Recombinant Human Olfactory receptor 2J3 (OR2J3)
Description
Recombinant Human Olfactory Receptor 2J3 (OR2J3): Structure, Function, and Genetic Variation
Olfactory receptor 2J3 (OR2J3) represents a crucial component of the human olfactory system with significant implications for individual differences in smell perception. Research has identified this receptor as particularly important for detecting specific plant-derived odorants, with genetic variations explaining substantial differences in olfactory sensitivity across populations. This report synthesizes current knowledge on the recombinant human OR2J3 protein, its structural properties, functional characteristics, and genetic polymorphisms that influence its activity.
Physiological Function
OR2J3 plays a specific role in the detection of cis-3-hexen-1-ol, a volatile organic compound typically described as having a "green grassy" odor or the smell of "cut grass" . This compound is naturally released by wounded plants, making OR2J3 particularly important for detecting plant damage in the environment . When cis-3-hexen-1-ol binds to OR2J3, it initiates a neuronal response cascade that ultimately triggers the perception of this distinctive smell . The receptor's interaction with odorant molecules represents the first critical step in translating chemical signals into neural impulses that the brain interprets as specific odors.
Recombinant Protein Technology
Recombinant human OR2J3 refers to artificially produced versions of the native receptor protein created using recombinant DNA technology. This approach involves isolating the OR2J3 gene sequence and inserting it into expression vectors, which are then introduced into host cell systems (typically bacterial, yeast, insect, or mammalian cells) programmed to produce the protein. The recombinant protein maintains the same amino acid sequence as the naturally occurring receptor but can be produced in controlled laboratory conditions, allowing for higher yields and purity for research applications.
Expression Systems and Challenges
The expression of recombinant olfactory receptors, including OR2J3, presents significant technical challenges due to their hydrophobic nature and complex folding requirements. Research has demonstrated successful expression of functional OR2J3 in specialized cell systems. For instance, the Hana3A cell line has been utilized for in vitro assays of OR2J3, with the receptor co-expressed with accessory proteins like RTP1S that facilitate proper membrane trafficking . These expression systems typically require careful optimization of conditions to ensure the receptor maintains its native conformation and functionality.
Polymorphisms and Their Functional Impact
Extensive research has identified significant genetic variation in the OR2J3 gene that influences individual differences in odor perception. In a study across an ethnically diverse population of 52 individuals, researchers sequenced the coding regions of OR2J3 and identified multiple polymorphisms . Among the most significant variations were single nucleotide polymorphisms (SNPs) that alter the amino acid sequence of the receptor protein, potentially affecting its functionality.
The SNP rs28757581, which results in a threonine to alanine substitution at position 113 (T113A) in the OR2J3 protein, shows strong association with variations in the ability to detect cis-3-hexen-1-ol . Another critical substitution identified is R226Q (arginine to glutamine at position 226). Laboratory investigations have demonstrated that both T113A and R226Q impair the receptor's ability to respond to cis-3-hexen-1-ol, with the combination of both substitutions effectively abolishing the response to this compound .
Haplotypes and Their Distribution
Research has identified five distinct haplotypes of OR2J3 across human populations, as summarized in Table 1 . These haplotypes differ in their functional properties regarding cis-3-hexen-1-ol detection sensitivity.
Table 1: OR2J3 Haplotypes and Their Functional Properties
| Haplotype | Key Amino Acid Positions | Functional Response to cis-3-hexen-1-ol | Population Distribution |
|---|---|---|---|
| Haplotype 1 | Reference sequence (wild type) | Strong response | Widely distributed |
| Haplotype 2 | T113A | Impaired response | Variable across populations |
| Haplotype 3 | R226Q | Impaired response | Variable across populations |
| Haplotype 4 | T113A + R226Q | No significant response | Explains 26.4% of detection variation |
| Haplotype 5 | Other variations | Variable responses | Less common |
The specific haplotype containing both the T113A and R226Q substitutions explains approximately 26.4% of the variation in cis-3-hexen-1-ol detection thresholds observed in study populations . This represents a remarkably high percentage for a single gene's contribution to a sensory perception trait, underscoring the importance of OR2J3 in human olfactory function.
In Vitro Assay Systems
Researchers have developed sophisticated in vitro systems to evaluate the functional properties of OR2J3 and its variants. These typically involve transfecting cells with the OR2J3 gene along with accessory proteins that support proper receptor function. A common experimental approach utilizes Hana3A cells transfected with OR2J3 variants along with 5 ng/well of RTP1S, 10 ng/well of CRE-luciferase, 5 ng/well of pRL-SV40, 2.5 ng/well of M3 muscarinic receptor, and appropriate transfection reagents like Lipofectamine2000 .
Following transfection, cells are exposed to varying concentrations of odorant compounds, particularly cis-3-hexen-1-ol, and receptor activation is measured through luciferase-based reporter systems that detect downstream signaling events. These assays have been instrumental in characterizing the functional properties of different OR2J3 variants and haplotypes.
Response Characteristics to cis-3-hexen-1-ol
Laboratory studies have demonstrated that OR2J3 responds to cis-3-hexen-1-ol in a concentration-dependent manner . Among the 13 intact receptors investigated in the chromosome 6 cluster, only three receptors showed responsiveness to this compound: OR2W1, OR2J2, and OR2J3 . This specificity highlights the specialized nature of olfactory receptors and their ligand preferences.
When comparing different OR2J3 variants, researchers observed significant differences in response profiles. The wild-type receptor shows robust activation when exposed to cis-3-hexen-1-ol, while variants containing the T113A or R226Q substitutions exhibit diminished responses. The variant containing both substitutions shows almost complete loss of function with regard to cis-3-hexen-1-ol detection .
Antibodies and Research Reagents
Commercial antibodies against OR2J3 are available for research applications, facilitating studies of this receptor's expression, localization, and function. Polyclonal antibodies targeting the human OR2J3 protein allow for detection of this receptor in various experimental systems . These antibodies are typically generated against specific epitopes of the receptor and may be conjugated with different detection molecules (such as biotin or fluorescent labels) for various applications including Western blotting, immunohistochemistry, and flow cytometry.
Recombinant Protein Applications
Recombinant OR2J3 proteins and expression systems have various research applications, including:
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Functional characterization studies
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Drug discovery and odorant screening
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Structure-function relationship investigations
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Development of biosensors and artificial olfactory systems
Additionally, knockdown gene products such as siRNA and shRNA targeting OR2J3 are commercially available, allowing researchers to study the effects of reducing OR2J3 expression in various model systems .
Phenotypic Consequences of OR2J3 Variation
The genetic variation in OR2J3 has significant implications for understanding individual differences in odor perception. Detection threshold concentrations for cis-3-hexen-1-ol have been shown to vary across almost two orders of magnitude (0.3–19.2 ppm) in cohorts of mixed sex and ethnicity . This substantial variation can be partially explained by different OR2J3 haplotypes, with the haplotype containing both T113A and R226Q substitutions accounting for 26.4% of this variation .
These findings suggest that individuals with different OR2J3 genotypes may experience the smell of cut grass and related green odors quite differently, potentially influencing food preferences, environmental perception, and other aspects of daily life affected by olfaction.
Future Research Directions
While significant progress has been made in understanding OR2J3's role in detecting cis-3-hexen-1-ol, several important questions remain for future research:
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How OR2J3 variations influence the perception of complex food flavors containing cis-3-hexen-1-ol
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Whether OR2J3 haplotypes explain variations in food preferences and consumption
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The potential evolutionary significance of OR2J3 variation across human populations
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The detailed structural mechanisms by which T113A and R226Q substitutions impair receptor function
Further research in these areas may provide deeper insights into the molecular basis of olfactory perception and the role of genetic variation in sensory experience.
Product Specs
Please note: We will prioritize shipping the format we have in stock. However, if you have any specific format requirements, please indicate them in your order remarks, and we will prepare accordingly.
Note: All proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance as additional charges will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
- Activation of OR2J3 induces apoptosis and inhibits cell proliferation and migration in long-term stimulus experiments with helional. PMID: 27939274
- Two amino acid substitutions, T113A and R226Q, impaired the ability of OR2J3 to respond to cis-3-hexen-1-ol, and these two substitutions combined effectively abolished the response to the compound. PMID: 22714804
- Observational study of gene-disease association. (HuGE Navigator) PMID: 19851445
Q&A
What is OR2J3 and what is its basic function?
OR2J3 (olfactory receptor family 2 subfamily J member 3) is a protein encoded by the OR2J3 gene in humans. As an olfactory receptor, it interacts with odorant molecules in the nose to initiate a neuronal response that triggers the perception of smell. OR2J3 belongs to the large family of G-protein-coupled receptors (GPCRs) that arise from single coding-exon genes. Like other olfactory receptors, OR2J3 shares a 7-transmembrane domain structure with many neurotransmitter and hormone receptors and is responsible for the recognition and G protein-mediated transduction of odorant signals .
What are the known ligands for OR2J3?
The most well-documented ligand for OR2J3 is cis-3-hexen-1-ol, a compound typically described as having a "green grassy" odor or the smell of "cut grass." In vitro assays have demonstrated that OR2J3 can respond to this compound, confirming its role as a functional receptor for this odorant . Researchers investigating OR2J3 should note that genetic variations in this receptor significantly affect sensitivity to cis-3-hexen-1-ol, making it an excellent model for studying structure-function relationships in olfactory receptors .
How does OR2J3 differ from other olfactory receptors?
OR2J3 is part of the Class II (tetrapod-specific) olfactory receptors. While it shares structural similarities with other olfactory receptors, its ligand specificity and genetic polymorphisms make it distinct. OR2J3 has been particularly well-studied for its genetic variations that affect odorant perception, with specific SNPs that strongly influence sensitivity to cis-3-hexen-1-ol . The gene has 5 SNPs and 6 haplotypes identified across study populations, with a dN/dS ratio of 4, suggesting it has been under positive selection during evolution .
What are the established methods for recombinant expression of OR2J3?
For recombinant expression of olfactory receptors like OR2J3, the Hana3A cell line has proven effective. These cells can be cultured in DMEM containing 10% fetal bovine serum, 5 mM Glutamax, and 100 units/ml penicillin/streptomycin in a 5% CO₂ humidified atmosphere at 37°C .
For expression vector construction:
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Use standard PCR methods to amplify the OR2J3 coding sequence
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Design expression constructs where OR2J3 is expressed as a fusion protein containing 20 amino acids of the N-terminal region of bovine rhodopsin (rhodopsin tag)
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For transfection, use Lipofectamine 2000 with a mixture containing 300 ng of the OR plasmid and 60 ng of the RTP1s plasmid, which enhances the transport of the OR into the cell membrane
How can OR2J3 expression be verified in recombinant systems?
Verification of OR2J3 expression can be achieved through several complementary techniques:
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Immunocytochemistry: Transfected cells can be fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 1% fish gelatin. Use specific antibodies against OR2J3 (commercially available) along with antibodies against the rhodopsin tag if the fusion protein approach is used. Visualization can be done with fluorescently labeled secondary antibodies and confocal microscopy .
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Western blotting: Use purified antibodies against OR2J3 to confirm protein expression at the expected molecular weight.
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RNA expression analysis: Confirm transcript expression through RT-PCR or RNA-Seq. Normalized counts can be analyzed using packages like DESeq2 to account for sequencing depth between samples .
What functional assays are recommended for measuring OR2J3 activity?
For measuring OR2J3 activity in response to ligands such as cis-3-hexen-1-ol:
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Calcium imaging assays: Measure changes in intracellular calcium upon receptor activation
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cAMP assays: Quantify cAMP production following receptor stimulation
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Luciferase reporter assays: Using constructs that express luciferase under the control of response elements activated by OR2J3 signaling
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Electrophysiological recordings: Patch-clamp techniques can directly measure electrical responses in cells expressing OR2J3
For threshold determination studies, prepare serial dilutions of the odorant (e.g., cis-3-hexen-1-ol) and measure detection thresholds, which can then be correlated with receptor genotypes .
What are the key SNPs in OR2J3 that affect olfactory function?
Several significant SNPs in OR2J3 have been identified that impact olfactory function:
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rs28757581: This SNP results in a T113A amino acid substitution that impairs the ability of OR2J3 to respond to cis-3-hexen-1-ol .
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R226Q substitution: This amino acid change also reduces receptor function .
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Combined effect: When both T113A and R226Q substitutions are present in the same haplotype, they effectively abolish the receptor's response to cis-3-hexen-1-ol .
The genetic variation in OR2J3 explains approximately 26.4% of the variation in cis-3-hexen-1-ol detection thresholds in studied populations, making these SNPs critical for understanding individual differences in olfactory perception .
How prevalent are OR2J3 genetic variants across populations?
Based on the available data from the 1000 Genomes Project, OR2J3 shows genetic diversity across human populations. The gene has 5 predictive haplotypes that have been identified across study populations . While specific population frequencies for each variant are not detailed in the provided search results, researchers can access this information through resources like the 1000 Genomes phase 1 data release .
The following table summarizes genetic information about OR2J3 compared to other olfactory receptor genes:
| Gene | Sites | SNPs (S) | Haplotypes | Haplotype Diversity (Hd) | Pi | dN/dS |
|---|---|---|---|---|---|---|
| OR2J3 | 936 | 5 | 6 | 0.695 | 0.001431 | 4 |
This data indicates that OR2J3 has moderate genetic diversity with a relatively high dN/dS ratio, suggesting positive selection has acted on this gene .
How can OR2J3 variants be used in structure-function relationship studies?
OR2J3 presents an excellent model for structure-function relationship studies in olfactory receptors due to its well-characterized genetic variants that affect function. Researchers can:
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Generate site-directed mutants with specific amino acid substitutions (such as T113A and R226Q) to study their individual and combined effects on receptor function
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Create chimeric receptors between OR2J3 and related ORs to identify domains critical for ligand binding and activation
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Use computational modeling based on the 7-transmembrane structure to predict how specific amino acid changes affect the receptor's binding pocket
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Perform molecular dynamics simulations to understand the conformational changes induced by ligand binding
These approaches can provide insights into both the specific mechanisms of OR2J3 function and broader principles of GPCR activation and ligand specificity .
What methodological challenges exist in studying OR2J3 function in native tissues?
Studying OR2J3 in its native context presents several methodological challenges:
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Low expression levels: Like most olfactory receptors, OR2J3 is expressed at relatively low levels in olfactory tissue, making detection challenging. RNA-Seq analysis typically requires normalization to account for sequencing depth between samples .
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Cell-specific expression: Olfactory receptors are expressed in specific subsets of olfactory sensory neurons, necessitating single-cell approaches or careful microdissection techniques.
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Functional redundancy: Multiple olfactory receptors may respond to similar odorants, complicating the isolation of OR2J3-specific responses in native tissues.
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Tissue accessibility: Human olfactory tissue samples are difficult to obtain, leading researchers to rely on model organisms or heterologous expression systems.
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Technical validation: When performing immunohistochemistry, careful validation of antibody specificity is essential, as demonstrated by control experiments with recombinant expression systems .
How can transcriptomic approaches be applied to study OR2J3 expression patterns?
Transcriptomic approaches offer powerful tools for studying OR2J3 expression:
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RNA-Seq analysis: This technique can quantify OR2J3 expression across different tissues, developmental stages, or disease states. On average, 83.85 ± 1.66% of total reads can be mapped uniquely to the genome in well-designed experiments .
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Single-cell RNA-Seq: This approach allows for the identification of specific cell populations expressing OR2J3 within heterogeneous tissues.
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Differential expression analysis: Using packages like DESeq2, researchers can identify conditions that alter OR2J3 expression. Key statistical parameters to report include:
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Cluster analysis: Hierarchical clustering (hclust function in R) can identify patterns of co-expression with other genes. Cluster stability can be evaluated using metrics such as average silhouette distance, average Pearson gamma, and within-between cluster ratio .
How does OR2J3 genetic variation correlate with perceptual differences in humans?
The genetic variation in OR2J3 has been directly linked to perceptual differences in humans, particularly regarding the detection of cis-3-hexen-1-ol. Research has demonstrated that:
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Individuals with different OR2J3 haplotypes show significant variation in their ability to detect cis-3-hexen-1-ol, with certain variants (particularly those containing both T113A and R226Q substitutions) showing severely impaired detection abilities .
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The haplotype of OR2J3 containing both T113A and R226Q explains 26.4% of the variation in cis-3-hexen-1-ol detection thresholds in study cohorts .
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This genetic basis for variation in odor perception provides a model system for understanding how genetic differences influence sensory experiences across individuals.
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Further research is needed to determine whether OR2J3 haplotypes explain variation in perceived flavor experiences and consumption preferences for foods containing cis-3-hexen-1-ol .
What are the potential applications of OR2J3 research in medical diagnostics?
While the search results don't directly address OR2J3 in medical diagnostics, research on olfactory receptors in general has shown potential diagnostic applications:
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Biomarker development: Other olfactory receptors (such as OR2B6) have been investigated as potential biomarkers in cancer research, particularly in breast carcinoma. Similar approaches could be applied to OR2J3 in specific disease contexts .
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Olfactory dysfunction assessment: Given the specific link between OR2J3 variants and cis-3-hexen-1-ol perception, testing responses to this compound could provide a targeted assessment of specific olfactory pathways in patients with smell disorders.
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Personalized medicine: Understanding a patient's OR2J3 genotype might help predict responses to certain medications with odorant-like structures or side effects related to smell perception.
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Genetic counseling: For families with hereditary olfactory disorders, OR2J3 genotyping could contribute to comprehensive genetic assessment.
What bioinformatic resources are available for advanced OR2J3 research?
Researchers studying OR2J3 can leverage several bioinformatic resources:
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Genomic databases: OR2J3 information is available through resources like OMIM (615016), GeneCards, and HomoloGene (128270) .
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Variation databases: The 1000 Genomes Project provides data on allele and haplotype frequencies across global populations .
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Analysis tools:
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Protein structure databases: While not mentioned specifically in the search results, resources like PDB and modeling tools like AlphaFold may provide structural insights for OR2J3.
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Orthology resources: OMA database contains OR2J3 ortholog information that can be valuable for comparative genomic studies .
What emerging technologies could advance OR2J3 functional studies?
Several emerging technologies hold promise for advancing OR2J3 research:
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CRISPR/Cas9 genome editing: This technology allows precise modification of OR2J3 in model organisms or cell lines to study the functional consequences of specific genetic variants.
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Organoid models: Olfactory epithelium organoids could provide more physiologically relevant systems for studying OR2J3 function than traditional cell lines.
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Advanced imaging techniques: Methods such as cryo-electron microscopy could potentially resolve the structure of OR2J3, providing unprecedented insights into its ligand-binding mechanisms.
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Biosensor development: Creating biosensors based on OR2J3 could enable high-throughput screening of potential ligands and modulators.
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Systems biology approaches: Integration of transcriptomic, proteomic, and metabolomic data could provide a more comprehensive understanding of OR2J3's role in the broader olfactory system.
How might OR2J3 research contribute to understanding evolutionary aspects of olfaction?
The study of OR2J3 can provide valuable insights into the evolution of olfaction:
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Comparative genomics: The dN/dS ratio of 4 for OR2J3 suggests it has been under positive selection during evolution, indicating potential adaptive significance .
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Ecological relevance: Understanding the specific ligands for OR2J3 across species could reveal how environmental factors have shaped olfactory receptor evolution.
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Hominid evolution: Comparing OR2J3 function and genetic variation between humans and closely related primates might illuminate changes in olfactory perception during human evolution.
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Population genetics: Patterns of OR2J3 variation across human populations may reveal selective pressures related to different environments, diets, or cultural practices.
What are the current gaps in our understanding of OR2J3 regulation and expression?
Despite progress in OR2J3 research, several knowledge gaps remain:
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Transcriptional regulation: The mechanisms controlling OR2J3 expression in olfactory sensory neurons versus other tissues are poorly understood.
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Post-translational modifications: How these modifications affect OR2J3 trafficking, stability, and function requires further investigation.
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Receptor trafficking: The specific molecular machinery involved in transporting OR2J3 to the cell membrane in olfactory sensory neurons needs clarification.
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Non-olfactory functions: Potential roles of OR2J3 in tissues outside the olfactory system remain largely unexplored, though other olfactory receptors have shown diverse functions in non-olfactory tissues.
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Ligand repertoire: While cis-3-hexen-1-ol is established as a ligand, the full range of compounds that activate OR2J3 is unknown, as are potential antagonists that might block its function.
