Recombinant Macaca mulatta C-C chemokine receptor-like 2 (CCRL2)

What is CCRL2 and how does it differ from classical chemokine receptors?

CCRL2 belongs to the family of atypical chemokine receptors (ACKRs) and differs fundamentally from classical chemokine receptors in its signaling mechanisms. Unlike conventional chemokine receptors, CCRL2 behaves differently from both ACKRs and classical chemotactic receptors in terms of its internalization properties . While conventional chemokine receptors typically signal through G-protein coupled pathways, CCRL2 exhibits distinct trafficking patterns and does not appear to trigger the same downstream signaling cascades. The degree of CCRL2 internalization is lower than observed for signaling chemotactic receptors like CXCR2 and CMKLR1, yet it does undergo constitutive internalization and recycling to the plasma membrane following the slow recycling pathway .

What is the primary ligand for CCRL2 and how does binding occur?

Chemerin is the only recognized ligand for CCRL2 . The binding mechanism is particularly interesting from a structural biology perspective, as CCRL2 binds chemerin at the N-terminus while leaving the C-terminus accessible. This unique binding pattern enables chemerin to simultaneously interact with its functional receptor, chemokine-like receptor 1 (CMKLR1), on other cells . This suggests that CCRL2 may function as a chemerin-presenting molecule at the surface of barrier cells rather than a traditional signaling receptor . This presenting function differentiates CCRL2 from other atypical chemokine receptors that typically internalize, recycle, and scavenge their ligands.

What expression systems are commonly used for producing recombinant Macaca mulatta CCRL2?

E. coli expression systems are commonly utilized for producing recombinant Macaca mulatta chemokine receptors, as demonstrated with similar receptors like CCR8 . The expression system typically employs an N-terminal His-tag for purification purposes, resulting in proteins with high purity (greater than 90% as determined by SDS-PAGE) . The recombinant protein is typically produced as a lyophilized powder and requires specific reconstitution protocols in deionized sterile water to a concentration of 0.1-1.0 mg/mL, often with the addition of 5-50% glycerol as a cryoprotectant for long-term storage .

What are the optimal storage and handling conditions for recombinant Macaca mulatta CCRL2?

Recombinant Macaca mulatta chemokine receptors require careful handling to maintain functional integrity. Based on protocols for similar receptors, the lyophilized protein should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . For short-term use, working aliquots can be stored at 4°C for up to one week . The optimal storage buffer typically consists of a Tris/PBS-based solution containing approximately 6% Trehalose at pH 8.0 .

For reconstitution, it is recommended that the vial be briefly centrifuged prior to opening to bring contents to the bottom. Following reconstitution in deionized sterile water, the addition of glycerol (typically to a final concentration of 50%) is recommended for long-term storage stability at -20°C/-80°C .

What functional assays are appropriate for studying CCRL2 activity in vitro?

Several functional assays can be employed to study CCRL2 activity:

• Internalization assays: Fluorescent labeling approaches can be used to track CCRL2 internalization and recycling to the plasma membrane. Unlike other receptors, CCRL2's internalization is not significantly modified by treatment with its ligand chemerin or other chemokines .

• Co-immunoprecipitation studies: These are valuable for investigating CCRL2's interactions with signaling molecules such as JAK2. CCRL2 has been shown to coprecipitate with JAK2 and potentiate JAK2-STAT interactions in human models, suggesting similar pathways may be investigated in Macaca mulatta systems .

• Cell growth and clonogenicity assays: CCRL2 knockdown experiments can be used to assess the receptor's influence on cell growth and clonogenicity both in vitro and in vivo .

• Phosphorylation analysis: Western blotting for phosphorylated JAK2/STAT3/STAT5 can provide insights into CCRL2's effects on downstream signaling pathways .

• Chemerin binding studies: Assays to evaluate the binding of chemerin to CCRL2 and subsequent presentation to CMKLR1-expressing cells can help elucidate the receptor's function in chemerin-mediated processes .

How can CCRL2 knockout or knockdown models be generated in Macaca mulatta systems?

Creating CCRL2 knockout or knockdown models in Macaca mulatta systems requires sophisticated genetic engineering approaches. While the search results don't specifically address this for Macaca mulatta, the following methodologies can be adapted from human cell studies:

• RNA interference (RNAi): Short hairpin RNA (shRNA) or small interfering RNA (siRNA) targeting CCRL2 mRNA can be delivered using viral vectors. This approach has been successful in human cell lines, where CCRL2 knockdown suppressed cell growth and clonogenicity both in vitro and in vivo .

• CRISPR-Cas9 genome editing: This technology can be used to create precise genetic modifications in the CCRL2 gene of Macaca mulatta cells. Design of guide RNAs must account for any sequence differences between human and Macaca mulatta CCRL2.

• Cell line models: Establishing stable Macaca mulatta cell lines with CCRL2 knockdown or knockout can provide valuable tools for studying receptor function. Similar approaches in human myeloid cell lines have demonstrated that CCRL2 knockdown increases apoptotic rates and alters cell cycle progression .

For validation of knockdown efficiency, quantitative RT-PCR and western blotting should be employed to confirm reduction in CCRL2 expression at both mRNA and protein levels.

What role does CCRL2 play in myeloid malignancies and how can Macaca mulatta models contribute to this research?

CCRL2 has been implicated in myeloid malignancies, particularly myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (sAML). Studies have shown that CCRL2 is up-regulated in primitive cells from patients with these conditions . CCRL2 appears to promote the growth and clonogenicity of MDS and sAML cells and activates JAK2/STAT signaling .

Macaca mulatta models could provide valuable insights into these pathological processes due to their close genetic similarity to humans. Rhesus macaques have been used extensively in cancer research , and therefore represent an excellent model for studying CCRL2's role in myeloid malignancies. Creating Macaca mulatta cell lines or primary cultures with manipulated CCRL2 expression could help elucidate:

• The mechanisms by which CCRL2 promotes cell growth in myeloid malignancies

• The specific interactions between CCRL2 and JAK2/STAT signaling pathways

• The potential of CCRL2 as a therapeutic target for myeloid malignancies

Research has shown that CCRL2 knockdown inhibits the growth of MDS and sAML cells and alters the effect of JAK2 inhibitors like fedratinib , suggesting complex interactions between CCRL2 and established therapeutic targets.

How does CCRL2's interaction with the JAK/STAT pathway influence experimental design in inflammation and cancer research?

CCRL2's interaction with JAK/STAT signaling has significant implications for experimental design in inflammation and cancer research. Studies have demonstrated that CCRL2 coprecipitates with JAK2 and potentiates JAK2-STAT interactions . This relationship affects several aspects of experimental design:

• Inhibitor studies: When using JAK2 inhibitors such as fedratinib, researchers must consider CCRL2 expression levels as they can alter inhibitor efficacy. CCRL2 knockdown has been shown to enhance the effects of JAK2 inhibitors in some contexts .

• Cell line selection: Cell lines expressing JAK2 mutations (e.g., JAK2V617F) show different responses to CCRL2 manipulation compared to wild-type lines , necessitating careful cell model selection.

• Pathway analysis: Experiments should include comprehensive analysis of JAK2/STAT3/STAT5 phosphorylation states when studying CCRL2 function, as these appear to be key downstream effectors .

• Combination approaches: Targeting CCRL2 in combination with JAK/STAT pathway inhibitors may yield synergistic effects, suggesting experimental designs should consider combination treatment arms.

How can Macaca mulatta CCRL2 studies be translated to human disease research?

Translating findings from Macaca mulatta CCRL2 studies to human disease research is facilitated by the significant genetic and physiological similarities between rhesus macaques and humans. Rhesus macaques have contributed to numerous medical breakthroughs, including the identification of the Rh factor in blood typing and the development of the polio vaccine .

For effective translation of CCRL2 research, consider:

• Comparative sequence analysis: While using Macaca mulatta CCRL2, researchers should conduct detailed sequence alignments with human CCRL2 to identify conserved domains and potential functional differences.

• Cross-species validation: Findings in Macaca mulatta systems should be validated in human cell lines or primary samples whenever possible.

• Tissue-specific expression: Compare the tissue distribution and expression patterns of CCRL2 between species to ensure relevance of the model system.

• Functional conservation: Verify that key functions, such as chemerin binding and JAK2 interaction, are conserved between Macaca mulatta and human CCRL2.

• Disease modeling: Rhesus macaques can be used to model human diseases in which CCRL2 plays a role, including inflammatory conditions and myeloid malignancies .

What are the technical challenges in studying the internalization and recycling properties of CCRL2?

Studying CCRL2 internalization and recycling presents several technical challenges:

How do post-translational modifications affect CCRL2 function and experimental outcomes?

Post-translational modifications (PTMs) of CCRL2 likely play crucial roles in its function, though specific information about PTMs of Macaca mulatta CCRL2 is limited in the provided search results. Based on studies of related receptors and general principles of membrane protein biology, researchers should consider:

• Glycosylation: N-linked glycosylation often affects chemokine receptor trafficking and ligand binding. Expression systems like E. coli do not perform mammalian-type glycosylation, potentially affecting protein functionality compared to naturally expressed receptors.

• Phosphorylation: While CCRL2 influences JAK2/STAT3/STAT5 phosphorylation , the phosphorylation status of CCRL2 itself may regulate its function. Mass spectrometry approaches can be used to map phosphorylation sites.

• Ubiquitination: This modification often regulates receptor degradation and trafficking. Differences in ubiquitination machinery between expression systems and native environments may affect experimental outcomes.

• Palmitoylation: This lipid modification can influence receptor localization and signaling capabilities. Expression systems should be chosen with consideration of their ability to perform appropriate lipid modifications.

• Disulfide bonds: Proper formation of disulfide bonds is critical for maintaining the tertiary structure of many receptors. Expression in E. coli may require specialized strains or refolding protocols to ensure correct disulfide bond formation.

When designing experiments with recombinant CCRL2, researchers should consider using multiple expression systems (bacterial, insect, and mammalian) to account for PTM-related differences in receptor functionality.

What are the current limitations in understanding CCRL2's role as a chemerin-presenting molecule and how can these be addressed?

Current limitations in understanding CCRL2's role as a chemerin-presenting molecule include:

• Mechanistic details: While CCRL2 is proposed to act as a chemerin-presenting molecule at the surface of barrier cells , the precise molecular mechanisms remain unclear. Advanced structural studies using techniques like cryo-electron microscopy could help elucidate the exact configuration of CCRL2-chemerin complexes.

• Functional consequences: The physiological significance of chemerin presentation by CCRL2 to CMKLR1-expressing cells requires further investigation. Co-culture systems with cells expressing CCRL2 and others expressing CMKLR1 could help elucidate this presenter-receptor relationship.

• Species differences: Potential differences in chemerin binding and presentation between human and Macaca mulatta CCRL2 need systematic investigation. Comparative binding studies using recombinant proteins from both species would address this limitation.

• Context-dependent functions: The function of CCRL2 may vary depending on cell type and physiological/pathological context. Comprehensive expression profiling across multiple tissues and disease states in both humans and Macaca mulatta would provide valuable insights.

• Integration with other signaling pathways: The relationship between CCRL2's chemerin-presenting function and its effects on JAK2/STAT signaling requires clarification. Systems biology approaches combining proteomics, transcriptomics, and phospho-proteomics could help integrate these seemingly distinct functions.

What emerging technologies might advance our understanding of Macaca mulatta CCRL2?

Several emerging technologies hold promise for advancing our understanding of Macaca mulatta CCRL2:

• Single-cell analysis: Single-cell RNA sequencing and CyTOF could reveal heterogeneous CCRL2 expression patterns across cell populations and states, providing insights into its context-specific functions.

• CRISPR-based technologies: Beyond knockout studies, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) could enable precise modulation of CCRL2 expression. CRISPR-based screens could identify genetic interactors of CCRL2.

• Organoid systems: Macaca mulatta-derived organoids could provide physiologically relevant environments for studying CCRL2 function in specific tissue contexts.

• Advanced imaging techniques: Super-resolution microscopy and intravital imaging could reveal the spatial and temporal dynamics of CCRL2-chemerin interactions in living cells and tissues.

• AlphaFold and other AI-driven structural prediction tools: These could generate structural models of Macaca mulatta CCRL2, facilitating rational design of inhibitors or modulators.

• Spatial transcriptomics and proteomics: These technologies could map CCRL2 expression and signaling networks within tissues, providing insights into its microenvironmental context.

How might comparative studies between human and Macaca mulatta CCRL2 advance translational research?

Comparative studies between human and Macaca mulatta CCRL2 could significantly advance translational research in several ways:

• Evolutionary insights: Understanding conserved and divergent features of CCRL2 across primates could reveal functionally critical domains and species-specific adaptations.

• Therapeutic development: Macaca mulatta models provide an essential step in translating findings from cell culture to human applications. Comparative studies could identify the best models for testing CCRL2-targeting therapeutics.

• Disease modeling: Rhesus macaques have been used extensively in biomedical research, including infectious disease, vaccine development, aging, cardiovascular disease, metabolic diseases, neurologic diseases, and cancer research . Comparative CCRL2 studies could reveal its role across these conditions.

• Predictive biomarkers: Identifying similarities and differences in CCRL2 expression patterns between species could help validate it as a biomarker for human diseases such as MDS and sAML .

• Drug efficacy and safety: Understanding species-specific differences in CCRL2 function and regulation could help predict and explain variations in drug responses between preclinical Macaca mulatta models and human clinical trials.