Additional file 4 of The mutational landscape of human olfactory G protein-coupled receptors
Ramón Cierco JimenezNil Casajuana‐MartinAdrián García‐RecioLidia AlcántaraLeonardo PardoMercedes CampilloÁngel González
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Additional file 4. The human OR mutation database table. The mutation data table is available at the human Olfactory Receptor Mutation database (hORMdb) website and contains information of 119,069 natural human OR nucleotide variants extracted from gnomAD v2 and annotated with genomic and structural information as described in the main text (resumed in the diagram of Fig. 1). A total of 78 descriptors were associated with each of the natural variants generating a total of 9,287,382 data points. More information about the data types at each column can be found at the HELP panel on the hORMdb web application.Neurophysiology
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Abstract This review intends to integrate recent data from the Drosophila olfactory system into an up‐to‐date account of the neuronal basis of olfaction. It focuses on (1) an electron microscopic study that mapped a large proportion of fruitfly olfactory sensilla, (2) large‐scale electrophysiological recordings that allowed the classification of the odor response spectra of a complete set of sensilla, (3) the identification and expression patterns of candidate odorant receptors in the olfactory tissues, (4) central projections of neurons expressing a given odorant receptor, (5) an improved glomerular map of the olfactory center, and (6) attempts to exploit the larval olfactory system as a model of reduced cellular complexity. These studies find surprising parallels between the olfactory systems of flies and mammals, and thus underline the usefulness of the fruitfly as an olfactory model system. Both in Drosophila and in mammals, odorant receptor neurons appear to express only one type of receptor. Neurons expressing a given receptor are scattered in the olfactory tissues but their afferents converge onto a few target glomeruli only. This suggests that in both phyla, the periphery is represented in the brain as a chemotopic map. The major difference between mammals and fruitflies refers to the numbers of receptors, neurons, and glomeruli, which are largely reduced in the latter, and particularly in larvae. Yet, if activated in a combinatorial fashion, even this small set of elements could allow discrimination between a vast array of odorants. Microsc. Res. Tech. 55:284–296, 2001. © 2001 Wiley‐Liss, Inc.
Antennal lobe
Glomerulus
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Introduction GPCRs and the Human Genome GPCR Architecture, Signaling and Drug Action Identification of GPCRs GPCRs in the Postgenomic Era: Orphan Receptors Ligand Hunting Reverse Pharmacology Approaches to oGPCRs In Silico Approaches Tissue Expression Expression of an oGPCR of Interest Screening Approaches Screening for oGPCR Ligands using Functional Assays GTPγS-binding Assays Measurements of cAMP Ca2+ Measurements The Cytosensor Microphysiometer Reporter Gene Assays GPCR–G-protein Coupling Ligand-independent GPCR Activity Novel Screening Strategies Future Prospects References
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G-protein coupled receptors (GPCRs) are popular biological targets for drug discovery and development. To date there are more than 140 orphan GPCRs, i.e. receptors whose endogenous ligands are unknown. Traditionally orphan GPCRs have been difficult to study and the development of therapeutic compounds targeting these receptors has been extremely slow although these GPCRs are considered important targets based on their distribution and behavioral phenotype revealed by animals lacking the receptor. Recent advances in several methods used to study orphan receptors, including protein crystallography and homology modeling are likely to be useful in the identification of therapeutics targeting these receptors. In the past 13 years, over a dozen different Class A GPCRs have been crystallized; this trend is exciting, since homology modeling of GPCRs has previously been limited by the availability of solved structures. As the number of solved GPCR structures continues to grow so does the number of templates that can be used to generate increasingly accurate models of phylogenetically-related orphan GPCRs. The availability of solved structures along with the advances in using multiple templates to build models (in combination with molecular dynamics simulations that reveal structural information not provided by crystallographic data and methods for modeling hard-to-predict flexible loop regions) have improved the quality of GPCR homology models. This, in turn, has improved the success rates of virtual ligand screens that use homology models to identify potential receptor binding compounds. Experimental testing of the predicted hits and validation using traditional GPCR pharmacological approaches can be used to drive ligand-based efforts to probe orphan receptor biology as well as to define the chemotypes and chemical scaffolds important for binding. As a result of these advances, orphan GPCRs are emerging from relative obscurity as a new class of drug targets.
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Abstract Two large families of olfactory receptors, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odor binding “tuning” OR receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco to function. The situation for IRs is more complex. Different tuning IR receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a coreceptor, but its function has not been systematically examined. This is particularly important as recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we report the effects of Drosophila olfactory co-receptor mutants on odor detection by coeloconic olfactory neurons and determine their broader impact on gene expression through RNASeq analysis. We demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses, whereas in ac1 sensilla, amine responses persist in the absence of Ir76b or Ir25a, but are lost in a double-mutant. Such responses do not require Ir8a. Conversely, acid-sensing ORNs require Ir8a, but not Ir76b or Ir25a. Using antennal transcriptional profiling, we find that the expression of acid-sensing IR receptors is significantly reduced in Ir8a mutants, but is unaffected by the loss of Ir25a or Ir76b. Similarly, select OR tuning receptors are also downregulated in Orco 2 mutants. In contrast, expression of amine-sensing IR receptors is mostly unchanged in Ir25a and Ir76b mutants. Together, our data reveal new aspects of co-receptor function in the olfactory system. Summary Insect vectors of human disease rely on their sense of smell to locate humans for blood meals. A critical first step in olfaction is the odorant-induced activation of receptors on olfactory neurons. There are two major olfactory receptor families in insects, with each species having dozens of different odorant-binding “tuning” receptors. The receptor complexes also contain non-tuning co-receptors, which are highly conserved across insect species and are required for function. Here we characterize co-receptor mutants with electrophysiological recordings and transcriptome analysis in Drosophila . Our findings resolve the differential co-receptor dependence of olfactory neuron responses to volatile amines and acids. We also report changes in antennal gene expression that result from the absence of these co-receptors. Most notably, the absence of some co-receptors leads to a selective loss of transcript expression for the tuning olfactory receptors whose function depends on the missing co-receptors. Together our data provide new insight into the roles of co-receptors in insect olfaction.
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Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.
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ABSTRACT Olfactory systems employ combinatorial receptor codes for odors. Systematically generating stimuli that address the combinatorial possibilities of an olfactory code poses unique challenges. Here, we present a stimulus method to probe the combinatorial code, demonstrated using the Drosophila larva. This method leverages a set of primary odorants, each of which targets the activity of one olfactory receptor neuron (ORN) type at an optimal concentration. Our setup uses microfluidics to mix any combination of primary odorants on demand to activate any desired combination of ORNs. We use this olfactory pattern generator to demonstrate a spatially distributed olfactory representation in the dendrites of a single interneuron in the antennal lobe, the first olfactory neuropil of the larva. In the larval mushroom body, the next processing layer, we characterize diverse receptive fields of a population of Kenyon cells. The precision and flexibility of the olfactory pattern generator will facilitate systematic studies of processing and transformation of the olfactory code.
Neuropil
Antennal lobe
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Stimulus (psychology)
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Caribbean spiny lobsters (Panulirus argus) rely on their sense of olfaction for many behaviours. Growth of their olfactory systems, and maintenance of olfactory function, is ensured by structural change that occurs continuously throughout life. In this paper, we review recent studies on postembryonic development in the olfactory system of P. argus and several other decapod species. Major structural change occurs in both the peripheral and central olfactory systems; it includes addition and loss of olfactory receptor neurons (ORNs), aesthetasc and other sensilla, and interneurons associated with the olfactory lobes of the brain. From these studies it is clear that continuous growth and turnover of olfactory tissue is a normal process in decapod crustaceans. In addition, we describe for the first time mechanisms that enable the peripheral olfactory system of spiny lobsters to regenerate after injury. We monitored the regeneration of olfactory tissue usingin vivo incorporation of the cell proliferation marker 5- bromo-2′-deoxyuridine (BrdU). Our results show that regeneration after partial antennular amputation, which reduces the length of the antennule and thereby the number of ORNs, occurs as a result of upregulation of the normal mode of ORN addition and down-regulation of loss. In contrast, localized injury to aesthetasc sensilla, which causes the associated ORNs to degenerate but does not reduce antennular length, is followed by local regeneration of olfactory tissue.
Panulirus argus
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