Recombinant Rat Substance-K receptor (Tacr2)
Description
Definition and Biological Role
Recombinant rat Tacr2 is a laboratory-produced version of the tachykinin receptor 2, a G protein-coupled receptor (GPCR) that binds neurokinin A (substance K). This receptor is involved in modulating physiological processes such as smooth muscle contraction, neurotransmission, and ovarian follicular dynamics . The recombinant form enables controlled in vitro and in vivo studies without requiring native tissue extraction.
Production Methods
Recombinant rat Tacr2 is typically expressed in heterologous systems:
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Host Systems:
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Purification:
Agonists and Antagonists
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Recombinant rat Tacr2 couples to Gq/11 proteins, activating phospholipase C and increasing intracellular calcium .
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Ligand affinity hierarchy: substance K > neuromedin-K > substance P .
Key Findings from Studies
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Ovarian Function: Tacr2 is highly expressed in mural granulosa cells, where it interacts with kisspeptin to regulate follicular development .
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Neurological Pathways: Modulates anxiety and depression pathways in preclinical models, though clinical trials for related drugs (e.g., saredutant) were discontinued .
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Cancer Research: Tacr2 inhibits migration and proliferation in prostate cancer cells via Wnt/β-catenin signaling .
Experimental Models
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In Vitro: Ligand-binding assays using radiolabeled neurokinin A .
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In Vivo: Transgenic rat models to study receptor knockout effects on reproductive and neural systems .
Challenges and Limitations
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Stability Issues: Membrane protein aggregation necessitates nanodisc or detergent stabilization .
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Species Specificity: Rat Tacr2 shows 85% homology to human TACR2, limiting cross-species extrapolation .
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Ligand Selectivity: Peptide agonists/antagonists (e.g., GR-64349) have short half-lives in vivo .
Product Specs
Note: We will prioritize shipping the format that we have in stock. However, if you have any specific format requirements, please include them in your order notes. We will strive to fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance. Additional fees will apply.
Generally, the shelf life for the liquid form is 6 months at -20°C/-80°C. Lyophilized forms have a shelf life of 12 months at -20°C/-80°C.
Target Background
- Research indicates that medial septal NK(2)-R play a crucial role in mediating acetylcholine release through the septal-hippocampal cholinergic projection and via direct or indirect pathways to the amygdala, but not the frontal cortex. PMID: 20882546
- Findings demonstrate that injection of vehicle alone into the medial septum impaired memory for temporal order and spatial location. The NK(2) receptor antagonist partially reversed this deficit by restoring memory for temporal order. PMID: 19854231
- Identification of a tachykinin NK(2) receptor splice variant. PMID: 12427486
- Data suggest that neurokinin-1 receptor (NK-1R) and neurokinin-2 receptor (NK-2R) contribute to intestinal damage and are implicated in the pathophysiological changes observed in acute necrotizing pancreatitis PMID: 12508374
- Evidence suggests a role for tachykinin NK-1 (and potentially NK-2) receptors in tachykinin-mediated stimulation of vasopressin secretion from the rat hypothalamo hypophyseal system in vitro. PMID: 16136007
- Activation of NK2 autoreceptors by NKs released from the peripheral afferent terminals or by mast cells during inflammatory responses may be a mechanism that sensitizes TRPV1 channels and enhances afferent excitability PMID: 18598261
Q&A
Experimental Validation of Recombinant TACR2 Activity
Q: What methodologies are most effective for validating the functional activity of recombinant rat TACR2 in vitro? A: Functional validation requires multi-tiered approaches:
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Ligand-binding assays: Use radioligand displacement (e.g., [³H]-neurokinin A) to confirm receptor affinity for tachykinins. Rat TACR2 exhibits a potency ranking of NKA > NKB > SP, with EC₅₀ values in the nanomolar range .
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Functional assays: Measure intracellular calcium mobilization or cAMP production in transfected cells (e.g., COS-7 or HEK293) using fluorometric or luciferase-based systems .
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Electrophysiological studies: Inject mRNA encoding TACR2 into Xenopus oocytes to observe depolarization responses to tachykinins .
| Method | Advantages | Limitations |
|---|---|---|
| Radioligand binding | High sensitivity for affinity | Requires radioactive materials |
| Calcium imaging | Real-time signal detection | Limited temporal resolution |
| cAMP assays | Direct readout of Gαs signaling | Nonspecific signals in complex media |
Key Data: Rat TACR2 mRNA encodes a 390-residue GPCR with seven transmembrane domains. Functional expression in oocytes confirmed its role in neuromedin K signaling .
Structural Determinants of Ligand Specificity
Q: How do structural features of TACR2 influence its selectivity for neurokinin A (NKA) over other tachykinins? A: TACR2’s selectivity arises from conserved residues in transmembrane domains (TMs):
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TM III and VII: Highly conserved regions between rat and bovine TACR2 receptors, critical for ligand binding .
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Extracellular loops: Variations in N-terminal sequences (e.g., alternative mRNA splicing) modulate receptor affinity .
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Cytoplasmic domains: Serine/threonine residues in intracellular loops enable G protein coupling and desensitization .
Contradictory Findings: While TACR2 and NK3R share >70% homology in TMs, distinct histidine residues in TMs V and VI confer differential agonist binding .
Challenges in Developing NK2R Therapeutics
Q: Why have NK2R antagonists faced clinical hurdles despite preclinical success? A: Key barriers include:
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Short half-life of endogenous ligands: NKA’s rapid degradation limits therapeutic utility, necessitating synthetic agonists .
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Cross-reactivity: Structural homology with NK1R/NK3R complicates selectivity, as seen with ibodutant and saredutant .
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Central vs. peripheral effects: Central appetite suppression must be balanced against peripheral gastrointestinal side effects .
| Compound | Mechanism | Clinical Outcome |
|---|---|---|
| GR-64349 | Agonist | Preclinical efficacy |
| Ibodutant | Antagonist | Failed Phase 3 (IBS) |
| Saredutant | Antagonist | Mixed results (depression) |
Recent Advances: Long-acting NK2R agonists (e.g., weekly dosing) show promise in preclinical obesity models by enhancing energy expenditure .
mRNA Splicing and TACR2 Isoforms
Q: How do alternative mRNA splicing events affect TACR2 function in vivo? A: Rat TACR2 transcripts exist in two isoforms differing in 5' untranslated regions (UTRs). These variants may regulate:
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Tissue-specific expression: Northern blotting revealed distinct mRNA patterns in brain vs. peripheral tissues .
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Receptor trafficking: N-terminal modifications could alter surface expression or ligand access .
Experimental Approach: Use isoform-specific qPCR primers targeting exon-intron boundaries to quantify splice variants in tissues of interest .
Cross-Reactivity in Heterologous Systems
Q: How can researchers minimize off-target effects when studying TACR2 in transfected cell lines? A: Implement these controls:
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Subcloning: Ensure TACR2 cDNA is free of sequence errors via Sanger sequencing .
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Antibody validation: Use TACR2-specific antibodies (e.g., CSB-PA050196 for human) to confirm membrane localization .
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Ligand specificity: Test with NK1R/NK3R antagonists (e.g., GR-159897) to exclude cross-reactivity .
Key Insight: Rat TACR2’s 95% purity in recombinant preparations minimizes contamination risks .
Thermal Stability and Storage Protocols
Q: What storage conditions preserve recombinant TACR2’s functional integrity? A:
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Short-term (1 month): Store lyophilized protein at 2–8°C. Reconstitute in PBS with 5% trehalose and 1 mM DTT to prevent aggregation .
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Long-term (12 months): Aliquot into single-use vials and freeze at -80°C. Avoid repeated freeze-thaw cycles, as thermal degradation exceeds 5% at 37°C .
Critical Note: Endotoxin levels (<1.0 EU/μg) must remain below thresholds to prevent nonspecific immune responses in cell-based assays .
Species-Specific Considerations
Q: How does rat TACR2 compare to human TACR2 in pharmacological studies? A:
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Structural homology: Rat and human TACR2 share >85% amino acid identity in TMs, but diverge in N-terminal regions .
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Ligand affinity: Rat TACR2 binds NKA with higher affinity than human TACR2, necessitating species-specific agonist/antagonist profiling .
Application: Use species-matched recombinant proteins (e.g., canine, murine TACR2 from Cusabio) for translational studies .
Data Contradictions in Receptor Signaling
Q: Why do some studies report TACR2 coupling to Gαs, while others suggest Gαq/11 involvement? A: Context-dependent signaling arises from:
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Cell-type variation: TACR2 may preferentially activate Gαs in neurons vs. Gαq in smooth muscle .
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Post-translational modifications: Phosphorylation of cytoplasmic domains could alter G protein specificity .
Resolution: Use G-protein inhibitors (e.g., pertussis toxin for Gαi/o) alongside calcium or cAMP assays to dissect pathways .
Applications in Metabolic Research
Q: How does TACR2 activation influence energy homeostasis? A: Recent studies highlight dual roles:
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Central effects: NK2R agonists suppress appetite via hypothalamic pathways, bypassing leptin resistance .
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Peripheral effects: Increased brown adipose tissue thermogenesis enhances energy expenditure .
Methodological Insight: Combine hyperinsulinaemic–euglycaemic clamps with indirect calorimetry to assess metabolic outcomes .
Future Directions in TACR2 Research
Q: What unresolved questions warrant further investigation in TACR2 biology? A:
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Crystallographic studies: Determine TACR2’s active-state conformation to guide drug design.
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Epigenetic regulation: Explore how environmental factors modulate TACR2 expression in metabolic diseases.
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Synthetic biology: Engineer TACR2 variants with prolonged half-lives for therapeutic use.
Critical Gaps: Limited data on TACR2’s role in immune modulation and neuroinflammation despite its expression in immune cells .
