Recombinant Mouse Probable G-protein coupled receptor 141 (Gpr141)

What is GPR141 and what is its classification within the GPCR family?

GPR141 is an orphan G protein-coupled receptor belonging to the class A receptor family. It functions primarily as a negative regulator of immune responses by controlling the functions of monocytes and dendritic cells. The receptor is expressed at high levels in myeloid-lineage cells, including neutrophils (CD11b+ Gr1+), monocytes (CD11b+ Gr1-Ly6C+ and CD11b+ Gr1-Ly6C-), macrophages (F4/80+), and dendritic cells (CD11c+). As a member of the GPCR superfamily, it shares structural features with other Class A receptors, though its ligand remains unidentified, thus its "orphan" designation .

What cellular processes does GPR141 regulate in normal physiological conditions?

Under normal physiological conditions, GPR141 plays a critical role in regulating immune responses through its expression in myeloid-lineage cells. Research indicates that GPR141 functions as a negative regulator of immune responses by controlling the functions of monocytes and dendritic cells. In steady-state conditions, Gpr141-/- mice display almost no abnormalities in myeloid cell differentiation and compartmentalization in the spleen and bone marrow, suggesting that while GPR141 is present in these cells, its primary function may become more apparent during inflammatory or pathological conditions rather than during homeostasis .

What are the major experimental models available for studying GPR141 function?

Several experimental models have been developed for studying GPR141 function, with the primary ones being:

• Knockout mouse models: Gpr141-/- mice have been independently generated to study the effects of GPR141 deficiency on immune responses and disease progression .

• Cell line overexpression and knockdown systems: Human breast cancer cell lines (MCF-7, MDA-MB-231, ZR-75-1) with GPR141 overexpression or siRNA-mediated knockdown have been used to study the role of GPR141 in cancer progression .

• Recombinant protein systems: Recombinant Mouse GPR141 Protein pre-coupled magnetic beads are available for immunoassay, in vitro diagnostics, cell sorting, immunoprecipitation/co-precipitation, and protein/antibody separation and purification .

• Experimental autoimmune encephalomyelitis (EAE) model: This model for multiple sclerosis has been used to study the role of GPR141 in autoimmune diseases of the central nervous system .

How does GPR141 deficiency affect experimental autoimmune encephalomyelitis progression and what are the cellular mechanisms involved?

GPR141 deficiency significantly exacerbates the disease conditions of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model for multiple sclerosis. The mechanistic pathway involves multiple cellular components and inflammatory processes:

• Enhanced inflammatory cell infiltration: Gpr141-/- mice show increased infiltration of CD11b+ Gr1+ neutrophils, CD11b+ Gr1- monocytes, CD11c+ dendritic cells, and CD4+ T cells into the EAE-induced spinal cord compared with littermate control mice .

• Increased pro-inflammatory cytokine production: Lymphocytes enriched from Gpr141-/- mice immunized with myelin oligodendrocyte glycoprotein 35-55 produce higher amounts of interferon-γ (IFN-γ), interleukin-17A (IL-17A), and interleukin-6 (IL-6) compared with those from wild-type mice .

• Enhanced dendritic cell function: CD11c+ dendritic cells purified from Gpr141-/- mice increased cytokine production of myelin oligodendrocyte glycoprotein 35-55-specific T cells, suggesting that GPR141 normally restrains DC-mediated T cell activation .

These findings collectively demonstrate that GPR141 functions as a negative regulator of immune responses by controlling the functions of monocytes and dendritic cells, particularly in the context of autoimmune inflammation in the central nervous system.

What signaling pathways are modulated by GPR141 in myeloid cells during inflammatory responses?

While the complete signaling cascade for GPR141 in myeloid cells has not been fully elucidated, current research indicates several key pathways that are modulated during inflammatory responses:

• Cytokine production regulation: GPR141 appears to suppress the production of pro-inflammatory cytokines including IFN-γ, IL-17A, and IL-6 in response to antigenic stimulation, suggesting involvement in cytokine signaling pathways .

• Dendritic cell activation pathways: GPR141 regulates the ability of dendritic cells to activate antigen-specific T cells, possibly through modulation of co-stimulatory molecule expression or antigen presentation machinery .

• Cell migration and infiltration mechanisms: Given the increased infiltration of myeloid cells into the CNS in Gpr141-/- mice during EAE, GPR141 likely regulates chemokine signaling or adhesion molecule expression that controls cell migration during inflammation .

Understanding these pathways is critical for developing therapeutic interventions targeting GPR141 for treating chronic inflammatory and autoimmune diseases.

How does aberrant GPR141 expression contribute to breast cancer progression and metastasis?

Aberrant expression of GPR141 in different breast cancer subtypes correlates with poor prognosis and contributes to cancer progression through several interconnected molecular mechanisms:

• Enhanced migratory behavior: Increased GPR141 expression enhances the migratory behavior of breast cancer cells, contributing to metastatic potential .

• Activation of epithelial-to-mesenchymal transition (EMT): GPR141 overexpression drives oncogenic pathways both in vitro and in vivo through activation of EMT, a critical process for cancer cell invasion and metastasis .

• Regulation of p-mTOR/p53 signaling axis: GPR141 overexpression leads to p53 downregulation and activation of p-mTOR1 and its substrates in breast cancer cells, accelerating tumorigenesis .

• Cell cycle dysregulation: GPR141 overexpression in breast cancer cells downregulates cell cycle inhibitors (p27, p21) and enhances G1 to S phase transition, promoting cellular proliferation as evidenced by increased expression of proliferative markers (CyclinD1, CyclinD3) .

• Enhanced colony formation: Breast cancer cells overexpressing GPR141 show increased colony formation capacity, demonstrating enhanced clonogenic potential .

These findings collectively identify GPR141 as a stimulator of breast tumorigenesis and metastasis, making it a potential target for breast cancer therapeutics .

What is the molecular mechanism by which GPR141 regulates p53 and mTOR signaling in cancer cells?

The molecular mechanism by which GPR141 regulates p53 and mTOR signaling in cancer cells involves a complex interplay between ubiquitin-mediated protein degradation and signaling pathway activation:

• E3 ubiquitin ligase involvement: In GPR141 overexpressed cells, an E3 ubiquitin ligase called Cullin1 partly mediates p53 degradation via the proteasomal pathway, leading to reduced p53 levels .

• Formation of protein complexes: Co-immunoprecipitation studies have shown that the phosphorylated form of 40S ribosome protein S6 (pS6, a p-mTOR1 substrate) forms a complex with Cullin1, suggesting a direct interaction between the mTOR signaling pathway and the ubiquitin-proteasome system .

• Interplay between Cullin1 and p-mTOR1: The interaction between Cullin1 and p-mTOR1 in GPR141 overexpressed cells leads to downregulation of p53 expression, thus inducing tumor growth .

• Pathway reversal upon GPR141 silencing: Importantly, GPR141 silencing restores p53 expression and attenuates p-mTOR1 signaling events, thereby impeding proliferation and migration in breast cancer cells. This reversal suggests a direct and specific role of GPR141 in regulating these signaling pathways .

This mechanism provides important insights into how GPR141 functions as an oncogenic driver and suggests potential therapeutic strategies targeting this pathway.

What are the optimal techniques for detecting and quantifying GPR141 expression in mouse tissue samples?

Several complementary techniques are recommended for optimal detection and quantification of GPR141 expression in mouse tissue samples:

• Enzyme-Linked Immunosorbent Assay (ELISA): Commercial mouse GPR141 ELISA kits are available with a test range of 0.156 ng/ml - 10 ng/ml, suitable for tissue homogenates, cell lysates, and other biological fluids. For accurate results, sample concentrations must be diluted to mid-range of the kit .

• Quantitative PCR (qPCR): This technique allows for sensitive detection of GPR141 mRNA levels. Research has shown high GPR141 messenger RNA levels in myeloid-lineage cells, making qPCR valuable for expression studies .

• Western Blotting: Immunoblotting with specific anti-GPR141 antibodies allows for protein-level detection. This technique has been used successfully to demonstrate changes in GPR141 expression following overexpression or knockdown experiments .

• Immunohistochemistry/Immunofluorescence: These techniques allow for visualization of GPR141 expression in tissue sections, providing spatial information about expression patterns.

• Flow Cytometry: For single-cell analysis of GPR141 expression in heterogeneous cell populations, particularly in immune cells where GPR141 is differentially expressed.

It's important to note that detection kits are optimized for native samples rather than recombinant proteins, which may have different sequences or tertiary structures compared to the native protein .

What experimental approaches are most effective for studying GPR141 function in immune cells?

To effectively study GPR141 function in immune cells, researchers should consider the following experimental approaches:

• Gene knockout and knockdown studies:

  • Generation of Gpr141-/- mice for in vivo studies

  • siRNA or shRNA-mediated knockdown in primary immune cells or cell lines

• Ex vivo functional assays using cells from Gpr141-/- mice:

  • Cytokine production assays (ELISA, flow cytometry)

  • Antigen presentation assays with dendritic cells

  • Migration assays for monocytes and neutrophils

  • Proliferation assays for T cell activation

• Disease models:

  • Experimental autoimmune encephalomyelitis (EAE) for studying autoimmune responses

  • Infection models to assess innate immune function

  • Inflammatory models to evaluate myeloid cell recruitment and function

• Molecular interaction studies:

  • Immunoprecipitation to identify binding partners

  • Signaling pathway analysis using phospho-specific antibodies

  • Receptor-ligand binding assays (once a ligand is identified)

• Single-cell analysis techniques:

  • Single-cell RNA sequencing to identify cell-specific expression patterns

  • Mass cytometry for high-dimensional analysis of GPR141-expressing cells

These approaches, used in combination, can provide comprehensive insights into GPR141 function in different immune cell populations and contexts.

How can researchers effectively design experiments to identify potential ligands for the orphan GPR141 receptor?

Designing experiments to identify potential ligands for orphan receptors like GPR141 requires a systematic approach:

• In silico approaches:

  • Homology modeling to predict receptor structure

  • Virtual screening of compound libraries against the predicted binding site

  • Phylogenetic analysis to identify related receptors with known ligands

• High-throughput screening methods:

  • Cell-based reporter assays (measuring calcium flux, cAMP, or β-arrestin recruitment)

  • Binding assays using recombinant GPR141 protein pre-coupled magnetic beads

  • Functional assays monitoring downstream signaling activation

• Tissue and context-specific screening:

  • Screening tissue extracts from sites of high GPR141 expression

  • Testing candidate ligands in inflammatory contexts where GPR141 function is evident

  • Examining metabolites or signaling molecules produced during immune responses

• Reverse pharmacology approaches:

  • Overexpression of GPR141 in heterologous systems

  • Testing responses to candidate ligands based on phenotypic observations

  • Validation of hits through competition binding assays

• Experimental validation:

  • Dose-response studies with candidate ligands

  • Structure-activity relationship analysis

  • Validation in Gpr141-/- systems to confirm specificity

This multi-faceted approach increases the likelihood of identifying physiologically relevant ligands for GPR141.

What are the critical controls and variables to consider when studying the role of GPR141 in breast cancer models?

When studying GPR141 in breast cancer models, researchers should consider these critical controls and variables:

Critical Controls:

• Gene expression manipulation validation:

  • Confirmation of GPR141 overexpression or knockdown at both mRNA and protein levels

  • Use of appropriate vector-only controls for overexpression studies

  • Non-targeting siRNA controls for knockdown experiments

• Cell line selection:

  • Include multiple breast cancer cell lines representing different molecular subtypes (e.g., MCF-7, MDA-MB-231, ZR-75-1)

  • Include non-tumorigenic breast epithelial cell lines as controls

• Pathway analysis controls:

  • Include positive and negative controls for p-mTOR and p53 pathway analyses

  • Use of pathway inhibitors (e.g., mTOR inhibitors) to validate specificity

• In vivo model controls:

  • Age and sex-matched animals

  • Appropriate sham treatments

  • Littermate controls when using transgenic models

Key Variables to Consider:

• GPR141 expression levels:

  • Baseline expression across different breast cancer subtypes

  • Correlation with clinical outcomes and prognostic markers

• Cell characteristics:

  • Proliferation rate (measured through multiple assays)

  • Migration and invasion capacity

  • Colony formation ability

  • Cell cycle distribution

• Signaling pathway indicators:

  • p53 expression and its downstream targets (p21, p27)

  • Phosphorylation status of mTOR and its substrates

  • EMT markers (E-cadherin, vimentin)

  • Proliferative markers (CyclinD1, CyclinD3, CDK4)

• Experimental conditions:

  • Serum concentrations and culture conditions

  • Duration of experiments

  • Cell density and passage number

By carefully controlling these variables and including appropriate controls, researchers can generate more reliable and reproducible data on GPR141's role in breast cancer.

What approaches can be used to reconcile contradictory findings about GPR141 function in different biological contexts?

Reconciling contradictory findings about GPR141 function requires systematic approaches:

• Context-dependent analysis:

  • Compare experimental conditions across studies (cell types, disease models)

  • Assess GPR141 expression levels and subcellular localization in different contexts

  • Consider tissue-specific factors that might influence receptor function

  • Examine the inflammatory state of the system (homeostatic vs. inflammatory conditions)

• Comprehensive signaling pathway analysis:

  • Map GPR141 signaling in different cell types and conditions

  • Investigate potential crosstalk with other signaling pathways

  • Examine time-dependent signaling responses to capture dynamic effects

• Meta-analysis and systematic review:

  • Categorize findings by experimental approach and biological context

  • Identify methodological differences that might explain discrepancies

  • Evaluate statistical power and reproducibility of conflicting studies

• Integrated multi-omics approaches:

  • Combine transcriptomic, proteomic, and metabolomic data

  • Analyze GPR141-associated protein complexes in different contexts

  • Identify context-specific interaction partners

• Collaborative validation studies:

  • Design experiments that directly test competing hypotheses

  • Use standardized protocols across different laboratories

  • Share biological materials (cell lines, mouse models) to eliminate source variation

The apparent contradiction between GPR141's role as a negative regulator in immune cells versus its pro-tumorigenic role in breast cancer might reflect genuine biological complexity rather than experimental inconsistency. GPR141 may activate different downstream pathways depending on the cellular context, leading to distinct functional outcomes.

How can recombinant mouse GPR141 protein pre-coupled magnetic beads be utilized in immunoprecipitation experiments?

Recombinant mouse GPR141 protein pre-coupled magnetic beads offer several advantages for immunoprecipitation experiments:

• Protein-protein interaction studies:

  • The uniform particle size (~2 μm) and narrow size distribution provide a large surface area for capturing target molecules with high specificity

  • The hydrophilic bead surface minimizes non-specific binding while maintaining high capacity (>200 pmol rabbit IgG/mg beads)

  • These properties make the beads ideal for co-immunoprecipitation experiments to identify novel binding partners of GPR141

• Protocol optimization considerations:

  • Sample preparation: Cell lysates should be prepared using non-denaturing conditions to preserve protein-protein interactions

  • Binding conditions: Optimization of buffer composition, incubation time, and temperature to maximize specific interactions

  • Washing steps: Balanced to remove non-specific binding while preserving genuine interactions

  • Elution methods: Different elution strategies (pH, ionic strength, competitive elution) may be necessary depending on the nature of the interaction

• Applications in GPR141 research:

  • Identification of signaling complexes associated with GPR141

  • Validation of predicted protein interactions

  • Investigation of dynamic changes in protein interactions under different stimulation conditions

  • Purification of GPR141-containing protein complexes for further analysis

• Technical advantages:

  • Compatibility with automation equipment for high-throughput operations

  • Stability for at least 6 months under proper storage conditions (2-8°C, avoiding freeze-thaw cycles)

  • Ready-to-use format eliminates variability introduced by manual coupling procedures

This methodology can significantly advance our understanding of GPR141's molecular mechanisms by identifying its interaction partners in different cellular contexts.

What are the best practices for validating GPR141 knockout or knockdown models before using them in functional studies?

Thorough validation of GPR141 knockout or knockdown models is essential for reliable functional studies:

• Genetic validation:

  • Confirm disruption of the GPR141 gene by DNA sequencing in knockout models

  • Verify targeting strategy (e.g., exon deletion, frameshift mutation)

  • For CRISPR-based models, check for potential off-target effects

• Transcriptional validation:

  • Quantify GPR141 mRNA levels using qPCR with primers spanning different exons

  • Perform RT-PCR to detect potential alternative splicing events

  • For partial knockouts, measure the extent of transcript reduction

• Protein-level validation:

  • Confirm absence or reduction of GPR141 protein using Western blotting

  • Use ELISA to quantify GPR141 protein levels in tissue homogenates or cell lysates

  • Consider immunohistochemistry to verify loss of expression in specific cell types

• Functional validation:

  • Assess known downstream signaling pathways affected by GPR141

  • For immune cell models, examine cytokine production and cell function

  • For cancer cell models, evaluate proliferation, migration, and colony formation

• Phenotypic comparison with existing models:

  • Compare phenotypes with published GPR141 knockout models

  • Assess whether basic cellular compartmentalization and differentiation are intact

  • Evaluate specific phenotypes only upon relevant stimulation or disease induction

• Controls to include:

  • Wild-type controls from the same genetic background

  • Littermate controls whenever possible

  • For knockdown models, include non-targeting controls

  • Consider rescue experiments by re-introducing GPR141 expression