Recombinant Human Probable G-protein coupled receptor 75 (GPR75)

What is GPR75 and what are its confirmed ligands?

GPR75 is a member of the G protein-coupled receptor family first identified and cloned from retinal cDNA libraries. Initially considered an orphan receptor, GPR75 has been "deorphanized" with the identification of two primary ligands:

• 20-Hydroxyeicosatetraenoic acid (20-HETE): Binds with high affinity (KD of 1.56 × 10⁻¹⁰ M) and functions as an agonist

• CCL5/RANTES: Binds with high affinity (KD of 5.85 × 10⁻¹⁰ M) but acts as a negative regulator rather than an agonist, inhibiting 20-HETE's activation of GPR75

The receptor is primarily coupled to heterotrimeric Gq proteins, and upon activation, it stimulates inositol trisphosphate production and intracellular calcium mobilization .

How do structural features of GPR75 differ from typical Class A GPCRs?

Cryo-EM studies of GPR75 reveal several structural differences compared to canonical Class A GPCRs:

• GPR75 lacks the highly conserved P5.50 residue (present in most Class A GPCRs), instead having a C214 at position 5.50, which is found in only 1.7% (5 out of 292) of human Class A GPCRs

• The absence of P5.50 results in a straight, rigid TM5 conformation with reduced flexibility for responding to ligand binding

• Several conserved motifs typical of Class A GPCRs are absent in GPR75, suggesting a unique conformational allosteric modulation mechanism

• The orthosteric ligand binding pocket comprises both polar and hydrophobic residues, with Thr212 being particularly critical—mutation of this residue abolishes 20-HETE's ability to activate GPR75

These structural distinctions likely contribute to GPR75's unique pharmacological properties and may influence drug discovery approaches targeting this receptor.

What phenotypes are observed in GPR75 knockout mouse models?

GPR75 knockout mice display multiple phenotypes across different tissue systems:

Metabolic Effects:

• Resistance to weight gain in high-fat diet models, which is allele-dose dependent (25% lower weight gain in heterozygous Gpr75+/- mice and 44% lower weight gain in homozygous Gpr75-/- mice compared to wild type)

• Improved glycemic control and insulin sensitivity

Retinal Effects:

• Age-dependent cone photoreceptor degeneration, suggesting GPR75's role in maintaining retinal health

• This aligns with human genetic studies where five different point mutations in GPR75 were identified in patients with age-related macular degeneration (AMD) but not in control patients

Neurological Effects:

• Altered behaviors in the adult hippocampus, as evaluated through histological, proteomic, and behavioral endpoints

These diverse phenotypes highlight GPR75's multifunctional role across different physiological systems and its potential as a therapeutic target for multiple conditions.

What is the relationship between GPR75 variants and human obesity?

Large-scale exome sequencing studies have revealed significant associations between GPR75 variants and obesity-related phenotypes:

The protective effect of GPR75 loss-of-function variants against obesity is further supported by mouse knockout studies showing resistance to weight gain on high-fat diets . The consistency between human genetic data and mouse models makes GPR75 a particularly promising target for anti-obesity therapeutic development.

What techniques are optimal for studying GPR75-ligand interactions?

Several complementary approaches have proven effective for characterizing GPR75-ligand interactions:

• Surface Plasmon Resonance (SPR):

  • Successfully used to determine binding affinities and kinetics between GPR75 and its ligands

  • Immobilize human full-length recombinant GPR75 onto high sensitivity carboxyl sensors

  • For small molecule binding (e.g., 20-HETE), immobilize ~8500-9300 response units (RU)

  • For protein ligand binding (e.g., CCL5), ~6000 RUs are recommended

  • Use HBSS (pH 7.4) as running buffer, adjust to pH 4.3 for CCL5 binding studies

  • Inject samples at 20 or 50 μl/min flow rate depending on experimental design

• Functional Assays:

  • FLIPR Calcium 6 assays to measure intracellular Ca²⁺ mobilization

  • Homogeneous time-resolved fluorescence (HTRF) IP-1 assays to quantify inositol phosphate accumulation

  • β-arrestin recruitment assays using PRESTO-Tango methodology

  • All three approaches have successfully demonstrated GPR75 activation by 20-HETE and inhibition by CCL5

• Computational Modeling:

  • Identification of putative ligand-binding pockets

  • Mutation of key residues (e.g., Thr212) to validate binding sites

These methodologies provide complementary data on both binding affinity and functional consequences of ligand-receptor interactions.

What approaches are effective for generating GPR75 knockout models?

Based on published studies, successful generation of GPR75 knockout models has employed CRISPR/Cas9 gene editing with the following considerations:

• Guide RNA Design:

  • Multiple guide RNA sets have been successfully used

  • Design should target early exons to ensure complete functional disruption

  • Validation of different founder lines is recommended to account for potential off-target effects

• Backcrossing Strategy:

  • Backcross founder mice to C57BL/6N genetic background through at least 3 successive generations of heterozygous intercrossing

  • This approach minimizes the impact of potential off-target CRISPR effects

  • Genotype verification by DNA sequencing is essential at each generation

• Validation Approaches:

  • Confirm knockout at both mRNA level (using RNAscope) and protein level

  • Evaluate multiple tissues to ensure complete knockout

  • Screen for compensatory changes in related GPCRs

The successful generation of both heterozygous and homozygous GPR75 knockout mice has been instrumental in revealing the receptor's physiological functions.

What are the critical considerations for expressing recombinant GPR75?

Successful expression and purification of functional recombinant GPR75 requires attention to several key factors:

• Expression Systems:

  • Wheat germ cell-free expression systems have been successfully used to produce full-length human GPR75 without tags

  • Mammalian expression systems (particularly HEK293) are preferable for functional studies due to proper post-translational modifications and trafficking

• Protein Stability Considerations:

  • Storage at -80°C with aliquoting to avoid repeated freeze-thaw cycles

  • Use of 25 mM Tris-HCl (pH 8.0) containing 2% glycerol as storage buffer

  • Avoid heating before electrophoresis as it may cause protein aggregation

• Functional Assessment:

  • Verify protein integrity by Western blotting

  • Confirm functional activity through ligand binding assays

  • GPR75 can be successfully used for antibody production, functional studies, and compound screening

The availability of properly folded, functional recombinant GPR75 is critical for structural studies, ligand screening, and antibody development.

How does the interaction between CCL5 and 20-HETE regulate GPR75 function?

The complex interplay between CCL5 and 20-HETE represents a unique regulatory mechanism for GPR75:

• Binding Properties:

  • Both ligands bind GPR75 with nanomolar affinity (20-HETE: KD = 1.56 × 10⁻¹⁰ M; CCL5: KD = 5.85 × 10⁻¹⁰ M)

  • Despite high-affinity binding, CCL5 fails to activate GPR75 signaling

• Functional Antagonism:

  • CCL5 inhibits 20-HETE's ability to activate GPR75 signaling cascades

  • This represents a unique example of ligand-ligand competition where a chemokine serves as a negative regulator of eicosanoid-mediated GPCR activation

• Physiological Implications:

  • The antagonistic relationship may serve as a biological "brake" on 20-HETE's pro-inflammatory and hypertensive signaling

  • This may have therapeutic implications for conditions where 20-HETE signaling is pathologically elevated

This complex regulatory mechanism suggests opportunities for developing biased ligands that could selectively modulate specific GPR75 signaling pathways.

What is the potential role of GPR75 in retinal diseases?

Evidence from both genetic and functional studies suggests GPR75 plays a critical role in retinal health:

• Genetic Association:

  • Five different point mutations in GPR75 were identified in patients with age-related macular degeneration (AMD) but not in control patients, suggesting GPR75 dysfunction may contribute to this degenerative disease

• Expression Pattern:

  • GPR75 is highly expressed in the retina, with levels among the highest in the human body

  • Initially discovered and cloned from human retina cDNA libraries

• Knockout Phenotype:

  • GPR75 knockout mice display age-dependent cone photoreceptor degeneration

  • This aligns with a potential neuroprotective role of GPR75 in retinal health

• Potential Mechanisms:

  • GPR75 has demonstrated neuroprotective properties in cultured brain cells, which may extend to retinal neurons

  • Signaling through GPR75 might support photoreceptor survival through activation of PI3, Akt, and MAP kinase pathways

These findings suggest GPR75 agonists might have therapeutic potential for retinal degenerative diseases, particularly age-related macular degeneration.

How can researchers effectively modulate GPR75 expression in experimental models?

Several approaches have proven effective for modulating GPR75 expression in various experimental systems:

• Gene Knockout:

  • CRISPR/Cas9-based germline knockout in mice

  • Should include appropriate controls and validation of knockdown efficiency

• RNA Interference:

  • siRNA-mediated knockdown in cell cultures

  • Has been successfully employed in human endothelial cells to nullify 20-HETE-stimulated intracellular Ca²⁺ responses

• Overexpression Systems:

  • PRESTO-Tango receptor-ome methodology has been successfully used for GPR75 overexpression in functional studies

  • Allows for analysis of downstream signaling events following ligand binding

• Expression Validation:

  • RNAscope has been successfully used to verify GPR75 expression levels

  • Western blotting with validated anti-GPR75 antibodies (multiple commercial sources available)

  • Functional assays to confirm changes in receptor-mediated signaling

These complementary approaches provide researchers with multiple tools to manipulate GPR75 expression and function across different experimental models.