Evolution of odorant receptors

Odorant receptors (ORs) located in the nasal epithelium, at the ciliated surface of olfactory sensory neurons, represent the initial step of a transduction cascade that leads to odor detection. ORs form the largest and most diverse family of G-protein-coupled receptors (GPCRs). They are encoded by a multigene family that has been partially characterized in cyclostomes, teleosts, amphibia, birds and mammals, as well as in Drosophila melanogaster and the nematode Caenorhabditis elegans. As new sequence data emerge, it is increasingly clear that OR primary structure can vary dramatically across phyla. Some chemoreceptors are encoded by genes with little sequence similarity to the prototypical ORs originally isolated in mammals. A large number of sequences are now available allowing a detailed study of the evolutionary implications of OR diversity across species. This review discusses the evolutionary implications of the divergent primary structures of chemoreceptors with identical functions.     

Ligand specificity of odorant receptors

Odorant receptors belong to class A of the G protein-coupled receptors (GPCRs) and detect a large number of structurally diverse odorant molecules. A recent structural bioinformatic analysis suggests that structural features are conserved across class A of GPCRs in spite of their low sequence identity. Based on this work, we have aligned the sequences of 29 ORs for which ligand binding data are available. Recent site-directed mutagenesis experiments on one such receptor (MOR174-9) provide information that helped to identify nine amino-acid residues involved in ligand binding. Our modeling provides a rationale for amino acids in equivalent positions in most of the odorant receptors considered and helps to identify other amino acids that could be important for ligand binding. Our findings are consistent with most of the previous models and allow predictions for site-directed mutagenesis experiments, which could also validate our model.     

Functional evolution of mammalian odorant receptors

The mammalian odorant receptor (OR) repertoire is an attractive model to study evolution, because ORs have been subjected to rapid evolution between species, presumably caused by changes of the olfactory system to adapt to the environment. However, functional assessment of ORs in related species remains largely untested. Here we investigated the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. We functionally characterized the in vitro responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that, while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.     

A new function for odorant receptors

Myofibers with an abnormal branching cytoarchitecture are commonly found in various neuromuscular diseases as well as after severe muscle injury. These aberrant myofibers are fragile and muscles containing a high percentage of these myofibers are weaker and more prone to injury. To date the mechanisms and molecules regulating myofiber branching have been obscure. Recent work analyzing the role of mouse odorant receptor 23 (MOR23) in muscle regeneration revealed that MOR23 is necessary for proper skeletal muscle regeneration in mice as loss of MOR23 leads to increased myofiber branching. Further studies demonstrated that MOR23 expression is induced when muscle cells were extensively fusing and plays an important role in controlling cell migration and adhesion. These data demonstrate a novel role for an odorant receptor in tissue repair and identify the first molecule with a functional role in myofiber branching.     

Using GEFs to deorphanize odorant receptors

Odorant perception is initiated when odorants activate uniquecombinations of odorant receptors (ORs) expressed in the ciliaof olfactory sensory neurons in the nose (Buck and Axel 1991;Malnic et al. 1999). One of the greatest challenges in the olfactoryscience is to correlate ORs with their ligands (to deorphanizethe ORs). The determination of OR–ligand pairs shouldreveal how the OR family is used to generate diverse odorantperceptions (Keller et al. 2007, Menashi et al. 2007). However,these studies are complicated     

Evolution of odorant receptors expressed in mammalian testes

About 10% of mammalian odorant receptors are transcribed in testes, and odorant-receptor proteins have been detected on mature spermatozoa. Testis-expressed odorant receptors (TORs) are hypothesized to play roles in sperm chemotaxis, but they might also be ordinary nasal odorant receptors (NORs) that are expressed gratuitously in testes. Under the sperm-chemotaxis hypothesis, TORs should be subject to intense sexual selection and therefore should show higher rates of amino acid substitution than NORs, but under the gratuitous-expression hypothesis, TORs are misidentified NORs and therefore should evolve like other NORs. To test these predictions, we estimated synonymous and nonsynonymous divergences of orthologous NOR and TOR coding sequences from rat and mouse. Contrary to both hypotheses, TORs are on average more highly conserved than NORs, especially in certain domains of the OR protein. This pattern suggests that some TORs might perform internal nonolfactory functions in testes; for example, they might participate in the regulation of sperm development. However, the pattern is also consistent with a modified gratuitous-expression model in which NORs with specialized ligand specificities are both more highly conserved than typical NORs and more likely to be expressed in testes.     

Heteromeric Anopheline odorant receptors exhibit distinct channel properties

Background

Insect odorant receptors (ORs) function as odorant-gated ion channels consisting of a conventional, odorant-binding OR and the Orco coreceptor. While Orco can function as a homomeric ion channel, the role(s) of the conventional OR in heteromeric OR complexes has largely focused only on odorant recognition.

Results

To investigate other roles of odorant-binding ORs, we have employed patch clamp electrophysiology to investigate the properties of the channel pore of several OR complexes formed by a range of different odorant-specific Anopheles gambiae ORs (AgOrs) each paired with AgOrco. These studies reveal significant differences in cation permeability and ruthenium red susceptibility among different AgOr complexes.

Conclusions

With observable differences in channel function, the data support a model in which the odorant-binding OR also affects the channel pore. The variable effect contributed by the conventional OR on the conductive properties of odorant-gated sensory channels adds additional complexity to insect olfactory signaling, with differences in odor coding beginning with ORs on the periphery of the olfactory system.     

Functional cloning and reconstitution of vertebrate odorant receptors

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 Ca2+-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.     

Axonal ephrin-As and odorant receptors: coordinate determination of the olfactory sensory map

Olfactory sensory neurons expressing a given odorant receptor (OR) project with precision to specific glomeruli in the olfactory bulb, generating a topographic map. In this study, we demonstrate that neurons expressing different ORs express different levels of ephrin-A protein on their axons. Moreover, alterations in the level of ephrin-A alter the glomerular map. Deletion of the ephrin-A5 and ephrin-A3 genes posteriorizes the glomerular locations for neurons expressing either the P2 or SR1 receptor, whereas overexpression of ephrin-A5 in P2 neurons results in an anterior shift in their glomeruli. Thus the ephrin-As are differentially expressed in distinct subpopulations of neurons and are likely to participate, along with the ORs, as one of a complement of guidance receptors governing the targeting of like axons to precise locations in the olfactory bulb.     

RTP family members induce functional expression of mammalian odorant receptors

Transport of G protein-coupled receptors (GPCRs) to the cell surface membrane is critical in order for the receptors to recognize their ligands. However, mammalian GPCR odorant receptors (ORs), when heterologously expressed in cells, are poorly expressed on the cell surface. Here we show that the transmembrane proteins RTP1 and RTP2 promote functional cell surface expression of ORs expressed in HEK293T cells. Genes encoding these proteins are expressed specifically in olfactory neurons. These proteins are associated with OR proteins and enhance the OR responses to odorants. Similar although weaker effects were seen with a third protein, REEP1. These findings suggest that RTP1 and RTP2 in particular play significant roles in the translocation of ORs to the plasma membrane as well as in the functioning of ORs. We have used this approach to identify active odorant ligands for ORs, providing a platform for screening the chemical selectivity of the large OR family.     

Synergism of accessory factors in functional expression of mammalian odorant receptors

The discovery of odorant receptors led to endeavors in matching them with their cognate ligands. Although it has been challenging to functionally express odorant receptors in heterologous cells, previous studies have linked efficient odorant receptor expression with N-terminal modifications and accessory proteins, including the receptor-transporting proteins (RTPs) and Ric8b. Here we have shown that a shorter form of RTP1, RTP1S, supports robust cell-surface and functional expression of representative odorant receptors. Using a combination of accessory proteins, including RTP1S, Ric8b, and G(alphaolf), a diverse set of untagged odorant receptors were successfully expressed heterologously due to the synergistic effects among the various accessory proteins. Furthermore, the addition of an N-terminal rhodopsin tag to the odorant receptors, along with the same set of accessory proteins, exhibits an additional level of synergism, inducing enhanced odorant receptor responses to odorants and thus defining a more efficient heterologous expression system. We then showed that the presence or absence of different N-terminal tags has little effect on the ligand specificity of odorant receptors, although the amount of receptor expressed can play a role in the ligand response profile. The accuracy of the odorant receptor heterologous expression system involving tagged odorant receptors and various accessory proteins promises success in high throughput de-orphaning of mammalian odorant receptors.     

Differences in (-)citronellal binding to various odorant receptors

To test the hypothesis that olfactory receptors (ORs) recognize different molecular features of odor molecules termed "odotypes", we studied receptor-ligand interactions of two human and two mouse ORs, recognizing (-)citronellal. Structurally similar receptors provide identical binding pockets (OLFR43, OR1A1, and OR1A2), and have comparable EC(50) values. Other ORs with lower sequence identity bind (-)citronellal in a different way, leading to different EC(50) values.     

Insect G protein-coupled receptors and signal transduction

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins (7-TM) that transduce extracellular signals into cellular physiological responses through the activation of heterotrimeric guanine nucleotide binding proteins (alpha beta gamma subunits). Their general properties are remarkably well conserved during evolution. Despite this general resemblance, a large variety of different signals are mediated via this category of receptors. Several GPCR-(sub)families have an ancient origin that is situated before the divergence of Protostomian and Deuterostomian animals. Nevertheless, an enormous diversification has occurred since then. The availability of novel sequence information is growing very rapidly as a result of molecular cloning experiments and of metazoan genome (Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens) and EST (expressed sequence tags) sequencing projects. The Drosophila Genome Sequencing Project will certainly have an important impact on insect signal transduction and receptor research. In parallel, convenient expression systems and functional assay procedures will be needed to investigate insect receptor properties and to monitor the effects of natural and artificial ligands. The study of the evolutionary aspects of G protein-coupled receptors and of their signaling pathways will probably reveal insect-specific features. More insight into these features may result in novel methods and practical applications. Arch.