Recombinant Mouse Probable G-protein coupled receptor 25 (Gpr25)

What is mouse Gpr25 and what is its molecular structure?

Mouse Gpr25 (UniProt No. P0C5I1) is an orphan G protein-coupled receptor that belongs to the GPCR superfamily. Like other GPCRs, it contains seven transmembrane domains (TMDs), with a short extracellular N-terminus and a short intracellular C-terminus . The receptor contains several conserved structural elements crucial for its function, including disulfide bonds formed between C112-C191 (connecting extracellular loop 2 to TMD2) and C30-C279 (connecting the N-terminal region to TMD7) . The orthosteric ligand binding pocket is formed by the seven TMDs and includes conserved residues W95, L92, W105, R178, and W257 that are likely responsible for ligand interactions .

What are the current methods for producing recombinant mouse Gpr25?

Recombinant mouse Gpr25 can be produced in E. coli expression systems, achieving a purity of >85% as determined by SDS-PAGE . For research applications, commercial recombinant mouse Gpr25 is available as partial proteins with specific product codes (e.g., CSB-EP009797MO1-B) . Alternatively, researchers can obtain mouse Gpr25 cDNA clones containing the complete open reading frame (ORF) along with a Kozak consensus sequence for optimal translation initiation, which can be transferred into desired expression vectors .

How should recombinant mouse Gpr25 be handled and stored in laboratory settings?

For optimal stability and activity, follow these research-validated protocols:

• Short-term storage: Keep working aliquots at 4°C for up to one week

• Long-term storage:

  • Liquid form: Store at -20°C/-80°C with a shelf life of approximately 6 months

  • Lyophilized form: Store at -20°C/-80°C with a shelf life of approximately 12 months

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (recommended: 50%) for long-term storage

• Critical precaution: Avoid repeated freezing and thawing cycles, as this significantly decreases protein stability and activity

What known ligands activate Gpr25 across different species?

The ligand profile of Gpr25 varies significantly between mammalian and non-mammalian vertebrates:

Notably, recent research has identified CXCL17 as a potent activator of human Gpr25, with an EC50 value of ~70 nM in β-arrestin 2 recruitment assays . This finding represents a significant advancement in understanding mammalian Gpr25 signaling.

How can researchers effectively assess Gpr25 activation in experimental settings?

Multiple experimental approaches can be employed to study Gpr25 activation, each providing different insights into receptor function:

• cAMP assays: For non-mammalian Gpr25, forskolin-stimulated cAMP production can be measured using the pGL3-CRE-luciferase reporter assay. Activation of zebrafish, spotted gar, and pigeon Gpr25 by Apelin/Apela leads to inhibition of cAMP production .

• β-arrestin recruitment assays: The NanoLuc Binary Technology (NanoBiT) provides a sensitive method for detecting Gpr25 activation:

  • Co-express Gpr25-LgBiT and SmBiT-ARRB2 (or SmBiT-ARRB1) in HEK293T cells

  • Measure bioluminescence after ligand addition

  • This approach successfully detected human Gpr25 activation by CXCL17, showing dose-dependent increases in bioluminescence

• Receptor internalization: Confocal microscopy can be used to visualize Gpr25 internalization following ligand binding. This approach demonstrated that zebrafish Gpr25 internalized after Apelin/Apela treatment .

• Mutational analysis: Strategic mutations of key residues (e.g., W95A, R178A) can confirm specific ligand-receptor interactions. These mutations abolished CXCL17-induced activation of human Gpr25 .

What is the molecular basis for ligand selectivity in Gpr25 across different species?

The evolutionary divergence in Gpr25 ligand selectivity presents an intriguing research area:

• Structural determinants: AlphaFold 3 predictions suggest that in human Gpr25, the conserved W95 forms hydrophobic interactions with P118 of CXCL17, while R178 forms a hydrogen bond with the carboxyl oxygen of L117 . These interactions are critical, as W95A and R178A mutations abolish CXCL17 activation.

• Species-specific differences: Human Gpr25 cannot be activated by Apelin/Apela under conditions where non-mammalian Gpr25 orthologs show strong responses . This suggests evolutionary changes in the ligand binding pocket structure.

• C-terminal importance: The C-terminal fragment of CXCL17 is essential for Gpr25 activation. Deletion of just three C-terminal residues completely abolished CXCL17's activity on human Gpr25 . This is consistent with the high conservation of C-terminal residues among CXCL17 orthologs.

• Research approach: Comparative analysis of Gpr25 sequences across species reveals conserved residues that are likely responsible for structural integrity, downstream signaling, and ligand binding specificity .

What are the downstream signaling pathways and cellular responses activated by Gpr25?

Current research indicates that Gpr25 activation influences multiple signaling pathways:

• G protein-mediated signaling: In non-mammalian vertebrates, Gpr25 activation inhibits forskolin-stimulated cAMP production , suggesting coupling to Gαi/o proteins.

• β-arrestin recruitment: Human Gpr25 activation by CXCL17 leads to recruitment of both β-arrestin 1 and β-arrestin 2, with calculated EC50 values of ~150 nM and ~70 nM, respectively . This indicates that Gpr25 can signal through β-arrestin-dependent pathways.

• Receptor internalization: Following activation, zebrafish Gpr25 undergoes internalization , a process typically associated with receptor desensitization and potentially β-arrestin-dependent signaling.

• Physiological implications: The expression of Gpr25 in various tissues including testes and intestine across vertebrate species suggests potential roles in multiple physiological processes . In humans, Gpr25 has been implicated in autoimmune diseases and blood pressure regulation .

How can researchers design experiments to characterize novel Gpr25 ligands?

Based on recent successful deorphanization approaches:

• Computational prediction: The AlphaFold 3 algorithm has proven effective in predicting protein-protein interactions for GPCRs. This approach successfully identified CXCL17 as a ligand for human Gpr25 .

• Validation workflow:

  • Express Gpr25 in HEK293T cells using complete ORF constructs

  • Employ NanoBiT-based β-arrestin recruitment assays to screen potential ligands

  • Confirm specificity by testing against multiple other GPCRs

  • Validate structure-function relationships through mutation of key residues (e.g., W95, R178)

  • Assess C-terminal importance through truncation studies

• Target residue identification: Focus mutations on conserved residues in the orthosteric ligand pocket:

  Residue	Position	Potential Function
  W95	Orthosteric pocket	Hydrophobic interactions with ligand
  L92	Orthosteric pocket	Structural integrity of binding pocket
  W105	Orthosteric pocket	Ligand binding
  R178	Orthosteric pocket	Hydrogen bonding with ligand
  W257	Orthosteric pocket	Ligand binding

What is the tissue expression profile of Gpr25 and its potential physiological roles?

RNA-seq analyses have revealed that Gpr25 exhibits a diverse expression pattern:

• Expression sites: Gpr25 is expressed in multiple tissues across vertebrate species, with notable expression in:

  • Testes

  • Intestine

  • Various immune cells (in humans)

• Evolutionary conservation: Gpr25 is widely present in vertebrates from fish to mammals, suggesting fundamental physiological roles .

• Potential functions:

  • Immune regulation: Human Gpr25 has been associated with autoimmune diseases

  • Blood pressure regulation: Human studies have implicated Gpr25 in blood pressure control

  • Mucosal immunity: The activation of Gpr25 by CXCL17, which is primarily expressed in mucosal tissues, suggests potential roles in mucosal defense

• Research direction: Investigating tissue-specific Gpr25 knockout models could provide valuable insights into the physiological roles of this receptor in different systems.