Extracellular leucine-rich repeat (LRR) structures are widely present in secreted proteins, extracellular matrix proteins, and transmembrane receptors and constitute an important structural basis for extracellular molecular recognition. Proteins such as LRG1, Slit, SLRPs, TLRs, and LGRs can use LRRs to participate in receptor regulation, matrix organization, cell migration, and immune signal transduction.

Keywords: leucine-rich repeat proteins; LRG1; SLRP; Decorin; Biglycan; Lumican; TLR; LGR

1 Structural Basis and Functional Types of Extracellular LRR Proteins

1.1 LRR Repeats and Protein-Binding Interfaces

LRRs consist of tandemly arranged repeat units enriched in Leu and other hydrophobic residues. These repeat units collectively form curved solenoid-like or horseshoe-shaped structures. The relatively regular inner β-sheet surface forms a continuous molecular-recognition interface, whereas the outer structure maintains overall conformational stability, making LRRs suitable for multipoint contacts with proteins, glycoproteins, or large extracellular structures. LRRs themselves do not possess a fixed catalytic function; their biological roles depend on the protein in which they are integrated, their spatial location, and the molecules they directly recognize.

1.2 Classification According to Extracellular Form

(1) Soluble Secreted LRR Proteins

LRG1 and Slit both contain LRR structures and are secreted extracellularly, but they function differently. LRG1 mainly alters existing signaling outputs by interacting with receptor systems or extracellular regulatory proteins, whereas Slit uses its LRR structures to directly bind Robo receptors and functions as a ligand in cell migration and axon-guidance signaling.

(2) Extracellular Matrix LRR Proteins

Decorin, Biglycan, Lumican, and Fibromodulin belong to the Small leucine-rich proteoglycan (SLRP) system. Their LRR cores can directly bind matrix components such as Collagen and regulate collagen-fiber formation, diameter, and organization. After release from the ECM, some members can also alter growth-factor receptor or pattern-recognition receptor signaling.

(3) Membrane Receptor LRR Proteins

The TLR family and LGR4/5/6 position LRRs directly within their extracellular receptor regions. TLRs use LRR structures to organize recognition of pathogen-associated molecules and transmit information to intracellular TIR domains, whereas LGRs bind R-spondin through extracellular LRRs and regulate RNF43/ZNRF3 and Wnt receptor stability.

Table 1 Functional Classification of Representative Extracellular LRR Proteins

2 Soluble Secreted LRR Proteins

2.1 LRG1

(1) Secreted Glycoprotein and LRR Recognition Surface

Leucine-rich alpha-2-glycoprotein 1 (LRG1) is a secreted glycoprotein. The mature protein contains multiple tandem LRRs and exhibits substantial glycosylation. The large protein-binding surface formed by the LRRs makes LRG1 more suitable as an extracellular interaction-regulatory protein than as a signaling molecule with intrinsic catalytic activity. Hepatocytes, granulocytes, and cells from certain diseased tissues can all contribute distinct sources of LRG1.

(2) LRG1–Endoglin and TGF-β Signaling Bias

In vascular endothelial cells, LRG1 can interact with the Endoglin-associated receptor system and enhance ALK1–SMAD1/5-related output in the presence of TGF-β. LRG1 is not a new TGF-β ligand and does not directly phosphorylate SMAD proteins. Instead, it acts at the receptor-complex level by altering the relationship among Endoglin, type I receptors, and TGF-β input, thereby biasing endothelial signaling toward a pro-angiogenic branch.

(3) Tissue Environment-Dependent Signaling Output

The effect of LRG1 is not invariably equivalent to SMAD1/5 activation. In renal tubular and certain fibrotic environments, LRG1 can enhance TGF-β–SMAD3-related responses and promote fibroblast activation and ECM deposition. Different SMAD outputs in endothelial cells and fibrotic tissues mainly reflect differences in receptor composition, cell type, and local microenvironment.

2.2 Slit Family

(1) LRR Domains and Robo Recognition

Slit1, Slit2, and Slit3 are large secreted proteins containing multiple LRR domains at their N termini, among which the second LRR domain is an important region for Robo receptor recognition. Unlike LRG1, which regulates receptor complexes, the LRRs of Slit directly constitute the ligand-side binding interface, allowing the Slit–Robo axis to form a relatively defined ligand–receptor recognition system.

(2) Robo Signaling and Directional Migration

After Slit binds Robo, it alters Rho-family small GTPases and cytoskeleton-related networks, thereby regulating growth-cone movement, cell polarity, and migration direction. In the nervous system, this mechanism mainly participates in axon guidance, whereas in vascular endothelial cells, immune cells, and tumor cells, it can further influence migration, tissue infiltration, and spatial relationships among cells.

3 Small Leucine-Rich Proteoglycans and ECM Organization

3.1 Structural Characteristics of SLRPs

(1) LRR Core and Glycosaminoglycan Modification

SLRPs generally contain a protein core composed of LRRs, and some members are additionally linked to glycosaminoglycan chains such as Dermatan sulfate, Chondroitin sulfate, or Keratan sulfate. The protein core mainly determines binding to Collagen and other proteins, whereas the glycan chains influence hydration, intermolecular spacing, and spatial distribution of proteins within the ECM, allowing SLRPs to participate simultaneously in matrix architecture and cellular signaling regulation.

(2) Collagen Fibril Formation

During fibril formation, Collagen molecules undergo alignment, nucleation, lateral aggregation, and maturation. SLRPs can bind specific regions on fibril surfaces and restrict disordered lateral aggregation. Their primary function is not to increase Collagen synthesis but to control how existing collagen assembles into mature fibrils with defined diameters, spacing, and orientation.

3.2 Decorin and Biglycan

(1) Decorin

Decorin can directly bind fibrillar Collagens such as type I Collagen and restrict lateral expansion of collagen fibrils, thereby maintaining relatively stable fibril diameter and organization. Decorin can also interact with receptor systems such as EGFR and MET as well as certain growth factors, giving it dual functions in ECM structural regulation and cell-surface signaling. When Decorin is reduced, marked abnormalities in fibril organization and tissue mechanics may occur even when total Collagen levels change only slightly.

(2) Biglycan

Within intact ECM, Biglycan mainly associates with Collagen and other matrix components, whereas after tissue injury or proteolytic release, it can convert into a soluble signaling molecule. Free Biglycan can engage TLR2/TLR4-associated receptor systems and activate inflammatory pathways including NF-κB and MAPK, allowing the same protein to perform different functions in its “matrix-bound state” and “soluble state.”

3.3 Lumican and Fibromodulin

(1) Lumican

Lumican interacts with fibrillar Collagen through its LRR core, restricting collagen-fibril diameter and maintaining regular organization. The cornea is highly sensitive to collagen-fibril spacing and alignment, and abnormalities in Lumican can disrupt this ordered structure and impair transparency. In tissues such as skin, its alterations are more commonly reflected by abnormalities in collagen organization and mechanical properties.

(2) Fibromodulin

Fibromodulin likewise binds fibrillar Collagen but is more prominently involved in fibril formation and maturation and has an important role in tissues rich in type I Collagen, such as tendons. Fibromodulin and Lumican have partially overlapping functions, and combined loss of both produces more pronounced collagen-fibril abnormalities, indicating that different SLRPs jointly constrain fibril assembly through distinct binding positions and temporal stages.

4 Membrane Receptor LRR Proteins

4.1 Toll-Like Receptors

(1) Extracellular LRR Recognition Structures of TLRs

TLRs are type I transmembrane receptors with extracellular regions composed of continuous LRRs and intracellular regions containing TIR domains. Differences in the shape and chemical properties of the LRR surfaces allow TLR3, TLR4, TLR2 heterodimers, and other receptors to recognize nucleic acids, lipopolysaccharide-associated complexes, or microbial lipids, respectively. Extracellular molecular recognition is subsequently transmitted to intracellular TIR domains through receptor clustering.

(2) TLR4–MD-2–LPS Complex

Recognition of LPS by TLR4 requires MD-2. After the lipid component of LPS enters the hydrophobic cavity of MD-2, LPS–MD-2 further contributes to the dimerization interface between two TLR4 complexes. Formation of the extracellular complex brings the two intracellular TIR domains into proximity, followed by recruitment of MyD88- or TRIF-associated adaptor proteins, converting LRR-mediated pathogen recognition into inflammatory signals including NF-κB, p38, JNK, and ERK.

4.2 LGR4, LGR5, and LGR6

(1) R-spondin–LGR Recognition

LGR4/5/6 are seven-transmembrane receptors containing large extracellular LRR domains. R-spondin can bind their LRR structures and form cell-surface regulatory complexes. In this process, LGRs differ from classical GPCRs that generate second messengers through G proteins; their primary function is concentrated on regulation of the Wnt receptor system.

(2) RNF43/ZNRF3 and Wnt Receptor Stability

RNF43 and ZNRF3 are E3 ubiquitin ligases that promote removal of Wnt receptors such as Frizzled from the plasma membrane. The R-spondin–LGR complex can limit this negative regulatory system, allowing Frizzled and related receptors to remain at higher levels on the cell surface. Cellular responses to the same concentration of Wnt ligand are thereby enhanced. LRG1 and LGR also illustrate a common functional mode of LRR proteins: rather than directly providing the major ligand input, they modify the responsiveness of an existing receptor system.

5 Functional Diversification of LRR Structures

5.1 Direct Ligand–Receptor Recognition

Slit and TLR both depend on LRRs for molecular recognition, but the LRRs are located on different sides of the interaction. Slit places LRRs on the secreted ligand side and uses them to bind Robo, whereas TLRs position LRRs on the extracellular side of membrane receptors to recognize microbial-associated molecules or corresponding coreceptor complexes. These systems demonstrate two different uses of the same structural module on the ligand and receptor sides.

5.2 Receptor-Complex Regulation

LRG1 and LGR share the characteristic of primarily modifying the operational state of other receptor systems. LRG1 regulates Endoglin and the TGF-β receptor environment to bias SMAD branch output, whereas R-spondin–LGR controls Frizzled membrane stability through RNF43/ZNRF3, thereby altering Wnt receptor abundance and ligand sensitivity.

5.3 Organization of ECM Supramolecular Structures

The LRRs of Decorin, Lumican, and Fibromodulin are mainly used for sustained contact with Collagen surfaces rather than for transmembrane signal transmission. Multiple SLRPs impose spatial constraints on lateral collagen aggregation, fibril diameter, and maturation, allowing different tissues to establish collagen structures that meet their mechanical or optical requirements.

5.4 Conversion Between Matrix-Bound and Soluble States

Biglycan illustrates direct control of function by protein spatial state: when immobilized within the ECM, it participates in matrix organization, whereas after release from the matrix, it can engage TLR2/4 and generate inflammatory signaling. ECM degradation caused by tissue injury not only disrupts structural integrity but also alters the availability of preexisting matrix proteins, allowing some structural molecules to convert into damage-associated signals.

Table 2 Molecular Modes of Action of Different LRR Proteins

6 Experimental Investigation and Interpretation of Extracellular LRR Proteins

6.1 Protein Expression and Actual Functional State

Total expression levels of LRR proteins do not directly represent their functional state because different members depend on secretion, matrix immobilization, or membrane localization. For LRG1, it is necessary to confirm whether the protein is actually released extracellularly; for Biglycan, the ECM-bound and soluble states need to be distinguished; and for TLRs/LGRs, membrane receptor and receptor-complex status should be confirmed. Measuring only total tissue or whole-cell lysates can lose the spatial information that determines function.

6.2 Secreted LRR Proteins

(1) LRG1

In endothelial models, LRG1 can be linked with Endoglin, ALK1-associated p-SMAD1/5, and angiogenic phenotypes. In fibrosis models, it is more appropriate to examine p-SMAD2/3, particularly SMAD3, together with α-SMA, Collagen, and ECM deposition. Using the same set of SMAD indicators across different tissues can lead to incorrect interpretation of the TGF-β branch actually connected to LRG1.

(2) Slit

Slit studies need to connect secretion levels, Robo receptor status, and directional cell movement. Increased Slit expression may not produce corresponding migration phenotypes when Robo is insufficient, whereas observing altered cell migration alone cannot demonstrate involvement of the Slit–Robo axis; ligand–receptor dependency needs to be validated.

6.3 Matrix SLRPs

(1) Collagen Content and Collagen Organization

Abnormalities in Decorin, Lumican, or Fibromodulin can cause marked changes in fibrillar architecture even when total Collagen levels change only slightly. Collagen-fibril diameter, organization, maturation, and tissue mechanics more directly reflect the core functions of these SLRPs than measurement of COL1A1 or total Collagen alone.

(2) State Conversion of Biglycan

Biglycan studies need to separately analyze matrix-associated and soluble fractions while simultaneously examining TLR2/4, NF-κB, and MAPK signaling. Increased total Biglycan may indicate ECM accumulation, matrix release following tissue injury, or increased Biglycan secretion under inflammatory conditions; elevation of soluble Biglycan more directly corresponds to TLR2/4-mediated inflammatory signaling.

6.4 Membrane Receptor LRR Proteins

(1) TLR

TLR studies should be interpreted stepwise according to “ligand recognition–receptor complex–adaptor protein–downstream signaling.” In the TLR4 system, MD-2 participates in proximal recognition, MyD88/TRIF represent intracellular signaling entry points, NF-κB and MAPKs reflect downstream transduction, and inflammatory mediators such as TNF-α and IL-6 represent final functional outputs.

(2) LGR

The core functions of LGR4/5/6 need to be evaluated in the context of R-spondin and Wnt. Frizzled membrane stabilization, β-Catenin accumulation, and changes in Wnt target genes can be measured. Conventional GPCR readouts such as cAMP do not represent the principal receptor-regulatory function of the R-spondin–LGR system.

Table 3 Experimental Interpretation Levels for Different LRR Proteins

7 Extracellular LRR Proteins and Related Research Products

7.1 Secreted LRR Proteins and Receptor-Regulation-Related Products

7.2 SLRP and Extracellular-Matrix-Related Products

7.3 TLR- and LGR-Related Products

Extracellular LRR proteins use similar repeat structures to form different types of extracellular recognition interfaces, but their ultimate functions depend on the molecular environment in which they reside, the molecules they directly bind, and the downstream signaling connections they establish.

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