Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons

Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human. The central insight utilized is that functional contact residues would be conserved among pairs of orthologous receptors, but considerably less conserved among paralogous pairs. Using judiciously selected subsets of 218 ortholog pairs and 518 paralog pairs, we have identified 22 sequence positions that are both highly conserved among the putative orthologs and variable among paralogs. These residues are disposed on transmembrane helices 2 to 7, and on the second extracellular loop of the receptor. Strikingly, although the prediction makes no assumption about the location of the binding site, these amino acid positions are clustered around a pocket in a structural homology model of ORs, mostly facing the inner lumen. We propose that the identified positions constitute the odorant binding site. This conclusion is supported by the observation that all but one of the predicted binding site residues correspond to ligand-contact positions in other rhodopsin-like GPCRs.     

Mammalian-specific OR37 receptors are differentially activated by distinct odorous fatty aldehydes

The capacity of the mammalian olfactory system to detect an enormous collection of different chemical compounds is based on a large repertoire of odorant receptors (ORs). A small group of these ORs, the OR37 family, is unique due to a variety of special features. Members of this subfamily are exclusively found in mammals, they share a high degree of sequence homology and are highly conserved during evolution. It is still elusive which odorants may activate these atypical receptors. We have reasoned that compounds from skin, hairs, or skin glands might be potential candidates. We have exposed mice to such compounds and monitored activation of glomeruli through the expression of the activity marker c-fos in juxtaglomerular cells surrounding ventrally positioned glomeruli in the olfactory bulb (OB). Employing this methodology it was found that stimulation with long-chain alkanes elicits activation in the ventral part of the OB, however, none of the OR37 glomeruli. Analyses of long-chain hydrocarbon compounds with different functional groups revealed that long-chain aliphatic aldehydes elicited an activation of defined OR37 glomeruli, each of them responding preferentially to an aldehyde with different chain lengths. These results indicate that OR37 receptors may be tuned to distinct fatty aldehydes with a significant degree of ligand specificity.     

Expression of odorant receptor family, type 2 OR in the aquatic olfactory cavity of amphibian frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions.     

Comparison of odorant specificity of two human olfactory receptors from different phylogenetic classes and evidence for antagonism

Humans are able to detect and discriminate myriads of odorants using only several hundred olfactory receptors (ORs) classified in two major phylogenetic classes representing ORs from aquatic (class I) and terrestrial animals (class II). Olfactory perception results in a combinatorial code, in which one OR recognizes multiple odorants and different odorants are recognized by different combinations of ORs. Moreover, recent data suggest that odorants could also behave as antagonists for other ORs, thus making the combinatorial coding more complex. Here we describe the odorant repertoires of two human ORs belonging to class I and class II, respectively. For this purpose, we set up an assay based on calcium imaging in which 100 odorants were screened using air-phase odorant stimulation at physiological doses. We showed that the human class I OR52D1 is functional, exhibiting a narrow repertoire related to that of its orthologous murine OR, demonstrating than this human class I OR is not an evolutionary relic. The class II OR1G1 was revealed to be broadly tuned towards odorants of 9-10 carbon chain length, with diverse functional groups. The existence of antagonist odorants for the class II OR was also demonstrated. They are structurally related to the agonists, with shorter carbon chain length.     

Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.     

The variable and conserved interfaces of modeled olfactory receptor proteins.

The accumulation of hundreds of olfactory receptor (OR) sequences, along with the recent availability of detailed models of other G-protein-coupled receptors, allows us to analyze the OR amino acid variability patterns in a structural context. A Fourier analysis of 197 multiply aligned olfactory receptor sequences showed an alpha-helical periodicity in the variability profile. This was particularly pronounced in the more variable transmembranal segments 3, 4, and 5. Rhodopsin-based homology modeling demonstrated that the inferred variable helical faces largely point to the interior of the receptor barrel. We propose that a set of 17 hypervariable residues, which point to the barrel interior and are more extracellularly disposed, constitute the odorant complementarity determining regions. While 12 of these residues coincide with established ligand-binding contact positions in other G-protein-coupled receptors, the rest are suggested to form an olfactory-unique aspect of the binding pocket. Highly conserved olfactory receptor-specific sequence motifs, found in the second and third intracellular loops, may comprise the G-protein recognition epitope. The prediction of olfactory receptor functional sites provides concrete suggestions of site-directed mutagenesis experiments for altering ligand and G-protein specificity.     

Primate evolution of an olfactory receptor cluster: diversification by gene conversion and recent emergence of pseudogenes.

The olfactory receptor (OR) subgenome harbors the largest known gene family in mammals, disposed in clusters on numerous chromosomes. We have carried out a comparative evolutionary analysis of the best characterized genomic OR gene cluster, on human chromosome 17p13. Fifteen orthologs from chimpanzee (localized to chromosome 19p15), as well as key OR counterparts from other primates, have been identified and sequenced. Comparison among orthologs and paralogs revealed a multiplicity of gene conversion events, which occurred exclusively within OR subfamilies. These appear to lead to segment shuffling in the odorant binding site, an evolutionary process reminiscent of somatic combinatorial diversification in the immune system. We also demonstrate that the functional mammalian OR repertoire has undergone a rapid decline in the past 10 million years: while for the common ancestor of all great apes an intact OR cluster is inferred, in present-day humans and great apes the cluster includes nearly 40% pseudogenes.     

Distinct evolutionary patterns between chemoreceptors of 2 vertebrate olfactory systems and the differential tuning hypothesis

Most tetrapod vertebrates have 2 olfactory systems, the main olfactory system (MOS) and the vomeronasal system (VNS). According to the dual olfactory hypothesis, the MOS detects environmental odorants, whereas the VNS recognizes intraspecific pheromonal cues. However, this strict functional distinction has been blurred by recent reports that both systems can perceive both types of signals. Studies of a limited number of receptors suggest that MOS receptors are broadly tuned generalists, whereas VNS receptors are narrowly tuned specialists. However, whether this distinction applies to all MOS and VNS receptors remains unknown. The differential tuning hypothesis predicts that generalist MOS receptors detect an overlapping set of ligands and thus are more likely to be conserved over evolutionary time than specialist VNS receptors, which would evolve in a more lineage-specific manner. Here we test this prediction for all olfactory chemoreceptors by examining the evolutionary patterns of MOS-expressed odorant receptors (ORs) and trace amine-associated receptors (TAARs) and VNS-expressed vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs) in 7 tetrapods (mouse, rat, dog, opossum, platypus, chicken, and frog). The phylogenies of V1Rs and V2Rs show abundant lineage-specific gene gains/losses and virtually no one-to-one orthologs between species. Opposite patterns are found for ORs and TAARs. Analysis of functional data and ligand-binding sites of ORs confirms that paralogous chemoreceptors are more likely than orthologs to have different ligands and that functional divergence between paralogous chemoreceptors is established relatively quickly following gene duplication. Together, these results strongly suggest that the functional profile of the VNS chemoreceptor repertoire evolves much faster than that of the MOS chemoreceptor repertoire and that the differential tuning hypothesis applies to the majority, if not all, of MOS and VNS receptors.     

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.     

Making sense of olfaction through predictions of the 3-D structure and function of olfactory receptors

We used the MembStruk first principles computational technique to predict the three-dimensional (3-D) structure of six mouse olfactory receptors (S6, S18, S19, S25, S46 and S50) for which experimental odorant recognition profiles are available for a set of 24 odorants (4-9 carbons aliphatic alcohols, acids, bromo-acids and diacids). We used the HierDock method to scan each predicted OR structure for potential odorant binding site(s) and to calculate binding energies of each odorant in these binding sites. The calculated binding affinity profiles are in good agreement with experimental activation profiles, validating the predicted 3-D structures and the predicted binding sites. For each of the six ORs, the binding site is located between trans-membrane domains (TMs) 3-6, with contributions from extracellular loops 2 and 3. In particular, we find six residue positions in TM3 and TM6 to be consistently involved in the binding modes of the odorants. Indeed, the differences in the experimental recognition profiles can be explained on the basis of these critical residues alone. These predictions are also consistent with mutation data on ligand binding for catecholamine receptors and sequence hypervariability studies for ORs. Based on this analysis, we defined amino acid patterns associated with the recognition of short aliphatic alcohols and mono-acids. Using these two sequence fingerprints to probe the alignment of 869 OR sequences from the mouse genome, we identified 34 OR sequences matching the fingerprint for aliphatic mono-acids and 36 corresponding to the recognition pattern for aliphatic alcohols. We suggest that these two sets of ORs might function as basic arrays for uniquely recognizing aliphatic alcohols and acids. We screened a library of 89 additional molecules against the six ORs and found that this set of ORs is likely to respond to aldehydes and esters with longer carbon chains than their currently known agonists. We also find that compounds associated with the flavor in foods are often among the best calculated binding affinities. This suggests that physiologic ligands for these ORs may be found among aldehydes and esters associated with flavor.     

Cyclic nucleotide-gated channel activation is not required for activity-dependent labeling of zebrafish olfactory receptor neurons by amino acids

The olfactory epithelium of fish is heterogeneous both with respect to the types of receptor cells (ORNs) present and the families of odorant receptors expressed in these cells. As a consequence of this diversity, the transduction cascade(s) activated by odorants has yet to be unambiguously established. In the current study, electrophysiological and activity-dependent labeling techniques were used to assess the role of the cyclic nucleotide-gated channel in zebrafish olfactory transduction. Both amino acid and bile salt odorants elicited robust electrophysiological responses, however, activity-dependent labeling of ORNs could be stimulated only by the amino acid odorants. An adenylate cyclase (AC) activator (forskolin) and a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) also elicited robust electrophysiological responses; generally larger than the responses elicited by either the amino acid or bile salt odorants. However, neither forskolin alone or a mixture of forskolin and IBMX stimulated activity-dependent labeling. Bathing the olfactory epithelium with forskolin, which presumably increased the intracellular concentration of cAMP, reduced the responses to bile salt odorants to a significantly greater extent than amino acid odorants. Collectively, these findings suggest that the transduction of amino acid input does not rely primarily on cyclic nucleotide-gated (CNG) channel activation and that CNG channel activation may be required for the transduction of bile salt input. Copyright Copyright 1999 S. Karger AG, Basel     

Olfactory receptor antagonism between odorants

The detection of thousands of volatile odorants is mediated by several hundreds of different G protein-coupled olfactory receptors (ORs). The main strategy in encoding odorant identities is a combinatorial receptor code scheme in that different odorants are recognized by different sets of ORs. Despite increasing information on agonist-OR combinations, little is known about the antagonism of ORs in the mammalian olfactory system. Here we show that odorants inhibit odorant responses of OR(s), evidence of antagonism between odorants at the receptor level. The antagonism was demonstrated in a heterologous OR-expression system and in single olfactory neurons that expressed a given OR, and was also visualized at the level of the olfactory epithelium. Dual functions of odorants as an agonist and an antagonist to ORs indicate a new aspect in the receptor code determination for odorant mixtures that often give rise to novel perceptual qualities that are not present in each component. The current study also provides insight into strategies to modulate perceived odorant quality.     

Molecular determinants of odorant receptor function in insects

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.     

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.     

The role of the coreceptor Orco in insect olfactory transduction

Insects sense odorants with specialized odorant receptors (ORs). Each antennal olfactory receptor neuron expresses one OR with an odorant binding site together with a conserved coreceptor called Orco which does not bind odorants. Orco is necessary for localization of ORs to dendritic membranes and, thus, is essential for odorant detection. It forms a spontaneously opening cation channel, activated via phosphorylation by protein kinase C. Thereafter, Orco is also activated via cyclic adenosine monophosphate (cAMP). Orco forms homo—as well as heteromers with ORs with unknown stoichiometry. Contradictory publications suggest different mechanisms of olfactory transduction. On the one hand, evidence accumulates for the employment of more than one G protein-coupled olfactory transduction cascade in different insects. On the other hand, results from other studies suggest that the OR–Orco complex functions as an odorant-gated cation channel mediating ionotropic signal transduction. This review analyzes conflicting hypotheses concerning the role of Orco in insect olfactory transduction. In conclusion, in situ studies in hawkmoths falsify the hypothesis that Orco underlies odorant-induced ionotropic signal transduction in all insect species. Instead, Orco forms a metabotropically gated, slow cation channel which controls odorant response threshold and kinetics of the sensory neuron.     

Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors (hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory repertoire of hORs remains a tremendous challenge. We therefore developed a chemical systems level approach based on protein-protein association network to investigate novel hOR-odorant relationships. Using this new approach, we proposed and validated new bioactivities for odorant molecules and OR2W1, OR51E1 and OR5P3. As it remains largely unknown how human perception of odorants influence or prevent diseases, we also developed an odorant-protein matrix to explore global relationships between chemicals, biological targets and disease susceptibilities. We successfully experimentally demonstrated interactions between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma (PPARγ). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome.     

Responses of Xenopus laevis water nose to water-soluble and volatile odorants.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.     

Functional identification of a goldfish odorant receptor.

The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding.     

Combinatorial receptor codes for odors

The discriminatory capacity of the mammalian olfactory system is such that thousands of volatile chemicals are perceived as having distinct odors. Here we used a combination of calcium imaging and single-cell RT-PCR to identify odorant receptors (ORs) for odorants with related structures but varied odors. We found that one OR recognizes multiple odorants and that one odorant is recognized by multiple ORs, but that different odorants are recognized by different combinations of ORs. Thus, the olfactory system uses a combinatorial receptor coding scheme to encode odor identities. Our studies also indicate that slight alterations in an odorant, or a change in its concentration, can change its "code," potentially explaining how such changes can alter perceived odor quality.