Recombinant Human Probable G-protein coupled receptor 141 (GPR141)

Where is GPR141 predominantly expressed in human and mouse tissues?

GPR141 demonstrates a tissue-specific expression pattern primarily concentrated in cells of the immune system. Recent research indicates high GPR141 messenger RNA levels are expressed predominantly in myeloid-lineage cells, including:

• Neutrophils (CD11b+ Gr1+)

• Monocytes (CD11b+ Gr1-Ly6C+ and CD11b+ Gr1-Ly6C-)

• Macrophages (F4/80+)

• Dendritic cells (CD11c+)

This expression profile suggests specialized functions in immune regulation and potential involvement in inflammatory or immune response pathways. Interestingly, beyond the immune system, aberrant expression of GPR141 has been identified in different breast cancer subtypes, with particularly high alteration frequency observed in breast invasive mixed mucinous carcinoma (4% altered out of 25 cases) and breast invasive ductal carcinoma (2.23% in 1660 cases) .

What experimental approaches are available for studying GPR141 function?

Multiple experimental systems and reagents are available for investigating GPR141:

Genetic Models:

• Gpr141-/- knockout mice have been independently generated and used to study the role of GPR141 in immune responses and experimental autoimmune encephalomyelitis

• siRNA-mediated knockdown of GPR141 in cancer cell lines (e.g., ZR-75-1 cells) has been employed to assess its role in proliferation and migration

Expression Systems:
For recombinant protein production, various expression systems have been validated:

Plasmid Resources:

• GPR141-Tango plasmid (#66318) available through Addgene for PRESTO-Tango assays to study GPCR activation via arrestin translocation

Reagents:

• Commercial antibodies (unconjugated, HRP-conjugated, FITC-conjugated, biotin-conjugated) for detection of human GPR141

• Recombinant proteins (full-length and partial) for both human and mouse GPR141

How does GPR141 regulate immune responses in myeloid cells?

Recent research has demonstrated that GPR141 functions as a negative regulator of immune responses by controlling the functions of monocytes and dendritic cells. In an experimental autoimmune encephalomyelitis (EAE) model, which mimics aspects of multiple sclerosis, GPR141 plays a significant immunomodulatory role.

Key findings from studies with Gpr141-/- mice include:

These findings collectively suggest that targeting GPR141 may represent a potential therapeutic approach for modulating chronic inflammatory diseases through its effects on myeloid cell function and subsequent T cell activation.

What is the relationship between GPR141 expression and cancer progression?

Recent studies have identified a previously unknown role for GPR141 in cancer biology, particularly in breast cancer. Increased GPR141 expression has been associated with enhanced migratory behavior and tumor growth through several mechanisms:

• Correlation with poor prognosis: Aberrant expression of GPR141 in different breast cancer subtypes corresponds with poorer clinical outcomes

• Effects on epithelial-to-mesenchymal transition (EMT): GPR141 overexpression stimulates migration through:

  • Accumulation of mesenchymal markers (N-cadherin, Snail)

  • Reduction in epithelial markers (E-cadherin)

  • These changes were observed in both MCF-7 (Estrogen receptor positive) and MDA-MB-231 (triple-negative) breast cancer cell lines

• Impact on cell cycle progression: GPR141 overexpression leads to:

  • Upregulation of proliferative markers (CyclinD1, CyclinD3)

  • Downregulation of CDK4 and p53 downstream targets p27 and p21

  • Enhanced G1 to S transition (S phase acquiring 54.1% of the cell cycle compared to 21.1% in control MDA-MB-231 cells)

• Molecular mechanism involving p-mTOR/p53 axis:

  • GPR141 promotes breast cancer cell tumorigenesis via the p-mTOR/p53 pathway

  • Increased activation of p-mTOR1 and reduced p53 expression levels

  • E3 ubiquitin ligase Cullin1 partly mediates p53 degradation via proteasomal pathway

  • Phosphorylated form of 40S ribosome protein S6 (ps6, a p-mTOR1 substrate) forms a complex with Cullin1

• Reversal of effects through GPR141 silencing:

  • siRNA-mediated knockdown of GPR141 in ZR-75-1 cells showed increased E-cadherin expression

  • Enhanced p53 expression both at protein and transcript level

  • Decreased phosphorylation levels of p-mTOR1 substrates

These findings suggest that modulating GPR141 expression could provide a novel therapeutic approach for regulating breast cancer progression and metastasis.

What signaling pathways are modulated by GPR141?

• p-mTOR/p53 Pathway:

  • GPR141 overexpression increases activation of p-mTOR1 while reducing p53 expression

  • Treatment with rapamycin (p-mTOR1 inhibitor) restores p53 expression in GPR141 overexpressed cells

  • This suggests GPR141 promotes cancer cell proliferation through p53 degradation mediated by Cullin1 interaction and elevated p-mTOR1 levels

• EMT Signaling:

  • GPR141 drives tumorigenesis by E-cadherin degradation and MMP7 activation

  • This promotes the transition from epithelial to mesenchymal phenotype, enhancing migratory capacity

• Cell Cycle Regulation:

  • GPR141 modulates expression of cell cycle regulators including CyclinD1, CyclinD3, CDK4, p27, and p21

  • This leads to enhanced G1 to S transition and increased proliferative capacity

• Immune Signaling in Myeloid Cells:

  • GPR141 negatively regulates cytokine production in myeloid cells

  • Loss of GPR141 results in increased production of pro-inflammatory cytokines including interferon-γ, interleukin-17A, and interleukin-6

The identification of these pathways provides potential targets for therapeutic intervention in conditions where GPR141 signaling is dysregulated, such as in cancer or inflammatory diseases.

What are the optimal expression systems for producing recombinant GPR141 protein?

Multiple expression systems have been successfully employed for producing recombinant GPR141, each with distinct advantages depending on the intended application:

For GPR141 specifically, commercially available recombinant proteins have been produced in several systems:

• E. coli expression: Utilized for producing both full-length and partial GPR141 proteins with His-tags

  • Example: Recombinant Human Probable G-protein coupled receptor 141(GPR141) (1-305aa) with N-terminal His tag

  • Applications: Antibody production, immunogen preparation

• Mammalian expression: HEK293T cells used to produce GPR141 with tags such as C-Myc/DDK

  • Example: Recombinant protein of human GPR141 from HEK293T cells

  • Applications: Functional assays, interaction studies

For optimal results in structural studies, insect cell or mammalian expression systems are generally preferred for full-length GPR141 due to the complex transmembrane topology of GPCRs.

How can I validate GPR141 knockdown/knockout effects in cellular assays?

When studying the effects of GPR141 deletion or suppression, a comprehensive validation approach is essential. Based on published research methodologies, the following techniques have proven effective:

1. Molecular Validation:

• Western blotting: To confirm reduction in GPR141 protein expression

• qRT-PCR: To verify decreased GPR141 transcript levels

• Genomic PCR: For confirmation of gene targeting in knockout models

2. Functional Validation Assays:

For Cancer Cell Studies:

• Colony formation assay: To assess proliferative capacity

• Cell viability assay: MTT or similar assays to measure metabolic activity

• Cell cycle analysis by flow cytometry: To evaluate cell cycle progression

• Transwell migration assay: To assess migratory capacity

• Wound healing assay: To measure collective cell migration

For Immune Cell Studies:

• Cytokine production measurement: ELISA or flow cytometry-based detection of IFN-γ, IL-17A, IL-6

• Immune cell infiltration analysis: Flow cytometry to assess cell populations in tissues

• T cell activation assays: Co-culture of GPR141-deficient dendritic cells with antigen-specific T cells

3. Signaling Pathway Analysis:

• Phosphorylation status of mTOR pathway components: Western blotting for p-mTOR and downstream targets like phosphorylated S6

• p53 expression and downstream targets: Analysis of p21, p27 levels

• EMT marker expression: E-cadherin, N-cadherin, Snail levels

4. In vivo Validation:

• Disease models: Such as experimental autoimmune encephalomyelitis for immune function studies

• Xenograft tumor models: For cancer-related studies

A comprehensive validation approach using multiple techniques provides more robust evidence for GPR141's role in the biological process under investigation.

What approaches can help identify potential ligands for the orphan receptor GPR141?

As an orphan receptor, identifying the endogenous ligand(s) for GPR141 represents a significant research challenge. Several complementary approaches can be employed:

1. Computational Methods:

• Homology modeling based on structurally characterized GPCRs

• Virtual screening of compound libraries targeting the predicted ligand-binding domain

• Phylogenetic analysis to identify related receptors with known ligands as starting points

2. High-throughput Screening Platforms:

• PRESTO-Tango system: The GPR141-Tango plasmid (available through Addgene, #66318) enables screening via a transcriptional output following arrestin translocation

• Calcium mobilization assays: Using GPR141-expressing cells loaded with calcium-sensitive dyes

• GTPγS binding assays: To measure G protein activation upon receptor stimulation

• β-arrestin recruitment assays: Using enzyme complementation or BRET-based approaches

3. Tissue/Cell-Specific Approaches:

• Extraction and fractionation of bioactive components from tissues with high GPR141 expression (myeloid cells)

• Testing supernatants from activated immune cells on GPR141-expressing reporter cells

• Screening cytokines and chemokines relevant to myeloid cell function

4. Candidate-Based Testing:

• Based on GPR141's role in immune regulation, testing known immunomodulatory molecules

• Given its expression in myeloid cells, examining myeloid cell-derived factors

• Testing molecules involved in the p-mTOR/p53 pathway given its downstream effects

5. Validation Approaches:

• Dose-response relationships for candidate ligands

• Competition binding assays

• Functional selectivity (biased signaling) assessment

• Knockout/knockdown studies to confirm specificity

The identification of GPR141's endogenous ligand(s) would significantly advance our understanding of its physiological role and potentially open new therapeutic avenues for inflammatory diseases and cancer.

What are the best methods for studying GPR141's role in immune regulation?

Based on recent publications, several effective methodological approaches have been established for investigating GPR141's immunoregulatory functions:

1. In Vivo Disease Models:

• Experimental autoimmune encephalomyelitis (EAE): A well-established model for multiple sclerosis that has successfully revealed GPR141's role in regulating neuroinflammation

  • Induction protocol: Immunization with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide

  • Assessment: Clinical scoring, histopathological analysis of CNS tissues, flow cytometric analysis of infiltrating immune cells

• Other potential disease models:

  • Collagen-induced arthritis for studying autoimmune joint inflammation

  • DSS-induced colitis for intestinal inflammation

  • LPS challenge for acute inflammatory responses

2. Ex Vivo Cellular Assays:

• Antigen-specific T cell stimulation: Co-culture of GPR141-deficient dendritic cells with antigen-specific T cells to measure:

  • T cell proliferation (CFSE dilution)

  • Cytokine production (ELISA or intracellular cytokine staining)

  • Activation marker expression (flow cytometry)

• Myeloid cell functional assays:

  • Phagocytosis capacity of neutrophils and macrophages

  • Oxidative burst activity

  • Cytokine/chemokine production profiles

  • Migration and chemotaxis assays

3. Cell-Type Specific Analysis:

• Flow cytometry panels for detailed immune profiling:

  • Neutrophils (CD11b+ Gr1+)

  • Monocyte subsets (CD11b+ Gr1-Ly6C+ and CD11b+ Gr1-Ly6C-)

  • Macrophages (F4/80+)

  • Dendritic cells (CD11c+)

  • T cell subsets (Th1, Th17, Tregs)

4. Molecular and Signaling Analysis:

• Transcriptomics: RNA-seq of isolated immune cell populations from wild-type vs. GPR141-deficient animals

• Proteomics: Phosphoproteomic analysis to identify signaling pathways affected by GPR141 deficiency

• Chromatin immunoprecipitation (ChIP): To identify transcriptional targets regulated by GPR141 signaling

5. Human Translational Studies:

• Analysis of GPR141 expression in patient samples from autoimmune diseases

• Correlation of expression levels with disease severity or therapeutic response

• Ex vivo studies using human peripheral blood mononuclear cells

These methodological approaches provide a comprehensive framework for investigating GPR141's role in immune regulation and identifying potential therapeutic applications.

What are the main challenges in expressing and purifying functional GPCRs like GPR141?

GPCRs, including GPR141, present several unique challenges for expression and purification:

1. Membrane Protein Expression Issues:

• Low expression levels: GPCRs often express poorly compared to soluble proteins

• Toxicity to host cells: Overexpression can disrupt membrane integrity

• Improper folding: The complex transmembrane topology requires specialized folding machinery

Solutions:

• Use of specialized expression vectors with inducible promoters

• Codon optimization for the expression host

• Fusion tags that enhance expression (e.g., BRIL, T4 lysozyme)

• Expression as truncated constructs focusing on specific domains

2. Purification Challenges:

• Detergent selection: Critical for maintaining protein stability and function

• Lipid requirements: Many GPCRs require specific lipids for stability

• Protein instability: GPCRs often rapidly denature once removed from the membrane

Solutions:

• Screening multiple detergents and lipid combinations

• Use of lipid nanodiscs or other membrane mimetics

• Addition of stabilizing ligands during purification

• Temperature control throughout purification process

3. Functional Validation Difficulties:

• Lack of known ligands: As an orphan receptor, functional validation is complicated

• Complex signaling outputs: GPCRs can couple to multiple G proteins and other effectors

Solutions:

• Use of constitutive activity assays

• Arrestin recruitment assays that don't require ligand binding

• GTPγS binding assays to measure basal activity

4. Specific Recommendations for GPR141:

• Based on commercial products, E. coli expression has been successful for producing partial constructs (e.g., amino acids 153-183)

• Full-length GPR141 (1-305 aa) has been successfully expressed in both E. coli and mammalian systems

• For functional studies, HEK293T expression appears most effective

• Reconstitution buffer recommendations: Tris/PBS-based buffer, 6% Trehalose, pH 8.0

How can I optimize experimental design when studying GPR141 in disease models?

When designing experiments to investigate GPR141 in disease models, several key considerations can enhance the quality and translational value of the research:

1. Model Selection and Validation:

• Choose appropriate disease models based on GPR141 expression patterns:

  • For immune regulation: EAE model has proven effective

  • For cancer studies: Consider orthotopic breast cancer models given GPR141's role in breast cancer

• Validate model relevance:

  • Confirm GPR141 expression in relevant tissues/cells in the chosen model

  • Compare expression patterns between human disease and animal model

2. Controls and Experimental Design:

• Use multiple control groups:

  • Wild-type littermates as genetic controls

  • Sham or vehicle-treated controls for intervention studies

  • Consider both positive and negative controls for pharmacological studies

• Statistical considerations:

  • Perform power calculations to determine appropriate sample sizes

  • Plan for stratified randomization when appropriate

  • Consider blinded assessment of outcomes

3. Phenotypic Analysis:

• Multi-parameter assessment:

  • Clinical scoring systems appropriate to the disease model

  • Histopathological evaluation with quantitative scoring

  • Molecular and cellular analyses (flow cytometry, immunohistochemistry, etc.)

• Temporal dynamics:

  • Assess outcomes at multiple time points

  • Consider both acute and chronic phases of disease

4. Molecular Mechanisms:

• Pathway analysis:

  • For GPR141 in cancer: Focus on p-mTOR/p53 axis and EMT markers

  • For GPR141 in immune regulation: Examine cytokine profiles and immune cell activation markers

• Rescue experiments:

  • Genetic rescue (re-expression of GPR141 in knockout models)

  • Pharmacological rescue (e.g., rapamycin for p-mTOR pathway in cancer models)

5. Translational Considerations:

• Correlate with human data when possible:

  • Expression of GPR141 in human disease samples

  • Genetic association studies (SNPs, expression QTLs)

• Consider therapeutic implications:

  • Test potential interventions targeting GPR141 or its downstream pathways

  • Evaluate biomarkers that could predict response to such interventions

By incorporating these considerations into experimental design, researchers can enhance the rigor and translational potential of studies investigating GPR141's role in disease processes.

What are the emerging research areas for GPR141 that show the most promise?

Based on recent findings and current gaps in knowledge, several promising research directions for GPR141 warrant further investigation:

1. Therapeutic Development:

• Immune modulation: Since GPR141 functions as a negative regulator of immune responses, developing agonists could potentially treat inflammatory and autoimmune conditions

• Cancer therapeutics: As GPR141 promotes breast cancer progression, antagonists or silencing strategies might represent novel anti-cancer approaches

2. Ligand Discovery:

• Using modern high-throughput screening approaches and the PRESTO-Tango system to identify the endogenous ligand(s) for this orphan receptor

• Investigating whether GPR141 functions constitutively or requires specific activating signals in different cell types

3. Signaling Network Mapping:

• Comprehensive characterization of GPR141 coupling preferences to different G protein subtypes

• Identification of the complete set of downstream effectors and signaling pathways in different cell types

• Understanding the interplay between GPR141 and other myeloid cell receptors

4. Structural Biology:

• Determination of GPR141's three-dimensional structure through crystallography or cryo-EM

• Structure-based design of selective modulators

• Investigation of potential dimerization or oligomerization behaviors

5. Expanded Disease Relevance:

• Beyond EAE and breast cancer, exploring GPR141's role in other inflammatory conditions and cancer types

• Investigating potential functions in metabolic diseases given the importance of GPCRs in metabolic regulation

• Examining possible roles in infectious diseases, particularly those involving myeloid cell responses

6. Translational Research:

• Development of biomarkers based on GPR141 expression or activity

• Patient stratification strategies for potential GPR141-targeting therapeutics

• Investigation of GPR141 polymorphisms and their association with disease susceptibility or progression

These emerging areas represent significant opportunities for advancing our understanding of GPR141 biology and developing novel therapeutic approaches for conditions involving dysregulated immune responses or aberrant cell proliferation.

How might integrating GPR141 research with other disciplines advance our understanding?

The integration of GPR141 research with other scientific disciplines offers promising opportunities for more comprehensive insights:

1. Systems Biology Integration:

• Network analysis incorporating GPR141 into broader signaling networks

• Multi-omics approaches (genomics, transcriptomics, proteomics, metabolomics) to understand GPR141's position in cellular systems

• Mathematical modeling of GPR141-mediated pathways to predict responses to perturbations

2. Immunology and Cancer Biology Interface:

• Exploration of GPR141's dual roles in immune regulation and cancer progression

• Investigation of GPR141's effects on tumor microenvironment and immune evasion

• Potential development of immunotherapeutic approaches targeting GPR141

3. Computational Biology and Artificial Intelligence:

• Machine learning approaches to predict GPR141 ligands based on structural modeling

• Network inference algorithms to identify hidden connections between GPR141 and other cellular systems

• Virtual screening for GPR141 modulators with desired functional profiles

4. Precision Medicine Applications:

• Development of GPR141 expression signatures as biomarkers for disease stratification

• Pharmacogenomic studies to identify genetic variants affecting response to potential GPR141-targeting therapies

• Integration of GPR141 data into clinical decision support systems

5. Drug Delivery and Medicinal Chemistry:

• Design of selective GPR141 modulators with optimal pharmacokinetic properties

• Development of targeted delivery systems for GPR141-modulating compounds to specific cell types

• Exploration of biased ligands that selectively activate beneficial signaling pathways

6. Evolutionary Biology:

• Comparative analysis of GPR141 across species to understand evolutionary conservation and divergence

• Identification of species-specific differences in function that might inform translational research

• Understanding of how GPR141's role evolved in immune regulation

By fostering collaborations across these disciplines, researchers can develop more comprehensive approaches to understanding GPR141's biology and leveraging this knowledge for therapeutic development.