Reporting recently in Cell, Dalton et al (2013) identify a central role for the unfolded protein response in the regulation of olfactory receptor expression, unveiling molecular players in an elaborate feedback loop that controls the stabilization and establishment of single olfactory receptor gene choice.
In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.
calreticulin; calreticulin4; calnexin; endoplasmic reticulum; chaperone; trafficking; receptor; channel; retention; vomeronasal; V2R; CFTR
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 coexpressed 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 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.
olfactory receptors; Xenopus oocytes; electrophysiology
Humans have approximately 400 intact odorant receptors, but each
individual has a unique set of genetic variations that lead to variation in
olfactory perception. We used a heterologous assay to determine how often
genetic polymorphisms in odorant receptors alter receptor function. We
identified agonists for 18 odorant receptors and found that 63% of the
odorant receptors we examined had polymorphisms that altered in
vitro function. On average, two individuals differ functionally at
over 30% of their odorant receptor alleles. To show that these
in vitro results are relevant to olfactory perception, we
verified that variations in OR10G4 genotype explain over
15% of the observed variation in perceived intensity and over
10% of the observed variation in perceived valence for the high affinity
in vitro agonist guaiacol, but do not explain phenotypic
variation for the lower affinity agonists vanillin and ethyl vanillin.
Genetic variations in olfactory receptors likely contribute to the diversity of odorant-specific sensitivity phenotypes. Our working hypothesis is that genetic variations in auxiliary olfactory genes, including those mediating transduction and sensory neuronal development, may constitute the genetic basis for general olfactory sensitivity (GOS) and congenital general anosmia (CGA). We thus performed a systematic exploration for auxiliary olfactory genes and their documented variation. This included a literature survey, seeking relevant functional in vitro studies, mouse gene knockouts and human disorders with olfactory phenotypes, as well as data mining in published transcriptome and proteome data for genes expressed in olfactory tissues. In addition, we performed next-generation transcriptome sequencing (RNA-seq) of human olfactory epithelium and mouse olfactory epithelium and bulb, so as to identify sensory-enriched transcripts. Employing a global score system based on attributes of the 11 data sources utilized, we identified a list of 1,680 candidate auxiliary olfactory genes, of which 450 are shortlisted as having higher probability of a functional role. For the top-scoring 136 genes, we identified genomic variants (probably damaging single nucleotide polymorphisms, indels, and copy number deletions) gleaned from public variation repositories. This database of genes and their variants should assist in rationalizing the great interindividual variation in human overall olfactory sensitivity (http://genome.weizmann.ac.il/GOSdb).
olfactory candidate genes; congenital general anosmia; RNA-seqIntroduction
The ability to detect many odors varies among individuals; however, the contribution of genotype to this variation has been assessed for relatively few compounds. We have identified a genetic basis for the ability to detect the flavor compound cis-3-hexen-1-ol. This compound is typically described as “green grassy” or the smell of “cut grass,” with variation in the ability to detect it linked to single nucleotide polymorphisms (SNPs) in a region on human chromosome 6 containing 25 odorant receptor genes. We have sequenced the coding regions of all 25 receptors across an ethnically mixed population of 52 individuals and identified 147 sequence variants. We tested these for association with cis-3-hexen-1-ol detection thresholds and found 3 strongly associated SNPs, including one found in a functional odorant receptor (rs28757581 in OR2J3). In vitro assays of 13 odorant receptors from the region identified 3 receptors that could respond to cis-3-hexen-1-ol, including OR2J3. This gene contained 5 predicted haplotypes across the 52 individuals. We tested all 5 haplotypes in vitro and several amino acid substitutions on their own, such as rs28757581 (T113A). Two amino acid substitutions, T113A and R226Q, impaired the ability of OR2J3 to respond to cis-3-hexen-1-ol, and together these two substitutions effectively abolished the response to the compound. The haplotype of OR2J3 containing both T113A and R226Q explains 26.4% of the variation in cis-3-hexen-1-ol detection in our study cohort. Further research is required to examine whether OR2J3 haplotypes explain variation in perceived flavor experience and the consumption of foods containing cis-3-hexen-1-ol.
cis-3-hexen-1-ol; genetic association; odor; odorant receptor; threshold of detection
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 mammalian odorant receptor repertoire has been subjected to significant gene duplication and gene loss between species, presumably to adapt to the environment of an organism. However, even in distantly related species, a clear orthologous relationship exists for many genes. While ligands have been identified for several ORs, many of these receptors remain uncharacterized, especially in species other than human and mouse. Due to this paucity of functional data, it is assumed that ORs with similar sequence share functional characteristics. Here we investigate the functional evolution of OR orthologs—genes related via speciation—and OR paralogs—genes related via a duplication event—to provide insight as to how this large gene family has evolved. We show that OR orthologs have similar ligand selectivity to a panel of odors but differ in response magnitude. Additionally, orthologs respond to a common ligand more often than human OR paralogs, but there are vast differences in the potency and efficacy of individual receptors. This result stresses the broad importance of combining evolutionary genomics and molecular biology approaches to study gene function.
Although odour perception impacts food preferences, the effect of genotypic variation of odorant receptors (ORs) on the sensory perception of food is unclear. Human OR7D4 responds to androstenone, and genotypic variation in OR7D4 predicts variation in the perception of androstenone. Since androstenone is naturally present in meat derived from male pigs, we asked whether OR7D4 genotype correlates with either the ability to detect androstenone or the evaluation of cooked pork tainted with varying levels of androstenone within the naturally-occurring range. Consistent with previous findings, subjects with two copies of the functional OR7D4 RT variant were more sensitive to androstenone than subjects carrying a non-functional OR7D4 WM variant. When pork containing varying levels of androstenone was cooked and tested by sniffing and tasting, subjects with two copies of the RT variant tended to rate the androstenone-containing meat as less favourable than subjects carrying the WM variant. Our data is consistent with the idea that OR7D4 genotype predicts the sensory perception of meat containing androstenone and that genetic variation in an odorant receptor can alter food preferences.
In mammals, odorants and pheromones are detected by hundreds of odorant receptors (ORs) and vomeronasal receptors (V1Rs and V2Rs) expressed by sensory neurons that are respectively located in the main olfactory epithelium and in the vomeronasal organ. Even though these two olfactory systems are functionally and anatomically separate, their sensory neurons show a common mechanism of receptor gene regulation: each neuron expresses a single receptor gene from a single allele. The mechanisms underlying OR and VR gene expression remain unclear. Here we investigated if OR and V1R genes share common sequences in their promoter regions.
We conducted a comparative analysis of promoter regions of 39 mouse V1R genes and found motifs that are common to a large number of promoters. We then searched mouse OR promoter regions for motifs that resemble the ones found in the V1R promoters. We identified motifs that are present in both the V1R and OR promoter regions. Some of these motifs correspond to the known O/E like binding sites while others resemble binding sites for transcriptional repressors. We show that one of these motifs specifically interacts with proteins extracted from both nuclei from olfactory and vomeronasal neurons. Our study is the first to identify motifs that resemble binding sites for repressors in the promoters of OR and V1R genes. Analysis of these motifs and of the proteins that bind to these motifs should reveal important aspects of the mechanisms of OR/V1R gene regulation.
A diverse repertoire of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) enables cells to sense their environment. Mammalian olfaction requires the activation of odorant receptors (ORs), the largest family of GPCRs; however, whether ORs functionally interact with other families of GPCRs is unclear. We show that the interaction of ORs with the type 3 muscarinic acetylcholine receptor (M3-R), which is found in olfactory sensory neurons (OSNs), modulated OR responses to cognate odorants. In human embryonic kidney–293T cells, ORs and the M3-R physically interacted, and the M3-R increased the potency and efficacy of odorant-elicited responses of several ORs. Selective M3-R antagonists attenuated odorant-dependent activation of OSNs, and, when the M3-R and ORs were expressed in transfected cells, OR activation was enhanced by muscarinic agonists and inhibited by muscarinic antagonists. Furthermore, M3-R–dependent potentiation of OR signaling synergized with that of receptor transporting protein 1S (RTP1S), an accessory factor required for the efficient membrane targeting of ORs. However, the M3-R did not enhance the abundance of ORs at the cell surface, suggesting that the M3-R acted through a distinct mechanism independent of RTP1S. Finally, the activation of ORs by cognate odorants transactivated the M3-R in the absence of its agonist. The cross talk between ORs and the M3-R suggests that the functional coupling of ORs and the M3-R is required for robust OR activation.
The polycystic kidney disease-like ion channel PKD2L1 and its associated
partner PKD1L3 are potential candidates for sour taste receptors. PKD2L1 is
expressed in type III taste cells that respond to sour stimuli and genetic
elimination of cells expressing PKD2L1 substantially reduces chorda tympani
nerve responses to sour taste stimuli. However, the contribution of PKD2L1
and PKD1L3 to sour taste responses remains unclear.
We made mice lacking PKD2L1 and/or PKD1L3 gene and investigated whole nerve
responses to taste stimuli in the chorda tympani or the glossopharyngeal
nerve and taste responses in type III taste cells. In mice lacking PKD2L1
gene, chorda tympani nerve responses to sour, but not sweet, salty, bitter,
and umami tastants were reduced by 25–45% compared with those
in wild type mice. In contrast, chorda tympani nerve responses in PKD1L3
knock-out mice and glossopharyngeal nerve responses in single- and
double-knock-out mice were similar to those in wild type mice. Sour taste
responses of type III fungiform taste cells (GAD67-expressing taste cells)
were also reduced by 25–45% by elimination of PKD2L1.
These findings suggest that PKD2L1 partly contributes to sour taste responses
in mice and that receptors other than PKDs would be involved in sour
The current consensus model in mammalian olfaction is that the detection of millions of odorants requires a large number of odorant receptors (ORs) and that each OR interacts selectively with a small subset of odorants, which are typically related in structure. Here, we report the odorant response properties of an OR that deviates from this model: SR1, a mouse OR that is abundantly expressed in sensory neurons of the septal organ and also of the main olfactory epithelium. Patch-clamp recordings reveal that olfactory sensory neurons (OSNs) that express SR1 respond to many, structurally unrelated odorants, and over a wide concentration range. Most OSNs expressing a gene-targeted SR1 locus that lacks the SR1 coding sequence do not show this broad responsiveness. Gene transfer in the heterologous expression system Hana3A confirms the broad response profile of SR1. There may be other mouse ORs with such broad response profiles.
In mammals, sweet taste is mediated largely by a single receptor. New work shows that polymorphisms in the promoter region of one subunit contribute to variation in sweet perception.
Humans have five basic taste modalities: sweet, bitter, sour, salt and umami (taste of l-amino acids). Among thirty-three genes related to transient receptor potential (TRP) channels, three including TRP-melastatin 5 (TRPM5), Polycystic kidney disease-1 like 3 (PKD1L3) and Polycystic kidney disease-2 like 1 (PKD2L1), are specifically expressed in taste receptor cells. TRPM5 is co-expressed with sweet, bitter and umami taste receptors, T1Rs and T2Rs, and functions as a common downstream component in taste signal transduction. In contrast, PKD1L3 and PKD2L1 are co-expressed in distinct subsets of taste receptor cells not expressing sweet, bitter and umami sensing cells. In the heterologous expression system, cells expressing both PKD1L3 and PKD2L1 responded to sour stimuli. Genetic ablation of PKD2L1-expressing cells resulted in elimination of gustatory nerve response to sour stimuli, indicating that cells expressing PKD2L1 function as sour taste detector. These results suggest that PKD1L3/PKD2L1 may play a significant role, possibly as taste receptors, in sour taste sensation.
taste; receptor; TRP; channel
Although mammalian odorant receptors (ORs) were identified over 15 years ago, we still do not understand how odorant molecules interact with ORs at a molecular level. Previous studies of mammalian ORs have tested small numbers of ORs against large numbers of odorants. Some fundamental properties of the olfactory system, however, require investigation of a wide panel of diverse ORs with a large number of chemically diverse odorants. Previously, we identified OR accessory proteins, RTP1 and RTP2. They are expressed specifically in olfactory neurons, are associated with OR proteins and facilitate the OR trafficking to the plasma membrane when coexpressed in mammalian cell lines. Using this approach, high-throughput screening using a large repertoire of mammalian ORs is now possible. The activation profiles can be used to develop a predictive model relating physicochemical odorant properties, receptor sequences, and their interactions, enabling us to predict a tested receptor's response to a novel odorant and a novel receptor's response to a tested odorant. This will provide a basis for understanding how structurally diverse odorant molecules activate the mammalian OR repertoire. Similarly, two families of vomeronasal receptors, V1Rs and V2Rs, are also notoriously difficult to functionally express in heterologous cells. However, coexpression of the RTP family members with V1Rs or V2Rs does not seem to facilitate trafficking of the receptor proteins. This suggests that the vomeronasal organ has a unique biosynthetic pathway for membrane proteins.
The chemical senses—smell and taste—are the most poorly understood sensory modalities. In recent years, however, the field of chemosensation has benefited from new methods and technical innovations that have accelerated the rate of scientific progress. For example, enormous advances have been made in identifying olfactory and gustatory receptor genes and mapping their expression patterns. Genetic tools now permit us to monitor and control neural activity in vivo with unprecedented precision. New imaging techniques allow us to watch neural activity patterns unfold in real time. Finally, improved hardware and software enable multi-neuron electrophysiological recordings on an expanded scale. These innovations have enabled some fresh approaches to classic problems in chemosensation.
A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigate odorant-OR relationship in mammals has been an inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein (RTP) family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput “deorphanization” of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently-expressed mammalian odorant receptors in HEK293T cells. The stages of odorant receptor cell-surface expression include cell culture preparation, transfer of cells, transfection, and immunocytochemistry/flow cytometry, odorant stimulation, and luciferase assay. This protocol can be completed in a period of 3 days from transfer of cells to cell-surface expression detection and/or measurement of functional activation.
The transient receptor potential (TRP) channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCβ2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1 positive cells are immunoreactive for NCAM, serotonin, PGP-9.5 (ubiquitin carboxy terminal transferase) and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about “sour”, but have little or no role in transmission of taste information of other taste qualities.
Polycystic-kidney disease-like ion channel (PKD2L1); sour taste; taste bud cells; gustation; mouse
Comparison of the olfactory systems in Drosophila and mouse uncovers clear differences and a few surprising similarities.
The olfactory systems of insects and mammals have analogous anatomical features and use similar molecular logic for olfactory coding. The molecular underpinnings of the chemosensory systems that detect taste and pheromone cues have only recently been characterized. Comparison of these systems in Drosophila and mouse uncovers clear differences and a few surprising similarities.