL): CRC Press/Taylor & Francis; 2010.

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The Neurobiology of Olfaction.

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Chapter 7Odorant Receptors

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7.1. THE IDENTIFICATION OF ODORANT RECEPTORS (ORs)

The receptors responsible for odorant discrimination were first cloned in 1991 by Linda Buck and Richard Axel (Buck and Axel 1991). A series of physiological and biochemical experiments performed during the mid-1980s indicated that odorant activation of olfactory sensory neurons was mediated by a G-protein-dependent pathway, which led to activation of adenylyl cyclase, increases in intracellular concentrations of cyclic adenosine monophosphate (cAMP), activation of cyclic nucleotide-gated channels, and neuron depolarization (Firestein et al. 1991; Lowe et al. 1989; Nakamura and Gold 1987; Pace et al. 1985; Sklar et al. 1986; see also Chapter 8). The subsequent cloning of olfactory-specific genes coding for a Gα protein (Gαolf) (Jones and Reed 1989) and for a cAMP-gated channel (Dhallan et al. 1990) further strengthened the involvement of cAMP in odorant signal transduction. These experiments strongly indicated that the odorant receptors (ORs) should be G-protein-coupled receptors (GPCRs).

About the same time, the polymerase chain reaction (PCR) technique was developed (Saiki et al. 1988), and the first GPCRs had been identified. Comparison between the sequences of rhodopsin and (β-adrenergic receptors indicated that receptors that couple to G-proteins showed related structures, with seven membrane-spanning regions (Dixon et al. 1986). Comparison of the sequences of a higher number of GPCRs (around 20 G-protein-compled receptor (GPCR) sequences were known by 1989) revealed that they all shared a related seven-transmembrane structure and they also shared limited sequence motifs. In 1989, it was shown for the first time that degenerate primers could be used in PCR reactions to identify new members of the GPCR family (Libert et al. 1989).

The approach used by Buck and Axel to isolate the Odorant receptor (OR) genes was based on the assumptions that the receptors should belong to a large family of GPCRs and their expression should be restricted to the olfactory epithelium (Buck and Axel 1991). Eleven degenerate primers that would allow amplification of all known GPCRs at the time were designed and all possible combinations were used in PCR reactions with rat olfactory epithelium cDNA. As a result, 64 bands of appropriate sizes were obtained in agarose gels. The next step was to screen these bands for the ones containing the OR genes. It was reasoned that if one of the bands contained cDNAs corresponding to multiple OR genes, four-cutter restriction enzymes would cleave the DNA into smaller fragments showing sizes that, when summed up, would produce a size greater than that of the original band. The 64 PCR bands were treated with the HinfI or HaeIII restriction enzymes, and for most of the bands, restriction digestion generated fragments with sizes that summed up to the original band size. Therefore, these PCR products contained a single DNA species. However, restriction digestion of one of the bands produced fragments with sizes that summed to a value far greater than that of the original PCR product. This PCR band contained a mixture of different DNA species, each of which was amplified by the same pair of degenerate primers. The band was cloned into plasmid, and individual recombinant plasmids were sequenced. All sequences were different, but they all showed a GPCR-like structure. Using Northern blot analysis, it was also demonstrated that these receptors are expressed in the olfactory epithelium, but not in a further eight tissues analyzed, including the brain, retina, and liver (Buck and Axel 1991). In addition, in order to estimate the approximate size of the OR gene family, rat genomic libraries were screened for OR genes using a mixture of the OR cDNAs as probes. It was estimated then that the rat haploid genome should contain at least 500–1000 OR genes (Buck and Axel 1991; Buck 1992).

Comparison of different rat OR amino acid sequences revealed that, even though they are extremely diverse, they share conserved motifs that are characteristic of the OR family, such as GN in transmembrane domain I, PMYF/LFL in transmembrane domain II, MAYDRYVAIC in transmembrane domain III, KAFSTCA/GSHLSVV in transmembrane domain 6, and PMLNPFIYSLRN in transmembrane domain VII (Buck and Axel 1991) (Figure 7.1). Additional members of the OR family were identified by using degenerate primers matching these OR motifs in PCR reactions with olfactory epithelium cDNA or genomic DNA (since the OR coding region is contained in one single exon). Degenerate primers matching to the highly conserved motifs in transmembrane III and VI were very efficient in amplifying a large fraction of the mouse OR genes (Malnic et al. 1999; Michaloski et al. 2006; Ressler et al. 1993).

FIGURE 7.1. Typical structure of an odorant receptor.

FIGURE 7.1

Typical structure of an odorant receptor. The diagram illustrates one odorant receptor in the plasmatic membrane (shown in gray), with its seven putative transmembrane domains. Amino acids that are highly conserved among the majority of the OR proteins (more...)

7.2. THE ODORANT RECEPTOR (OR) GENE FAMILY

7.2.1. Class I and Class II Odorant Receptors (ORs)

The ORs can be classified into two distinct classes, based on their amino acid sequences and phylogenetic distribution. The class I ORs were first identified in fish (Ngai et al. 1993) and in frog (Freitag et al. 1995), and it was later shown that teleost fish, including the goldfish, have only class I OR genes (Freitag et al. 1998). Semiaquatic animals, such as frogs, have both class I and class II OR genes (see also Chapter 4), and initially it was believed that mammals lacked functional class I ORs, and only contained class II ORs (Freitag et al. 1998). These findings suggested that the class I ORs (also denominated fishlike ORs) must be specialized in recognizing water-soluble odorants, while class II ORs (mammalianlike ORs) must recognize volatile odorants. However, recent analysis of genome sequences has shown that there are relatively large numbers of class I ORs in the genomes of human (Glusman et al. 2001; Malnic et al. 2004; Zozulya et al. 2001), mouse (Godfrey et al. 2004; Young et al. 2002; Zhang and Firestein 2002), and other mammalian species (Niimura and Nei 2007). Even though the majority of OR genes belong to class II, between 10% and 20% of the ORs in mammals are class I ORs (Glusman et al. 2001; Niimura and Nei 2007; Zhang and Firestein 2002), indicating that class I ORs may also have important roles in mammalian olfaction.

7.2.2. The Size of the Odorant Receptor (OR) Gene Family

The recent availability of the complete genome sequences for several different species allows for the rapid identification of their OR genes. The fact that OR genes have intronless coding regions facilitates their identification. Typically, conserved amino acid sequences corresponding to known OR genes can be used as queries in TBLASTN searches of the genome sequences, to obtain new sequences that are related to OR genes (Glusman et al. 2001; Godfrey et al. 2004; Malnic et al. 2004; Niimura and Nei 2003; Zozulya et al. 2001). The retrieved nucleotide sequences are then translated into amino acid sequences and analyzed. A protein is considered an OR if it is encoded by a coding region of around 1 kb and if it contains the OR sequence motifs (or its variants) located at the appropriate positions (Figure 7.1).

In this way, the complete repertoires of OR genes have been identified for a large number of species. The OR repertoires vary in size and probably reflect the specific olfactory requirements of each one of these species. Some species have high numbers of intact (and potentially functional) OR genes, such as mouse (~1000), dog (~800), and opossum (~1200), while others have comparatively lower numbers of intact OR genes, such as human (~370), chimpanzee (~370), and platypus (~300) (Figure 7.2). The numbers of pseudogenes, which do not express functional ORs, also vary among species, although not as dramatically: while humans and chimpanzees have around 460–480 pseudogenes, dogs, mice, and opossum have around 250–330 pseudogenes (Figure 7.2). The OR gene repertoires also reflect the habitats of the different species. It has been shown, for example, that marine mammals, which evolved from terrestrial ancestors and have adapted to the aquatic environment, have higher numbers of pseudogenes: in dwarf sperm whales and minke whales 77 and 58% of the OR genes are pseudogenes (Kishida et al. 2007). Dolphins completely lack class I OR genes, and their class II OR genes are all pseudogenes (Freitag et al. 1998).

FIGURE 7.2. The size of the OR gene family in different mammalian species.

FIGURE 7.2

The size of the OR gene family in different mammalian species. The numbers of intact OR genes and pseudogenes were determined from platypus (Ornithorhynchus anatinus) (Niimura, Y. and Nei, M. 2007; Warren, W. et al. 2008), opossum (Monodelphis domestica (more...)

The number of intact OR genes does not always correlate well with the olfactory abilities of a given species, indicating that other factors must also be involved. For example, dogs, which have a rich sense of smell, have a smaller number of OR genes than mice or rats (Figure 7.2), however, it is well known that they have larger surfaces of olfactory epithelia. Even though it is generally believed that primates have a poor sense of smell, behavioral studies have shown that primates, including humans, have a surprisingly good sense of smell (Laska et al. 2000). Humans have a smaller number of intact OR genes when compared to dogs or rodents (Figure 7.2). Interestingly, it was recently demonstrated that humans have an unexpectedly high number of glomeruli in their olfactory bulbs: while mice, which have around 1000 intact OR genes, have around 1800 glomeruli, humans, who have around 400 intact OR genes, have on average 5500 glomeruli per olfactory bulb (Maresh et al. 2008). Combined with the fact that the regions in the human brain that are involved in olfactory processing are expanded when compared to other species, these anatomical differences may explain why humans have a good olfactory sensitivity despite having a small repertoire of OR genes (Shepherd 2004).

The expression in the olfactory epithelium has been confirmed for around 400 mouse OR genes through the screening of an olfactory cDNA library with degenerate olfactory receptor probes (Young et al. 2003). Using quantitative RT-PCR, it was also demonstrated that some OR genes are expressed at higher levels than others. It was observed that the expression levels can vary by 10- to 300-fold between genes. These same differences were found in three different mice that were examined, although there was a variation in the expression level of some OR genes between mice (Young et al. 2003). Differences may be due to increased numbers of expressing neurons, or to increased levels of OR gene transcripts per expressing neuron.

A high-throughput microarray analysis detected the specific expression of ~800 mouse OR genes in the olfactory epithelium (Zhang et al. 2004). Very few OR genes were expressed in the nonolfactory tissues that were analyzed, such as testis, liver, heart, cerebellum, and muscle, showing that although there might be a small number of OR genes expressed in other tissues, very few are exclusively expressed in nonolfactory epithelium tissues. Microarray analysis was also used to analyze the expression of human OR genes (Zhang et al. 2007). This study detected the expression of 437 OR genes, including pseudogenes, in the human olfactory epithelium.

7.2.3. Comparative Genomics of Odorant Receptors (ORs)

Analysis of the composition of the OR gene families in different species has revealed several interesting points regarding the olfactory sensory function in these animals. In one study, a random group of 221 ORs was cloned from 10 different primate species, from prosimian lemur to human. Analysis of these OR gene sequences showed that the percentage of functional OR genes decreases, from New World monkeys to hominoids: while New World monkeys (like the squirrel monkey and marmoset) lack pseudogenes, Old World monkeys (macaque and baboon) have around 27% pseudogenes, and hominoids (chimpanzee, gorilla, orangutan, and human) have around 50% pseudogenes (Rouquier et al. 2000). These numbers may reflect the evolution of the olfactory sensory function in primates, which shows reduced olfactory abilities, when compared to other species, such as rodents and dogs. The recent availability of the complete sequence of the chimpanzee genome allowed for the comparison between the entire human and chimpanzee OR gene repertoires (Gilad et al. 2005; Gimelbrant et al. 2004; Go and Niimura 2008). While one study showed that humans have a significantly higher percentage of pseudogenes than chimpanzees (Gilad et al. 2005), another study, where an updated version of the chimpanzee genome sequence was analyzed, showed that the numbers of pseudogenes and intact OR genes are approximately the same between the two species (Go and Niimura 2008). However, this same study showed that 25% of the intact ORs are nonorthologous between human and chimpanzees (Go and Niimura 2008), indicating that the OR repertoires of these two species are somewhat different. Differences in OR repertoire composition may be responsible for species-specific abilities of odorant detection, and have also been observed when other species are compared. For instance, comparison of the human and mouse genome sequences identified 29 human ORs that have no counterpart in the mouse, and 177 mouse ORs with no counterpart in humans (Godfrey et al. 2004). Comparison of the canine and human OR genes showed that the canine repertoire has expanded relative to that of humans, leading to the emergence of specific canine OR genes (Quignon et al. 2003). For the class I ORs, no human or dog OR genes appeared to be species-specific, but for class II, one group of 26 ORs was considered to be dog-specific, as no counterparts were found in humans (Quignon et al. 2003).

7.2.4. Chromosomal Distribution of the Odorant Receptor (OR) Genes

Early analysis of the chromosomal distribution of the mouse OR genes revealed that they are broadly distributed in the genome (Sullivan et al. 1996). In these experiments, the chromosomal locations of 21 mouse OR genes were experimentally determined using genetic crosses, and it was shown that these OR genes are clustered within multiple loci located in seven different chromosomes. Another study used fluorescence in situ hybridization (FISH) and fluorescence-activated cell sorter (FACS) experiments to determine the genomic locations of a large number of human OR genes (Rouquier et al. 1998). Degenerate oligonucleotide primers matching conserved sequences in OR genes were used to amplify OR genes directly from chromosomes separated by flow sorting. OR genes were found in different loci located in all human chromosomes, except for chromosomes 20 and X (Rouquier et al. 1998).

The chromosomal locations of the complete set of OR genes in different species have now been determined using bioinformatics analysis of the genomic sequences. The human OR genes are distributed in clusters located in all chromosomes, except for chromosomes 20 and Y (Glusman et al. 2001; Malnic et al. 2004; Niimura and Nei 2003). Similarly, the mouse OR genes are distributed in several loci located in all chromosomes, except for chromosomes 18 and Y (Niimura and Nei 2005; Zhang et al. 2004). Although the majority of the OR genes are concentrated in clusters, a few solitary OR genes have also been identified (Godfrey et al. 2004; Malnic et al. 2004).

7.3. EXPRESSION OF THE ODORANT RECEPTOR (OR) GENES

Even though the OR genes are broadly distributed all over the genome, their expression is tightly regulated by a still undeciphered mechanism. Basically, there are three levels of OR gene expression. First, even though OR gene expression was reported in nonolfactory tissues, principally in the testis (Mombaerts 1999; Parmentier et al. 1992; Spehr et al. 2003), the vast majority of the OR genes are exclusively expressed in the olfactory epithelium (Zhang et al. 2004). Second, each OR gene is expressed in only one out of four OR expression zones in the olfactory epithelium. Third, each olfactory sensory neuron expresses one single OR gene allele, while the other genes remain silent.

7.3.1. Zonal Expression of Odorant Receptors (ORs) in the Olfactory Epithelium

A series of in situ hybridization experiments using ORs as molecular probes showed that the olfactory epithelium is divided into four distinct spatial zones in which different groups of OR genes are expressed (Ressler et al. 1993; Vassar et al. 1993). The zones are symmetrically distributed along the dorsal-ventral axis of the epithelium, with zone 1 localized in the dorsal region and zone 4 in the ventral region of the epithelium (according to the nomenclature of Sullivan et al. [1996]). Each zone is likely to express hundreds of OR genes, and the olfactory sensory neurons that express a given receptor are randomly dispersed within its expression zone. The class I OR genes are mostly expressed in zone 1 (Tsuboi et al. 2006; Zhang et al. 2004). Some class II OR genes are also expressed in zone 1, but the majority is expressed in zones 2–4 (Miyamichi et al. 2005; Zhang et al. 2004).

In another study, 80 class II OR genes were analyzed for their expression pattern in the olfactory epithelium (Miyamichi et al. 2005). This analysis showed that, with the exception of the zone 1 OR genes, the OR genes did not fit in one of the previously described four expression zones, but their expression areas are distributed in an overlapping and continuous manner along the dorsal-ventral axis of the olfactory epithelium, such that no clear borders are present between the neighboring zones.

It has been expected that each zone in the olfactory epithelium should express about one-quarter of the OR gene repertoire. The spatial distribution of OR gene expression in the olfactory epithelium was analyzed using a high-throughput microarray analysis (Zhang et al. 2004). Regions corresponding to zone 1 and zones 2–4 were microdissected from the olfactory epithelium and tested on the microarray for OR gene expression. Interestingly, zone 1 contained more than one-third of all OR genes expressed in the olfactory epithelium. Together with the fact that class I ORs are specifically expressed in zone 1, these results indicate that zones 1 and 2–4 may play distinct roles in olfaction.

The functional implications of the zonal organization of OR gene expression in the olfactory epithelium are still unclear. The axonal projection from the olfactory epithelium to the olfactory bulb is also organized along the dorsal-ventral axis, that is, zone 1 neurons project their axons to the dorsal region of the bulb, while zone 4 neurons project their axons to the ventral region of the bulb. This pattern of projection suggests that the zonal segregation of ORs, and consequently of the sensory information, in the nasal cavity is maintained in the olfactory bulb. However, it is not yet known whether ORs in different zones respond to different classes of odorants.

7.3.2. One Olfactory Sensory Neuron One Odorant Receptor and (OR)

Olfactory sensory neurons select, from over a thousand possible choices, one single OR gene allele to express (Chess et al. 1994; Malnic et al. 1999; Ressler et al. 1993; Serizawa et al. 2000; Vassar et al. 1993). Axons of neurons that express one same given OR converge onto two or a few glomeruli at two specific sites in the olfactory bulb (Ressler et al. 1994; Vassar et al. 1994). Interestingly, specific glomeruli show approximately the same locations in different individuals. These results indicate that the information provided by different ORs in the nose is organized into a stereotyped sensory map in the olfactory bulb.

The receptor type that is chosen will determine the range of odorants to which this neuron will respond, and it has been shown that it is also required for axonal targeting to specific glomeruli in the olfactory bulb (Mombaerts et al. 1996; Wang et al. 1998). OR gene choice is, therefore, fundamental for the functional organization of the olfactory system.

Different mechanisms have been proposed for the control of OR gene expression. One possibility considered was that OR gene choice could be controlled by specific DNA rearrangements in the olfactory neurons (Kratz et al. 2002). However, it was demonstrated that mice cloned from olfactory sensory nuclei, despite having originated from a neuron expressing a single OR type, showed no irreversible DNA changes in the OR genes and exhibited a normal range of OR gene expression (Eggan et al. 2004; Li et al. 2004). It has also been considered that each OR gene could be selected by a unique combination of transcription factors. However, the fact that OR transgenes and their corresponding endogenous OR genes are not coexpressed in the same neuron (Serizawa et al. 2000) argues against this possibility.

It has been demonstrated that the monoallelic expression of an OR gene is regulated by a negative feedback mechanism that requires a functional OR protein (Lewcock and Reed 2004; Serizawa et al. 2003). In addition, it was shown that immature olfactory neurons expressing a given OR can switch receptor expression at a low frequency, while neurons expressing a mutant (nonfunctional) OR can switch expression with a greater probability (Shykind et al. 2004). These results indicate that after an OR gene is stochastically selected for expression by a limiting factor, its corresponding OR protein product mediates a feedback signal that results in the maintenance of the receptor choice (Serizawa et al. 2004; Shykind 2005).

Little is known about the role of cis-regulatory sequences in the regulation of OR gene expression. In studies using transgenic mice, different sizes of genomic DNA segments containing OR genes were tested for their ability to drive an OR expression similar to that of the endogenous gene. It was demonstrated that short pieces of DNA located upstream of the coding region, ranging from 460 to 6.7 kb, are sufficient for expression of the ORs M4, M71, and MOR23 (Qasba and Reed 1998; Vassalli et al. 2002). However, large segments of around 200 kb are required to obtain expression of MOR28 (Serizawa et al. 2000). Sequence comparison of the mouse and human genome revealed a 2 kb conserved sequence located ~75 kb upstream of the MOR28 cluster. This region, denominated H region or H enhancer, was proposed to work as a cis-acting locus control region (LCR), which would activate the expression of one single OR gene member from within the MOR28 cluster (Serizawa et al. 2003).

A detailed analysis of the minimal proximal promoter of OR M71 showed that it contains homeodomain and O/E-like binding sites (Nishizumi et al. 2007; Rothman et al. 2005). Mutations in these binding sites abolish its ability to drive OR gene expression in transgenic animals, indicating that homeodomain and olf-1 (O/E-like) transcription factors are involved in OR gene expression. Consistent with this finding, homeodomain and O/E-like binding sites have been identified in a large number of OR gene promoters (Hoppe et al. 2006; Michaloski et al. 2006). O/E-like binding sites were also identified in the promoters of several other olfactory specific genes, such as Golf, adenylyl cyclase III (ACIII), olfactory cyclic nucleotide-gated channel (OcNC), and olfactory marker protein (OMP) (Kudrycki et al. 1993; Wang et al. 1993). Interestingly, the H region also contains homeodomain and O/E-like binding sites (Hirota and Mombaerts 2004), and it was shown that mutations in these sites abolish the ability of the H region to drive expression of OR genes in transgenic animals (Nishizumi et al. 2007).

So far, two different homeodomain transcription factors have been implicated in OR gene expression. Lhx2, a LIM-homeodomain protein, was shown to bind to the MOR71 promoter region (Hirota and Mombaerts 2004). Lhx2-deficient mice lack mature olfactory sensory neurons, indicating that this homeodomain protein is required for olfactory sensory neuron development (Hirota and Mombaerts 2004; Kolterud et al. 2004). In these mutant mice, the expression of class II OR genes is abolished, while most class I OR genes are still expressed in a few OMP-positive neurons located in the dorsal region (corresponding to zone 1) of the olfactory epithelium (Hirota et al. 2007). These results indicate that Lhx2 is directly involved in class II OR gene expression, but is not required for class I OR gene expression. The results also suggest that class I and class II OR gene expression is regulated by distinct mechanisms.

Recently, the Emx2 homeobox transcription factor has also been implicated in OR gene regulation (McIntyre et al. 2008). Emx2 was shown to bind to the mouse OR71 gene promoter (Hirota and Mombaerts 2004) and to be expressed in the olfactory epithelium (Nedelec et al. 2004). Emx2-mutant mice develop a normal olfactory epithelium, except that they have a reduced number of mature olfactory sensory neurons (McIntyre et al. 2008). The expression of many OR genes is reduced greater than the 42% reduction in mature olfactory sensory neurons, indicating that the absence of Emx2 is not altering OR gene expression only because of a general defect in olfactory sensory neuron development. Altogether, these results indicate that Emx2 acts directly on OR gene promoters to regulate gene transcription. Interestingly, a few OR genes show increased expression, when compared to wildtype mice (McIntyre et al. 2008). It is possible that these OR genes do not depend on Emx2 to be transcribed.

The olf1 (O/E) transcription factors are specifically expressed in the olfactory neurons and in B-lymphocytes (Hagman et al. 1993; Wang and Reed 1993). The roles of O/E-like proteins in OR gene expression are still unclear. Disruption of olf-1-like genes does not alter OR gene expression (Lin and Grosschedl 1995; Wang et al. 2003), possibly due to the functional redundancy of the multiple O/E family members expressed in the olfactory epithelium (O/El, O/E2, O/E3, and O/E4; Wang et al. 1997, 2002). However, it was demonstrated that O/E2- and O/E3-mutant mice show defects in the projection of olfactory neurons to the olfactory bulb, indicating that the O/E genes function may not be completely redundant (Wang et al. 2003).

Experiments using the chromosome conformation capture (3C) technique showed that in the nuclei of olfactory sensory neurons, the H region, which is located on chromosome 14, associates with OR gene promoters located in different chromosomes (Lomvardas et al. 2006). DNA and RNA FISH analysis demonstrated that the H region is associated with the single OR gene that is transcribed in a given neuron. Also, in the olfactory sensory neurons, one of the two H alleles is methylated, and therefore inactive. Based on these results, a model for OR gene choice was proposed, where one single trans-acting H enhancer element allows stochastic activation of one single OR gene allele per olfactory sensory neuron. However, it was subsequently shown that mice that have the H region deleted show regular expression of OR genes, except for some of the OR genes that are located within the MOR28 cluster. In this case, the expression of the three OR genes located proximal to the H region, MOR28, MOR10, and MOR83, was abolished (Fuss et al. 2007; Nishizumi et al. 2007). These results indicate that the H region acts in cis to promote expression of these three genes, but is not an essential trans-acting enhancer that regulates monoallelic expression of OR genes in olfactory sensory neurons.

In order to obtain expression of a particular OR gene in a large number of olfactory sensory neurons, transgenic mice were constructed, where the full length of the OR coding sequence is placed under the control of the promoter of genes that are abundantly expressed in these neurons, such as the OMP or Gγ8 genes (Nguyen et al. 2007). However, these constructs did not result in transgenic expression of the OR gene. When the OR coding sequence is replaced by a different unrelated GPCR, like the human taste receptor hT2R16 or the opioid receptor RASSL, these GPCRs are expressed in the vast majority of the OMP or Gy8 positive neurons, indicating that the suppression of OR gene expression in the olfactory sensory neurons is not extended to GPCRs in general, but is specific to OR. In addition, when the OR coding sequence is replaced by an OR coding sequence containing a mutation at the highly conserved DRY sequence, a motif known to be essential for G-protein activation and signal transduction, the OR gene expression is still suppressed, showing that OR function is not required for OR silencing. These results are consistent with other experiments that showed that the mechanism of negative feedback regulation may not require G-protein-mediated signaling (Imai et al. 2006).

The inhibition of the OR transgene expression could be part of the normal process that controls endogenous OR gene expression, so that one single type of OR gene is expressed, while the remaining OR genes are repressed. Interestingly, OR gene expression was achieved only when the OR coding sequence was separated from the promoter sequences: for example, a transgenic line where the OMP (or Gy8) promoter sequence drives the expression of the tetracycline transactivator is crossed with a transgenic mouse carrying a TetO promoter driving the expression of the OR gene (Nguyen et al. 2007). These results suggest that both the OR coding sequence and the promoter driving its expression, must be involved in OR gene regulation.

7.4. ODORANT SIGNAL TRANSDUCTION THROUGH ODORANT RECEPTORS (ORs)

Antibodies recognizing distinct ORs have been used to determine their cellular distribution. These experiments showed that the receptor proteins are localized in the cilia of olfactory sensory neurons, the site of odorant signal transduction (Barnea et al. 2004; Menco et al. 1997; Schwarzenbacher et al. 2005; Strotmann et al. 2004). Odorant signal transduction is initiated by the binding of odorants to ORs and the activation of the associated heterotrimeric G-protein, Golf. Once activated, Gαolf exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP), the GTP-bound Gαolf subunit dissociates from the Gβ/y complex and activates ACIII, leading to increased intracellular levels of cAMP and opening of cyclic nucleotide-gated channels. The resulting influx of Na+ and Ca2+ ions ultimately leads to the generation of an action potential in the olfactory neuron axon (Firestein 2001; Mombaerts 2004; Ronnett and Moon 2002; see also Chapter 8).

Initially, it was believed that two separate types of intracellular signaling pathways could be activated by different classes of odorants: the cAMP pathway and the IP3 pathway (Boekhoff et al. 1990; Huque and Bruch 1986; Ronnett et al. 1993; Sklar et al. 1986). However, because mice that are knockout for components of the cAMP pathway do not respond to odorants of any class (Belluscio et al. 1998; Brunet et al. 1996; Wong et al. 2000), it is believed that olfactory transduction is exclusively mediated by the cAMP pathway, although it is possible that the IP3 pathway plays a modulatory role (Spehr et al. 2002), or is involved in signaling in different types of cells in the olfactory epithelium (Elsaesser et al. 2005; Gold 1999; Liberles and Buck 2006; Lin et al. 2007; see also Chapter 9).

Recently, additional proteins that are likely to be involved in OR function have been identified. The receptor transporting proteins, RTP1, RTP2, and REEP1, which are specifically expressed in the olfactory sensory neurons in the olfactory epithelium, were shown to associate with ORs when coexpressed in HEK293T cells (Saito et al. 2004). It was also shown that they promote cell surface expression of ORs in HEK293T cells. In vivo, it is possible that they work as chaperones that aid in OR folding and/or trafficking to the plasma membrane (Saito et al. 2004).

The guanine nucleotide exchange factor (GEF) Ric-8B, interacts with Gαolf (Von Dannecker et al. 2005). Ric-8B is specifically expressed in olfactory sensory neurons and in a few regions in the brain where Gαolf is also expressed, such as the striatum, nucleus accumbens, and olfactory tubercle (Von Dannecker et al. 2005). Guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP to generate an activated form of Gα, which is then able to activate a variety of effectors. Consistent with this potential function, Ric-8B is able to amplify dopamine receptor and OR signaling through Gαolf (Von Dannecker et al. 2005, 2006). It was recently demonstrated that Ric-8B, besides interacting with Gαolf, also interacts with Gγl3, which is also expressed in mature olfactory sensory neurons (Kerr et al. 2008). In addition, it was shown that GβT is the Gβ subunit that is predominantly expressed in the mature olfactory sensory neurons and that the GpT protein is localized to the cilia of olfactory sensory neurons, together with Gyl3 and Ric-8B (Kerr et al. 2008).

The physiological roles of the RTPs and Ric-8B in OR function should be clarified through the analysis of mice that are knockout for these proteins.

7.5. ODORANT RECEPTORS (ORs) AND AXONAL TARGETING IN THE OLFACTORY BULB

The experiments using antibodies against ORs showed that the receptors are also localized in the axonal processes of the olfactory sensory neurons (Barnea et al. 2004; Menco et al. 1997; Schwarzenbacher et al. 2005; Strotmann et al. 2004), consistent with the role they play in axonal targeting to specific glomeruli in the olfactory bulb (Feinstein et al. 2004; Wang et al. 1998). The mechanisms through which the ORs regulate axonal projection are not completely understood. ORs in the axonal terminals could recognize guidance molecules in the olfactory bulb to form specific glomeruli (Mombaerts 2006), or, alternatively, axons could coalesce into a glomerulus independently of the presence of a target in the bulb, but as a consequence of homophilic interactions between axons containing the same OR type (Feinstein et al. 2004; Feinstein and Mombaerts 2004). Recent studies have demonstrated that OR-derived cAMP signals are essential for axonal targeting in the bulb (Imai et al. 2006; Serizawa et al. 2006). In this model, each OR type generates a unique level of cAMP The levels of cAMP define the expression levels of guidance molecules, which determine the anterior-posterior topography of axonal projection in the olfactory bulb. It is not yet clear whether ORs present in the cilia or in the axonal terminals, or both, are involved in the generation of the cAMP signals that regulate the formation of the OR-specific glomerular map.

7.6. ODORANT DISCRIMINATION BY ODORANT RECEPTORS (ORs)

7.6.1. Combinatorial Receptor Codes for Odorants

Even though mammals have only 100s of functional ORs, they can discriminate a much higher number (several thousands) of odorants. In order to understand how the olfactory system utilizes the OR gene family to discriminate odorants, one should determine the odorant specificities of individual ORs. However, to date, only a few ORs have been linked to odorants they recognize because ORs cannot be efficiently expressed in heterologous cells (Malnic 2007). They are usually retained in the endoplasmatic reticulum and cannot reach the plasmatic membrane (Gimelbrant et al. 1999, 2001; Katada et al. 2004; Lu et al. 2003, 2004). In order to circumvent this problem, a combination of Ca2+ imaging and single-cell RT-PCR was used to identify the ORs expressed by olfactory neurons that responded to different aliphatic odorants (Malnic et al. 1999), to lyral (Touhara et al. 1999), or to eugenol (Kajiya et al. 2001). In these experiments, dissociated olfactory sensory neurons are loaded with the Ca2+ sensitive dye, fura-2, and exposed to a panel of odorants. The increases in Ca2+ concentration are recorded as fluorescence decreases in the intensity of the emitted light (510 nm) of neurons excited at 380 nm (Malnic et al. 1999). The neurons that respond to the odorants are individually transferred to micro tubes and a two-step, single-cell RT-PCR/PCR procedure is used to identify the OR genes expressed by each neuron. In a primary PCR reaction, cDNAs derived from all of the mRNAs expressed by a neuron are amplified. In a secondary PCR reaction, the primary PCR products are used as template with degenerate primers that specifically amplify members of the OR family. In this way, we can identify the OR expressed by the recorded neuron.

These experiments showed that one OR can recognize multiple odorants, but that different odorants are recognized by different combinations of receptors (Malnic et al. 1999). Thus, the olfactory receptor family is used in a combinatorial manner to discriminate odorants. Given that there are around 1000 OR genes, this combinatorial receptor-coding scheme should permit the detection of a vast number of odorants. It should also permit the olfactory system to discriminate between odorants that have very similar structures, such as aliphatic odorants with different carbon chain lengths (Malnic et al. 1999). These results are consistent with previous observations that single olfactory sensory neurons (Firestein et al. 1993; Sato et al. 1994; Sicard and Holley 1984) and individual glomeruli in the olfactory bulb (Adrian 1950; Friedrich and Korsching 1997; Leveteau and MacLeod 1966; Mori et al. 1992) can be stimulated by multiple odorants.

7.6.2. Functional Expression of Odorant Receptors (ORs) in Heterologous Cells

As explained above, it is believed that the major reason for the inefficient functional expression of ORs in heterologous cells is the fact that the receptors do not reach the plasma membrane. However, recent advances have improved the expression of ORs in heterologous cell lines. Some of the techniques being used to deorphanize ORs in heterologous cells are based on strategies that should contribute to increased amounts of receptors on the cell surface. It has been demonstrated that fusion of the 20 N-terminal amino acids of rhodopsin to the N-terminal region of ORs facilitates cell surface expression of at least some ORs (Krautwurst et al. 1998). Using cotransfection, ORs with an N-terminal segment of rhodopsin ("rho-tagged ORs") can be expressed in heterologous cells together with the Gα15/16 subunits, which can promiscuously couple receptors to the phospholipase C pathway (Krautwurst et al. 1998). Receptor activation by odorants results in increased intracellular Ca2+, which can be measured at the single-cell level using Ca2+ sensitive dyes.

ORs expressed in heterologous cells can also couple to Gαolf (the natural partner of ORs), leading to odorant-induced increases in cAMP (Kajiya et al. 2001; Shirokova et al. 2005). A cell line that stably expresses the olfactory signal transduction molecules Gαolf and cyclic nucleotide-gated channel subunit A2 (CNGA2) (named HeLa/Olf cell line), has also been used to functionally express ORs (Shirokova et al. 2005). Importantly, it was observed that the use of nonolfactory G-proteins may alter the OR responses to particular odorants, indicating that heterologous systems that use endogenous olfactory transduction molecules are more likely to reproduce OR physiological responses (Krautwurst 2008; Shirokova et al. 2005).

It was also demonstrated that coexpression with the olfactory-specific RTPs in HEK293T cells promotes OR functional surface expression (Saito et al. 2004). The RTPs are transmembrane proteins and were shown to directly interact with ORs in coimmunoprecipitation assays (Saito et al. 2004). It was demonstrated that cotransfection of RTP1 and OR also enhances surface expression of RTP1; it is possible that they work as coreceptors with ORs. They could also be involved in different functions, such as OR folding, export from the endoplasmic reticulum, or vesicle transport (Saito et al. 2004). HEK293T cells stably expressing Gαolf, RTP1, RTP2, and REEP1 were established (named Hana3A cell line) and can now be used to investigate the specificities of a large number of ORs (Saito et al. 2004).

In a different approach, it was demonstrated that coexpression with the GEF Ric-8B and Gαolf results in functional expression of ORs in HEK293T cells (Von Dannecker et al. 2006). Importantly, it was shown that Ric-8B promotes functional expression of untagged (without a rho tag) ORs, which is advantageous because it is possible that receptor protein modifications interfere with the ligand affinities. GEFs are considered to work as positive regulators of GPCR signaling. Therefore, in this case, functional expression of ORs is not mediated by an increase in the amount of receptors on the cell surface, but instead, results from the amplification of the OR signaling through the G-protein.

Interestingly, it was recently demonstrated that the use of a combination of Ric-8B, RTP1S (a short form of RTP1), and rho tags results in an improved heterologous expression of ORs (Von Dannecker et al. 2006; Zhuang and Matsunami 2007). The use of these methods in the future should facilitate the deorphanization of mammalian ORs.

7.7. HUMAN ODORANT RECEPTORS (ORs)

The fact that almost half of the human ORs repertoire is apparently nonfunctional (Go and Niimura 2008; Rouquier and Giorgi 2007) suggests that during the process of evolution, olfaction may have lost importance for primates. Even though the number of functional OR genes is smaller when compared to other species, humans have a very sensitive sense of smell, which is important for the detection of odorants that are essential for life, such as the smell of smoke (detection of fire) and the smell of rotten food (to avoid its ingestion). Smells are also intimately related to how humans taste food (Shepherd 2004; see also Chapter 16).

Comparison between the human and mice OR gene repertoires showed that, despite the smaller number of intact human ORs, the vast majority of human OR subfamilies have counterparts in the mouse repertoire (Godfrey et al. 2004). These results suggest, in principle, that the majority of odorant features detectable by one species may also be recognized by the other. However, mice may have a better ability to discriminate between similar odorants than humans (Godfrey et al. 2004).

7.7.1. Deorphanized Human Odorant Receptors (ORs)

There are approximately 400 functional ORs in humans (Glusman et al. 2001; Malnic et al. 2004; Niimura and Nei 2003; Zozulya et al. 2001). Analysis of the amino acid sequences of all intact human ORs shows that they share the typical OR motifs (Figure 7.3). To date, only a few human ORs have been linked to odorants they recognize. Some examples are shown in Figure 7.4. Two of these (OR51E1 and OR52D1) are class I ORs, and the remaining are class II ORs. Several approaches, which use coexpression with different Gα subunits, have been used to deorphanize these ORs. For instance, receptors OR1A1 and OR1A2 were functionally expressed in Hela/Olf cells (via Gαolf) and can specifically detect citronellic terpenoid odorants (Schmiedeberg et al. 2007). In a previous study, Shirokova and colleagues demonstrated that the mouse orthologue, Olfr43, also responds to this agonist using the same system (Shirokova et al. 2005). Some human ORs were analyzed by using HEK293 cells that coexpress ORs and the promiscuous Gαl6 subunit, which couples the receptor to the IP3-mediated pathway (Krautwurst et al. 1998). OR1G1 was shown to preferentially respond to 9-carbon aliphatic molecules, and can be inhibited by some odorants with structures that are related to that of the agonists (Sanz et al. 2005). OR52D1, a class I OR, was shown to respond to methyl octanoate (Sanz et al. 2005).

FIGURE 7.3. Motif patterns found in the human ORs.

FIGURE 7.3

Motif patterns found in the human ORs. Weblogo representation for 397 human OR amino acid sequences. Sequences were aligned using Clustalw.

FIGURE 7.4. Phylogenetic relationships of deorphanized human ORs and their ligands.

FIGURE 7.4

Phylogenetic relationships of deorphanized human ORs and their ligands. OR1A1 and OR1A2 (Schmiedeberg, K. et al. 2007), OR1D2 (Spehr, M. et al. 2003), OR1G1 (Sanz, G. et al. 2005), OR3A1 (Jaquier, V. et al. 2006; Wetzel, C.H. et al. 1999), OR7D4 (Keller, (more...)

7.7.2. Odorant Receptors (ORs) and Pheromones

Pheromones are intraspecific chemical signals that regulate a series of innate behaviors, such as reproduction and aggression (Brennan and Zufall 2006; see also Chapter 6). The detection of pheromones is mediated by an accessory olfactory system, the vomeronasal system, which is anatomically segregated from the main olfactory system. Two different families of vomeronasal receptors, the VlRs and V2Rs, which are also GPCRs, are expressed in the vomeronasal neurons and are responsible for the recognition of pheromones (Dulac and Torello 2003). Humans, however, do not have a functional vomeronasal organ (Brennan and Zufall 2006). In addition, the vast majority of human VlRs and all V2Rs are pseudogenes (Young et al. 2005; Young and Trask 2007). Therefore, it is possible that the main olfactory system is the organ that detects pheromones in humans (Brennan and Zufall 2006).

The comparison between the OR repertoires in humans and mice has also revealed the presence of species-specific subfamilies of OR genes. These ORs are strong candidates to be involved in the detection of pheromones, or maybe of odorant classes that are detected by only one species (Godfrey et al. 2004).

The OR repertoires of humans and a closer species in terms of evolution to humans, the chimpanzee, were also compared (Gilad et al. 2005). Although their different habitats should result in different odorant detection needs, these species share the majority of OR genes. However, two subfamilies that are specific to chimpanzees and three subfamilies that are specific to humans were identified. The ORs that constitute these species-specific subfamilies show 99% amino acid sequence identity among themselves, with the exception of one human subfamily, which is composed of ORs with 70% identity among themselves (Gilad et al. 2005). Also, as described above, another study showed that 25% of the intact ORs are nonorthologous between humans and chimpanzees (Go and Niimura 2008). The agonists of species-specific ORs, which may have acquired species-specific functions, are still unknown, but their identification should be of interest.

A second family of GPCRs, known as trace amine-associate receptor (TAARs) and expressed in the olfactory epithelium, was recently described (Liberles and Buck 2006). The term “trace-amines” refers to α-phenylethylamine, p-tyramine, tryptamine, and octopamine, which are present at very low concentrations (nanomolar range) in mammalian tissues. While mice have 15 TAARs, only 6 TAARS were identified in humans (Liberles and Buck 2006). These receptors were shown to bind to volatile amines found in urine, which are linked to stress or are differentially concentrated in male vs female urine, and therefore are believed to be involved in pheromone detection. The role of TAARs in humans remains unknown.

7.7.3. Odorant Receptors (ORs) and Perception

An interesting feature of the human OR repertoire is that it is highly polymorphic. From pioneering perception studies, it is known that the ability to detect some odorants can vary greatly between individuals. Individuals that only detect some odorants when present in high concentrations or individuals that do not detect some odorants at all are relatively common in the human population (Amoore 1967, 1977; Amoore and Steinle 1991). Heterologous systems can now be used to functionally express polymorphic variants of human ORs to correlate differences in the structure of ORs in a population and their agonists.

A small number of polymorphisms in human OR genes have been described so far, but with the increasing availability of genomic sequences and single nucleotide polymorphisms (SNPs) from different individuals, new ones should be identified. For example, analysis of the 17 human OR genes present in the chromosomic region 17pl3.3 revealed the existence of polymorphisms in the coding region of 14 of the OR genes, which show a total of 26 SNPs; from these, 21 are cSNPs (coding SNPs), that is, modifications that result in amino acid changes in the structure of the protein (Sharon et al. 2000).

In another study, 51 OR gene loci that are potential pseudogenes were analyzed in 189 individuals from several ethnic origins. The results revealed a high level of interindividual variability (Menashe et al. 2003). Interestingly, it was observed that non-African individuals had fewer functional OR genes than African American individuals. These results suggest that different evolutionary pressures may have shaped the OR repertoire in different human populations (Menashe et al. 2003).

Recently, Keller and colleagues showed that SNP variations in OR7D4 (Figure 7.4) correlate to differences in the perception of two substances that bind to this OR: androstenone and androstadienone (Keller et al. 2007). Individuals containing one or two nonfunctional alleles from gene OR7D4, that is, with two SNPs that result in two amino acid substitutions, are less sensitive to the abovementioned agonists. Another recent study identified a single SNP in the gene OR11H7P This mutation in some individuals changes their sensitivity to the OR agonist, isovaleric acid (Menashe et al. 2007). These results support a relationship between genotypic and phenotypic variability in human olfaction.

Recent studies show that most of the human genome variation is not only due to SNPs, but also to structural variations of the genome, such as deletion of kilo- or megabase pairs, duplications, insertions, and inversions (Kidd et al. 2008; Korbel et al. 2007; Redon et al. 2006). Structural variations that affect the number of copies of a given region larger than 1 kb are called copy number variants (CNVs) (Feuk et al. 2006). Recently, the impact of CNVs on the individual OR gene content has been analyzed. It was shown that ~30% of the human OR genes, including pseudogenes, are polymorphic with respect to copy number (Hasin et al. 2008; Nozawa et al. 2007; Young et al. 2008). Experimental validation of some CNV ORs in 50 individuals demonstrated that some ORs are deleted in some individuals and not in others, while others are duplicated in a subset of individuals (Young et al. 2008). The combination of SNPs and CNVs in the OR gene family among different individuals must have a significant impact on our olfactory abilities.

Recent work by Saito and colleagues identified agonists for 10 human and 56 mouse ORs by using a high-throughput screening (Saito, H., Chi, Q., Zhuang, H., Matsunami, H. and Mainland, J.D. (2009) Odor coding by a Mammalian receptor repertoire. Sci. Signal. 2(60): ra9).

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