Recombinant Mouse Probable G-protein coupled receptor 135 (Gpr135)

What are the basic signaling properties of mouse GPR135?

GPR135 demonstrates constitutive activity with specific signaling pathway preferences. Unlike some other orphan receptors, GPR135 does not show constitutive activation of the G𝑞/IP1 pathway in HEK293T cells, as measured through inositol phosphate (IP1) production assays . This distinguishes it from receptors like GPR62, which shows approximately 40% constitutive activation of this pathway compared to control conditions .

Instead, GPR135 exhibits strong constitutive β-arrestin recruitment capabilities. When expressed in HEK293 cells stably expressing β-arrestin1-EA or β-arrestin2-EA, GPR135 demonstrates spontaneous and dose-dependent recruitment of both β-arrestin1 and β-arrestin2 in an agonist-independent manner . This recruitment can be competitively reduced by approximately 40-50% through the additional transfection of β-arrestin2-YFP, confirming the specificity of this interaction .

At physiologically relevant expression levels matching those observed in human pancreatic EndoC-βH1 cells, GPR135 maintains significant β-arrestin recruitment, suggesting it functions primarily as a β-arrestin-biased receptor under normal conditions . This signaling profile positions GPR135 as an interesting target for research into non-canonical GPCR signaling mechanisms.

How does GPR135 compare to other orphan GPCRs in its family?

Unlike GPR62, which demonstrates constitutive activity on the G𝑞/IP1 pathway, GPR135 shows no detectable constitutive activation of this signaling cascade . This indicates divergent G protein coupling preferences among these related orphan receptors. Additionally, all three receptors have been demonstrated to be unable to bind melatonin, despite showing a regulatory interaction with melatonin MT₂ receptors .

The following table compares key features of these related orphan receptors:

These differences suggest distinct physiological functions despite structural similarities and provide important considerations for experimental design when studying these receptors.

What are the optimal expression systems for recombinant mouse GPR135 studies?

For reliable expression of recombinant mouse GPR135, mammalian expression systems are strongly preferred over bacterial or insect cell systems. HEK293T cells have been successfully used for expression studies, showing robust surface expression of epitope-tagged GPR135 constructs . When planning expression studies, researchers should consider the following methodological approaches:

• Vector selection: Mammalian expression vectors with strong promoters (like CMV) yield efficient expression. The GPR135-Tango plasmid system (Addgene #66317) provides a validated expression construct with a CMV promoter and necessary features for detection and functional studies . This vector backbone includes a size of 6632 bp without the insert and contains neomycin selection markers for generating stable cell lines .

• Epitope tagging strategy: N-terminal epitope tagging has been successfully employed for GPR135 detection. HA-tagging allows for efficient immunodetection without compromising receptor function . For studies requiring β-arrestin recruitment detection, C-terminal PK2 tags have proven effective when used in conjunction with enzyme complementation assays .

• Transfection optimization: For transient expression studies, carefully optimize DNA amounts since GPR135 demonstrates dose-dependent effects on downstream signaling. Successful expression has been achieved with as little as 1 ng of expression vector in functional assays .

• Sample preparation considerations: When analyzing GPR135 by western blot, standard denaturation conditions (95°C for 10 minutes) are suitable, as GPR135 is detectably expressed at the predicted size of the receptor monomer under these conditions, unlike some related orphan receptors that require modified denaturation protocols .

What are the most effective assays for measuring GPR135 signaling activity?

Given GPR135's signaling profile, certain assays are particularly effective for measuring its activity. Based on the constitutive and β-arrestin-biased nature of GPR135, the following methodological approaches are recommended:

• β-arrestin recruitment assays: The PathHunter™ enzyme complementation assay has proven highly effective for measuring GPR135 activity . This approach requires expressing GPR135 with a C-terminal PK2 tag in cells stably expressing β-arrestin1/2-EA. The assay directly measures the physical association between the receptor and β-arrestin through enzyme complementation, providing a quantitative readout of receptor activity .

• β-arrestin competition assays: To confirm the specificity of β-arrestin recruitment, additional transfection of β-arrestin2-YFP in the PathHunter system causes competitive reduction in recruitment signal (typically 40-50% reduction) . This serves as an important control for validating the specificity of the interaction.

• G protein activation assays: Despite the apparent β-arrestin bias of GPR135, researchers should still assess potential G protein coupling using assays like BRET-based sensors for different G protein subtypes. While IP1 accumulation assays showed no constitutive activation of Gq pathways by GPR135 , other G protein families should be investigated.

• Transcriptional reporter assays: The GPR135-Tango system enables measurement of receptor activity through transcriptional output following arrestin translocation . This approach is particularly useful for high-throughput screening applications.

When designing these experiments, it's critical to include appropriate positive controls (like known GPCR agonists such as angiotensin II for AT1R) and to carefully titrate receptor expression levels to avoid artifacts from overexpression.

How can researchers design siRNA knockdown experiments for GPR135?

RNA interference approaches provide powerful tools for investigating GPR135 function through targeted knockdown. When designing siRNA experiments targeting mouse GPR135, consider the following methodological guidelines:

• siRNA selection and modification: Commercial siRNAs targeting mouse Gpr135 are available with ribo-modifications that enhance stability, specificity, and reduce immunogenicity . These modifications are particularly important for in vivo applications or extended experiments. The reference sequence NM_181752.1 provides the target for validated siRNA designs .

• Delivery optimization: Transfection efficiency varies significantly between cell types. For hard-to-transfect cells, consider viral vector-based shRNA delivery systems or electroporation approaches. Lipid-based transfection typically works well for easily transfected lines like HEK293.

• Validation controls: Always include the following controls:

  • Scrambled sequence control siRNA with the same chemical modifications

  • Untransfected cells

  • Positive control siRNA targeting a housekeeping gene

• Knockdown verification: Quantify knockdown efficiency using:

  • qRT-PCR to measure mRNA reduction (typically 24-48 hours post-transfection)

  • Western blot or functional assays to confirm protein reduction (typically 48-72 hours post-transfection)

• Functional readouts: After confirming successful knockdown, measure relevant phenotypes such as changes in β-arrestin recruitment capabilities, alterations in downstream signaling cascades, or cell-type specific functional outcomes.

The timing between siRNA delivery and functional assays is critical, as it must allow sufficient time for protein turnover while avoiding potential compensatory mechanisms that may emerge with prolonged knockdown.

What are the known tissue expression patterns and physiological roles of GPR135?

The tissue distribution and physiological functions of GPR135 remain partially characterized, with evidence pointing to specific expression patterns and potential roles in metabolic regulation. Current research indicates the following:

GPR135 is expressed in human pancreatic beta cells, suggesting a potential role in glucose homeostasis or insulin secretion pathways . This expression pattern is significant given the importance of GPCR signaling in pancreatic function and metabolic regulation. Additionally, experimental evidence shows that cold exposure increases GPR135 expression specifically in mouse liver but not in adipose tissue . This selective upregulation implies a potential role in temperature-dependent metabolic adaptation and liver-specific functions.

Epigenetic studies have identified GPR135 as one of the methylated placental genes associated with maternal cigarette smoking during pregnancy . This observation is particularly noteworthy since maternal smoking increases risks for numerous pregnancy complications including miscarriage, low birth weight, preterm birth, and childhood obesity . This suggests GPR135 may play a role in placental function or development.

GPR135's subcellular localization provides further clues to its function. The receptor is found in both endosome and plasma membrane compartments , suggesting potential functions in both cell surface signaling and endosomal signaling pathways. This dual localization pattern is characteristic of receptors involved in complex signaling regulation.

What mechanistic insights have emerged from GPR135 signaling studies?

Molecular characterization of GPR135 signaling has revealed several distinctive features that provide insight into its functional capabilities. Key mechanistic findings include:

• Constitutive β-arrestin recruitment: GPR135 demonstrates robust constitutive recruitment of both β-arrestin1 and β-arrestin2 without requiring ligand stimulation . This spontaneous activity occurs at physiologically relevant expression levels, suggesting it represents a genuine property of the receptor rather than an artifact of overexpression .

• Agonist independence: Unlike many GPCRs, GPR135's association with β-arrestins is not further modulated by potential ligands like melatonin . This suggests the receptor may function primarily through ligand-independent mechanisms or requires yet-unidentified endogenous ligands.

• Signaling bias: GPR135 exhibits a signaling profile consistent with β-arrestin bias, as it constitutively recruits β-arrestins while showing no detectable constitutive activity on the G𝑞/IP1 pathway in experimental systems . This bias may direct GPR135's cellular functions toward β-arrestin-mediated outcomes rather than classical G protein signaling.

• Regulatory interactions: Despite not binding melatonin directly, GPR135 shows reciprocal regulatory interactions with melatonin MT₂ receptors . This suggests GPR135 may function as part of receptor complexes or signaling networks rather than as an isolated signaling unit.

These mechanistic insights provide important considerations for experimental design when studying GPR135 and may help guide future research into identifying its physiological functions and potential as a therapeutic target.

What strategies can researchers employ to identify potential endogenous ligands for GPR135?

Identifying endogenous ligands for orphan receptors like GPR135 remains one of the most significant challenges in GPCR research. The following methodological approach represents a comprehensive strategy for ligand identification:

• Bioinformatic prediction: Utilize structural modeling of GPR135 based on crystal structures of related GPCRs to predict potential binding pockets. This can be combined with in silico screening of compound libraries to identify candidate ligands with high predicted binding affinity.

• Tissue extract fractionation: Prepare extracts from tissues with high GPR135 expression (such as pancreatic beta cells or liver tissue) and fractionate them using high-performance liquid chromatography. Test fractions for activity using β-arrestin recruitment assays, which have been established as effective readouts for GPR135 activity .

• High-throughput screening approaches: The GPR135-Tango system enables screening via transcriptional output following arrestin translocation . This platform allows for efficient screening of compound libraries against GPR135.

• Candidate molecule approach: Based on GPR135's tissue distribution and response to conditions like cold exposure , test metabolites and signaling molecules associated with these physiological states.

• Reverse pharmacology: Use the constitutive β-arrestin recruitment property of GPR135 to screen for compounds that modulate this activity, either enhancing or inhibiting it . Compounds that specifically inhibit the constitutive activity may represent inverse agonists.

• Genetic correlation studies: Analyze gene expression datasets to identify molecules whose production correlates with GPR135 activity or expression patterns across tissues or physiological conditions.

When performing these experiments, the established β-arrestin recruitment assays provide reliable readouts for receptor activation or inhibition .

What are the emerging techniques for investigating orphan receptor function beyond ligand identification?

Research on orphan receptors like GPR135 is increasingly moving beyond the traditional focus on ligand identification to explore alternative approaches for understanding function. Advanced methodological strategies include:

• CRISPR/Cas9 genome editing: Generate GPR135 knockout models in relevant cell lines or animal models to investigate physiological roles. This approach allows for precise deletion or mutation of the receptor and can reveal phenotypes that provide clues to function. Given the evidence of GPR135 expression in pancreatic beta cells , CRISPR-mediated knockout in beta cell lines could reveal potential roles in insulin secretion or glucose metabolism.

• Proximity labeling proteomics: Techniques like BioID or APEX2 fused to GPR135 can identify proximal interacting proteins in living cells, revealing the receptor's signaling network. This approach is particularly valuable for understanding the signaling complexes formed by constitutively active receptors like GPR135.

• Single-cell transcriptomics: Analyze transcriptional changes in cells expressing different levels of GPR135 to identify downstream gene expression patterns associated with receptor activity. This can provide insights into signaling pathways and cellular functions influenced by GPR135.

• Resonance energy transfer techniques: Advanced BRET or FRET approaches can investigate conformational changes in GPR135 and its interactions with signaling partners like β-arrestins in real-time in living cells . These techniques allow monitoring of protein-protein interactions and receptor dynamics with high temporal resolution.

• Phosphoproteomics: Global phosphoproteomic analysis comparing wild-type cells to those with GPR135 knockdown can reveal downstream signaling events triggered by the receptor's constitutive activity, particularly given its strong β-arrestin recruitment capabilities .

• Receptor trafficking studies: Investigation of GPR135's endosomal localization using live-cell imaging can reveal whether this subcellular distribution contributes to unique signaling properties or regulation mechanisms.

These approaches can provide valuable functional insights even in the absence of identified endogenous ligands, advancing our understanding of GPR135's physiological roles.