RARRES2 Human, Sf9

What is RARRES2 and what are its primary functions?

RARRES2 (Retinoic Acid Receptor Responder Protein 2) is a 14 kDa protein encoded by the RARRES2 gene that is upregulated by the synthetic retinoid tazarotene. It's also known as chemerin or tazarotene-induced gene 2 protein (TIG2). RARRES2 functions primarily as a growth inhibitory and cell differentiation protein, making it valuable for treating hyperproliferative dermatological diseases .

RARRES2 acts as a ligand for multiple G protein-coupled receptors, including CMKLR1, GPR1, and CCRL2. It stimulates chemotaxis of dendritic cells and macrophages to sites of inflammation. Additionally, RARRES2 plays important roles in lipolysis, adipocyte differentiation, and glucose uptake, earning classification as an adipokine. Studies have demonstrated its significance in the pathogenesis of obesity and insulin resistance .

How is RARRES2 structurally organized and processed?

RARRES2 is initially synthesized as a 163-amino acid preprotein. It undergoes proteolytic processing to generate bioactive forms. The most active form results from cleavage of the N-terminal 20-amino acid signal peptide and the C-terminal 6-amino acid segment, producing the chemerin(21-157) isoform. This processed form serves as the endogenous ligand for activating receptors, particularly CMKLR1 .

Multiple isoforms of chemerin exist due to cleavage at different sites in the C-terminal region. These isoforms have been detected in vivo with varying potencies in activating CMKLR1 compared to the 21-157 isoform . The C-terminal nonapeptide (YFPGQFAFS, referred to as chemerin9 or C9) retains significant biological activity and is frequently used in research .

Which receptors interact with RARRES2 and how do they differ functionally?

Three chemerin receptors have been identified that interact with RARRES2:

• CMKLR1 (ChemR23): Responds to both full-length chemerin and C9 peptide by activating the Gi protein pathway and the β-arrestin pathway. CMKLR1 belongs to a family of Gi-coupled chemoattractant G protein-coupled receptors in the γ-subgroup of Class A GPCRs .

• GPR1: Capable of Gi signaling but exhibits very weak β-arrestin signaling when activated by chemerin or C9. It was initially identified as an orphan receptor before being recognized as a chemerin receptor .

• CCRL2: Binds chemerin but does not mediate transmembrane signaling, suggesting a different biological role than the other receptors .

These functional differences indicate distinct physiological roles for each receptor in mediating chemerin's effects.

What structural insights have been gained about RARRES2-receptor interactions using Sf9 expression systems?

Cryo-EM studies using Sf9-expressed proteins have revealed critical structural insights about RARRES2-receptor interactions. High-resolution cryo-EM structures of GPR1-Gi complexes bound to both full-length chemerin and the C9 peptide demonstrated distinct binding mechanisms :

• C9 peptide inserts directly into a transmembrane binding pocket

• Full-length chemerin uses its N-terminal globular core for extensive interaction with the N-terminus of GPR1

For CMKLR1, a cryo-EM structure of the CMKLR1-Gi-scFv16 complex with chemerin9 was determined to 2.94 Å resolution. This structure allowed unambiguous modeling of all 9 amino acids of chemerin9 (Tyr149 to Ser157), providing molecular details about the receptor's binding pocket .

How does chemerin9 affect macrophage phenotype in research models?

Chemerin9 induces complex phenotypic changes in macrophages that don't align with the traditional M1/M2 classification paradigm. When stimulated by chemerin9, primary human macrophages showed:

• Significantly decreased levels of CD206 and CD163 (similar to IFNγ+LPS effects)

• No up-regulation of HLA-DR or CD86 (unlike typical pro-inflammatory responses)

• Slightly lowered levels of HLA-DR and CD86 compared to IL-10 stimulation (suggesting some anti-inflammatory properties)

These findings indicate that the chemerin9-CMKLR1 signaling axis induces a unique macrophage phenotype requiring further investigation to fully understand its role in regulating inflammation.

What therapeutic potential does RARRES2/chemerin research demonstrate?

Chemerin9 has shown positive therapeutic effects in several animal disease models:

• Cardiovascular diseases

• Memory impairment

• Diabetes

Recent studies provide evidence that CMKLR1 activation mediates the protective effects of ω3-polyunsaturated fatty acids (PUFAs) in conditions including:

• Aortic valve stenosis

• Atherosclerosis

• Pulmonary hypertension

• Depression

These effects appear to involve resolvin E1 (RvE1), a specialized pro-resolvin lipid mediator suggested to act on CMKLR1 to promote the resolution of inflammation. The structural insights gained from studying these receptor-ligand complexes could facilitate the development of synthetic small molecule agonists mimicking the action of chemerin9 .

What is the optimal protocol for expressing human RARRES2 and its receptors in Sf9 cells?

Based on published protocols, an effective approach for expressing human RARRES2 and its receptors in Sf9 cells involves:

• Vector preparation:

  • Clone human RARRES2 into a pFastBac vector (excluding the last six amino acids for optimal activity)

  • For receptors like GPR1, include an N-terminal HA signal peptide, FLAG tag, HRV-3C protease cleavage site, and BRIL fusion protein

  • For G-protein complex components, use dominant negative Gαi1 (DNGαi1) with G203A and A326S substitutions, along with His-tagged Gβ1 and Gγ2

• Expression conditions:

  • Culture Sf9 cells in SIM SF Expression Medium to a density of 3.5 × 10^6 cells/mL

  • Co-infect cells with baculoviruses at optimized ratios:

    • 1:4:2 (receptor:G-protein:β/γ subunits) for C9-receptor-Gi complexes

    • 1:2:4:2 (receptor:chemerin:G-protein:β/γ subunits) for chemerin-receptor-Gi complexes

  • Harvest cells after 60 hours of infection by centrifugation at 2,000 × g for 15 minutes

  • Store frozen at -80°C until purification

What purification strategy yields high-quality RARRES2-receptor complexes for structural studies?

The following purification strategy has proven effective for obtaining high-quality RARRES2-receptor complexes:

• Membrane protein extraction:

  • Solubilize membrane proteins using lauryl maltose neopentyl glycol (LMNG) with cholesterol hemisuccinate (CHS)

• Affinity purification:

  • Use anti-FLAG M1 resin for immunoaffinity chromatography

  • Apply sequential washes with decreasing detergent concentrations while maintaining ligand presence (e.g., 4 μM C9 peptide)

  • Use specific wash buffers with precise component ratios:

    • W1: 20 mM HEPES pH 7.5, 0.5% LMNG, 0.05% CHS, 100 mM NaCl, 2 mM CaCl2

    • W2: 20 mM HEPES pH 7.5, 0.2% LMNG, 0.02% CHS, 100 mM NaCl, 2 mM CaCl2

  • Mix W1 and W2 in specific ratios: 5:5, 2:8, 1:9, 0.5:9.5, and 0:10

• Elution and final preparation:

  • Elute with buffer containing 20 mM HEPES pH 7.5, 0.01% LMNG, 0.002% CHS, 100 mM NaCl, 4 μM C9 peptide, 5 mM EDTA, and 0.2 mg/ml FLAG peptide

How should researchers optimize cryo-EM for structural analysis of RARRES2-receptor complexes?

Optimizing cryo-EM for RARRES2-receptor complexes involves several critical steps:

• Sample preparation:

  • Stabilize the complex by including antibody fragments (e.g., scFv16) to support the G-protein heterotrimer

  • Maintain ligand presence throughout sample preparation

• Data collection parameters:

  • Magnification: 105,000×

  • Voltage: 300 kV

  • Electron exposure: 56 e^-/Å^2

  • Defocus range: -1.0 to -1.8 μm

  • Pixel size: 0.848 Å

• Image processing workflow:

  • Apply patch motion correction and patch CTF estimation

  • Auto-pick particles (2-3 million initial particles)

  • Perform 2D classification

  • Conduct ab initio reconstruction

  • Execute multiple rounds of heterogeneous refinements

  • Select highest quality particles (100,000-300,000)

  • Perform non-uniform refinement and local refinement

• Model building and refinement:

  • Use predicted structures from AlphaFold as initial models

  • Apply coordinates from related complexes (e.g., CMKLR1-Gi-Scfv16) as templates

  • Dock models into EM density maps using UCSF Chimera

  • Perform iterative manual building in Coot and refinement in Phenix

What functional assays can validate RARRES2-receptor interactions in research?

Several complementary functional assays can validate RARRES2-receptor interactions:

• BRET (Bioluminescence Resonance Energy Transfer) assays:

  • Transfect HEK293T cells with receptor and NanoLuc-tagged Gαi

  • Load cells with coelenterazine H (50 μM)

  • Measure baseline using a multimode plate reader

  • Add ligands (full-length chemerin or C9 peptides) at various concentrations

  • Record luminescence signals 15 minutes after ligand addition

  • Calculate fold changes normalized to PBS-treated signals

  • Determine EC50 values based on concentration-response curves

• Macrophage phenotyping assays:

  • Treat primary human macrophages with chemerin9

  • Include appropriate controls (IFNγ+LPS for M1-like, IL-10 for M2-like phenotypes)

  • Measure expression of cell surface markers by flow cytometry:

    • HLA-DR and CD86 (pro-inflammatory markers)

    • CD206 and CD163 (anti-inflammatory markers)

  • Compare phenotypic changes to established paradigms

How do the cryo-EM parameters for RARRES2-receptor complexes compare to other GPCR studies?

The parameters used for the Chemerin9-CMKLR1-Gi complex analysis fall within optimal ranges for high-resolution GPCR structural studies, allowing detailed molecular insights into receptor-ligand interactions .

What are the challenges of working with RARRES2 in Sf9 expression systems?

When working with RARRES2 in Sf9 expression systems, researchers face several challenges:

• Protein processing differences: The proteolytic processing of RARRES2 may differ between insect and mammalian cells, potentially affecting the generation of bioactive forms.

• Complex stabilization: Maintaining the stability of RARRES2-receptor-G protein complexes during expression and purification requires careful optimization of conditions, including detergent selection and ligand presence.

• Co-expression ratios: Finding the optimal ratios for co-expressing multiple proteins (RARRES2, receptors, G-proteins) is critical for complex formation but requires extensive empirical testing.

• Post-translational modifications: While Sf9 cells can perform many eukaryotic post-translational modifications, some human-specific modifications may be absent or altered, potentially affecting protein function.

• Functional validation: Ensuring that Sf9-expressed RARRES2 and its receptors retain proper functionality requires comprehensive validation using multiple complementary assays .

What unexplored aspects of RARRES2 biology could benefit from Sf9 expression studies?

Several promising research directions could leverage Sf9 expression systems to advance RARRES2 biology:

• Structure-based drug design: The high-resolution structures obtained from Sf9-expressed proteins provide templates for designing small molecule modulators of RARRES2-receptor interactions.

• Receptor selectivity mechanisms: Further structural studies could elucidate the molecular basis for the differential signaling observed between CMKLR1, GPR1, and CCRL2.

• Isoform-specific activities: Expression and structural characterization of different RARRES2 isoforms could help understand their distinct biological roles.

• Resolvin E1 interaction mechanisms: Clarifying whether RvE1 directly binds to and activates CMKLR1 to induce Gi signaling would advance understanding of inflammation resolution.

• Small molecule agonist development: As noted in the research, "no small-molecule agonists of CMKLR1 have been reported so far," making this an important avenue for therapeutic development .