Recombinant Mouse G-protein coupled receptor 182 (Gpr182)

What is mouse Gpr182 and what functional role does it play in murine physiology?

Mouse Gpr182 is a G-protein coupled receptor that functions as an atypical chemokine receptor (ACKR), recently proposed to be designated as ACKR5. Unlike canonical chemokine receptors, Gpr182 acts primarily as a scavenger that binds and internalizes chemokines without inducing typical G-protein mediated signaling.

Methodologically, researchers have demonstrated that Gpr182:

• Is predominantly expressed in the endothelium of multiple organs including spleen, liver, and lymph nodes

• Exhibits high constitutive activity regarding β-arrestin recruitment and rapidly internalizes in a ligand-independent manner

• Negatively regulates definitive hematopoiesis by inhibiting hemogenic endothelium/hematopoietic stem cell (HE/HSC) formation

• Controls chemokine levels in serum and interstitial spaces by scavenging activity

Importantly, Gpr182 shares conserved elements with other ACKRs, such as a modified DRYLAIV motif (DRYVTLV in Gpr182) and the NPXXY sequence at the end of helix VII, though these modifications prevent typical G-protein coupling .

What is the chemokine binding profile of mouse Gpr182 and how does it differ from human GPR182?

Mouse Gpr182 exhibits a broad chemokine binding spectrum with varying affinities. Key methodological findings include:

High-affinity ligands for mouse Gpr182 (Ki ≤ 10 nM):

• CXCL10

• CXCL12

• CXCL13

• CCL28

Intermediate-affinity ligands (Ki 10-100 nM):

• CCL20 (Ki = 120 nM)

• CCL22, CCL24, CCL25, CCL27

Species-specific differences exist between mouse and human GPR182:

• Mouse XCL1, CCL1, CCL19, CXCL3, and CXCL16 do not bind mouse Gpr182, unlike in humans

• Human CCL28 has significantly lower affinity for human GPR182 than mouse CCL28 has for mouse Gpr182

Research techniques to determine binding profiles include competition binding assays at 4°C and scavenging assays at 37°C with fluorescently labeled chemokines .

What experimental models and tools are available for studying mouse Gpr182?

Several experimental models and tools have been developed for Gpr182 research:

Mouse models:

• Constitutive GPR182 knockout mice (GPR182-/-)

• GPR182 mCherry knock-in reporter mice (hemizygous maintains one functional allele)

• Inducible endothelium-specific GPR182-deficient mice

• GPR182-ACKR4 double knockout mice to study cooperative relationships

Cell lines and expression systems:

• Mouse 300.19 pre-B cells stably expressing Gpr182

• HEK293T cells for overexpression studies

• Tango assay system for studying β-arrestin recruitment

Molecular tools:

• GPR182-Tango plasmid (#66341) for expression in mammalian cells

• Fluorescently labeled chemokines for binding and internalization assays

• ACKR3 competitive agonist CCX771 for comparative studies

For specific methodological applications, researchers can use the PRESTO-Tango system for parallel receptor expression and screening via transcriptional output with activation following arrestin translocation .

How does Gpr182 deficiency affect hematopoiesis and myeloid cell development in mice?

Gpr182 functions as a negative regulator of definitive hematopoiesis, with knockout studies revealing:

Hematopoietic stem cell (HSC) effects:

• Gpr182-/- zebrafish and mice exhibit increased HSC formation during development

• Time-lapse confocal imaging of Tg(cmyb:EGFP); Tg(kdrl:Hras-mChrry) zebrafish showed increased numbers of HSCs in gpr182-/- embryos during endothelial-to-hematopoietic transition

• Global and endothelium-specific Gpr182-deficient mice display decreased HSCs in bone marrow and increased hematopoietic progenitors in blood and spleen

Myeloid lineage effects:

• GPR182 KO mice show increased myeloid cells, especially neutrophils (32.52% vs. 18.57% in wildtype)

• Corresponding decrease in lymphocytes in GPR182 KO mice (61.1% vs. 76.98% in wildtype)

• Transcriptomic analysis of gpr182-/- endothelial cells reveals upregulation of genes associated with definitive hematopoiesis and myeloid cell differentiation

Methodologically, these findings were established through flow cytometry of blood samples, whole-mount in situ hybridization (WISH), confocal imaging, and transcriptomic analysis .

What molecular mechanisms underlie Gpr182's function as an atypical chemokine receptor?

Gpr182 functions through distinct molecular mechanisms that differentiate it from canonical chemokine receptors:

Signaling properties:

• Lacks typical G-protein signaling upon ligand binding

• Fails to recruit mini Gαi-protein or activate heterotrimeric G-proteins upon chemokine stimulation

• Does not induce BRET signal reduction indicative of Gα-Gβγ dissociation

• Shows high constitutive β-arrestin recruitment independent of agonist stimulation

Internalization and trafficking mechanisms:

• Spontaneously traffics between plasma membrane and endosomes in a β-arrestin-dependent manner

• β-arrestin recruitment, internalization, and scavenging do not entirely rely on the C-terminus of the receptor

• Rapidly internalizes bound chemokines and targets them for degradation, effectively removing them from the extracellular space

Research techniques for studying these mechanisms include:

• Split luciferase luminescence assays for G-protein recruitment

• BRET assays for G-protein activation

• Confocal microscopy for visualization of receptor trafficking

• Co-immunoprecipitation for protein interaction studies

How does Gpr182 cooperate with other atypical chemokine receptors to regulate chemokine homeostasis?

Research has revealed important cooperative relationships between Gpr182 and other ACKRs:

Gpr182 and ACKR3 cooperation:

• Combined deficiency of Gpr182 and inhibition of ACKR3 produces an additive ~4-fold increase in serum CXCL12 levels, compared to 2-fold increase with either alone

• Treatment of GPR182-/- mice with the ACKR3 competitive agonist CCX771 demonstrates synergistic effects on CXCL12 levels

Gpr182 and ACKR4 cooperation:

• GPR182-/-ACKR4-/- double knockout mice show further increased CCL19 serum levels compared to single knockouts

• Gpr182 and ACKR4 differentially regulate CCL20: Gpr182 has higher affinity (120 nM) than ACKR4 (800 nM)

• CCL21 is primarily regulated by ACKR4, with double knockout showing no difference from ACKR4 single knockout

Methodologically, these relationships were investigated through:

• Serum chemokine measurements using ELISA

• Interstitial chemokine measurements in tissues

• Comparative analysis of single and double knockout mouse models

• In vivo administration of ACKR3 inhibitors

What is the relationship between Gpr182 and the leukotriene biosynthesis pathway?

Gpr182 has been linked to regulation of the leukotriene biosynthesis pathway, particularly leukotriene B4 (LTB4):

Molecular connections:

• Transcriptomic analysis of gpr182-/- endothelial cells revealed upregulation of the leukotriene biosynthesis pathway

• Cell-based small molecule screening and transcriptomic analyses identified a connection between Gpr182 and arachidonic acid metabolism

• Both cyclooxygenase and leukotriene biosynthesis (lipoxygenase) pathways are downstream of arachidonic acid metabolism and linked to inflammatory responses

Functional interactions:

• LTB4 treatment of HEK293T cells overexpressing GPR182 resulted in β-arrestin-1 recruitment at high concentrations (>1 μM)

• COX inhibition promotes GPR182-TANGO activation, suggesting an interaction between these pathways

• Loss of GPR182 function promotes upregulation of leukotriene biosynthesis

Methodological approaches included:

• DAVID pathway analysis of transcriptomic data

• β-arrestin recruitment assays

• Comparison of gene expression in bone marrow and spleen from GPR182 KO vs. wildtype mice

What emerging roles does Gpr182 play in cancer biology and tumor immunology?

Recent research has begun to uncover potential roles for Gpr182 in cancer, particularly in the context of tumor immunology:

Cancer-related expression patterns:

• Gpr182 is selectively upregulated in peritumoral lymphatics in melanoma models

• The receptor is expressed in tumor-associated lymphatics, suggesting involvement in cancer microenvironment regulation

Effects on tumor immunity:

• Ablation of Gpr182 in mice leads to increased effector T cell infiltration in tumors

• This results in retardation of tumor growth in multiple mouse melanoma models

• The mechanism appears to involve CXCR3, as blockade of this receptor completely abolished improved antitumor immunity in Gpr182-deficient mice

Potential therapeutic approaches:

• Gpr182 may represent a novel target for converting "cold" (non-T cell-inflamed) tumors to "hot" (T cell-inflamed) tumors

• Peptides blocking the interaction between Gpr182 and chemokines are being investigated for antitumor effects

• Targeting Gpr182 could potentially enhance immune checkpoint inhibitor therapy efficacy

These findings suggest Gpr182 inhibits anti-tumor immune responses by limiting chemokine availability in the tumor microenvironment, positioning it as a potential therapeutic target for cancer immunotherapy .

What expression systems and purification methods are optimal for producing recombinant mouse Gpr182?

For researchers working with recombinant mouse Gpr182, several expression and purification approaches have proven effective:

Mammalian expression systems:

• Mouse 300.19 pre-B cells using Amaxa Nucleofector transfection (Lonza) with self-cleaving peptide linked to GFP for monitoring expression levels

• HEK293T cells for overexpression studies and functional assays

• HeLa cells for G-protein and β-arrestin recruitment studies

Expression constructs:

• pME-Cas9-T2A-GFP plasmid system (#63155 - Addgene) allows receptor and GFP to be post-translationally split, enabling tracking of receptor expression via GFP fluorescence intensity

• GPR182-Tango plasmid (#66341 - Addgene) uses a CMV promoter with an N-terminal FLAG tag for detection and purification

Culture conditions:

• For mouse 300.19 pre-B cells: RPMI-1640 supplemented with 10% FBS, 1% PenStrep, 1% nonessential amino acids, 1% Glutamax, and 50 μM β-mercaptoethanol

• Selection with G418 for stable transfectants when using neomycin resistance marker

When designing expression systems, researchers should consider that both human and mouse Gpr182 have divergent extracellular N-termini (binding site 1) while the helical transmembrane domains (binding site 2) are mostly conserved, which may affect chemokine recognition and binding affinities .

What methodological approaches are most effective for studying Gpr182-chemokine interactions?

Several complementary methodological approaches can be used to characterize Gpr182-chemokine interactions:

Binding assays:

• Competition binding at 4°C: Incubate cells expressing Gpr182 with a fixed concentration of fluorescent chemokine (3 nM) and increasing concentrations of unlabeled competitor

• Calculate Ki values to determine binding affinities for different chemokines

Internalization/scavenging assays:

• Scavenging at 37°C: Measure uptake of fluorescently labeled chemokines (200 nM) after 45-minute incubation

• Flow cytometry to quantify internalization using GFP-expressing cells as a reference

In vivo studies:

• I.V. injection of fluorescently labeled chemokines into GPR182 heterozygous vs. knockout mice

• Analysis of chemokine localization in specific cell types (e.g., spleen endothelial cells)

• Co-injection of control chemokines (e.g., CXCL11_12 chimera) that bind other ACKRs for comparison

Serum and tissue chemokine measurements:

• ELISA-based quantification of chemokine levels in serum

• Measurement of interstitial chemokine levels in tissues (normalized to total protein content)

• Comparison between wildtype, heterozygous, and knockout animals

For optimal results, researchers should combine multiple approaches to establish both binding affinities and functional scavenging capabilities of Gpr182.

What genetically engineered mouse models are most informative for Gpr182 research?

Several genetically engineered mouse models have provided valuable insights into Gpr182 function:

Constitutive knockout models:

• GPR182-/- (complete knockout): Exhibits increased serum levels of multiple chemokines and altered hematopoiesis

• GPR182+/- (heterozygous): Important control showing that one copy of the gene is often sufficient for normal function

Reporter models:

• GPR182 mCherry knock-in reporter mouse: In hemizygous mice, mCherry replaces one GPR182 allele, enabling visualization of expression while maintaining function

• Tg(kdrl:Hsa.HRASmCherry): Labels endothelial cells for isolation and transcriptomic analysis

Conditional models:

• Inducible endothelium-specific GPR182-deficient mice: Allow temporal control of GPR182 deletion specifically in endothelial cells

Compound knockout models:

• GPR182-/-ACKR4-/- double knockout: Reveals cooperative relationships between different atypical chemokine receptors

• Can be combined with pharmacological inhibition (e.g., ACKR3 inhibitor CCX771) to study intersecting pathways

For developmental studies, zebrafish models have also proven valuable:

• gpr182-/- zebrafish

• Tg(cmyb:EGFP); Tg(kdrl:Hras-mChrry) for visualization of endothelial-to-hematopoietic transition

These models can be analyzed using flow cytometry, histology, confocal microscopy, and transcriptomic approaches to comprehensively assess Gpr182 function in different contexts.

What are the emerging therapeutic potentials of targeting mouse Gpr182 in disease models?

Several promising therapeutic applications for targeting Gpr182 are emerging:

Cancer immunotherapy:

• Gpr182 ablation increases effector T cell infiltration in melanoma models

• Blockade of Gpr182-chemokine interactions could convert "cold" tumors to "hot" tumors, potentially enhancing immunotherapy efficacy

• Peptide-based inhibitors that block Gpr182-chemokine interactions are being developed and optimized

Hematopoietic disorders:

• Given Gpr182's role as a negative regulator of hematopoiesis, targeting it could potentially enhance blood cell production in certain blood disorders

• Modulation of Gpr182 might affect the balance between myeloid and lymphoid differentiation

Inflammatory conditions:

• The connection between Gpr182 and leukotriene biosynthesis suggests potential applications in inflammatory diseases

• Targeting Gpr182 could modulate chemokine gradients and leukocyte trafficking during inflammation

Methodological approaches for exploring these therapeutic applications include:

• In vivo administration of peptide inhibitors in disease models

• Combination therapy with existing treatments (e.g., immune checkpoint inhibitors)

• Development of small molecule modulators based on structure-activity relationships

• Engineered biologics targeting Gpr182-expressing cells

What cutting-edge techniques are advancing our understanding of Gpr182 biology?

Several cutting-edge techniques are driving progress in Gpr182 research:

Advanced imaging techniques:

• Time-lapse confocal imaging of fluorescent reporter lines to track cellular processes in real-time (e.g., endothelial-to-hematopoietic transition)

• Visualization of chemokine uptake and trafficking in specific cell types in vivo

Single-cell technologies:

• Single-cell RNA sequencing to define cell-specific expression patterns and responses to Gpr182 modulation

• Single-cell proteomics to understand Gpr182's impact on cellular signaling networks

Structural biology approaches:

• Cryo-EM and X-ray crystallography to determine the three-dimensional structure of Gpr182 alone and in complex with chemokines

• Structure-based drug design to develop specific modulators

Advanced genetic engineering:

• CRISPR-Cas9 gene editing for precise manipulation of Gpr182 and related genes

• Site-specific mutagenesis to determine functional domains critical for chemokine binding and scavenging

Signaling pathway analysis:

• PRESTO-Tango system for parallel receptor screening via transcriptional output

• BRET and split luciferase assays to measure protein-protein interactions and signaling events

• Phosphoproteomics to map signaling networks downstream of Gpr182