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Gene name - Odorant receptor 83b
Synonyms - Cytological map position - 83A2 Function - G-protein coupled receptor Keywords - odorant receptor, seven-transmembrane proteins |
Symbol - Or83b
FlyBase ID: FBgn0037324 Genetic map position - 3R Classification - odorant receptor Cellular location - surface transmembrane |
Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. The results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants (Larsson, 2004).
The olfactory system has evolved the capacity to recognize and discriminate an inordinate number of chemically distinct odors that signal the presence of food, predators, or mating partners. The initial steps in odor detection involve the binding of a volatile odor to odorant receptor (OR) proteins displayed on ciliated dendrites of specialized olfactory sensory neurons (OSNs) that are exposed to the external environment. The OR genes that mediate odor detection in Drosophila fruit flies are expressed in subpopulations of OSNs and are members of a rapidly diverging superfamily of insect chemosensory genes that encode receptors with no homology to nematode or vertebrate ORs. Genetic analysis coupled with electrophysiology has demonstrated that the characteristic odor response profile of a given OSN is governed by the selective expression of one or more members of the family of 61 OR genes in that neuron (Dobritsa, 2003; Elmore, 2003; Hallem, 2004a; Hallem, 2004b). One member of the OR gene family, Or83b, is strikingly different from the other OR genes. Unlike the conventional ORs, it has clear homologs in other insect species that share nearly 70% amino acid identity with Or83b. Or83b and its homologs in other insects are coexpressed with conventional ORs in a large proportion of OSNs (Vosshall, 1999: Vosshall, 2000; Elmore, 2003; Hill, 2002; Krieger, 2003; Pitts, 2004; Larsson, 2004 and references therein).
Based on these observations, two models could account for Or83b function in insect olfaction. Or83b could bind distinct ligands independent of the other OR genes coexpressed with it in a given OSN, or it could act in concert with conventional ORs to recognize a wide variety of odors (Larsson, 2004).
In the first model, Or83b might act in most OSNs to recognize and report the presence in the environment of an important odor signifying danger or a particularly rich food source. Insects have evolved a diversity of food preferences and inhabit many different ecological niches. However, the vast majority of insects have an important relationship to plants, either as food sources, sites for egg-laying, habitat for prey, or shelter. Green leaf volatiles such as E2-hexenal and linalool are produced by many different plants and elicit physiological and behavioral responses in insects as varied as moths, mosquitoes, and Drosophila. Therefore, Or83b might interact selectively with various plant volatiles and transmit the same information to insects of diverse taxonomy; i.e., that they are in the presence of plants. Alternatively, Or83b could interact with a single odorant that has different meanings to different insects. For instance, isoamyl acetate is a key component of rotting fruit and signifies food to Drosophila, while the same odorant is produced by honeybees as an alarm and aggregation pheromone. In both cases, the stimulus is of great importance to these insects, and dedicating a broadly expressed receptor like Or83b to detecting it might be adaptive for each insect. In either variation of this first model, animals lacking Or83b would show essentially normal responses to general odors detected by conventional ORs but would be nonresponsive to the putative important odors of special meaning (Larsson, 2004).
In the second model, Or83b would not function independently as a ligand binding OR but would play a more general role in concert with the conventional ORs with which it is coexpressed. Such alternative roles could include interacting with conventional ORs to produce a receptor complex competent for ligand binding, acting as a protein chaperone that directs ORs to the dendrite, the OSN, or a combination of all of these functions. In this scenario, severe deficits in olfactory function to a wide range of odors would be expected in animals lacking Or83b (Larsson, 2004).
Despite the remarkable progress in recent years in elucidating the molecular logic of olfaction in Drosophila, existing data do not explicitly rule out either model. Electrophysiological analysis of the basiconic class OSNs reveal a diversity of responses to odorants that included activation and inhibition of a given OSN by different odorants. OSNs were identified with specialist and generalist properties, and a number of identified neurons did not respond to any of the approximately 50 odorants tested. Available data on the response properties of the remaining OSNs in the antenna associated with trichoid and coeloconic sensilla are less complete. The hypothesized odorant of special importance that activates Or83b independent of the other ORs might lie outside of collection of odorants used in these studies or might emerge from analysis of the trichoid and coeloconic OSNs. Elegant genetic and functional analysis of Drosophila ORs produced the clear result that the response properties of an identified OSN require the expression of its cognate OR and that replacing this OR with a novel OR alters the response properties of the neuron in an OR-dependent manner. While these results clearly implicate the conventional OR in the response properties of the OSN, they do not rule out the possibility that these ORs function in concert with Or83b, which is coexpressed with all of these ORs (Elmore, 2003). They also do not rule out an independent function for Or83b. Or22a/b and Or43b mutant neurons were not found to respond to any of the odorants tested, but the relevant odorant might not have been part of the stimulus panel (Larsson, 2004).
To distinguish between these competing models for Or83b function, gene targeting was used to delete the Or83b gene, and cell biological, electrophysiological, and behavioral techniques were used to characterize the mutant phenotype. Clear support for the second model is found: in Or83b mutants, the normal localization of OR proteins in distal chemosensory dendrites is disrupted in both larval and adult olfactory systems. Or83b mutant larvae fail to chemotax to most odors tested, and adult flies show severe deficits in odor-evoked electrophysiology and behavior. These data therefore imply that olfactory function in response to a broad range of odorants in Drosophila requires expression of a conventional OR along with Or83b in most olfactory neurons. These findings have important implications for control of medically and economically relevant insect pests, because clear homologs of Or83b exist in malaria mosquitoes and a variety of important agricultural pests (Krieger, 2003; Pitts, 2004). These data suggest a strategy in which olfactory host-seeking behavior of pest insects could be disrupted by small molecule inhibitors of Or83b homologs (Larsson, 2004).
This study shows, by cellular, physiological, and behavioral analysis, that Or83b is essential for olfaction in Drosophila. Or83b is an atypical member of the OR gene family because it is highly conserved across insect species and is expressed in a large number of OSNs with different odor specificities (Dobritsa, 2003; Elmore 2003; Hallem, 2004a). This receptor is selectively expressed only in OSNs throughout all four stages of Drosophila development, and no expression is detected in gustatory neurons or any other cell type. Fly Or83b is expressed in all OSNs of the larval dorsal organ and adult maxillary palp and in a large subset of adult antennal neurons. The onset of Or83b expression in both larval and adult olfactory systems is late, effectively ruling out any developmental role for this protein in patterning axonal connections of these neurons (Larsson, 2004).
An Or83b mutant was constructed by 'ends-out' gene targeting and shown to be null for both mRNA and protein expression. Conventional OR proteins fail to accumulate in both adult and larval OSN dendrites in Or83b mutants and are restricted to the cell body. This suggests a role for Or83b in regulating the proper subcellular localization of the conventional ORs. The OR localization defect was shown to be specific to Or83b and not a secondary effect of a sick neuron. The distribution of a general membrane marker in larvae and the futsch microtubule marker is unaffected in the mutant, as is the stability of Or22a-expressing axonal connections to the DM2 glomerulus in the antennal lobe. Electrical silencing of wild-type neurons does not produce the same OR localization defect as the Or83b mutation (Larsson, 2004).
Or83b mutant antennae show no odor-evoked potentials to a panel of eight odorants that elicit robust responses in wild-type antenna. Mutant ab1A, B, and D neurons fail to respond to their cognate stimuli and show little or no spontaneous electrical activity. In contrast, the carbon dioxide-sensitive ab1C neuron is normal in the Or83b mutant. Finally, both larval and adult Or83b mutants have severe deficits in odor-evoked behavioral responses. Taken together, these data support a model in which Or83b acts in concert with conventional ORs to respond to many different odorants and argue against an independent function for Or83b in recognizing a particular odorant (Larsson, 2004).
The number of ORs expressed in a given OSN is an important determinant of the coding logic of the olfactory system. In the nematode, C. elegans, 16 pairs of chemosensory neurons express an estimated 500 different chemosensory receptor genes. Of necessity, each chemosensory neuron expresses a large number of receptors, but each receptor functions independently within a neuron to recognize ligands that activate the chemosensory neuron to elicit either attractive or aversive behavioral responses according to behavioral output, which is regulated by the activation of the sensory neuron: the animal retains great discriminatory power in the face of a severely constricted number of sensory neurons and no central chemosensory processing circuits. In contrast, each mammalian olfactory neuron expresses a single odorant receptor gene that bestows upon that neuron a restricted receptive range for odors. In part because the odorant receptor itself is an important determinant in glomerular target selection, mammalian olfactory neurons have evolved elaborate regulatory mechanisms to suppress the expression of more than one functional odorant receptor gene per neuron. Unlike the situation in C. elegans, each sensory neuron contributes information about a small fraction of the odor universe, and significant olfactory processing must occur in higher order olfactory cortical regions to decode the salience of the odor stimulus and produce an appropriate behavioral response (Larsson, 2004).
Initial analysis of the Drosophila OR gene family suggested that the fly olfactory system was organized according to the vertebrate one receptor:one neuron:one glomerulus model. OR genes were found in nonoverlapping subpopulations of OSNs, and all OSNs expressing a given OR were found to converge upon a distinct and dedicated antennal lobe glomerulus. However, further analysis has somewhat complicated this initial view. Or22a and Or22b are coexpressed in the ab3a neuron, that Or22a functions independently of Or22b, and that Or22b does not discernibly contribute to the odor code of this neuron. This same neuron expresses Or83b in addition to these two conventional odorant receptors. If each of these three ORs hypothetically interacts with distinct ligands, this would substantially alter the view of how the fly olfactory system is organized. In such a multireceptor OSN model diverse stimuli would activate the same neuron, but all would lead to activation of the DM2 glomerulus to which the neuron projects. The animal would thus have no means to determine which of the three receptors was activated, resulting in a possible loss of odor discrimination. The data strongly argue against an independent requirement of a single receptor protein for the function for Or83b in odor detection and instead suggest that it acts as an essential cofactor for localizing conventional ORs in chemosensory dendrites. Therefore, the fly is likely to retain an organizational logic similar to that employed in vertebrates, despite expressing more than one OR in each olfactory neuron (Larsson, 2004).
This study demonstrates a genetic requirement for Or83b in dendritic localization of conventional ORs in vivo and would be consistent with a model in which Or83b interacts with the conventional odorant receptors to form a heteromeric receptor complex. Conclusive evidence for direct association between Or83b and conventional ORs, as well as more detailed biochemical insights into the function of Or83b, awaits the development of an expression system that can be used to examine the interactions of biologically active Drosophila ORs (Larsson, 2004).
Functional interaction between chemosensory receptors has been described in mammalian taste, where the assembly of different heterodimeric receptors determines whether a neuron responds to sweet or amino acid stimuli: the T1R2/3 heterodimer encodes a sweet taste receptor, while T1R1/2 encodes the umami or amino acid receptor. Homodimeric T1R3 receptors detect only high concentrations of natural sweeteners such as sucrose. Therefore, in the mouse three different classes of gustatory receptor cells express T1R3 but the functional specificity of the cell is determined by whether a given cell expresses T1R2 (regular sweet), T1R1 (umami), or neither (low-affinity sweet) (Larsson, 2004).
Broad expression of atypical chemosensory receptor genes, such as described in this study for Or83b, has also been seen. In the rodent vomeronasal organ, members of the V2R2 pheromone receptor subfamily are broadly expressed along with other more selectively expressed receptors (see the PDF file Molecular Detection of Pheromone Signals in Mammals: From Genes To Behaviour), but biochemical evidence that they interact functionally is lacking. Amino acid detection in fish relies in part on a broadly expressed receptor of the same structural class as the T1R taste receptors and V2R pheromone receptors, but it is not known whether this 5.24 receptor functions in concert with other fish receptors with which it is coexpressed (Larsson, 2004).
The data on Or83b point to a unique and unprecedented requirement of a single receptor protein for the function for functionality of an extremely diverse family of receptors. These results are unexpected because the Drosophila odorant receptor genes are unrelated to other receptor superfamilies that have been shown to heterodimerize through conserved protein-protein interaction domains. Further, this receptor family is undergoing rapid evolutionary change with virtually no direct homologs recognized between the fly and mosquito genomes. In contrast, Or83b is extremely conserved and has clear homologs in distant insect species. If these homologs are performing the same function in these divergent insect species, it would follow that Or83b must recognize a conserved feature of all OR proteins with which it is expressed, either directly by heterodimerizing with the ORs or through the action of accessory proteins (Larsson, 2004).
The investment of Drosophila in a single protein for odor detection suggests that Or83b performs a key function in all insects that cannot be diversified or made redundant. The alternative to maintaining strong selection pressure on Or83b would be to require parallel selection on a diversity of other genes that each interact with a partner OR protein. Such a coevolution of two large gene families is suggested by the work on the M10 MHC family that associates with a family of V2R receptors in the vomeronasal organ (Ishii, 2003; Loconto, 2003). There may be barriers to the generation of large numbers of new genes in Drosophila, which maintains a compact genome with few pseudogenes (Larsson, 2004).
Insects are the primary vectors for the infectious diseases malaria, dengue fever, yellow fever, and West Nile encephalitis, and they locate human hosts largely through their exquisitely sensitive olfactory systems. Host-seeking behavior is thought to require a number of different sensory stimuli to provide the maximum likelihood that the human host is near. For instance, mosquitoes are attracted to general odors along with carbon dioxide and body heat. The finding that the Or83b mutation disrupts Drosophila behavioral responses to many odors suggests a potential chemical strategy to disrupt the function of homologous genes in pest insects. Small molecule inhibitors that mimic the effects of this mutation may blunt or eliminate olfactory responses in pest insects, ultimately controlling the damaging olfactory-mediated behaviors that result in the spread of disease (Larsson, 2004).
In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. This study provides evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes show extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment (Sato, 2008).
Taken together, these data provide compelling evidence that a heteromeric complex of a conventional insect OR and the highly conserved Or83b family co-receptor has the characteristics of a cation non-selective ion channel directly gated by odour or pheromone ligands. It is concluded that G-protein-mediated signalling is negligible in producing the current elicited by odour activation of insect OR heteromultimers. These findings provide insight into long-argued insect olfactory transduction mechanisms and may explain the lack of clear consensus on the role of second messengers in this process. The insect ORs share no homology with any previously described ion channel and do not contain any known ion selectivity filter motifs. Insect OR activity is not inhibited by Gd3+, a lanthanide that is a broad-spectrum ion channel inhibitor. Therefore, although the ionic permeability reported in this study for Na+, K+ and Ca2+ would be consistent with the properties of non-selective cation channels, a molecular basis for this novel ionotropic activity remains to be elucidated. The spontaneous activity of the OR complex found in this study seems to account for previous observations that olfactory sensory neurons exhibit bipolar electrical activity and become electrically negative on the deletion of Or83b in vivo. Given that there are 62 and 79 potential ligand-binding OR subunits in Drosophila and Anopheles, respectively, the insect ORs may represent the largest single family of ion-channel-like proteins in any organism. This work also raises the intriguing possibility that the insect gustatory system, which senses bitter and sweet tastants as well as carbon dioxide, shares this ionotropic coupling mechanism with the insect ORs. In fact, an ionotropic sugar-gated channel in fleshfly taste cells has previously been reported. This finding offers the caveat that other orphan receptors classified as G-protein-coupled receptors merely because of their putative seven-transmembrane topology may instead possess ligand-gated channel activities, as has been shown previously for light-activated channelrhodopsin. This work has important implications for worldwide efforts to identify specific inhibitors for the insect ORs, which may prove useful in controlling host-seeking behaviours of disease-vector insects such as mosquitoes (Sato, 2008).
From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors1 that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila6. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. This study shows that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca2+ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling (Wicher, 2008).
See an alignment of Or83b-related proteins in Pitts, 2004.
Candidate olfactory receptors of the moth Heliothis virescens were found to be extremely diverse from receptors of the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae, but there is one exception. The moth receptor type HR2 shares a rather high degree of sequence identity with one olfactory receptor type both from Drosophila (Dor83b) and from Anopheles (AgamGPRor7); moreover, in contrast to all other receptors, this unique receptor type is expressed in numerous antennal neurons. This study describes the identification of HR2 homologues in two further lepidopteran species, the moths Antheraea pernyi and Bombyx mori, which share 86%-88% of their amino acids. In addition, based on RT-PCR experiments HR2 homologues were discovered in antennal cDNA of the honey bee (Apis mellifera; Hymenoptera), the blowfly (Calliphora erythrocephala; Diptera) and the mealworm (Tenebrio molitor; Coleoptera). Comparison of all HR2-related receptors reveals a high degree of sequence conservation across insect orders. In situ hybridization of antennal sections from the bee and the blowfly support the notion that HR2-related receptors are generally expressed in a very large number of antennal cells. This, together with the high degree of conservation suggests that this unique receptor subtype may fulfill a special function in chemosensory neurons of insects (Krieger, 2003).
Anopheles gambiae is a highly anthropophilic mosquito responsible for the majority of malaria transmission in Africa. The biting and host preference behavior of this disease vector is largely influenced by its sense of smell, which is presumably facilitated by G protein-coupled receptor signaling. Because of the importance of host preference to the mosquitoes' ability to transmit disease, studies have been intended to elucidate the molecular mechanisms underlying olfaction in An. gambiae. In the course of these studies, a number of genes were identified potentially involved in signal transduction, including a family of candidate odorant receptors. One of these receptors, encoded by GPRor7 (hereafter referred to as AgOr7), is remarkably similar to an odorant receptor that is expressed broadly in olfactory tissues and has been identified in Drosophila melanogaster and other insects (See alignment in Pitts, 2004). AgOr7 expression is observed in olfactory and gustatory tissues in adult An. gambiae and during several stages of the mosquitoes' development. Within the female adult peripheral chemosensory system, antiserum against the AgOR7 polypeptide labels most sensilla of the antenna and maxillary palp as well as a subset of proboscis sensilla. Furthermore, AgOR7 antiserum labeling is observed within the larval antenna and maxillary palpus. These results are consistent with a role for AgOr7 in both olfaction and gustation in An. gambiae and raise the possibility that AgOr7 orthologs may also be of general importance to both modalities of chemosensation in other insects (Pitts, 2004).
date revised: 27 February 2005
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