Recombinant Mouse G-protein coupled receptor 35 (Gpr35)

What is GPR35 and what are its primary functions in mice?

GPR35 is a G protein-coupled receptor with significant expression in the gastrointestinal tract, particularly in colonic epithelial cells, as well as in various immune cells including neutrophils. In mice, GPR35 plays crucial roles in maintaining colonic epithelial barrier integrity, regulating neutrophil recruitment during inflammation, and responding to bacterial toxins such as the Bacteroides fragilis toxin (BFT) .

The receptor demonstrates both constitutive (agonist-independent) activity and ligand-mediated signaling, predominantly through Gα12/Gα13 pathways leading to RhoA activation . Mouse GPR35 also contributes to efficient bacterial clearance during infection and participates in immune cell migration across endothelial barriers .

How does mouse GPR35 differ from human GPR35?

The most notable difference between mouse and human GPR35 is in their pharmacological profiles. The human GPR35 inverse agonist CID-2745687 has no significant affinity for mouse GPR35, creating important challenges when translating findings between species . This pharmacological distinction has led researchers to develop transgenic mouse models expressing human GPR35a to better study human-relevant mechanisms.

Unlike human GPR35, which has two isoforms (GPR35a and GPR35b, with the latter having a 31-amino acid N-terminal extension), mouse GPR35 exists as a single isoform . Additionally, while both human and mouse GPR35 can couple to similar G proteins, their affinity for various endogenous ligands may differ, which necessitates careful consideration when designing cross-species studies.

What are the known endogenous ligands for mouse GPR35?

Several potential endogenous activators have been identified for GPR35, with varying degrees of evidence for the mouse ortholog:

• Kynurenic acid: The most well-established endogenous agonist, shown by multiple research groups to activate GPR35 in various experimental settings .

• 5-hydroxyindoleacetic acid (5-HIAA): A serotonin metabolite derived from platelets that has been identified as a nanomolar-potency GPR35 ligand, particularly relevant for neutrophil recruitment .

• Lysophosphatidic acid (LPA): Particularly 2-acyl LPA has been suggested as a GPR35 ligand, though with some debate in the literature .

• CXCL17: Proposed as a potential activator, though less extensively characterized for mouse GPR35 .

How does GPR35 signal transduction differ between constitutive and ligand-induced activity?

GPR35 demonstrates significant biased constitutive signaling that is not equivalent across all potential signaling pathways. Research has shown that GPR35 exhibits substantial constitutive activation of heterotrimers containing Gα12 or Gα13, even at low receptor expression levels . In contrast, no constitutive interactions with arrestin-adaptor proteins or activation of Gαo-containing G protein heterotrimers were detected .

The level of receptor expression significantly influences the balance between constitutive and ligand-induced activity. At high receptor expression levels, constitutive activation of Gα12 or Gα13 can mask agonist-induced effects, while low expression levels with minimal constitutive activity permit measurement of agonist-induced responses . This creates an important methodological consideration for research design when studying GPR35 function.

What G protein pathways are activated by mouse GPR35?

The coupling profile appears to be biased, with stronger constitutive activation of Gα12/Gα13 pathways compared to Gαo pathways. This G protein coupling profile is important for GPR35's physiological roles, as Gα12/Gα13 signaling contributes to regulation of epithelial barrier function, while potential Gi/o coupling may be relevant for chemotactic responses in immune cells .

How can researchers distinguish between different signaling outcomes in GPR35 experiments?

To properly characterize GPR35 signaling, researchers should employ multiple complementary approaches:

• BRET (Bioluminescence Resonance Energy Transfer) assays using Nanoluciferase-tagged Gα subunits and fluorescent protein-tagged Gγ subunits can measure G protein heterotrimer dissociation in response to constitutive activity or ligand stimulation .

• Pertussis toxin pre-treatment can be used to distinguish Gi/o-mediated from G12/13-mediated effects .

• For studying arrestin recruitment, various protein-protein interaction assays including BRET or enzyme complementation approaches can be utilized .

• Downstream readouts such as RhoA activation (for G12/13 signaling) can be measured using FRET-based biosensors or pulldown assays for GTP-bound RhoA .

• Expression level control is critical - researchers should use inducible expression systems or careful titration of transfected DNA to examine how receptor density affects the balance between constitutive and ligand-induced signaling .

What are the optimal expression systems for studying recombinant mouse GPR35?

For controlled expression of recombinant mouse GPR35, several systems have proven effective:

• Inducible expression systems such as Flp-In T-REx are particularly valuable as they allow controlled titration of receptor expression levels, which is crucial given GPR35's expression-dependent constitutive activity .

• For stable expression, HEK293 cells have been widely used and characterized for GPR35 studies, though physiologically relevant cell types like colonocyte lines may provide more contextually appropriate backgrounds .

• For studying tissue-specific effects, transgenic mouse models are available, including those where mouse GPR35 is replaced with human GPR35a, allowing human-focused pharmacological studies in a physiological context .

When designing expression constructs, N-terminal epitope tags (such as FLAG) have been successfully used without significantly altering receptor function, while C-terminal modifications should be carefully validated to ensure they don't interfere with G protein coupling .

What pharmacological tools are available for studying mouse GPR35?

The pharmacological toolkit for specifically studying mouse GPR35 is more limited than for human GPR35:

• Agonists:

  • Lodoxamide has been used as an agonist in multiple studies

  • Zaprinast activates both human and mouse GPR35, though with different potencies

• Antagonists/Inverse agonists:

  • A significant limitation is that CID-2745687, a well-characterized inverse agonist at human GPR35, lacks affinity for mouse GPR35

  • ML145 has been used as a GPR35 antagonist in some studies, though species selectivity should be confirmed

When studying mouse models, this pharmacological species selectivity creates challenges for validating on-target effects. Researchers have addressed this by creating transgenic mice expressing human GPR35a instead of mouse GPR35, allowing the use of human-selective compounds in vivo .

How should researchers control for GPR35 expression levels in experimental systems?

Given that GPR35 activity is highly dependent on expression level, controlling and measuring receptor density is crucial:

• Use inducible expression systems with titrated inducer concentrations to achieve controlled expression levels .

• Quantify surface expression using cell-surface ELISA with N-terminal epitope tags, flow cytometry with anti-GPR35 antibodies, or radioligand binding when suitable radioligands are available .

• Include experimental conditions that span from low to high expression to capture the full range of receptor behaviors, particularly the transition from predominantly ligand-responsive to predominantly constitutively active states .

• When possible, compare experimental expression levels to physiological expression levels in target tissues to ensure biological relevance .

• For in-cell experiments, immunoblotting can be used to quantify total receptor expression across different experimental conditions .

How does GPR35 contribute to intestinal barrier function and inflammatory bowel disease models?

GPR35 plays a critical role in maintaining intestinal epithelial barrier integrity. In colonic organoids from human GPR35a-expressing transgenic mice, the GPR35 inverse agonist CID-2745687 significantly increased barrier permeability, demonstrating that constitutive activity of GPR35 contributes to maintaining epithelial barrier function .

In inflammatory bowel disease contexts, GPR35 appears to mediate responses to bacterial toxins. When the enterotoxigenic Bacteroides fragilis toxin (BFT) was studied, blocking GPR35 function in colonic epithelial cells using the antagonist ML145, or through genetic approaches (shRNA knockdown or CRISPR-Cas9 knockout), resulted in reduced cellular responses to BFT as measured by E-cadherin cleavage, β-arrestin recruitment, and IL-8 secretion .

Importantly, GPR35-deficient mice showed reduced mortality and disease severity compared to wild-type C57Bl6 mice in ETBF-induced colitis models, highlighting GPR35's role in sensing bacterial toxins and mediating inflammatory responses in the colon .

What is the role of GPR35 in neutrophil recruitment and how can this be studied?

GPR35 is upregulated in activated neutrophils and plays a significant role in promoting their migration. GPR35-deficient neutrophils show impaired recruitment from blood vessels into inflamed tissues, and GPR35-knockout mice demonstrate reduced efficiency in clearing peritoneal bacteria .

This role can be studied through several approaches:

• Neutrophil transmigration assays across endothelial monolayers, particularly with platelet-coated endothelium, as GPR35 function in neutrophil recruitment is strongly dependent on platelets .

• In vivo imaging of neutrophil trafficking in inflamed tissues using intravital microscopy in wild-type versus GPR35-deficient mice .

• Bacterial clearance assays in peritonitis models comparing wild-type and GPR35-knockout mice .

The platelet-derived serotonin metabolite 5-HIAA has been identified as a GPR35 ligand that may mediate these effects, suggesting that serotonin metabolism may link platelet activation to neutrophil recruitment via GPR35 .

How can biased signaling of GPR35 be leveraged for therapeutic development?

The biased signaling profile of GPR35—constitutive activation of Gα12/Gα13 but not Gαo or arrestin pathways—presents opportunities for developing pathway-selective therapeutics . Researchers interested in this area should consider:

• Performing comprehensive signaling pathway analysis using BRET-based G protein activation assays and arrestin recruitment assays to characterize the signaling fingerprint of candidate compounds .

• Developing cell-based functional assays that measure disease-relevant endpoints downstream of specific signaling pathways, such as epithelial barrier function for Gα12/Gα13 signaling .

• Utilizing transgenic mouse models expressing human GPR35 to test compounds with human selectivity in physiologically relevant in vivo models .

• Considering the expression level-dependent effects of GPR35, as compounds that modulate constitutive activity might have different effects depending on receptor expression in target tissues .

The high constitutive activity of GPR35 suggests that inverse agonists might be particularly valuable for conditions where excessive GPR35 activity contributes to pathology .

How can researchers address species differences when studying GPR35?

The species differences between mouse and human GPR35, particularly in pharmacological profiles, present significant challenges. Strategies to address these include:

• Creating "humanized" mouse models where mouse GPR35 is replaced with human GPR35a, allowing human-selective compounds to be studied in a physiological context .

• Conducting parallel studies with both species orthologs to identify conserved versus divergent mechanisms.

• When possible, validating findings from mouse models in human tissues or primary cells.

• Using genetic approaches (knockout, knockdown, or overexpression) rather than pharmacological approaches when studying mouse GPR35, since the pharmacological toolkit for mouse GPR35 is more limited .

• Being cautious when interpreting results from studies using human-selective compounds such as CID-2745687 in mouse systems, as effects are likely off-target .

What are common pitfalls in measuring GPR35 constitutive activity?

Several methodological considerations are critical when studying GPR35 constitutive activity:

• Expression level effects: At high receptor expression levels, constitutive activity can mask agonist effects, while at very low levels, constitutive activity may be difficult to detect .

• Time-dependent effects: The response to inverse agonists may increase over time as G protein heterotrimers reassociate, requiring extended measurement timeframes .

• Basal activity reference: Proper negative controls (mock-transfected or untreated cells) are essential to distinguish receptor-specific constitutive activity from system background .

• Pathway selection: Since GPR35 shows biased constitutive activity (Gα12/Gα13 but not Gαo or arrestin), selecting the appropriate signaling pathway to measure is critical .

• Antagonist versus inverse agonist effects: Distinguishing between neutral antagonists (blocking only agonist effects) and inverse agonists (reducing constitutive activity) requires careful experimental design .

What methods exist for functional validation of GPR35 in primary cells and tissues?

For researchers working with primary cells and tissues rather than recombinant systems, several approaches can validate GPR35 function:

• Genetic approaches: CRISPR-Cas9 knockout or shRNA knockdown of GPR35 in primary cells or organoids can help establish receptor-specific effects .

• Ex vivo tissue assays: Colonic organoids from wild-type versus GPR35-deficient mice can be used to measure functional endpoints such as barrier permeability .

• Primary immune cell assays: Neutrophil migration assays comparing cells from wild-type and GPR35-knockout mice can validate GPR35's role in immune cell trafficking .

• In vivo validation: Using GPR35-knockout mice or humanized GPR35 mice in disease models provides the strongest functional validation .

• For human samples, correlation of GPR35 expression levels (by qPCR or immunohistochemistry) with functional readouts can provide circumstantial evidence for receptor involvement, particularly in conditions like inflammatory bowel disease where GPR35 genetic variants have been identified .