Recombinant Rat Receptor expression-enhancing protein 6 (Reep6)

What is Receptor Expression-Enhancing Protein 6 (REEP6) and what are its primary functions?

REEP6 belongs to the receptor expression-enhancing protein family that plays a crucial role in G-protein-coupled receptor (GPCR) signaling. Unlike many accessory proteins that primarily affect membrane trafficking, REEP6 appears to be involved in receptor-mediated endocytosis following ligand binding rather than initial membrane expression. Research indicates that REEP6 enhances interleukin-8 (IL-8)-stimulated CXCR1 activation and participates in downstream signaling events .

Methodologically, researchers can study REEP6 function through:

• Co-immunoprecipitation assays with target receptors

• Reporter gene assays comparing wild-type and REEP6-deficient cells

• Fluorescence microscopy to track receptor localization

• Western blotting to detect protein expression levels

How do I express recombinant rat REEP6 in mammalian cells?

For optimal expression of recombinant rat REEP6 in mammalian cells, consider these methodological approaches:

• Vector selection: Use mammalian expression vectors containing strong promoters (CMV or EF1α)

• Regulatory elements: Include Kozak sequence and appropriate leader sequences upstream of the REEP6 gene to enhance expression

• Cell line selection: Chinese Hamster Ovary (CHO) cells have proven effective for recombinant protein expression

• Transfection method: Lipid-based transfection reagents or electroporation can be used, with optimization required for each cell type

• Selection: For stable expression, include appropriate antibiotic resistance genes (puromycin, hygromycin, or G418)

For highest yield, consider using an optimized vector containing regulatory elements upstream of the target gene, which has been shown to significantly increase recombinant protein expression compared to control vectors .

What are the structural characteristics of rat REEP6 compared to other species?

Rat REEP6 shares significant homology with mouse and human orthologs, with highest conservation in the transmembrane domains. The protein contains:

• N-terminal cytoplasmic domain

• Multiple transmembrane domains

• C-terminal region important for protein interactions

Comparative sequence analysis shows that the N-terminal region is particularly important for receptor interactions, as demonstrated by chimeric receptor studies with CXCR1 and CXCR2 .

Note: Rat REEP6 exhibits multiple bands on Western blots due to potential post-translational modifications .

How does REEP6 interact with G-protein coupled receptors (GPCRs) at the molecular level?

The molecular interaction between REEP6 and GPCRs, particularly CXCR1, involves specific binding domains that demonstrate receptor selectivity. Research indicates that:

• REEP6 binds to CXCR1 but not CXCR2, despite their structural similarities

• The N-terminal region of CXCR1 is critical for interaction with REEP6, as demonstrated through chimeric receptor studies

• The interaction persists throughout receptor activation and internalization

Methodologically, researchers should approach this question using:

• Site-directed mutagenesis to identify critical residues

• FRET/BRET assays to study real-time interactions

• Advanced microscopy techniques to visualize co-localization

• Structural biology approaches including cryo-EM

The co-immunoprecipitation data shows that both REEP5 and REEP6 co-precipitate with CXCR1 but not with CXCR2, demonstrating specificity in these interactions .

What signaling pathways are affected by REEP6 depletion in cellular models?

REEP6 depletion significantly impacts several signaling pathways downstream of CXCR1 activation:

• ERK Phosphorylation: IL-8-stimulated ERK phosphorylation is markedly reduced in REEP6-deficient cells, although not completely abolished

• Calcium Signaling: Intracellular calcium release following IL-8 stimulation is shortened and diminished in cells lacking REEP6

• β-arrestin Recruitment: REEP6 depletion impairs ligand-stimulated β-arrestin clustering and receptor internalization

• Phospholipase C Activation: Reduced inositol phosphate production in REEP6-deficient cells indicates compromised PLC pathway activation

For methodological investigation, researchers should:

• Use shRNA or CRISPR/Cas9 to generate REEP6-knockout cell lines

• Employ calcium imaging with fluorescent indicators

• Conduct phospho-specific western blotting

• Use reporter gene assays for downstream transcriptional activity

How can I optimize co-expression of REEP6 with its interacting GPCRs for functional studies?

For optimal co-expression of REEP6 with interacting GPCRs like CXCR1, implement these methodological strategies:

• Vector optimization:

  • Include Kozak sequences upstream of both genes

  • Utilize a bicistronic vector with IRES or 2A peptide sequences

  • Consider dual promoter systems for balanced expression

• Cell line selection and modification:

  • Use CHO cells with CRISPR/Cas9 modified apoptotic pathways for enhanced protein production

  • Consider stable cell lines derived from single clones for consistency

• Expression validation:

  • Confirm co-expression using co-immunoprecipitation

  • Verify subcellular localization with confocal microscopy

  • Assess functional expression through ligand-binding assays and downstream signaling readouts

What are the phenotypic consequences of REEP6 knockout in in vivo models?

REEP6 knockout in cellular and animal models reveals significant phenotypic consequences with important implications for physiological functions:

• Cellular proliferation: Cells deficient in REEP6 show reduced growth rates, particularly evident in IL-8-dependent growth assays

• Metastatic potential:

  • Lung cancer cells lacking REEP6 exhibit decreased invasion capacity

  • In xenograft models, REEP6-deficient tumor cells show:

    • Reduced primary tumor growth

    • Markedly decreased metastatic burden in lungs

• Gene expression profiles:

  • REEP6 deficiency blocks IL-8-induced expression of metastasis-related genes

  • Affected genes include uPA, uPAR, MMP2, MMP9, and inflammatory cytokines

Methodologically, researchers should approach in vivo studies through:

• CRISPR/Cas9 knockout in cell lines prior to xenografting

• Conditional knockout models for tissue-specific analysis

• Comprehensive histological and molecular analysis of affected tissues

• Quantitative assessment of tumor volume, weight, and metastatic nodules

Experimental data from NOD/SCID mice models demonstrated that A549 cells lacking REEP6 produced significantly smaller tumors with reduced metastatic capacity compared to control cells .

What are the optimal conditions for expressing and purifying recombinant rat REEP6?

For optimal expression and purification of recombinant rat REEP6, implement this methodological workflow:

• Expression system selection:

  • Mammalian: CHO cells are recommended for proper post-translational modifications

  • Bacterial: E. coli systems may be suitable for structural studies of soluble domains

• Vector optimization:

  • Include Kozak sequence and leader peptide for enhanced expression

  • Consider adding purification tags (His, FLAG, GST) at N or C-terminus

• Cell culture optimization:

  • Use engineered cell lines with apoptotic pathway modifications for increased yield

  • Implement fed-batch culture systems with optimized media formulations

• Purification strategy:

  • Membrane extraction using appropriate detergents (DDM, CHAPS, etc.)

  • Affinity chromatography using tag-specific resins

  • Size exclusion chromatography for final polishing

• Quality control:

  • Western blot for identity confirmation

  • Mass spectrometry for integrity analysis

  • Functional assays to confirm biological activity

Vector optimization studies have demonstrated that adding regulatory elements upstream of target genes can significantly increase recombinant protein expression in CHO cells .

How can I detect protein-protein interactions between REEP6 and GPCRs in living cells?

To effectively detect and measure protein-protein interactions between REEP6 and GPCRs in living cells, employ these methodological approaches:

• Resonance energy transfer techniques:

  • FRET (Fluorescence Resonance Energy Transfer): Tag REEP6 and GPCR with compatible fluorophores (CFP/YFP or GFP/RFP pairs)

  • BRET (Bioluminescence Resonance Energy Transfer): Use Renilla luciferase-tagged REEP6 with YFP-tagged GPCRs

• Proximity ligation assays (PLA):

  • Label proteins with primary antibodies

  • Use DNA-conjugated secondary antibodies that generate fluorescent signal when in proximity

• BiFC (Bimolecular Fluorescence Complementation):

  • Split fluorescent protein fragments fused to REEP6 and GPCR

  • Protein interaction brings fragments together to reconstitute fluorescence

• Live-cell imaging techniques:

  • Confocal microscopy for co-localization analysis

  • TIRF microscopy for membrane-specific interactions

  • Single-molecule tracking for dynamic interaction analysis

Experimental evidence shows that REEP6 partially co-localizes with CXCR1-GFP after IL-8 treatment, suggesting involvement in receptor trafficking .

What strategies can overcome common challenges in functional studies of recombinant REEP6?

When conducting functional studies with recombinant REEP6, researchers encounter several challenges that can be addressed through these methodological solutions:

• Protein solubility and stability issues:

  • Optimize detergent selection for membrane protein extraction

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • Use glycerol or specific stabilizing additives in buffers

• Low expression levels:

  • Implement vector optimization with regulatory elements

  • Consider codon optimization for the expression system

  • Explore engineered cell lines with reduced apoptotic pathways

• Functional assessment challenges:

  • Develop specific activity assays measuring GPCR signaling

  • Implement cell-based reporter systems with SRE-luc constructs

  • Use inositol phosphate production assays to measure downstream signaling

• Interacting partner identification:

  • Use proximity-dependent biotinylation (BioID, TurboID)

  • Implement crosslinking strategies followed by mass spectrometry

  • Conduct systematic screening with receptor chimeras to identify binding domains

For downstream signaling analysis, studies have successfully used SRE-luc reporter assays and measurement of ERK phosphorylation to quantify REEP6 enhancement of CXCR1 signaling .

How do I troubleshoot inconsistent results in REEP6 expression experiments?

When encountering inconsistent results in REEP6 expression experiments, implement this systematic troubleshooting approach:

• Vector-related issues:

  • Verify sequence integrity through sequencing

  • Confirm promoter activity with reporter assays

  • Add regulatory elements (Kozak, leader sequences) to enhance expression

• Cell line considerations:

  • Monitor cell health and passage number

  • Evaluate transfection efficiency with reporter genes

  • Consider engineering cell lines to reduce apoptotic responses

• Expression detection problems:

  • Validate antibody specificity with positive/negative controls

  • Use multiple detection methods (Western blot, immunofluorescence)

  • Consider epitope tag addition if antibody detection is problematic

• Functional variability:

  • Standardize ligand concentrations and treatment times

  • Control for endogenous REEP expression with RT-PCR screening

  • Implement clonal selection for stable cell lines

Research has demonstrated that even in control conditions, REEP5 and REEP6 are endogenously expressed in many cell lines, which may contribute to baseline variability .

How can I differentiate between the roles of REEP6 and other REEP family proteins in GPCR signaling?

To effectively differentiate between the functions of REEP6 and other REEP family members in GPCR signaling, employ these methodological approaches:

• Selective knockdown/knockout strategies:

  • Use specific shRNAs targeting individual REEP family members

  • Implement CRISPR/Cas9 to generate single and combined knockouts

  • Apply rescue experiments with shRNA-resistant constructs to confirm specificity

• Interaction specificity analysis:

  • Conduct systematic co-immunoprecipitation with different REEPs and GPCRs

  • Create chimeric REEP proteins to map interaction domains

  • Implement competitive binding assays to assess relative affinities

• Functional assessment:

  • Compare receptor-specific signaling (SRE-luc, ERK phosphorylation, calcium flux)

  • Analyze receptor trafficking patterns with different REEPs

  • Evaluate ligand binding dynamics in cells expressing different REEPs

• Expression profiling:

  • Conduct RT-PCR to determine tissue-specific expression patterns of REEP family members

  • Use qPCR to quantify relative expression levels in different cell types

Research has demonstrated functional cooperation between REEP5 and REEP6, with evidence that they bind to each other, suggesting they work together in receptor regulation .

What controls are essential when studying REEP6 effects on receptor trafficking and signaling?

When investigating REEP6 effects on receptor trafficking and signaling, include these essential controls to ensure experimental validity:

• Expression controls:

  • Empty vector transfection to control for transfection effects

  • Irrelevant protein expression (GFP alone) as negative control

  • Western blot confirmation of REEP6 knockdown/overexpression

• Receptor specificity controls:

  • Include CXCR2 (non-REEP6 interacting) alongside CXCR1 experiments

  • Test multiple GPCRs to determine interaction range

  • Use receptor chimeras to confirm binding domain specificity

• Signaling pathway controls:

  • Direct G-protein activators (e.g., AlF4-) to bypass receptor

  • Pathway-specific inhibitors to confirm signaling specificity

  • Dose-response curves for ligands to assess potency and efficacy

• Trafficking controls:

  • Use temperature blocks to synchronize trafficking

  • Include non-internalizing receptor mutants

  • Employ known trafficking inhibitors (dynamin inhibitors, etc.)

In experimental studies, CXCR2 serves as an excellent negative control, as it does not interact with REEP6 despite structural similarities to CXCR1, allowing clear differentiation of REEP6-specific effects .

How might REEP6 be leveraged for improving recombinant GPCR production systems?

REEP6 presents significant potential for enhancing recombinant GPCR production systems through these implementable approaches:

• Co-expression strategies:

  • Develop dual expression vectors containing both REEP6 and target GPCRs

  • Implement stable cell lines constitutively expressing optimal REEP6 levels

  • Create expression systems with tunable REEP6 expression to optimize GPCR:REEP6 ratios

• Cell line engineering:

  • Combine REEP6 overexpression with apoptotic pathway modifications

  • Engineer cells with optimized secretory pathways

  • Implement cell lines with enhanced post-translational modification capacity

• Fusion protein approaches:

  • Design REEP6-GPCR fusion constructs for certain applications

  • Create split constructs allowing reversible association

• Application-specific optimizations:

  • For structural biology: Focus on enhancing proper folding rather than signaling

  • For functional screening: Optimize signal-to-noise in receptor activation assays

  • For drug discovery: Enhance surface expression and stability

Research indicates that REEP6 enhances functional expression without significantly altering membrane localization of receptors, suggesting a role in optimizing receptor conformation or coupling efficiency rather than trafficking .

What potential therapeutic applications might target the REEP6-GPCR interaction?

The REEP6-GPCR interaction presents several potential therapeutic applications based on current understanding:

• Cancer therapeutics:

  • Studies show REEP6 depletion reduces tumor growth and metastasis

  • REEP6 inhibition could potentially sensitize tumors to conventional therapies

  • Targeting REEP6-CXCR1 interaction might reduce IL-8-driven cancer progression

• Anti-inflammatory approaches:

  • Given the role in CXCR1 signaling, REEP6 modulation could affect neutrophil recruitment

  • Potential applications in inflammatory conditions where IL-8 plays a role

• Precision medicine strategies:

  • Cancer profiling for REEP6 expression to predict metastatic potential

  • Personalized therapy selection based on REEP6-dependent signaling pathways

• Drug discovery enhancement:

  • Development of screening systems incorporating REEP6 for more physiologically relevant GPCR drug discovery

  • Creation of cell-based assays with optimized signal-to-noise ratios

Experimental evidence from xenograft models demonstrated that A549 lung cancer cells lacking REEP6 showed significantly reduced primary tumor growth and dramatically decreased metastatic spread to lungs, highlighting the potential therapeutic value of targeting this pathway .

How do post-translational modifications affect REEP6 function in different cellular contexts?

The impact of post-translational modifications (PTMs) on REEP6 function represents an important area for investigation:

• Potential phosphorylation:

  • REEP6 may be regulated by kinases downstream of receptor activation

  • Phosphorylation could modulate protein-protein interactions or subcellular localization

  • Time-course studies might reveal dynamic regulation during receptor signaling

• Ubiquitination and protein stability:

  • REEP6 turnover may be regulated by the ubiquitin-proteasome system

  • Stability might influence the duration of receptor signaling enhancement

• Glycosylation considerations:

  • Expression systems must maintain appropriate glycosylation for functional studies

  • CHO cells provide suitable mammalian glycosylation patterns for recombinant expression

• Methodological approaches:

  • Mass spectrometry to identify PTM sites

  • Site-directed mutagenesis of potential modification sites

  • Treatment with kinase/phosphatase inhibitors to assess regulatory roles

  • Pulse-chase experiments to determine protein half-life