Recombinant Macaca mulatta (Rhesus macaque) C-C chemokine receptor type 1 (CCR1)

What is the molecular classification of rhesus macaque CCR1?

Rhesus macaque CCR1 (C-C motif chemokine receptor 1) is a protein-coding gene with Entrez Gene ID 574188. It belongs to the G-protein coupled receptor family and functions as a chemokine receptor. The full-length protein is encoded by an open reading frame (ORF) of 1068 base pairs . Multiple transcript variants have been identified, including the standard C-C chemokine receptor type 1 (NM_001032858.1) and an alternative isoform X1 (XM_015131075.1/XM_015131075.2) . CCR1 plays a crucial role in immune cell trafficking and has been implicated in various inflammatory processes and viral infection mechanisms.

How do rhesus macaque and human CCR1 sequences compare structurally?

Rhesus macaque CCR1 exhibits notable differences from its human counterpart, particularly in the extracellular domains. While the seven transmembrane domains are highly conserved between species, significant variations exist in the predicted extracellular surfaces (residues 1-39, 94-108, 167-203, and 259-286) . The most pronounced divergence occurs in two clusters of completely divergent triplets at positions 177-179 and 192-194 on the predicted second extracellular loop . These structural differences may reflect:

• Potential divergence in ligand specificity between species

• Functional adaptations specific to each species

• Regions that may not critically impact ligand binding or receptor activation

The divergence in extracellular domains suggests careful consideration when extrapolating human CCR1 binding data to rhesus models, particularly when designing inhibitors or studying ligand interactions.

What are the recommended methods for cloning rhesus macaque CCR1?

Based on established protocols, researchers should consider the following methodological approach for cloning rhesus macaque CCR1:

• Source material selection: Total RNA isolated from rhesus macaque peripheral blood mononuclear cells (PBMCs) has proven effective as starting material .

• Isolation technique: Commercial RNA isolation kits (e.g., Purescript by Gentra Systems) have been successfully employed for high-quality RNA extraction .

• Amplification approach: RT-PCR is the recommended method for amplifying CCR1 from PBMC-derived RNA .

• Alternative approach: For researchers facing difficulties with RNA quality, genomic DNA from rhesus macaque PBMCs can serve as an alternative template, as the CCR1 coding sequence lacks introns .

• Verification strategy: Following cloning, sequence verification through alignment with reference sequences (e.g., NM_001032858.1) is essential to confirm successful isolation of the target gene .

This established workflow has proven reliable for obtaining functional CCR1 clones suitable for further characterization and recombinant expression studies.

What expression systems yield functional rhesus macaque CCR1 protein?

For optimal expression of rhesus macaque CCR1, researchers have successfully employed human embryonic kidney (HEK293) cells as the preferred expression system . This mammalian expression platform offers several advantages:

• Post-translational modifications: HEK293 cells provide appropriate glycosylation and membrane insertion critical for proper CCR1 folding and function.

• Protein tagging options: Multiple tagging strategies have been validated, including His-tag, Fc-fusion, and Avi-tag combinations to facilitate purification and detection .

• Yield and purity: When optimized, expression in HEK293 cells typically yields recombinant CCR1 with ≥85% purity as determined by SDS-PAGE analysis .

• Quality control considerations: Recombinant preparations should be assessed for endotoxin contamination, with acceptable levels being <1.0 EU per μg of protein as determined by the LAL method .

The expression vector selection should complement the chosen system, with pcDNA3.1-based vectors having demonstrated efficacy for mammalian expression of rhesus macaque CCR1 .

What are the optimal storage conditions for maintaining recombinant rhesus macaque CCR1 stability?

Maintaining the stability and functionality of recombinant rhesus macaque CCR1 requires careful attention to storage conditions:

• Buffer composition: PBS (phosphate-buffered saline) has been validated as an effective storage buffer for purified recombinant CCR1 protein .

• Temperature requirements:

  • Short-term storage (≤1 month): 2-8°C

  • Long-term storage: -20°C to -80°C in small aliquots to prevent repeated freeze-thaw cycles

• Stability duration: Under proper storage conditions, recombinant rhesus macaque CCR1 maintains stability for at least 6 months from the date of preparation .

• Critical caution: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided through proper aliquoting practices .

• Thawing protocol: When retrieving stored protein, rapid thawing at room temperature followed by immediate use or maintenance on ice is recommended to preserve functionality.

How does rhesus macaque CCR1 interact with its chemokine ligands?

The interaction between rhesus macaque CCR1 and its chemokine ligands exhibits both similarities and differences compared to the human system:

• Primary ligands: Rhesus macaque CCR1 binds several chemokines, including:

  • RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted)

  • MIP-1α (Macrophage Inflammatory Protein-1 alpha)

  • MCP-1 (Monocyte Chemoattractant Protein-1)

• Binding interface: The primary binding determinants are located within the extracellular domains of CCR1, particularly in the N-terminal region and extracellular loops. The notable sequence divergence in these regions between rhesus and human CCR1 suggests potential differences in binding kinetics or specificity .

• Functional implications: Three possible explanations for the observed sequence divergence have been proposed:

  • The ligands themselves (RANTES, MIP-1α, MCP-1) may differ significantly between rhesus macaques and humans

  • Rhesus macaque and human CCR1 may have similar but not identical functions in their respective hosts

  • The variations in CCR1 extracellular domains may not substantially impact ligand binding, suggesting functional conservation despite sequence divergence

Experimental approaches for studying these interactions should account for these species-specific considerations to generate physiologically relevant data.

What methodological approaches are recommended for studying CCR1's role in SIV infection?

Given the established role of chemokine receptors in SIV entry and pathogenesis, the following methodological approaches are recommended for investigating rhesus macaque CCR1 in SIV infection contexts:

• Viral entry assays: Cell lines expressing recombinant CCR1 (with or without CD4) can be used to assess SIV entry efficiency and tropism .

• Population variation analysis: Consideration of CCR1 genetic variations across different macaque populations (Indian-origin vs. Chinese-origin) is critical, as these may contribute to observed differences in disease progression rates .

• Comparative receptor studies: Since multiple chemokine receptors potentially serve as SIV co-receptors, parallel analysis of CCR1 alongside other receptors (CCR2b, CCR3, CCR5, CCR8, CXCR4, STRL33, GPR1, GPR15, APJ, and CRAM-A/B) provides more comprehensive insights .

• Ligand competition studies: Assessing how natural chemokine ligands compete with SIV for receptor binding offers insights into potential protective mechanisms and therapeutic approaches.

• Sequence-function correlation: Given the significant extracellular domain variation between human and rhesus CCR1, mutagenesis studies targeting these regions can help identify determinants of SIV interaction specificity .

These approaches collectively enable a comprehensive assessment of CCR1's contribution to SIV pathogenesis and its potential as a therapeutic target.

How do variations in CCR1 sequence affect experimental model selection?

When designing experiments involving rhesus macaque CCR1, researchers should consider several factors related to sequence variation:

• Population-specific variations: Significant genetic differences exist between Indian-origin and Chinese-origin rhesus macaques. While this has been extensively documented for CCL3L copy number variation affecting SIV progression , similar considerations may apply to CCR1 variants.

• Impact on disease models: These genetic variations may contribute to different disease progression rates observed between animal populations, with Chinese-origin macaques typically showing slower progression to simian-AIDS compared to Indian-origin animals .

• Experimental controls: When designing studies:

  • Document the specific origin of study animals

  • Consider genotyping CCR1 and related genes in experimental subjects

  • Account for genetic background when interpreting results across different studies

• Translational considerations: The significant differences between human and rhesus macaque CCR1, particularly in extracellular domains, necessitate caution when extrapolating findings from animal models to human applications .

By accounting for these variations, researchers can design more robust experiments and better interpret inter-study variations in results involving CCR1 function or targeting.

What techniques are available for studying CCR1 signaling pathways in rhesus macaque systems?

Several technical approaches can be employed to investigate CCR1 signaling in rhesus macaque systems:

• Calcium flux assays: For measuring immediate signaling responses following ligand binding, using fluorescent calcium indicators in cells expressing recombinant or native CCR1.

• Phosphorylation studies: Western blotting with phospho-specific antibodies to detect activation of downstream signaling components (ERK1/2, AKT, etc.) following CCR1 stimulation.

• Receptor internalization assays: Flow cytometry or immunofluorescence microscopy to track CCR1 surface expression changes following ligand exposure.

• Migration assays: Transwell or wound healing assays using rhesus macaque primary cells (e.g., PBMCs) to assess CCR1-mediated chemotactic responses.

• Gene expression analysis: RNA-seq or qPCR to identify transcriptional changes downstream of CCR1 activation in relevant cell types.

• BRET/FRET approaches: Bioluminescence/fluorescence resonance energy transfer techniques to study CCR1 interactions with G-proteins and other signaling partners.

These methodologies should be adapted to account for the specific characteristics of rhesus macaque CCR1, particularly when antibodies or other detection reagents designed for human CCR1 are employed.

What are the critical considerations for producing functional G-protein coupled receptors like CCR1 in recombinant systems?

Producing functional GPCRs such as CCR1 presents several technical challenges that researchers should address:

• Expression system selection: Mammalian expression systems (particularly HEK293 cells) are strongly recommended over bacterial or insect cell systems for rhesus macaque CCR1 production . This ensures proper:

  • Post-translational modifications

  • Membrane insertion

  • Protein folding

• Purification strategy optimization:

  • Use of appropriate detergents to solubilize membrane-embedded CCR1

  • Inclusion of stabilizing agents during purification

  • Affinity purification using validated tags (His, Fc, Avi)

• Functional verification:

  • Ligand binding assays

  • G-protein coupling assessments

  • Signaling activation measurements

• Storage and stability considerations:

  • Aliquoting to prevent freeze-thaw cycles

  • Addition of stabilizing agents

  • Proper buffer composition (typically PBS)

• Quality control metrics:

  • Purity assessment (≥85% by SDS-PAGE)

  • Endotoxin testing (<1.0 EU per μg)

  • Functional activity verification

By addressing these considerations, researchers can produce recombinant rhesus macaque CCR1 preparations suitable for structural studies, drug screening, and functional characterization.

How do rhesus macaque chemokine receptors compare with those of other primate species?

Comparative analysis of chemokine receptors across primate species provides valuable evolutionary insights:

• Conservation patterns: While the search results don't specifically address CCR1 across all primate species, related research on CCL3L genes demonstrates extensive variability across Old World monkeys and apes, suggesting this region has been subject to evolutionary pressures for at least 25 million years .

• Functional implications: The conservation of certain domains (particularly transmembrane regions) across species suggests fundamental functional requirements, while variations in extracellular domains may reflect species-specific adaptations to different ligands or pathogens .

• Evolutionary significance: The significant divergence observed in rhesus macaque CCR1 extracellular domains compared to human CCR1 suggests potential adaptations to:

  • Different pathogen pressures

  • Species-specific immune requirements

  • Coevolution with species-specific chemokine ligands

This evolutionary perspective is crucial when interpreting functional differences between human and rhesus macaque systems and helps establish the appropriate context for translational research.

What is the relationship between CCR1 and viral infection susceptibility in rhesus macaques?

The relationship between CCR1 and viral infection susceptibility is complex:

• SIV co-receptor functionality: While CCR5 is the primary co-receptor for SIV entry, CCR1 and other chemokine receptors can potentially serve as alternative entry portals, either with or without CD4 .

• Population-level variation: Different rhesus macaque populations show variable progression rates to simian-AIDS, which has been linked to genetic factors including chemokine system genes. For example, Chinese-origin macaques typically progress more slowly than Indian-origin animals .

• Mechanistic considerations: The role of CCR1 may involve:

  • Direct viral entry facilitation

  • Modulation of immune cell trafficking

  • Influence on inflammatory responses during infection

• Research applications: Understanding CCR1's contribution to viral pathogenesis provides:

  • Potential therapeutic targets

  • Insights into disease progression variability

  • Improved animal model selection criteria

Researchers should consider these factors when designing studies involving CCR1 and viral infection models in rhesus macaques.

What are the major challenges in studying rhesus macaque CCR1 and how can they be addressed?

Researchers face several technical challenges when studying rhesus macaque CCR1:

• Limited reagent availability: Unlike human CCR1, fewer commercial antibodies and detection reagents exist for rhesus macaque CCR1.

  • Solution: Cross-reactivity testing of human CCR1 antibodies or custom antibody development against rhesus-specific epitopes

• Expression system optimization: As a GPCR, CCR1 is challenging to express in functional form.

  • Solution: Use validated mammalian expression systems like HEK293 cells with appropriate vector systems

• Stability concerns: Purified CCR1 protein may lose activity during storage.

  • Solution: Proper buffer formulation (PBS), aliquoting to prevent freeze-thaw cycles, and storage at -20°C to -80°C

• Functional assay development: Assessing CCR1 activity requires specialized approaches.

  • Solution: Adaptation of calcium flux, migration, or receptor internalization assays specifically for rhesus macaque cells

• Genetic variation: Population differences between Indian-origin and Chinese-origin macaques may confound results.

  • Solution: Careful documentation of animal origin and consideration of genetic background in experimental design and interpretation

By anticipating and addressing these challenges, researchers can generate more robust and reproducible data when working with rhesus macaque CCR1.

How can researchers verify the functionality of recombinant rhesus macaque CCR1?

Verifying the functionality of recombinant rhesus macaque CCR1 requires multiple complementary approaches:

• Ligand binding assays:

  • Radiolabeled ligand binding studies with known CCR1 ligands (RANTES, MIP-1α, MCP-1)

  • Competition binding assays to determine specificity and affinity constants

• Signaling activation assessment:

  • Calcium mobilization assays in cells expressing recombinant CCR1

  • ERK1/2 phosphorylation following ligand stimulation

  • Measurement of cAMP levels to assess G-protein coupling

• Surface expression confirmation:

  • Flow cytometry to verify membrane localization

  • Cell surface biotinylation followed by pull-down assays

• Chemotactic activity:

  • Transwell migration assays with CCR1-expressing cells

  • Competitive inhibition using CCR1 antagonists to confirm specificity

• Comparative analysis:

  • Side-by-side testing with native CCR1 from rhesus macaque PBMCs or tissue

  • Functional comparison with human CCR1 to identify species-specific differences

These complementary approaches provide a comprehensive assessment of recombinant CCR1 functionality and ensure that the protein retains its native biological properties.

What emerging technologies might advance rhesus macaque CCR1 research?

Several emerging technologies hold promise for advancing research on rhesus macaque CCR1:

• CRISPR-Cas9 genome editing:

  • Generation of CCR1 knockout macaque models

  • Introduction of specific mutations to study structure-function relationships

  • Creation of reporter systems for in vivo tracking of CCR1 expression

• Single-cell RNA sequencing:

  • Characterization of CCR1 expression patterns across immune cell populations

  • Identification of cell-specific responses to SIV infection

  • Mapping of CCR1-dependent signaling networks at single-cell resolution

• Cryo-electron microscopy:

  • Determination of rhesus macaque CCR1 structure at atomic resolution

  • Visualization of CCR1-ligand and CCR1-SIV interactions

  • Comparative structural analysis with human CCR1

• Organoid technologies:

  • Development of rhesus macaque tissue-specific organoids for ex vivo CCR1 studies

  • Testing of CCR1-targeted interventions in physiologically relevant systems

• Systems biology approaches:

  • Integration of genomic, transcriptomic, and proteomic data to build comprehensive models of CCR1 function

  • Network analysis to identify novel CCR1 interaction partners

These technologies will enable more sophisticated investigations of CCR1 biology and potentially reveal new therapeutic targets for diseases involving chemokine signaling.

What are the translational implications of rhesus macaque CCR1 research for human health?

Research on rhesus macaque CCR1 has several important translational implications:

• HIV/AIDS therapeutics:

  • Understanding the role of CCR1 in SIV infection models may reveal new strategies for HIV intervention

  • Differences between rhesus and human CCR1 may highlight critical factors in viral pathogenesis

• Inflammatory disease models:

  • CCR1 signaling contributes to various inflammatory conditions

  • Rhesus macaque models can help assess the efficacy and safety of CCR1-targeted therapeutics

• Drug development considerations:

  • The significant differences in extracellular domains between human and rhesus CCR1 suggest that:

    • Drug screening results may not directly translate between species

    • Species-specific optimization of CCR1-targeted compounds may be necessary

    • Careful interpretation of preclinical results is essential

• Immunomodulatory approaches:

  • Insights into CCR1 regulation in rhesus macaques may inform strategies for modulating immune cell trafficking in humans

  • Understanding population-level variations in chemokine systems may help explain individual differences in disease susceptibility

By carefully considering the similarities and differences between rhesus macaque and human CCR1, researchers can maximize the translational value of their findings and develop more effective therapeutic strategies.