Functional Expression of a Mammalian Odorant Receptor (original) (raw)
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Identification of ligands for olfactory receptors by functional expression of a receptor library
Cell, 1998
neurons in the olfactory epithelium, is generated by the cAMP pathway (Brunet et al., 1996; Belluscio et al., 1998). Summary ies with in situ hybridization have suggested that each olfactory receptor neuron may express only one, or at The recognition of odorants by olfactory receptors most a few, of these olfactory receptor proteins (Ressler represents the first stage in odor discrimination. Here, et al., 1993; Vassar et al., 1993). Thus, odorant discrimiwe report the generation of an expression library connation for a given receptor neuron should depend on taining a large and diverse repertoire of mouse olfacthe ligand specificity of the one or few receptor proteins tory receptor sequences in the transmembrane II-VII it expresses. At the same time, electrophysiological region. From this library, 80 chimeric receptors were studies on single, dissociated olfactory neurons have tested against 26 odorants after transfection into HEKsuggested that individual cells can detect a broad spec-293 cells. Three receptors were identified to respond trum of odorants, in that any given cell has a high probato micromolecular concentrations of carvone, (Ϫ) citbility of responding to a small, arbitrary set of odorants ronellal, and limonene, respectively. We also found (Firestein et al., 1993). Taken together, these results that the mouse I7 receptor, unlike the rat I7 receptor, suggest that each olfactory receptor protein responds prefers heptanal instead of octanal, as a result of a to a large number of odorants.
Proceedings of the National Academy of Sciences, 2015
Significance Mammalian odorant receptors (ORs) comprise the largest family of G protein-coupled receptors, which are frequent drug targets. While many ORs respond specifically to select odorants, recent studies have identified a small number of ORs that respond to a large set of diverse odorants. Up to date, little is known about the molecular and structural features that shape the OR response properties. Our study reveals that broadly responsive ORs show elevated basal activity, suggesting a lower activation barrier. Surprisingly, a single amino acid mutation is sufficient to confer high basal activity and broad responsiveness to an OR, which originally shows weak responses to few odorants.
Receptor cell responses to odorants: Similarities and differences among odorants
Brain Research, 1984
Key words: olfactory receptor nerve --odorant space --odour discrimination --frog An extensive exploration of the discriminating properties of olfactory receptors cells has been conducted in our laboratory through the statistical processing of single cell responses recorded in the frog's olfactory epithelium. Similarities between odorant stimulating properties could be demonstrated by comparing the complex response profiles of receptor cells, resulting in the concept of odorant groups which depicts consistent relationships found between some odorous compounds. The recording technique limits the number of odorants which can be tested and compared within the same series of experiments. Thus, based upon previous studies we have chosen a specific set of 20 odorants whose characteristics are known but, up to this time, have not been compared in the same experimental system. These odorants were: acetophenone, anisole, n-butanol, OL-camphor, cyclodecanone, 1,8-cineole, p-cymene, D-citronellol, n-heptanol, isoamyl acetate, isovaleric acid, D-limonene, methyl amylketone, L-menthol, phenol, thiophenol, pyridine, thymol, cyclohexanol, cyclohexanone. The pattern of similarities between these odorants, as delineated with the aid of correlation coefficient computation, factor analysis and non-hierarchical taxonomy, confirms the reality of several odorant groups previously suggested and describes their cross-relationships. The receptor mechanisms underlying these odorant groups are discussed. Because reliable prominent features of the olfactory stimulus space can be established on an objective basis, the findings are proposed as a reference for future studies on other aspects of olfactory discrimination.
Journal of Biological Chemistry, 2005
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 OR 1 is complicated by their combinatorial odorant coding, and thus requires large scale odorant and receptor screening, and establishing of receptor-specific odorant profiles. We report here on the stable reconstitution of OR-specific signaling in HeLa/Olf cells, via G protein αolf and adenylyl cyclase type-III, to the Ca 2+ -influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Gαolf in odorant-specific signaling of OR: employment of the non-typical G protein α15 dramatically altered the odorant specificities of 3 out of 7 receptors, that had been characterrized 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 FLIPR experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal out of a 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 Gα15 or Gαolf. We finally established an EC 50ranking 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.
Functional analysis of a mammalian odorant receptor subfamily
Journal of Neurochemistry, 2006
Phylogenetic analysis groups mammalian odorant receptors into two broad classes and numerous subfamilies. These subfamilies are proposed to reflect functional organization. Testing this idea requires an assay allowing detailed functional characterization of odorant receptors. Here we show that a variety of Class I and Class II mouse odorant receptors can be functionally expressed in Xenopus laevis oocytes. Receptor constructs included the N-terminal 20 residues of human rhodopsin and were co-expressed with Gαolf and the cystic fibrosis transmembrane regulator to allow electrophysiological measurement of receptor responses. For most mouse odorant receptors tested, these conditions were sufficient for functional expression. Co-expression of accessory proteins was required to allow functional surface expression of some mouse odorant receptors. We used this assay to examine the receptive ranges of all members of the mouse odorant receptor 42 (MOR42) subfamily. MOR42-1 responded to dicarboxylic acids, preferring a 10–12 carbon chain length. MOR42-2 responded to monocarboxylic acids (7–10 carbons). MOR42-3 responded to dicarboxylic acids (8–10 carbons) and monocarboxylic acids (10–12 carbons). Thus, the receptive range of each receptor was unique. However, overlap between the individual receptive ranges suggests that the members of this subfamily form one contiguous subfamily receptive range, suggesting that odorant receptor subfamilies do constitute functional units.
Functional identification and reconstitution of an odorant receptor in single olfactory neurons
Proceedings of the National Academy of Sciences, 1999
The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand-receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca 2؉ -imaging and reverse transcriptioncoupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca 2؉ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green f luorescent protein as a marker. The infected neurons elicited external Ca 2؉ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest.
European Journal of Biochemistry, 2000
After characterization of a novel odorant-binding protein (OBP) variant isolated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR. Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a concentration of 80 mg´L 21 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD. We observed that, in contrast with porcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disulfide bonds (C44±C48 and C63±C155), a pairing close to that of hamster aphrodisin. OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) with a dissociation constant of 0.6^0.3 mm. Fluorescence experiments revealed that 1-AMA was displaced efficiently by molecules including usual solvents such as EtOH and dimethylsulfoxide. Owing to the large OBP-1F amounts expressed, we set up a novel biomimetic assay (volatileodorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions. The assay permitted observations on the binding of airborne odorants of different chemical structures and odors (2-isobutyl-3methoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl disulfide and methyl thiobutyrate). Uptake of airborne odorants in nearly physiological conditions strengthens the role of OBP as volatile hydrophobic odorant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells.
Concentration-dependent recruitment of mammalian odorant receptors
eneuro
A fundamental challenge in studying principles of organization used by the olfactory system to encode odor concentration information has been identifying comprehensive sets of activated odorant receptors (ORs) across a broad concentration range inside freely behaving animals. In mammals, this has recently become feasible with high-throughput sequencing-based methods that identify populations of activated ORs in vivo. In this study, we characterized the mouse OR repertoires activated by the two odorants, acetophenone and 2,5-dihydro-2,4,5trimethylthiazoline, from 0.01% to 100% (v/v) as starting concentrations using phosphorylated ribosomal protein S6 capture followed by RNA-Seq. We found Olfr923 to be one of the most sensitive ORs that is enriched by acetophenone. Using a mouse line that genetically labels Olfr923-positive axons, we provided evidence that acetophenone activates the Olfr923 glomeruli in the olfactory bulb. Through molecular dynamics stimulations, we identified amino acid residues in the Olfr923 binding cavity that facilitate acetophenone binding. This study sheds light on the active process by which unique OR repertoires may collectively facilitate the discrimination of odorant concentrations. 4 Significance Statement The ability of animals to discriminate odors over a range of odor concentrations while recognizing concentration-invariant odor identity presents an encoding challenge for the olfactory system. To further our understanding on how animals sense odors at different concentrations, it is important to describe how odor concentration information is represented at the receptor level. Here, we establish a sensitive in vivo approach to screen populations of odorant receptors enriched in the odor-activated sensory neurons in mice. We identified comprehensive lists of enriched odorant receptors against a 10,000-fold concentration range for two odorants. Describing the concentration-dependent activation for unique populations of odorant receptors is fundamental for future studies in determining how individual odorant receptors contribute to olfactory sensitivity and odor intensity coding.
Chemistry & Biology, 2008
Olfactory receptors (ORs) form a large family of G protein-coupled receptor proteins (GPCRs) responsible for sensing the ambient chemical environment. The molecular recognition strategies used by ORs to detect and distinguish odorant molecules are unclear. Here, we investigated the variable of odorant carbon chain conformation for an established odorant-OR pair: n-octanal and rat OR-I7. A series of conformationally restricted octanal mimics were tested on live olfactory sensory neurons (OSNs). Our results support a model in which unactivated OR-I7 binds aliphatic aldehydes indiscriminately, and then applies conformational and length filters to distinguish agonists from antagonists. Specific conformers are proposed to activate OR-I7 by steric buttressing of an OR activation pocket. Probing endogenously expressed rat OSNs with octanal and constrained mimics furnished evidence that odorant conformation contributes to an odorant's unique olfactory code signature.
Olfactory receptor cells: recognition and transduction of chemical signals
Cytotechnology, 1993
The reception of odorous molecules occurs in chemosensory neurons which encode the strength, duration and quality of odorant stimuli into distinct patterns of afferent neuronal signals which travel along the cells' axons towards the brain where decoding of the olfactory message is initiated. Our understanding of the molecular mechanisms mediating the primary events of odor detection has lagged behind the knowledge about other sensory modalities.