Recombinant Human Olfactory receptor 2L13 (OR2L13)
Description
Introduction to Recombinant Human Olfactory Receptor 2L13
Recombinant Human Olfactory receptor 2L13 (OR2L13) is an odorant receptor protein-coding gene belonging to the extensive family of G-protein-coupled receptors (GPCRs). The term "recombinant" refers to the artificially produced form of this protein, typically created for research purposes through molecular cloning and expression in host cells. OR2L13's primary function involves the interaction with odorant molecules in the nose to initiate neuronal responses that trigger smell perception . This receptor belongs to a remarkably large gene family, as olfactory receptors constitute the largest gene family in the human genome, with each receptor arising from single coding-exon genes .
The nomenclature "OR2L13" follows the standardized classification system for olfactory receptors, where "OR" denotes an olfactory receptor, "2" indicates the family, "L" specifies the subfamily, and "13" identifies the individual member within that subfamily. Alternative nomenclature includes OR2L14, as noted in some research literature . While initially studied primarily for its role in olfaction, recent research has uncovered unexpected functions of OR2L13 in platelets and vascular health, representing a significant expansion of our understanding of this receptor's physiological relevance.
Molecular Structure and Characteristics
OR2L13 exhibits the characteristic structural architecture typical of the olfactory receptor family and the broader G-protein-coupled receptor superfamily. The protein features a distinctive 7-transmembrane domain structure, comprising seven alpha-helical regions that span the cell membrane . This structural arrangement is shared with many neurotransmitter and hormone receptors, highlighting the evolutionary conservation of this functional architecture across diverse signaling systems .
The human OR2L13 protein has a molecular weight of approximately 36 kDa, as consistently demonstrated through western blot analyses . The protein is encoded by UniProt ID Q8N349 and is classified under the designation "OR2LD_HUMAN" in the UniProt database . Like most olfactory receptors, OR2L13 is encoded by a single exon gene, a characteristic feature that distinguishes this gene family.
Functional Role in Olfaction
The canonical function of OR2L13, like other olfactory receptors, involves the detection of specific odorant molecules and the initiation of signal transduction cascades that ultimately lead to the perception of smell. Within the olfactory epithelium, OR2L13 operates through G-protein-coupled mechanisms, where binding of an appropriate odorant to the receptor triggers conformational changes that activate associated G proteins .
Upon activation, OR2L13 initiates a signaling cascade that involves the activation of adenylyl cyclase, leading to increased intracellular cyclic adenosine monophosphate (cAMP) levels. This elevation in cAMP opens cyclic nucleotide-gated ion channels, causing membrane depolarization and generating action potentials that are transmitted to the brain, where they are interpreted as specific odors . This mechanism is fundamental to the functioning of the olfactory system and represents the primary evolutionary role of OR2L13.
The specificity of OR2L13 for particular odorant molecules remains an area of ongoing research. Like most olfactory receptors, OR2L13 likely recognizes a range of structurally related odorant molecules rather than a single compound, contributing to the remarkable ability of the olfactory system to distinguish thousands of different odors with a relatively limited number of receptors. This combinatorial coding strategy allows the olfactory system to detect and discriminate an extraordinarily diverse range of volatile compounds .
OR2L13 Expression in Human Tissues
While traditionally associated with olfactory epithelium in the nasal cavity, research has revealed that OR2L13 expression extends beyond olfactory tissues, a phenomenon known as ectopic expression that has been observed for several olfactory receptors. This ectopic expression suggests additional physiological functions beyond odor perception, representing a significant paradigm shift in our understanding of olfactory receptors .
Notably, OR2L13 has been detected in human platelets through both immunoblotting and confocal microscopy techniques. Using human brain lysate as a positive control, healthy platelets have been confirmed to express full-length OR2L13 protein . Confocal microscopy has further revealed that OR2L13 is expressed on the platelet membrane, suggesting a potential role in platelet function .
Detailed subcellular localization studies using co-staining techniques have shown that OR2L13 is primarily co-expressed with P-selectin in platelets, suggesting alpha granule storage and trafficking to the plasma membrane . This specific localization pattern provides insights into the potential functional role of OR2L13 in platelet physiology, particularly in relation to granule release and membrane dynamics during platelet activation.
Interestingly, research has demonstrated that platelet expression of OR2L13 and anoctamin 7 is increased in patients with abdominal aortic aneurysms (AAA) compared to healthy controls . This observation suggests a potential role for OR2L13 in the pathophysiology of vascular disorders, particularly those involving abnormal platelet function or vascular remodeling.
Research Findings on OR2L13 in Platelet Function
Recent research has uncovered compelling evidence for the role of OR2L13 in modulating platelet function, representing a significant departure from its canonical role in olfaction. Studies have identified six ligands specific for OR2L13 that function as receptor agonists, leading to the suppression of platelet aggregation and α-granule exocytosis . These effects appear to be mediated through various platelet receptors, including P2Y12, PAR1, Thromboxane Receptor (TxR), and glycoprotein VI (GPVI), suggesting that OR2L13 activation affects common downstream mediators in platelet signaling pathways .
Table 2: Research Findings on OR2L13 Agonists in Platelet Function
Mechanistically, the lead OR2L13 agonist has been shown to phosphorylate heat shock protein 27 (HSP27) and depolymerize the platelet filamentous actin cytoskeleton . These molecular events likely contribute to the observed inhibition of platelet aggregation and clot retraction. The functional significance of these changes has been confirmed in a clot retraction assay, where treatment with the OR2L13 agonist CCF0054500 resulted in a significantly larger clot area (70.6) compared to vehicle control (5.2), indicating reduced platelet contractile force (P<0.0001) .
The importance of HSP27 in mediating the effects of OR2L13 activation is further supported by the observation that an HSP27 inhibitor can reverse the anti-thrombotic effects of the OR2L13 agonist, reducing the clot area to 3.6 (P<0.0001) . This finding establishes HSP27 as a critical downstream effector in the OR2L13 signaling pathway in platelets.
In vivo studies using a murine cremaster arterial injury model have provided further evidence for the physiological relevance of OR2L13 in thrombosis. Treatment with the lead OR2L13 agonist reduced platelet accumulation at the injury site by 88.9% compared to vehicle control (P<0.0003), without altering fibrin generation in vivo . This observation suggests that OR2L13 agonists may have potential as anti-thrombotic agents with a favorable safety profile, as they appear to inhibit platelet accumulation without compromising fibrin formation, which is essential for hemostasis.
Therapeutic Potential of OR2L13 Agonists
The discovery of OR2L13's role in regulating platelet function has opened up promising avenues for therapeutic intervention in platelet-related disorders. The identification of specific OR2L13 agonists with demonstrated efficacy in suppressing platelet aggregation through various activation pathways represents a significant advancement in this field .
The lead OR2L13 agonist, identified as CCF0054500, has shown particularly promising results in experimental settings. In clot retraction assays, this compound significantly increased clot area compared to vehicle control, indicating reduced platelet contractile force . This effect was found to be dependent on HSP27, as HSP27 inhibition reversed the anti-thrombotic effects, confirming the mechanistic role of HSP27 in OR2L13-mediated platelet inhibition .
The therapeutic potential of OR2L13 agonists is further supported by in vivo studies in a murine cremaster arterial injury model, where the lead agonist reduced platelet accumulation at the injury site by 88.9% compared to vehicle control . Importantly, this potent anti-thrombotic effect occurred without altering fibrin generation, suggesting a favorable safety profile with potentially reduced bleeding risk compared to conventional antiplatelet agents that often compromise both platelet function and fibrin formation .
Beyond acute thrombotic events, OR2L13 has been implicated in the pathophysiology of abdominal aortic aneurysms (AAA), with increased expression observed in platelets from patients with this condition . This observation suggests that OR2L13-targeted therapies might have applications in vascular disorders characterized by abnormal platelet function or vascular remodeling. The association between OR2L13 expression and AAA pathophysiology highlights the potential broader implications of OR2L13-based therapeutic strategies beyond acute thrombotic disorders.
Methods for Studying OR2L13
Various methodological approaches have been developed to study OR2L13 expression, localization, and function, facilitating both basic research and potential clinical applications. These methods span molecular, cellular, and functional techniques, enabling comprehensive investigation of this receptor in different experimental contexts.
Table 3: Methods for Studying OR2L13
For detecting OR2L13 protein expression, immunological techniques such as western blotting using specific OR2L13 antibodies have proven effective. Commercial polyclonal antibodies are available, with recommended dilutions ranging from 1:1000 to 1:5000 for western blotting applications . These antibodies typically recognize specific epitopes of the human OR2L13 protein and consistently detect a band of approximately 36 kDa in western blot analyses, corresponding to the expected molecular weight of the protein .
Immunofluorescence microscopy has been successfully employed to determine the subcellular localization of OR2L13, with recommended dilutions ranging from 1:50 to 1:200 . This technique has been applied to various cell types, including HepG2 cells and platelets, revealing the membrane localization of OR2L13 . Co-staining with markers for specific platelet granules, particularly P-selectin for alpha granules, has provided valuable insights into the subcellular distribution and trafficking of OR2L13 in platelets .
For quantitative measurements of OR2L13 protein levels, enzyme-linked immunosorbent assay (ELISA) kits have been developed. These assays allow for the quantitative determination of OR2L13 in various biological samples, including tissue homogenates, cell lysates, and biological fluids, with detection ranges typically between 0.156 and 10 ng/ml . This quantitative approach complements the qualitative information obtained from western blotting and immunofluorescence studies.
Functional studies of OR2L13 have employed various approaches, including clot retraction assays to evaluate the effects of OR2L13 agonists on platelet contractile force and the murine cremaster arterial injury model to assess in vivo thrombosis . These functional assays provide crucial information about the physiological relevance of OR2L13 and the potential therapeutic applications of OR2L13-targeted interventions.
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Target Background
- RNA-Seq evidence indicates testis-specific monoallelic expression of OR2L13, located 419 kb downstream of the GCSAML somatic (secondary), imprinted differentially methylated region (iDMR). OR2L13 has a predicted CGI-bearing allelically methylated region that overlaps the promoter region in blood, spleen, lung, liver, esophagus, and brain, but is hypomethylated in gamete, blastocyst, placenta, thymus, and spinal cord methylomes. PMID: 29545821
- Clinical trial evidence explores gene-disease association and gene-environment interaction. (HuGE Navigator) PMID: 20379614
Q&A
What is OR2L13 and what are its basic molecular characteristics?
OR2L13 (Olfactory Receptor 2L13, also known as OR2L14) is a G protein-coupled receptor (GPCR) belonging to the large family of olfactory receptors. It has a molecular weight of approximately 35,634 Da and is encoded by a single-exon gene with a cDNA size of 939bp . Like other olfactory receptors, OR2L13 contains a 7-transmembrane domain structure and functions primarily through G-protein-mediated signal transduction pathways .
The protein is classified as an integral membrane protein that traditionally functions in olfaction by detecting and responding to specific odorants. OR2L13's UniProt code is Q8N349, and its NCBI Gene ID is 284521 . The receptor's primary sequence contains regions responsible for ligand binding and interaction with downstream signaling components.
Where is OR2L13 expressed in human tissues?
While traditionally associated with olfactory neurons, OR2L13 demonstrates a broader expression pattern beyond the nasal epithelium. Research has confirmed OR2L13 protein expression in human platelets through immunoblotting, with confocal microscopy revealing membrane expression . Co-staining experiments with platelet granule markers indicate that OR2L13 is primarily co-expressed with P selectin, suggesting alpha granule storage and trafficking to the plasma membrane .
Additionally, OR2L13 expression has been observed in various tissues involved in pathological conditions. Notably, its expression is altered in platelets from patients with abdominal aortic aneurysm (AAA) compared to healthy controls . OR2L13 has also been detected in brain tissues, with expression patterns that differ between normal brain tissue, low-grade gliomas, and glioblastomas .
What signaling pathways does OR2L13 activate?
OR2L13, like other olfactory receptors, primarily activates the canonical GPCR signaling pathway. Upon activation, OR2L13 stimulates adenylyl cyclase to increase intracellular cAMP levels from ATP . This has been demonstrated using reporter cell lines that evaluate OR2L13 odorant ligands based on postreceptor cAMP production .
The signaling cascade continues through downstream effectors including anoctamin 7, a calcium-sensitive chloride channel. In platelets, this pathway appears to be functionally conserved, with OR2L13 activation generating cAMP coincident with changes in platelet chloride concentrations . The generation of cAMP is particularly significant in platelets, as it serves as a second messenger that suppresses platelet reactivity .
How can researchers effectively express recombinant OR2L13 in mammalian cell systems?
To successfully express recombinant OR2L13 in mammalian cells, researchers should consider the following methodology:
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Vector selection: Use a mammalian expression vector such as pCMV3-untagged that contains appropriate promoters for high expression in mammalian cells .
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Co-expression with chaperones: For efficient membrane localization, co-express OR2L13 with chaperone proteins such as receptor transport protein 1 subunit (RTP1s). A bicistronic vector encoding both OR2L13 and RTP1s has been successfully employed to enhance proper membrane trafficking .
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Cell line selection: HEK293 cells have been validated for OR2L13 expression studies. These cells can be stably transduced with lentiviral vectors containing OR2L13 and RTP1s constructs .
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Reporter system integration: For functional studies, incorporate a cAMP response element in the 5′ position in-frame with a reporter gene such as luciferase to monitor receptor activation .
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Verification of expression: Confirm successful expression through Western blotting using validated antibodies such as the OR2L13 Polyclonal Antibody (PACO38238) .
This approach has been successfully used to create a reporter cell line that allows for rapid evaluation of potential OR2L13 odorant ligands based on postreceptor cAMP production .
What methods are recommended for detecting OR2L13 protein expression in various tissues?
For effective detection and quantification of OR2L13 in research samples, consider these validated methodologies:
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Western blotting: Use polyclonal antibodies such as the OR2L13 Antibody (PACO38238) at dilutions of 1:1000-1:5000. Human brain lysate can serve as a positive control . The expected band size is approximately 36 kDa .
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Immunofluorescence microscopy: Employ confocal microscopy with OR2L13-specific antibodies at dilutions of 1:50-1:200 for subcellular localization studies . This approach has been successful in visualizing OR2L13 membrane expression in platelets and determining co-localization with granule markers .
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Co-immunostaining: To assess OR2L13 trafficking and subcellular localization, co-stain with markers of specific cellular compartments. In platelets, co-staining with P selectin has revealed alpha granule storage and trafficking patterns .
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ELISA: For quantitative detection of OR2L13, ELISA can be performed using the anti-OR2L13 antibody at dilutions of 1:2000-1:10000 .
When analyzing tissues with potential pathological changes, include appropriate healthy controls and consider sex-dependent differences, although current research suggests similar expression patterns of platelet olfactory receptors in healthy men and women .
How does OR2L13 function in platelets and what is its role in thrombotic diseases?
OR2L13 serves as a novel regulator of platelet function through its ability to generate cAMP, a well-established inhibitor of platelet activation. Research has established that:
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Expression patterns: OR2L13 protein expression is increased in platelets from patients with abdominal aortic aneurysm (AAA) compared to healthy controls, suggesting a potential compensatory mechanism .
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Signal transduction: Upon activation, platelet OR2L13 generates cAMP endogenously, which suppresses platelet reactivity. This indicates that the canonical olfactory signal transduction pathway is functionally conserved in platelets .
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Pharmacological activation: The terpene derivative (–) carvone, an active ingredient in spearmint, has been identified as a potent platelet OR2L13 agonist. This compound activates OR2L13 in a dose-dependent manner compared to forskolin (a positive control for endogenous adenylyl cyclase activation) .
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Functional outcomes: OR2L13 activation limits both biochemical and biomechanical platelet activation as well as AAA growth. The OR2L13 agonist (–) carvone suppresses thrombosis and platelet reactivity both ex vivo and in vivo .
These findings position OR2L13 activators as potential foundational compounds for a new class of antiplatelet agents for thrombotic diseases . The mechanistic link between platelet reactivity and AAA progression identifies OR2L13 as a druggable target with therapeutic potential.
What is the role of OR2L13 in glioblastoma and other cancer types?
Recent integrated transcriptomic analyses have revealed intriguing connections between OR2L13 and brain tumors:
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Expression in glioblastoma: OR2L13 is significantly down-regulated in glioblastoma (GBM) compared to normal brain tissue and low-grade gliomas (LGG) .
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Association with synaptic activity: OR2L13 expression is correlated with synaptic activity in recurrent tumors, potentially mediating treatment-induced neuronal adaptations .
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Potential role in recurrent disease: The specific correlation of OR2L13 with synaptic activity in recurrent GBM suggests it may be involved in adaptive mechanisms following treatment, which could contribute to therapy resistance .
This emerging research suggests that OR2L13 may serve as a potential therapeutic target in GBM, offering new insights into regulatory mechanisms in tumor progression and treatment resistance. The specific functions of OR2L13 in modulating synaptic activity in the context of GBM warrant further investigation to fully understand its potential as a biomarker or therapeutic target.
What challenges arise when studying membrane localization of OR2L13, and how can they be addressed?
Olfactory receptors, including OR2L13, present significant challenges for membrane localization studies due to their hydrophobic nature and complex trafficking requirements. Researchers can overcome these challenges through:
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Co-expression with chaperone proteins: The use of receptor transport protein 1 subunit (RTP1s) as a chaperone has been demonstrated to enhance membrane localization of OR2L13. Implementing a bicistronic vector system encoding both OR2L13 and RTP1s facilitates efficient trafficking to the plasma membrane .
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Cell system optimization: HEK293 cells have proven effective for OR2L13 expression studies. When establishing stable cell lines, consider lentiviral transduction systems that allow for consistent and long-term expression .
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Subcellular fractionation: For biochemical verification of membrane localization, perform subcellular fractionation followed by Western blotting to quantify OR2L13 in membrane versus cytosolic fractions.
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Advanced microscopy techniques: Employ high-resolution confocal microscopy with appropriate membrane markers to visualize OR2L13 localization. Z-stack imaging can provide three-dimensional information about membrane versus intracellular receptor pools.
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Surface biotinylation assays: These can be used to specifically label and quantify the proportion of OR2L13 that reaches the cell surface, distinguishing between total expression and functional surface expression.
When studying OR2L13 in native tissues such as platelets, additional considerations include appropriate fixation protocols that preserve membrane structures while maintaining antibody epitope accessibility.
How can researchers effectively identify and validate ligands for OR2L13?
Identifying and validating ligands for olfactory receptors presents unique challenges due to their diverse binding properties. For OR2L13, consider this methodological approach:
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Reporter system development: Establish a cAMP-responsive reporter system in cells stably expressing OR2L13. The approach using HEK293 cells with a cAMP response element linked to luciferase provides a sensitive and quantifiable readout of receptor activation .
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Screening strategy:
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Dose-response validation: For identified hits, perform dose-response experiments to determine potency and efficacy. The response to (–) carvone has been characterized as dose-dependent in OR2L13-expressing cells .
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Secondary assays: Confirm ligand specificity through:
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Direct measurement of cAMP production using ELISA or other biochemical methods
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Calcium imaging if the receptor couples to calcium signaling
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Competitive binding assays with known ligands
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Validation in native systems: Test identified ligands in systems naturally expressing OR2L13, such as platelets, to confirm physiological relevance of the interaction .
This systematic approach has successfully identified (–) carvone as an OR2L13 agonist that reproducibly activates the receptor to generate endogenous cAMP in a dose-dependent manner .
How might OR2L13's role in platelet function be exploited for novel therapeutic approaches?
The identification of OR2L13 as a functional receptor in platelets that generates cAMP and limits platelet reactivity opens several therapeutic possibilities:
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Development of selective OR2L13 agonists: Building on the discovery of (–) carvone as an OR2L13 activator, structure-activity relationship studies could yield more potent and selective compounds. These could serve as novel antiplatelet agents with potentially fewer side effects than current therapies .
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Combination therapy approaches: OR2L13 agonists might synergize with existing antiplatelet agents that work through different mechanisms, potentially allowing for dose reductions of current drugs while maintaining efficacy.
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Targeted delivery strategies: Developing platelet-specific delivery systems for OR2L13 modulators could enhance therapeutic efficacy while reducing off-target effects in other tissues expressing this receptor.
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Biomarker applications: The observed upregulation of OR2L13 in platelets from AAA patients suggests potential utility as a biomarker for vascular diseases . Longitudinal studies could determine whether OR2L13 expression correlates with disease progression or treatment response.
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Personalized medicine applications: Given that OR2L13 expression changes in disease states, profiling individual patients' OR2L13 levels might help predict responsiveness to conventional antiplatelet therapies and guide treatment selection.
The mechanistic link established between platelet reactivity and AAA progression provides a strong rationale for pursuing OR2L13-targeted therapies, particularly for thrombotic conditions associated with vascular diseases .
What are the implications of OR2L13's altered expression in glioblastoma for understanding tumor biology?
The recent discovery of OR2L13's correlation with synaptic activity in recurrent glioblastoma opens new avenues for understanding tumor-neuron interactions:
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Neuron-tumor communication: OR2L13's association with synaptic activity suggests potential roles in mediating communication between neurons and tumor cells, possibly contributing to the phenomenon of tumor neurogenesis .
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Treatment resistance mechanisms: The specific association of OR2L13 with recurrent tumors points to potential involvement in treatment-induced adaptations that might contribute to therapy resistance. This could inform strategies to prevent or overcome such resistance .
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Tumor microenvironment modulation: Changes in OR2L13 expression may reflect or influence alterations in the tumor microenvironment, particularly in relation to neuronal components that are increasingly recognized as important in GBM progression.
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Biomarker potential: The significant down-regulation of OR2L13 in GBM compared to normal tissue and LGG suggests potential utility as a diagnostic or prognostic biomarker .
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Integration with other olfactory receptors: Comprehensive analysis of OR expression patterns in GBM has revealed tissue-specific profiles, with different ORs potentially mediating distinct aspects of tumor biology. Understanding how OR2L13 functions within this broader network could reveal new regulatory mechanisms in GBM .
This emerging area represents a novel intersection between sensory biology and cancer research, potentially opening new diagnostic and therapeutic opportunities for this aggressive brain tumor.
What are the optimal conditions for handling and storing recombinant OR2L13 expression plasmids?
For researchers working with OR2L13 expression constructs, proper handling and storage are critical for maintaining plasmid integrity and experimental reproducibility:
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Storage recommendations:
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Resuspension protocol:
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Quality control checks:
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Transformation recommendations:
These handling procedures will help ensure consistent results when working with OR2L13 expression constructs for functional studies and protein production.
What considerations should be made when designing antibodies against OR2L13 for various applications?
When developing or selecting antibodies for OR2L13 detection across different experimental applications, researchers should consider:
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Epitope selection:
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Target unique regions of OR2L13 to avoid cross-reactivity with other olfactory receptors
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For detection of native protein, target extracellular loops that are accessible in non-denatured conditions
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For western blotting, target regions that remain antigenic after denaturation
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Validated applications and dilutions:
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Species reactivity:
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Controls and validation:
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Storage and handling:
Careful consideration of these factors will help ensure reliable and specific detection of OR2L13 across different experimental contexts and applications.
