Recombinant Macaca fascicularis C-C chemokine receptor type 3 (CCR3)

What is Macaca fascicularis CCR3 and what is its homology to human CCR3?

C-C chemokine receptor type 3 (CCR3) is a G protein-coupled receptor that serves as the principal chemotactic receptor for eosinophils. Macaque CCR3 shares approximately 92% amino acid identity with its human homologue, making it a valuable model for studying human inflammatory diseases . The cloned Cynomolgus monkey (Macaca fascicularis) CCR3 demonstrates even greater similarity (99.4%) to Rhesus macaque (Macaca mulatta) CCR3 than to some previously reported Cynomolgus CCR3 sequences (98.0%) . This high degree of homology supports the use of Macaca fascicularis CCR3 as a relevant alternative species model system for studying CCR3-mediated processes.

What expression systems can be used for recombinant production of Macaca fascicularis CCR3?

Several expression systems have been successfully employed for the recombinant production of Macaca fascicularis CCR3:

• E. coli expression systems: Researchers have successfully expressed functional MBP-fused recombinant CCR3 in E. coli, which maintains binding activity with its ligands . This system allows for relatively straightforward production of the protein for structural and functional studies.

• Mammalian cell expression: Functional expression has been achieved in mammalian cell lines such as the murine pre-B L1-2 cell line, which allows for proper folding and post-translational modifications essential for CCR3 function .

• Stable transfection: Stable transfection approaches have been used to create cell lines expressing Cynomolgus CCR3 for ligand binding studies and functional assays .

The choice of expression system depends on the specific research application, with each system offering distinct advantages for different experimental approaches.

How can researchers verify the functionality of recombinant Macaca fascicularis CCR3?

Functional verification of recombinant Macaca fascicularis CCR3 can be performed using multiple complementary approaches:

• Ligand binding assays: Stably-transfected Cynomolgus CCR3 has been shown to bind human eotaxin (CCL11) with similar kinetics (Kd 240 pM) to human CCR3 . Radioligand binding assays using purified receptor can confirm proper folding and ligand recognition.

• Calcium mobilization assays: Functional CCR3 triggers Ca²⁺ flux in response to chemokine binding. This can be measured using calcium-sensitive fluorescent dyes in CCR3-expressing cells exposed to ligands such as eotaxin, eotaxin-2, and MCP4 .

• Chemotaxis assays: Migration assays measuring cell movement in response to CCR3 ligands provide a physiologically relevant confirmation of receptor functionality .

• GTP hydrolysis assays: The addition of cholesterol to proteoliposomes containing CCR3 leads to greater signal transduction as measured by Gαi3 activation and subsequent GTP hydrolysis .

• Gated autofluorescence forward scatter: This novel signaling assay has been used to characterize β-chemokines on native macaque CCR3 on eosinophils through eotaxin-induced shape change in whole blood .

How does cholesterol influence the signaling properties of Macaca fascicularis CCR3?

Recent research has uncovered a direct correlation between bilayer cholesterol and increased agonist-triggered CCR3 signal transduction. This relationship has significant implications for understanding CCR3 function in different membrane environments:

• Conformational selection: Cholesterol induces site-specific conformational restraint of extracellular loop (ECL) 2 and conserved motion in transmembrane helices and ECL3, which are not observed in simulations of bilayers containing only phosphatidylcholine lipids .

• Orthosteric pocket bias: Residue-residue contact scoring shows that cholesterol biases the conformational selection of the orthosteric pocket involving key residues Y41 1.39, Y113 3.32, and E287 7.39 .

• Activation pathway remodeling: Cholesterol presence leads to contact remodeling in activation pathway residues centered on the initial transmission switch, Na⁺ pocket, and R 3.50 in the DRY motif .

• Signal transduction enhancement: Experimental verification confirms that adding cholesterol to proteoliposomes containing CCR3 leads to greater signal transduction as measured by Gαi3 activation and subsequent GTP hydrolysis .

These findings suggest that cholesterol functions as an allosteric regulator of CCR3 signal transduction, directly interacting with the receptor to constrain it to conformations more likely to bind ligand, thereby influencing ligand affinity.

What approaches can be used to develop antagonists against Macaca fascicularis CCR3?

Development of antagonists against Macaca fascicularis CCR3 is critical for therapeutic applications targeting inflammatory diseases. Several approaches have proven successful:

• Monoclonal antibody development: Researchers have raised monoclonal antibodies (mAbs) against macaque CCR3 using two different immunogens:

  • A 30-amino acid synthetic peptide derived from the predicted NH₂ terminus of macaque CCR3

  • Intact macaque CCR3-transfected cells

• Antagonist screening: These anti-macaque CCR3 monoclonal antibodies have exhibited potent antagonist activity in receptor binding and functional assays .

• Small molecule antagonist development: The characterization of the macaque eotaxin/CCR3 axis and development of antagonistic anti-macaque CCR3 monoclonal antibodies facilitates the development of CCR3 small molecule antagonists .

• Structure-guided design: Recent structural studies of CCR3, including the V6.40A mutant Cryo-EM structure (PDB 7x9y), provide templates for rational design of small molecule antagonists .

The high degree of homology between macaque and human CCR3 (92% identity) suggests that antagonists developed against macaque CCR3 may have translational potential for human therapeutic applications.

What is known about the relationship between CCR3 and bone metabolism?

Recent research has revealed an unexpected relationship between CCR3 and bone metabolism that has significant implications for understanding inflammatory conditions affecting bone:

• Increased osteoclast activity: CCR3 deficiency is associated with increased osteoclast activity, suggesting CCR3 may play a regulatory role in bone homeostasis .

• Bone formation rate: Studies show significantly increased bone formation rate (BFR) in both trabecular (43% increase) and cortical (32% increase) bone in CCR3-deficient mice .

• Mineral apposition rate: Cortical mineral apposition rate (MAR) was 35% higher in CCR3-deficient mice, with the endosteal compartment showing a particularly notable increase of 56% .

• Bone volume changes: µCT analyses revealed that CCR3-deficient mice had significantly higher total tissue volume at the metaphysis and higher medullary volume at both metaphysis and diaphysis .

• Gene expression alterations: In bone marrow macrophage (BMM) cultures, CCR3 deficiency was associated with significantly altered expression of related chemokine receptors and ligands, including higher levels of CCR2, CCL2, and CCR5 mRNAs .

These findings suggest that CCR3 plays a previously unrecognized role in bone remodeling, which may have implications for understanding bone pathologies associated with inflammatory conditions where CCR3 is involved.

What are the key considerations for designing ligand binding studies with recombinant Macaca fascicularis CCR3?

When designing ligand binding studies using recombinant Macaca fascicularis CCR3, researchers should consider the following methodological factors:

• Receptor preparation: Stably-transfected Cynomolgus CCR3 has been shown to bind human eotaxin (CCL11) with specific kinetics (Kd 240 pM) . Ensuring proper receptor folding and membrane integration is critical for accurate binding measurements.

• Ligand selection: Macaca fascicularis CCR3 responds to multiple human CCR3 ligands, including eotaxin (CCL11), eotaxin-2 (CCL24), and MCP4 (CCL13) . Selection of appropriate ligands depends on the specific research question.

• Binding conditions: Studies have shown that macaque CCR3 expressed in murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (Kd = 0.1 nM) . Optimizing binding conditions including temperature, pH, and buffer composition is essential.

• Membrane composition: Cholesterol content significantly affects CCR3 ligand binding due to its allosteric effects on receptor conformation . Controlling membrane composition is therefore crucial for reproducible binding studies.

• Detection methods: Radioligand binding, fluorescence-based approaches, and surface plasmon resonance have all been successfully employed for CCR3 binding studies. The choice depends on the specific research aims and available resources.

How can solid-state NMR spectroscopy be used to study Macaca fascicularis CCR3 structure and dynamics?

Solid-state NMR spectroscopy provides valuable insights into CCR3 structure and dynamics in different membrane environments:

• Sample preparation: Uniformly ¹³C-¹⁵N-labeled samples of CCR3 can be prepared in phospholipid bilayers with and without cholesterol to study lipid-dependent conformational changes .

• 2D correlation spectroscopy: ¹³C-¹³C 2D correlation spectroscopy using techniques such as DARR (Dipolar-Assisted Rotational Resonance) can reveal differences in protein dynamics in different lipid environments .

• Spectral analysis: Analysis of chemical shifts and peak intensities can provide information about site-specific conformational changes. For CCR3, this has revealed that cholesterol induces site-specific conformational restraint of extracellular loop (ECL) 2 and conserved motion in transmembrane helices and ECL3 .

• Molecular dynamics validation: COMPASS (Comparative, Objective Measurement of Protein Architectures by Scoring Shifts) methodology can be used to grade model structures from molecular dynamics simulations against experimental NMR data .

• Temperature optimization: Acquiring spectra at reduced temperatures (e.g., -20°C) can reduce the rate of conformational sampling and improve spectral resolution .

What methods can be used to assess CCR3-mediated signaling in different experimental systems?

Several complementary approaches can be used to assess CCR3-mediated signaling:

• Ca²⁺ mobilization assays: CCR3 activation leads to intracellular calcium release, which can be measured using calcium-sensitive fluorescent dyes in CCR3-expressing cells .

• GTP hydrolysis assays: Measuring Gαi3 activation and subsequent GTP hydrolysis in proteoliposomes containing CCR3 provides a direct measure of receptor signaling efficacy .

• Chemotaxis assays: Quantifying cell migration in response to CCR3 ligands provides a functional readout of receptor signaling .

• Gated autofluorescence forward scatter: This technique measures eotaxin-induced shape change in whole blood, allowing characterization of β-chemokines on native macaque CCR3 on eosinophils .

• Molecular dynamics simulations: Combined with experimental validation, MD simulations can reveal how structural changes, such as those induced by cholesterol, affect receptor signaling pathways .

How should researchers interpret species-specific differences when using Macaca fascicularis CCR3 as a model for human CCR3?

When using Macaca fascicularis CCR3 as a model for human CCR3, researchers should consider these factors when interpreting their data:

• Sequence homology: Macaque CCR3 is 92% identical at the amino acid level to human CCR3 . While this represents high conservation, the 8% difference may affect specific ligand interactions or signaling properties.

• Ligand binding characteristics: Stably-transfected Cynomolgus CCR3 binds human eotaxin (CCL11) with similar kinetics (Kd 240 pM) to human CCR3 , but subtle differences may exist for other ligands.

• Cross-reactivity: Human CCR3 ligands (eotaxin [CCL11], eotaxin-2 [CCL24], and MCP4 [CCL13]) are effective at activating Macaca fascicularis CCR3 . This cross-reactivity supports the use of this model for studying human ligands.

• Functional conservation: Macaque CCR3 expressed in cell lines exhibits robust eotaxin-induced Ca²⁺ flux and chemotaxis , indicating functional conservation of key signaling pathways.

• Pharmacological responses: While antagonists developed against macaque CCR3 may have translational potential, species-specific differences in pharmacological responses should be carefully evaluated before extrapolating to human applications.

What are the implications of cholesterol-dependent CCR3 regulation for experimental design and data interpretation?

The discovery that cholesterol modulates CCR3 signaling has important implications for experimental design and data interpretation:

• Membrane composition control: Experiments should carefully control membrane cholesterol content since variations can significantly affect receptor behavior .

• Experimental variability: Differences in cholesterol levels between experimental preparations may contribute to variability in CCR3 functional assays and should be considered when comparing data across studies.

• Physiological relevance: The cholesterol-dependence of CCR3 signaling suggests that changes in membrane composition in vivo (e.g., in disease states) could alter receptor function .

• Drug screening considerations: Screening for CCR3 antagonists should account for cholesterol-dependent conformational changes in the receptor, as these may affect ligand binding pockets and drug efficacy .

• Model system selection: When designing experiments, researchers should consider whether their model system recapitulates the appropriate membrane environment for physiologically relevant CCR3 function .

What are promising areas for future research on Macaca fascicularis CCR3?

Several promising research directions emerge from current understanding of Macaca fascicularis CCR3:

• Structural studies: Further characterization of CCR3 structure in different activation states and membrane environments would enhance understanding of its function .

• Therapeutic development: The development of CCR3 small molecule antagonists based on the macaque model could lead to novel therapeutics for chronic inflammatory diseases in humans .

• CCR3-bone metabolism connection: Further investigation of the unexpected relationship between CCR3 and bone metabolism could reveal new therapeutic targets for bone disorders .

• Allosteric modulation: Deeper understanding of how cholesterol and other membrane components allosterically modulate CCR3 function could lead to development of novel allosteric modulators .

• Species-specific signaling differences: Comparative studies of signaling pathways between macaque and human CCR3 could reveal evolutionary adaptations in chemokine receptor function and improve translation of findings from animal models.

How might advanced molecular dynamics simulations contribute to understanding CCR3 function?

Molecular dynamics simulations offer powerful approaches for understanding CCR3 function:

• Conformational dynamics: COMPASS methodology combined with molecular dynamics simulations has already revealed how cholesterol influences CCR3 conformational selection and dynamics .

• Ligand binding prediction: Advanced simulations could predict binding modes of novel ligands and antagonists, facilitating drug design.

• Allosteric network mapping: Identifying networks of residues involved in transmitting conformational changes from cholesterol binding sites to the orthosteric pocket could reveal new targets for allosteric modulation .

• Membrane composition effects: Simulations with varied membrane compositions could provide insights into how lipid environment influences receptor function beyond cholesterol effects.

• Species comparison: Comparative simulations of human and macaque CCR3 could identify key differences in dynamics that influence ligand binding or signaling properties.