Recombinant Mouse Endothelin B receptor-like protein 2 (Gpr37l1)

What is GPR37L1 and what are its structural characteristics?

GPR37L1 (G Protein-Coupled Receptor 37-Like 1), also known as Endothelin B receptor-like protein 2, is an orphan G protein-coupled receptor belonging to the G-protein coupled receptor 1 family. It is encoded by the human GPR37L1 gene, containing an open reading frame of 1443 base pairs that encodes a 481 amino acid protein with seven transmembrane domains . GPR37L1 shows 68% similarity and 48% identity to GPR37 .

The protein structure includes critical features typical of GPCRs with notable post-translational modifications:

When heterologously expressed in cell lines such as HEK293 or U87 glioblastoma cells, GPR37L1 appears as two distinct cell surface species of approximately equivalent abundance, with the larger form corresponding to the full-length receptor . This processing appears to be functionally significant, as mutations lacking the N-terminus show altered signaling capability .

Where is GPR37L1 expressed in the mammalian nervous system?

GPR37L1 exhibits a highly specific expression pattern primarily restricted to the central nervous system, with distinct cell-type specificity:

GPR37L1 ranks among the most highly expressed GPCR transcripts in both mouse and human dorsal root ganglia (DRGs) and is selectively expressed in satellite glial cells (SGCs) . Unlike its related receptor GPR37, which is predominantly found in oligodendrocytes and specific neuronal populations like dopaminergic neurons, GPR37L1 shows preferential expression in astrocytes and OPCs .

The receptor's highly restricted expression pattern suggests specialized functions in glial cells that may be critical for neuron-glia communication and homeostasis in the nervous system.

What are the proposed ligands for GPR37L1 and how is receptor activity regulated?

Several mechanisms appear to regulate GPR37L1 activity:

• Metalloprotease-dependent proteolytic processing of the N-terminus occurs both in cultured cells and in rodent cerebellar tissue . This processing may serve as a negative regulatory mechanism for GPR37L1 signaling, as a mutant lacking the entire N-terminus shows impaired signaling via Gαs .

• Some evidence suggests GPR37L1 may be constitutively active, or that its activity is controlled by signals that regulate metalloprotease activity in tissue rather than through direct ligand binding .

• Unlike Protease-Activated Receptors (PARs), synthetic peptides corresponding to the N-terminus of GPR37L1 do not activate the receptor, suggesting a distinct regulatory mechanism .

How does GPR37L1 contribute to neurological disorders?

GPR37L1 has been implicated in several neurological conditions through both genetic association studies and functional analyses:

Migraine:

Using sequence kernel association testing on 51,289 whole-exome sequences from the DiscovEHR cohort, rare GPR37L1 coding variants were significantly associated with migraine diagnostic codes . Functional characterization of these variants revealed altered signaling properties, with variants showing different abilities to reduce cAMP levels, activate MAPK signaling, and upregulate receptor expression in response to prosaptide .

Epilepsy:

Rare GPR37L1 variants were also significantly associated with epilepsy . A point mutation in GPR37L1 has previously been identified in a consanguineous family with progressive myoclonus epilepsy . Both migraine and epilepsy are characterized by neuronal hyperexcitability, suggesting common pathophysiological mechanisms involving GPR37L1 .

Neuropathic Pain:

Transgenic mice with GPR37L1 deficiency exhibited impaired resolution of neuropathic pain symptoms following paclitaxel and streptozotocin-induced peripheral neuropathy . Conversely, overexpression of GPR37L1 in mouse DRGs reversed pain symptoms, suggesting a protective role in pain regulation .

Anxiety-Related Disorders:

While GPR37L1 knockout mice did not show acute migraine phenotypes, they displayed sex-specific changes in anxiety-related behaviors that are often comorbid with chronic migraine . This suggests GPR37L1 may contribute to the neuropsychiatric aspects of pain disorders.

What signaling pathways are modulated by GPR37L1?

GPR37L1 engages multiple signaling pathways with complex downstream effects:

The signaling repertoire of GPR37L1 suggests it functions as a multifunctional regulator with context-dependent effects. For example, TX14A-induced activation of the MAPK pathway appears to mediate acute regulation of cellular cholesterol levels , while interaction with potassium channels in SGCs may contribute to regulation of neuronal excitability and pain sensitivity .

How do post-translational modifications affect GPR37L1 function?

The N-terminus of GPR37L1 undergoes proteolytic processing that significantly impacts its function:

• Evidence for proteolytic processing includes the presence of GPR37L1 N-terminal fragments in human cerebrospinal fluid and the appearance of two distinct species when expressed in cell lines .

• This processing is metalloprotease-dependent, occurring both in heterologous expression systems and in native tissues such as rodent cerebellum .

• Functional consequences of N-terminal processing include altered signaling capability. Using a yeast G protein chimera assay, full-length GPR37L1 was shown to signal via Gαs, while a mutant lacking the entire N-terminus lost this ability .

• Unlike Protease-Activated Receptors where the cleaved peptide serves as a tethered ligand, synthetic peptides corresponding to the GPR37L1 N-terminus did not activate the receptor .

• These findings suggest that GPR37L1 signaling is negatively regulated by metalloprotease-mediated N-terminal processing, providing a potential mechanism for context-dependent modulation of receptor activity .

This regulatory mechanism represents a potential therapeutic target, as compounds that inhibit the relevant metalloproteases might enhance GPR37L1 signaling in conditions where this would be beneficial.

What methodological approaches are most effective for studying GPR37L1?

Researchers investigating GPR37L1 have employed diverse methodological approaches:

Genetic Models:

• Knockout Strategies: CRISPR-Cas9 gene editing has been used to create GPR37L1 knockout cell lines . For guide RNA design, the Optimized CRISPR Design online tool has been employed to identify target sites with minimal off-target effects .

• Animal Models: GPR37L1 knockout mice have been generated to study behavioral, cardiovascular, and pain-related phenotypes . These models are particularly valuable for examining sex-specific effects .

• Overexpression Models: Viral vector-mediated overexpression of GPR37L1 in specific tissues (e.g., dorsal root ganglia) has been used to assess potential therapeutic effects .

Functional Assays:

• G Protein Coupling: The yeast G protein chimera assay has proven effective for investigating GPR37L1 coupling to G proteins .

• Second Messenger Assays: Measurement of cAMP levels provides insights into Gαs and Gαi signaling downstream of GPR37L1 .

• MAPK Signaling: Assays for ERK1/2 phosphorylation have revealed MAPK pathway activation by GPR37L1 .

• Cholesterol Assays: Given the emerging role of GPR37L1 in cholesterol homeostasis, measurement of cellular cholesterol levels is valuable .

Protein Analysis:

• Fluorescent Tagging: GPR37L1 has been tagged with fluorescent proteins (e.g., eYFP, GFP) to monitor localization and trafficking .

• Co-immunoprecipitation: This approach has identified interaction partners such as DNMT1 and mTOR .

• Western Blotting: Differential migration patterns can distinguish processed forms of GPR37L1 .

Clinical/Translational:

• Variant Analysis: Sequence kernel association testing (SKAT) has been employed to identify disease-associated variants , using tools such as the SKAT R package.

• Classification Algorithms: Algorithms that classify variants as benign (VUSB), likely benign (VUSLB), likely pathogenic (VUSLP) or pathogenic (VUSP) have facilitated identification of functionally significant variants .

What phenotypes are observed in GPR37L1 knockout models?

GPR37L1 knockout models exhibit diverse phenotypes across multiple physiological systems:

Neurological/Behavioral Phenotypes:

• Sex-specific changes in anxiety-related behaviors resembling those seen in chronic migraine patients

• No acute migraine phenotype, suggesting compensatory mechanisms or a role in chronic rather than acute migraine

• Impaired resolution of neuropathic pain symptoms following paclitaxel and streptozotocin-induced peripheral neuropathy

Cardiovascular Effects:

• Altered baseline blood pressure regulation in female mice

• Deficits in cardiovascular compensatory responses in male mice

• The restriction of GPR37L1 expression to the brain (absence from peripheral cardiovascular tissues) suggests these effects are centrally mediated

Cellular/Molecular Phenotypes:

• Altered cholesterol homeostasis

• Changes in potassium channel expression and function in satellite glial cells

The sex-specific nature of many of these phenotypes highlights the importance of considering sex as a biological variable in GPR37L1 research. These differences may reflect interactions between GPR37L1 signaling and sex hormones, or sex-specific compensatory mechanisms.

How does GPR37L1 interact with ion channels and regulate neuronal excitability?

GPR37L1 has significant interactions with ion channels, particularly potassium channels in satellite glial cells:

• Coexpression Analysis: GPR37L1 is coexpressed with potassium channels, including KCNJ10 (Kir4.1) in mouse SGCs and both KCNJ3 (Kir3.1) and KCNJ10 in human SGCs .

• Surface Expression Regulation: GPR37L1 regulates the surface expression of these potassium channels, potentially affecting potassium buffering capacity .

• Neuroprotective Effects: GPR37L1 has demonstrated ability to protect neurons during ischemia, possibly by modulating extracellular glutamate concentration and NMDA receptor activation .

• Disease Relevance: Both migraine and epilepsy, conditions associated with GPR37L1 variants, are characterized by neuronal hyperexcitability , suggesting GPR37L1 may normally function to dampen excitation.

• Maresin 1 Signaling: The proposed ligand maresin 1 may signal through GPR37L1 to regulate potassium homeostasis in SGCs, potentially affecting local neuronal excitability .

The regulation of potassium channels by GPR37L1 represents a potential mechanism by which this receptor could modulate neuronal excitability and contribute to neurological disorders characterized by aberrant excitation. This interaction provides a promising target for therapeutic intervention in conditions such as migraine, epilepsy, and neuropathic pain.