Recombinant Macaca fascicularis N-arachidonyl glycine receptor (GPR18)

What is GPR18 and what is its relationship to the endocannabinoid system?

GPR18 is an orphan G protein-coupled receptor that belongs to the class A family. Despite sharing low sequence homology with cannabinoid receptors CB1R and CB2R, growing evidence suggests GPR18 is related to the endocannabinoid system . This relationship is established through:

• Recognition of cannabinoid ligands including Δ9-THC

• Expression patterns that overlap with cannabinoid receptors

• Ability to heteromerize with cannabinoid receptors

GPR18 has been proposed as a potential "third cannabinoid receptor" due to these associations, though its precise classification remains debated. The receptor is widely expressed in lymphoid tissues, with moderate expression in lungs, brain, testis, and ovary . In the Macaca fascicularis respiratory tract, the pattern of peptide distribution is similar to that previously reported in human postmortem material, suggesting the cynomolgus monkey may be a useful model for examining the pathophysiological role of peptides in human respiratory disease .

What are the known ligands for GPR18 and how do they compare?

Several endogenous and synthetic compounds have been identified as potential GPR18 ligands:

N-arachidonyl glycine (NAGly) was initially identified as the first endogenous GPR18 agonist , though subsequent studies have yielded contradictory results regarding its efficacy . More recently, synthetic ligands with higher potency and selectivity have been developed , providing better tools for investigating GPR18 function.

What cellular signaling pathways are associated with GPR18 activation?

GPR18 has been reported to couple to several G protein subtypes and activate various signaling pathways:

• Gαi/o pathway: Initially identified as the primary coupling mechanism , leading to inhibition of adenylyl cyclase and decreased cAMP levels

• Gαq/11 pathway: Subsequent findings have implicated this pathway , leading to calcium mobilization

• Gαs pathway: Some studies report coupling through this pathway , though less consistently

Downstream effects of GPR18 activation include:

• MAPK pathway activation (p44/42 MAPK phosphorylation)

• Regulation of cellular migration

• Modulation of intraocular pressure

• Effects on cellular proliferation and apoptosis

How is GPR18 expressed in different tissues and what physiological roles has it been associated with?

GPR18 exhibits distinctive expression patterns across various tissues:

GPR18 has been implicated in numerous physiological processes including:

• Resolution of inflammation through interaction with Resolvin D2

• Regulation of intraocular pressure independent of CB1, CB2, or GPR55

• Pain modulation through enhancement of inhibitory glycinergic transmission

• Regulation of microglial cell migration and neuroinflammation

• Vasodilation and hypotensive effects

What methodological approaches are most effective for studying GPR18 function, and what are their limitations?

Several methodological approaches have been employed to study GPR18, each with distinct advantages and limitations:

Receptor Expression Systems:

Functional Assays:

• cAMP accumulation assays show variable responses to proposed GPR18 ligands

• ERK phosphorylation assays have been used with mixed results

• Calcium mobilization assays demonstrate responses in some studies but not others

• β-arrestin recruitment assays have become preferred for screening novel ligands

• Receptor trafficking assays reveal constitutive trafficking but often fail to detect ligand-induced effects

Limitations:

• Inconsistent results between labs and assay systems

• Challenges in achieving sufficient surface expression of recombinant GPR18

• High constitutive activity complicates detection of ligand-mediated effects

• Limited availability of highly selective ligands (though improving with recent developments )

• Species differences in receptor pharmacology

For more reliable results, researchers should consider employing multiple complementary assays and validating findings in physiologically relevant cell types that endogenously express GPR18.

How can molecular modeling approaches be applied to study GPR18 structure and ligand binding?

Molecular modeling has emerged as a valuable approach for studying orphan receptors like GPR18:

Homology Model Development:
Several approaches have been employed to develop GPR18 models:

• Template-based homology modeling using evolutionarily closest homologous templates:

  • Human P2Y1 Receptor crystal structure has been used as a template

  • GPR18 inactive form models can predict ligand binding properties

• Template-free modeling approaches:

  • RoseTTAFold and trRosetta have been used to generate ab initio models

  • AlphaFold2-derived models have shown promising results despite sometimes lower protein structure qualitative estimations

Model Evaluation Criteria:

Applications of Molecular Modeling:

• Identification of key binding site residues to guide selective GPR18 compound design

• Virtual screening to identify novel GPR18 ligands

• Understanding the structural basis for ligand selectivity between GPR18 and related receptors

• Investigation of receptor activation mechanisms

An important finding was that a wide range of freely available software and academic licenses allow for meaningful molecular modeling/docking studies in this field , making these approaches accessible to more research groups.

What explains the contradictory findings regarding NAGly as a GPR18 ligand, and how should researchers address this controversy?

The literature contains significant discrepancies regarding NAGly's role as a GPR18 ligand. Understanding these contradictions is crucial for researchers in the field:

Evidence Supporting NAGly as a GPR18 Ligand:

• NAGly induces intracellular Ca2+ mobilization in GPR18-expressing cells

• NAGly decreases cAMP accumulation in GPR18-CHO cells

• NAGly activates p44/42 MAPK in GPR18-expressing cells

• NAGly induces migration of GPR18-expressing cells

• NAGly reduces intraocular pressure in mice, an effect blocked by the GPR18 antagonist O-1918

Evidence Against NAGly as a GPR18 Ligand:

• NAGly fails to recruit β-arrestin in some GPR18 expression systems

• NAGly does not inhibit Ca2+ currents or modulate cAMP in rat sympathetic neurons expressing GPR18

• NAGly doesn't activate G protein-coupled inwardly rectifying potassium channels in GPR18 systems

• NAGly shows no ligand-mediated effects on GPR18 trafficking

Possible Explanations for Discrepancies:

• Methodological differences:

  • Different cell types and expression systems

  • Varying receptor expression levels and surface localization

  • Diverse functional assays measuring different pathways

• Biased signaling:

  • NAGly may be a biased ligand that activates specific pathways but not others

  • Evidence suggests NAGly might be a biased-Gαi/o coupled GPR18 ligand that signals via G-protein only

• Species differences:

  • Recent research has identified significant species-dependent pharmacology for GPR18

  • For example, THC shows marked differences in potency between human and mouse GPR18

• Receptor constitutive activity:

  • GPR18 exhibits high constitutive activity and trafficking

  • This may mask or complicate detection of ligand-mediated effects

Recommended Research Approaches:

• Employ multiple complementary assay systems

• Include appropriate positive and negative controls

• Use the newly developed selective GPR18 ligands for validation

• Consider species differences when interpreting results

• Explore the possibility of biased signaling

• Investigate receptor heteromerization, which may affect ligand responses

How does GPR18 interact with other receptors, and what are the functional implications of these interactions?

GPR18 has been found to form heteromeric complexes with other receptors, which can significantly alter signaling properties and physiological responses:

GPR18-CB2R Heteromerization:

• Direct physical interaction demonstrated by bioluminescence resonance energy transfer (BRET) studies

• Heteromerization confirmed in microglial cells using proximity ligation assays (PLA)

• Functional consequences include:

  • Cross-talk in signaling pathways

  • Negative cross-talk and/or cross-antagonism effects

  • Altered responses to ligands

  • Changes in downstream signaling cascades

No Evidence for GPR18-CB1R Heteromerization:

• BRET studies showed an unspecific linear signal, indicating lack of direct interaction

• This differential interaction pattern is significant as it suggests specific, rather than general, receptor interactions

Physiological Significance:

• GPR18-CB2R heteromers are expressed in microglial cells in both resting conditions and upon activation with LPS and IFN-γ

• Microglial cells from transgenic Alzheimer's disease mouse models express CB2R-GPR18 receptor complexes that behave like those in cells from wild-type animals treated with LPS and IFN-γ

• These heteromeric complexes may play crucial roles in:

  • Modulating neuroinflammation

  • Regulating microglial cell migration

  • Balancing neurodegeneration and neuroprotection processes

  • Potentially serving as novel therapeutic targets for neurological disorders

Methodological Approaches to Study Receptor Interactions:

• Immunocytochemistry to assess receptor co-localization (though co-localization alone doesn't prove direct interaction)

• Bioluminescence resonance energy transfer (BRET) to demonstrate physical interactions

• Proximity ligation assays (PLA) to identify receptor complexes in native tissues

• Functional cross-talk assays to detect alterations in signaling responses

These heteromeric interactions highlight the complexity of GPR18 signaling and suggest that the functional consequences of GPR18 modulation may depend on the cellular context and the expression of interacting receptors.

What is the current understanding of the constitutive activity of GPR18, and how does it impact experimental design?

GPR18 exhibits remarkable constitutive activity, particularly in terms of receptor trafficking, which has significant implications for experimental approaches:

Characteristics of GPR18 Constitutive Activity:

• Rapid constitutive receptor membrane trafficking—several-fold faster than CB1R, which is itself considered highly constitutively active

• Predominantly intracellular localization in stably transfected cell lines

• The A108N mutant (alanine to asparagine at position 108) shows increased surface expression, potentially indicating reduced constitutive activity

Impact on Experimental Design:

Physiological Relevance:
The constitutive trafficking of GPR18 likely has functional consequences in vivo, potentially affecting:

• Receptor availability at the cell surface

• Tonic signaling in cells expressing GPR18

• Response dynamics to endogenous ligands

• Steady-state activation of downstream pathways

Understanding and accounting for this constitutive activity is essential for correctly interpreting experimental results and developing effective modulators of GPR18 function.

What therapeutic opportunities does modulation of GPR18 present, and what are the key challenges in developing selective GPR18-targeted compounds?

GPR18 modulation offers promising therapeutic potential across multiple disease areas, although significant challenges remain:

Therapeutic Opportunities:

Key Challenges in Developing Selective GPR18 Compounds:

• Pharmacological Challenges:

  • Contradictory findings regarding ligand efficacy and potency

  • High constitutive activity complicating detection of agonist effects

  • Need for truly selective ligands to delineate GPR18-specific effects

  • Species differences in ligand response requiring careful translation

• Structural Challenges:

  • Limited structural information despite recent modeling advances

  • Incomplete understanding of binding site residues crucial for selectivity

  • Challenges in distinguishing GPR18 activity from effects on related receptors

• Signaling Complexity:

  • Multiple G-protein coupling pathways with variable reporting

  • Potential biased signaling adding complexity to pharmacological profiling

  • Receptor heteromerization altering signaling properties

Recent Advances:

• Development of potent and selective GPR18 agonists like PSB-KK1415 (EC50 19.1 nM) and PSB-KK1445 (EC50 45.4 nM)

• PSB-KK1445 shows >200-fold selectivity versus CB receptors, GPR55, and GPR183

• These compounds exhibit minimal species differences, while THC acts as a weak partial agonist at mouse GPR18

Future Directions:

• Further structural characterization of GPR18 through crystallography or cryo-EM

• Development of selective antagonists and allosteric modulators

• Investigation of tissue-specific effects of GPR18 modulation

• Better understanding of the interplay between GPR18 and cannabinoid receptors

• Clinical translation of preclinical findings to validate GPR18 as a therapeutic target

The recent development of potent and selective GPR18 agonists represents a significant advance that will facilitate further investigation of this promising receptor as a therapeutic target across multiple disease areas.