Recombinant Mouse Probable G-protein coupled receptor 22 (Gpr22)
Description
Production and Purification
Recombinant mouse Gpr22 is produced using multiple expression systems:
Host Systems and Yields
| Host | Advantages | Applications |
|---|---|---|
| E. coli | Cost-effective, high yield | Structural studies, antibody production |
| Mammalian Cells (HEK-293) | Proper folding, post-translational modifications | Functional assays, signaling studies |
| Cell-Free Synthesis | Rapid production, no host contaminants | High-throughput screening |
Purification Methods
Functional and Signaling Properties
Gpr22 constitutively couples to Gαi/Gαo proteins, leading to inhibition of adenylate cyclase and reduced cAMP production . Key functional findings include:
Research Findings
Cardiac Pathophysiology
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Pressure Overload Models: Gpr22-deficient mice exhibit worsened cardiac function after aortic banding, indicating its role in stress adaptation .
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Ischemic Injury: Overexpression in cardiomyocytes reduces infarct size via Akt-mediated survival signaling .
Cilia Regulation
Knockdown or overexpression in zebrafish disrupts cilia structure in Kupffer’s vesicle, altering left-right patterning during embryogenesis .
Limitations and Future Directions
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order remarks for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
- Demonstrates GPR22 expression in the mouse heart and identifies a protective role for GPR22 in response to hemodynamic stress from increased afterload. PMID: 18539757
Q&A
How should researchers prepare cardiac tissue samples for GPR22 analysis?
Cardiac tissue homogenization must preserve GPR22’s labile structure:
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Use ice-cold RIPA buffer with protease inhibitors (e.g., 1 mM PMSF) to avoid degradation.
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Centrifuge at 16,000 × g for 20 min at 4°C to isolate soluble fractions .
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Validate lysate quality via β-actin Western blotting to ensure equal loading .
| Parameter | Recommendation | Rationale |
|---|---|---|
| Homogenization | Mechanical disruption with glass beads | Prevents GPCR denaturation |
| Storage | Aliquot at −80°C; avoid freeze-thaw cycles | Stability loss <5% over 6 months |
What controls are essential for GPR22 knockout studies?
When using GPR22−/− mice:
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Confirm germline deletion via Southern blotting with XbaI digestion and 990-bp 5′/930-bp 3′ probes .
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Include heterozygous (GPR22+/−) and wild-type littermates to assess gene dosage effects.
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Monitor cardiac hypertrophy markers (e.g., ANP, BNP) due to GPR22’s constitutive Gi coupling in myocytes .
How can researchers resolve contradictory signaling data for GPR22 in heterologous systems?
Constitutive Gi coupling of GPR22 is detectable only under optimized expression conditions :
Problem: Weak/no signal in transiently transfected cells.
Solution:
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Use synthetic GPR22 (sGPR22) with codon-optimized sequence (59% G/C vs. 36% in wild type) .
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Validate mRNA stability via Northern blotting (30-min exposure post-transfection) .
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Measure [35S]GTPγS binding in membrane fractions to confirm basal Gi activity .
Experimental Workflow:
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Transfect COS7 cells with sGPR22 under CMV promoter.
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Assess membrane localization via HA-tag immunocytochemistry.
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Quantify cAMP suppression (20–30% reduction vs. vector control) with/without pertussis toxin .
What strategies mitigate false negatives in recombinant GPR22 detection?
Commercial ELISAs show limited cross-reactivity with recombinant GPR22 due to structural differences :
How to interpret tissue-specific mRNA/protein expression discrepancies?
GPR22 mRNA levels exceed β1-adrenergic receptor levels in human heart tissue, yet protein abundance may not correlate :
Troubleshooting Framework:
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Technical factors:
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Biological factors:
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Assess post-transcriptional regulation (e.g., miR-328 targeting 3′UTR).
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Profile protein half-life using cycloheximide chase assays.
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What in vivo models best recapitulate GPR22’s cardiac pathophysiology?
Germline GPR22−/− mice show no overt phenotype, necessitating conditional models :
| Model | Utility | Limitations |
|---|---|---|
| Cardiomyocyte-specific KO (αMHC-Cre) | Assess contractile dysfunction | Neonatal lethality if deleted embryonically |
| Coronary artery endothelial KO (Tie2-Cre) | Study vascular remodeling | Off-target effects in bone marrow |
