LRG1 was associated with disease, but its receptor was unknown

LRG1 is a serum glycoprotein produced mainly by hepatocytes and neutrophils. Previous research had associated elevated LRG1 levels with inflammatory conditions, angiogenesis, vascular dysfunction and diabetes.

LRG1 was also known to influence transforming growth factor beta, or TGF-β, signalling. However, the researchers observed effects that could not be explained by TGF-β signalling alone.

This raised an important question: Does LRG1 bind to another receptor on the cell surface?

Finding that receptor was essential for understanding how LRG1 affects endothelial cells and nerve tissue. Without a defined receptor, it remained difficult to explain the mechanism, confirm the relevant signalling pathways or assess the interaction as a possible therapeutic target.

Identifying a receptor directly on living cells

To search for an unknown LRG1 receptor, the researchers used the LRC-TriCEPS ligand-receptor capture platform.

LRC-TriCEPS is designed to identify glycosylated cell-surface proteins that interact with a protein, peptide, antibody, virus or another ligand of interest. The experiment takes place on living cells, where receptors remain embedded in the plasma membrane and retain their natural cellular environment.

The approach does not require genetic modification of the cells or an existing hypothesis about the identity of the receptor.

For the LRG1 experiment, the researchers coupled recombinant LRG1 to the TriCEPS chemoproteomic reagent and incubated it with living HEK293T cells. A TriCEPS-coupled transferrin sample served as a control.

After the ligand-receptor interactions had been captured, the membrane proteins were isolated and analysed using liquid chromatography-tandem mass spectrometry, or LC-MS/MS.

The analysis identified three prospective LRG1-binding proteins:

• Galectin-3
• NADH dehydrogenase 1 alpha subcomplex subunit 5
• Latrophilin-2

LPHN2 was the only cell membrane protein among the three candidates, making it the strongest candidate for an LRG1 receptor.

From receptor candidate to validated interaction

Receptor discovery provides candidates that require independent confirmation. The research group therefore used several additional methods to examine the interaction between LRG1 and LPHN2.

Reducing LPHN2 expression through lentiviral-mediated knockdown significantly decreased the binding of LRG1 to the cell surface. Co-immunoprecipitation experiments also supported a specific association between LRG1 and LPHN2 on endothelial cell membranes.

Further binding studies showed that LRG1 interacts with the olfactomedin-like domain of LPHN2. The researchers also tested the closely related receptors LPHN1 and LPHN3. Despite their high sequence similarity, LRG1 did not bind to the corresponding domains of these receptors.

These experiments supported the conclusion that LPHN2 is a specific, TGF-β-independent receptor for LRG1.

What happens when LRG1 activates LPHN2?

After establishing the receptor interaction, the researchers examined its biological function.

Under high-glucose conditions, LRG1 promoted angiogenic and neurotrophic processes in endothelial cells and mouse tissue explants. These processes included microvessel sprouting, endothelial tube formation and neurite outgrowth.

When LPHN2 expression was reduced, the effects of LRG1 also decreased. This indicated that LPHN2 was required for the observed vascular and neurological responses.

The study identified PI3K, AKT and NF-κB p65 as important components of the intracellular signalling pathway activated through the LRG1-LPHN2 axis.

The researchers then studied the pathway in a diabetic mouse model of erectile dysfunction. Diabetes-related erectile dysfunction can involve both endothelial damage and peripheral neuropathy, making it a useful model for studying vascular and nerve repair simultaneously.

Local administration of LRG1 improved vascular and neurological abnormalities and restored erectile function in the diabetic mice. These results were preclinical and do not demonstrate therapeutic efficacy in humans, but they provided evidence that the newly identified receptor pathway had a measurable biological function.

A structural study revealed the role of LRG1 glycosylation

One year after the receptor study, the same research group published the three-dimensional crystal structure of LRG1.

The structure showed that LRG1 forms a horseshoe-like solenoid and contains four N-glycosylation sites at N79, N186, N269 and N325.

Glycosylation is the attachment of carbohydrate structures, known as glycans, to a protein. These glycans can affect protein folding, stability, localisation and interactions with other molecules.

The researchers investigated whether the glycans attached to LRG1 influenced its ability to bind to LPHN2.

Their experiments showed that deglycosylated LRG1 bound more strongly to LPHN2 than the native glycosylated protein. Deglycosylated LRG1 also promoted endothelial cell activity and neurite outgrowth under normal glucose conditions, where native LRG1 had shown limited activity.

Why the N325 glycan matters

To determine which glycosylation site had the greatest influence, the researchers created individual LRG1 variants in which the relevant asparagine residue was replaced with aspartic acid. This change was used to mimic the deglycosylated form of the protein.

Among the four variants, the N325D mutant produced the clearest effect.

The LRG1 N325D variant showed stronger binding to LPHN2 and promoted endothelial tube formation and neurite outgrowth under normal glucose conditions. Reducing LPHN2 expression diminished these effects, confirming that the activity still depended on the identified receptor.

The results suggest that the glycan attached to N325 creates steric hindrance that limits close interaction between LRG1 and LPHN2. Removing this glycan, or modifying the site, appears to allow tighter receptor binding and stronger activation of the downstream pathway.

The deglycosylated protein and the N325D variant were also studied in diabetic mice. Local administration improved vascular and neurological abnormalities and restored several measured parameters of erectile function.

These findings provide a structural starting point for exploring modified LRG1 variants. Considerable additional research would still be required to evaluate their pharmacology, safety, delivery and potential clinical application.

Why receptor identification changed the research direction

Before LPHN2 was identified, LRG1 was associated with several biological and disease-related processes, but the mechanism behind some of its effects remained unclear.

Identifying the receptor made several new lines of research possible:

• The ligand-receptor interaction could be validated using independent methods.
• The relevant binding domain could be investigated.
• Downstream intracellular signalling could be mapped.
• The effects of reducing receptor expression could be measured.
• Protein structure and glycosylation could be connected to receptor affinity.
• Modified ligand variants could be designed and compared.

This is the practical value of receptor discovery. A biological observation becomes a defined molecular interaction that can be tested, modified and assessed in disease models.

What the studies show and what remains open

The studies provide evidence that LPHN2 is a receptor for LRG1 and that the LRG1-LPHN2 axis can support angiogenic and neurotrophic processes in the experimental systems used.

They also show that glycosylation at N325 influences the strength and functional consequences of the interaction.

The results do not yet establish LRG1, deglycosylated LRG1 or the N325D variant as treatments for people with diabetes or diabetic complications. The findings are based on biochemical experiments, cell cultures, mouse tissue explants and diabetic mouse models.

Further research is needed to determine whether the pathway can be targeted safely and effectively in humans.

Receptor discovery with LRC-TriCEPS

LRC-TriCEPS enables the unbiased identification of cell-surface receptors and off-targets directly on living cells.

The platform can be used for a wide range of ligands, including:

• Proteins
• Peptides
• Antibodies
• Engineered affinity binders
• Viruses
• Virus-like particles
• Extracellular vesicles
• Other biologics

Because the interaction takes place on living cells, the target proteins remain within their natural membrane environment. Researchers can also select cell types and experimental conditions that reflect the biological context of their project.

The workflow combines ligand-receptor capture with mass spectrometry, statistics and bioinformatics to identify enriched cell-surface proteins for subsequent validation.