FLRT3 Human

What is the molecular structure of human FLRT3?

Human FLRT3 is a type I transmembrane glycoprotein synthesized as a 649 amino acid precursor with four distinct domains: a 28 amino acid signal sequence, a 500 amino acid extracellular domain (ECD), a 21 amino acid transmembrane segment, and a 100 amino acid cytoplasmic region. The ECD contains 10 N-terminal leucine-rich repeats (LRRs) flanked by cysteine-rich regions and a juxtamembrane fibronectin type III domain. The LRR domain facilitates protein-protein interactions, while the fibronectin domain mediates binding to FGF receptors . When working with recombinant FLRT3, researchers typically use the ECD portion (Lys29-Pro528) with a C-terminal 6-His tag to investigate binding interactions .

Where is FLRT3 expressed in human tissues?

FLRT3 exhibits a tissue-specific expression pattern. It is highly expressed in brain, kidney, lung, and skeletal muscle tissues, with lower expression levels detected in pancreas, heart, placenta, and liver . During embryonic development, FLRT3 is localized in somitic regions on dermatomyotomal muscle precursors and at the midbrain/hindbrain boundary . In cultured human endothelial cells (HUVECs), FLRT3 has very low basal expression but is rapidly upregulated following VEGF stimulation . For tissue-specific expression studies, immunofluorescent staining with anti-FLRT3 antibodies can detect both membrane-localized and internalized cytoplasmic FLRT3 protein.

What are the primary binding partners of human FLRT3?

FLRT3 engages with multiple protein partners through its leucine-rich repeat domain, including:

Immunofluorescent double-staining experiments have confirmed significant co-localization between FLRT3 and UNC5B in HUVECs, particularly after VEGF-A stimulation . This co-localization occurs both at the cell surface and in intracellular vesicles near the nucleus, suggesting functional coupling in signaling pathways.

How is FLRT3 expression regulated in endothelial cells?

FLRT3 expression in endothelial cells is primarily regulated by VEGF signaling through VEGFR-2. Stimulation with VEGFR-2-binding ligands (VEGF-A, VEGF-F, and VEGF-D) significantly upregulates FLRT3 mRNA, while VEGFR-1-binding ligand PlGF has no effect . The induction begins rapidly, within 30 minutes of VEGF-A stimulation, peaking at 1-1.5 hours post-treatment . VEGFR-2 inhibition with SU1498 completely abolishes FLRT3 upregulation, confirming this receptor's critical role . RNA polymerase II ChIP-Seq analysis shows rapid induction at the promoter and gene body 1 hour after VEGF-A stimulation, indicating release of paused polymerase into productive elongation .

What methods are effective for studying FLRT3 function in endothelial cells?

Several methodological approaches have proven effective for investigating FLRT3 function in endothelial cells:

• siRNA-mediated knockdown: Transfection of HUVECs with FLRT3-specific siRNA significantly reduces FLRT3 expression and allows assessment of functional consequences on survival, migration, and tube formation .

• Recombinant protein studies: Using carrier-free recombinant human FLRT3 protein (typically the ECD portion, Lys29-Pro528) for stimulation experiments to investigate direct effects on cellular functions .

• Live-cell imaging: Systems like IncuCyte S3 can continuously monitor cellular behaviors (migration, wound healing) after FLRT3 manipulation over extended time periods .

• Immunofluorescent co-localization: Double-staining with antibodies against FLRT3 and potential interaction partners (e.g., VEGFR-2, UNC5B) to visualize spatial relationships in subcellular compartments .

• In vitro angiogenesis assays: Tube formation and angiogenesis inhibition assays following FLRT3 manipulation provide insights into its role in vascular network formation .

How does FLRT3 influence VEGF-induced endothelial cell functions?

FLRT3 demonstrates complex and sometimes opposing effects on different VEGF-induced endothelial cell functions:

These apparently contradictory effects suggest FLRT3 may function as a contextual regulator, promoting stable vessel formation by enhancing survival and tube formation while limiting excessive migration . Future studies should investigate the molecular mechanisms underlying these differential effects, potentially through identification of signaling pathway components unique to each function.

How should researchers interpret the relationship between FLRT3 and UNC5B signaling in vascular biology?

The relationship between FLRT3 and UNC5B requires careful interpretation in vascular contexts. While both proteins are upregulated following VEGF stimulation, their temporal patterns differ: FLRT3 shows rapid induction (0.5-1.5 hours), whereas UNC5B demonstrates delayed upregulation (3-4 hours) . Immunofluorescence studies confirm their co-localization in both membrane and cytoplasmic compartments after VEGF stimulation . Notably, siRNA knockdown of either FLRT3 or UNC5B produces similar phenotypes regarding cell migration, suggesting functional coupling .

When designing experiments to investigate this relationship, researchers should:

• Consider the temporal dynamics of expression

• Examine both proteins simultaneously through co-immunoprecipitation or proximity ligation assays

• Employ domain-specific mutations to identify interaction regions

• Use conditional knockout models to dissect tissue-specific functions

• Explore downstream signaling pathways activated by the FLRT3-UNC5B interaction

What are the critical considerations for working with recombinant human FLRT3 protein?

When using recombinant human FLRT3 protein for research, several critical factors must be considered:

• Protein formulation: Standard preparations contain bovine serum albumin (BSA) as a carrier protein to enhance stability and shelf-life. For applications where BSA might interfere (such as certain binding assays or mass spectrometry), carrier-free (CF) versions should be used .

• Reconstitution protocol: Lyophilized protein should be reconstituted at 200 μg/mL in sterile PBS. Improper reconstitution may affect protein activity and experimental outcomes .

• Storage conditions: Use a manual defrost freezer and avoid repeated freeze-thaw cycles that can degrade protein quality. Upon receipt, store immediately at recommended temperatures .

• Domain selection: The full ECD (Lys29-Pro528) contains all functional domains, but some experiments may benefit from domain-specific constructs to isolate particular interactions .

• Concentration optimization: Optimal dilutions should be determined empirically for each application, as effective concentrations may vary substantially between different experimental systems .

What might cause inconsistent results when studying FLRT3 in endothelial cells?

Several factors can contribute to inconsistent results when studying FLRT3 in endothelial cells:

• Baseline expression variations: The extremely low basal expression of FLRT3 in unstimulated HUVECs means minor variations in culture conditions can significantly affect baseline levels .

• VEGF preparation differences: Different VEGF isoforms and preparations have varying potencies in inducing FLRT3. Researchers should standardize VEGF source, concentration (50-250 ng/ml is typically effective), and exposure time (peak induction at 1-1.5 hours) .

• Cell density effects: FLRT3 accumulates at cell-cell contacts, so experiments conducted at different cell densities may yield varying results due to differences in homotypic FLRT3-FLRT3 interactions .

• Passage number influence: Higher expression of FLRT3 is detected in proliferating HUVECs grown in high serum conditions, suggesting passage number and proliferation status affect expression levels .

• Non-specific antibody binding: When performing immunofluorescence or Western blot analysis, validate antibody specificity using appropriate controls (e.g., Hela cells expressing low endogenous FLRT3) .

How can researchers effectively measure FLRT3-mediated effects on cell migration versus survival?

To effectively distinguish between FLRT3's effects on cell migration versus survival, researchers should:

• Employ complementary assays:

  • For migration: Use both wound healing assays with live-cell imaging (e.g., IncuCyte) and transwell migration assays

  • For survival: Combine MTS/MTT assays with direct apoptosis measurements (Annexin V/PI staining, caspase activation)

• Control for confounding factors:

  • In migration assays, use mitomycin C to inhibit proliferation, ensuring observed effects reflect true migration

  • In survival assays, normalize to cell number at experiment initiation to account for attachment differences

• Implement time-course analyses: FLRT3's effects may vary temporally, with different outcomes at early (0-6h) versus late (24-48h) timepoints following VEGF stimulation .

• Consider pathway-specific inhibitors: Determine whether FLRT3's effects on migration versus survival operate through distinct signaling pathways by using targeted inhibitors during functional assays.

• Rescue experiments: Perform rescue experiments with recombinant FLRT3 following siRNA knockdown to confirm specificity of observed phenotypes.

What are the potential roles of FLRT3 in pathological angiogenesis?

Given FLRT3's newly discovered role in regulating endothelial functions, several promising research directions could explore its involvement in pathological angiogenesis:

• Tumor angiogenesis: Investigate FLRT3 expression in tumor vasculature and its correlation with tumor progression, metastasis, and response to anti-angiogenic therapies.

• Diabetic retinopathy: Examine FLRT3 levels in retinal vessels during diabetic retinopathy progression and test whether modulation affects abnormal vessel formation.

• Wound healing disorders: Study FLRT3's role in impaired angiogenesis associated with chronic wounds, particularly in diabetic patients.

• Inflammatory conditions: Explore how inflammatory mediators interact with VEGF-induced FLRT3 expression and function during inflammatory angiogenesis.

• Therapeutic targeting: Develop and test FLRT3-targeted antibodies or peptides that could modulate specific aspects of angiogenesis for therapeutic applications.

How might FLRT3 interact with other angiogenic signaling pathways beyond VEGF?

While FLRT3's relationship with VEGF signaling is now established, future research should investigate potential crosstalk with other angiogenic pathways:

• FGF pathway: Given FLRT3's known interaction with FGF receptors during development , explore whether similar interactions occur in endothelial cells and how this affects angiogenic responses to FGF stimulation.

• Notch signaling: Investigate potential interactions between FLRT3 and Notch pathway components, which are critical for tip/stalk cell determination during angiogenesis.

• Angiopoietin/Tie2 axis: Examine whether FLRT3 influences vessel maturation and stability through effects on angiopoietin signaling.

• Hypoxia response: Determine if hypoxia directly regulates FLRT3 expression independently of VEGF and whether FLRT3 affects cellular responses to hypoxic conditions.

• Mechanotransduction: Study whether FLRT3's localization at cell-cell contacts makes it sensitive to mechanical forces and a potential transducer of biomechanical signals during angiogenesis.