FLRT2 Human

What is the molecular structure of human FLRT2 and how does it compare to other FLRT family members?

Human FLRT2 is an 85 kDa type I transmembrane glycoprotein synthesized as a 660 amino acid (aa) precursor. Its structure includes a 35 aa signal sequence, a 506 aa extracellular domain (ECD), a 21 aa transmembrane segment, and a 98 aa cytoplasmic region. The ECD contains 10 N-terminal leucine-rich repeats (LRRs) flanked by cysteine-rich areas, and a juxtamembrane fibronectin type III domain .
Human FLRT1 and FLRT3 extracellular domains share approximately 47% amino acid identity with FLRT2, indicating significant structural conservation while maintaining distinct functions. The human FLRT2 ECD exhibits remarkable conservation across species, sharing 97%, 96%, 99%, 96%, and 95% amino acid sequence identity with mouse, rat, equine, canine, and bovine FLRT2 ECDs respectively . This high conservation suggests essential evolutionary functions for this protein.

What experimental methods are most effective for detecting FLRT2 expression in human tissues?

For detecting FLRT2 in human tissues and cell lines, multiple complementary approaches yield optimal results:
Western blotting has been successfully employed to detect FLRT2 in human large cell lung carcinoma (NCI-H460) cell line, with a specific band detected at approximately 80 kDa under reducing conditions . For Western blot analysis, PVDF membrane probed with 1 μg/ml of Goat Anti-Human FLRT2 Antigen Affinity-purified Polyclonal Antibody followed by HRP-conjugated Anti-Goat IgG Secondary Antibody provides reliable detection .
Simple Western analysis offers an alternative approach, where a specific band for FLRT2 appears at approximately 139 kDa when using 10 μg/mL of Goat Anti-Human FLRT2 Antigen Affinity-purified Polyclonal Antibody . The difference in molecular weight between these methods likely reflects post-translational modifications.
For mRNA expression analysis, quantitative PCR remains effective, particularly when investigating expression patterns across different tissues. In adult humans, FLRT2 mRNA is most abundant in pancreas, but is also present in skeletal muscle, brain, and heart tissues .

How does FLRT2 expression vary during human development and across adult tissues?

FLRT2 exhibits distinct spatiotemporal expression patterns that suggest tissue-specific functions. During embryonic development, FLRT2 shows highest expression in specific brain regions, stomach, and posterior to the developing heart . This expression pattern differs notably from FLRT1 and FLRT3, indicating non-redundant developmental roles .
In adult human tissues, FLRT2 mRNA is most abundant in pancreas, with significant expression also detected in skeletal muscle, brain, and heart . This differential expression suggests tissue-specific functions that may include roles in cell adhesion, tissue maintenance, and cell-cell communication.
When designing experiments to study developmental expression, immunohistochemistry with specific anti-FLRT2 antibodies combined with developmental stage markers provides valuable spatiotemporal information. For quantitative assessment across multiple tissues, qPCR and Western blot analysis of tissue lysates offer complementary approaches to establish expression profiles.

What roles does FLRT2 play in neuronal development and migration?

FLRT2 plays critical roles in neuronal development, particularly in conjunction with FLRT3. Studies in mouse models demonstrate that FLRT2 and FLRT3 cooperatively maintain the tangential migration of cortical interneurons during development . Mechanistically, these proteins function in a non-cell-autonomous manner, likely through repulsive signaling mechanisms .
Double deletion of FLRT2 and FLRT3 genes in mouse embryos results in abnormal distribution of interneurons within migratory streams during development, subsequently affecting the layering of somatostatin-positive interneurons postnatally . This indicates FLRT2's importance in establishing proper neuronal circuits.
The repulsive activity of FLRT2 appears to be mediated through interaction with Unc5 receptors. Supporting this mechanism, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, display interneuron defects during development similar to those observed in FLRT2/FLRT3 mutants . In vitro experiments confirm that FLRT proteins function as chemorepellent ligands for developing interneurons, an effect partially dependent on FLRT-Unc5 interaction .

How does FLRT2 interact with FGF receptors and what are the functional consequences?

The fibronectin domain of FLRT2, like other FLRT family members, can bind to Fibroblast Growth Factor (FGF) receptors . This interaction is thought to regulate FGF signaling during development, providing a modulatory mechanism for this important growth factor pathway .
For researchers investigating this interaction, co-immunoprecipitation experiments using tagged versions of FLRT2 and FGF receptors can confirm physical association. Functional assays measuring FGF-dependent cellular responses (such as ERK phosphorylation or cell proliferation) in the presence or absence of FLRT2 can elucidate the regulatory effects of this interaction.
The binding affinity between FLRT2 and different FGF receptor isoforms may vary, potentially conferring tissue-specific modulation of FGF signaling. Systematic binding studies using surface plasmon resonance or similar techniques would help characterize these interactions quantitatively.

What is the structural basis for FLRT2-Unc5D interaction and how can it be experimentally manipulated?

Crystal structure studies have revealed the binding interface between FLRT2 and Unc5D. Based on these structures, key residues in the binding interface have been identified. In FLRT2, histidine 170 (H170) plays a central role in the interaction . Experimental mutations replacing this histidine with either a negatively charged residue (H170E) or an N-linked glycosylation site (H170N) disrupt binding to Unc5D, confirming the essential nature of this binding site .
Similarly, mutations in Unc5D at residues E88, W89, and H91 (E88A+W89A+H91A or W89N+H91T) significantly reduce binding to FLRT2 . In contrast, mutations at sites involved in minor crystal interactions (FLRT2 D248N+P250T, Unc5D L101N+E103T) do not affect binding, suggesting these sites are not physiologically relevant .
For researchers seeking to manipulate this interaction experimentally, these site-directed mutations provide valuable tools to disrupt FLRT2-Unc5D binding without affecting other functions of either protein. This approach enables precise investigation of the specific consequences of FLRT2-Unc5D interaction in various biological contexts.

What evidence supports FLRT2's role as a tumor suppressor in cancer and what are the underlying mechanisms?

Evidence suggests FLRT2 functions as a tumor suppressor, particularly in breast cancer cells . Gene expression analysis reveals that FLRT2 regulates multiple pathways associated with cellular growth, proliferation, adhesion, and movement .
Mechanistically, the tumor-suppressive activity of FLRT2 appears to operate through regulation of several genes involved in cell proliferation. When FLRT2 is downregulated, expression of proliferation-enhancing genes such as TLR3 and IRS2 increases. Conversely, expression of cell proliferation-inhibiting genes including PTEN and PPARG decreases when FLRT2 is downregulated . This pattern of gene expression changes aligns with FLRT2's proposed function in suppressing cellular proliferation.
Research approaches to investigate FLRT2's tumor suppressor activity should include:

• Gene expression profiling before and after FLRT2 knockdown or overexpression

• Cell proliferation assays with FLRT2-modulated cancer cell lines

• In vivo tumor formation studies using xenograft models with manipulated FLRT2 expression

• Analysis of clinical cancer samples for FLRT2 expression correlation with patient outcomes

How does FLRT2 contribute to tumor-specific endothelial interactions in cancer progression?

FLRT2 appears to mediate tumor-specific inter-endothelial adhesion, which may facilitate cancer aggressiveness . Studies by Ando et al. suggest that FLRT2-mediated interactions between endothelial cells within the tumor microenvironment can influence tumor progression .
For researchers exploring this aspect, co-culture systems of endothelial cells with cancer cells expressing varying levels of FLRT2 would help elucidate the mechanisms involved. Techniques such as transwell migration and invasion assays can assess the functional impact of FLRT2-mediated cellular interactions on cancer cell behavior.
Additionally, in vivo models with endothelial-specific FLRT2 knockout or overexpression would provide valuable insights into how FLRT2-mediated endothelial interactions affect tumor angiogenesis, growth, and metastasis.

What are the optimal expression systems for producing recombinant human FLRT2 for structural and functional studies?

For producing recombinant human FLRT2, researchers typically express the extracellular domain (ECD) consisting of amino acids Cys36-Ser539 . The choice of expression system depends on the intended application:
For structural studies requiring large quantities of pure protein, mammalian expression systems such as HEK293 cells are preferred due to their ability to properly fold complex proteins and add appropriate post-translational modifications, particularly glycosylation. Secreted protein can be purified from conditioned media using affinity chromatography approaches.
For functional studies, the expression construct should be carefully designed to include all relevant domains. The CF (carrier-free) format is suitable for cell culture applications to avoid interference from stabilizing proteins . When designing constructs for specific binding studies, researchers should consider excluding the signal sequence (amino acids 1-35) while maintaining the entire functional ECD.

What strategies can resolve discrepancies in molecular weight detection of FLRT2 in experimental systems?

Researchers have reported varying molecular weights for FLRT2 detection across different experimental systems. Western blot analysis typically detects FLRT2 at approximately 80 kDa, while Simple Western analysis shows a band at approximately 139 kDa . These discrepancies likely reflect differences in post-translational modifications, particularly glycosylation.
To resolve these discrepancies, researchers should:

• Employ deglycosylation enzymes (PNGase F, Endo H) to determine the contribution of N-linked glycosylation to apparent molecular weight

• Use multiple detection methods in parallel (Western blot, Simple Western, immunoprecipitation)

• Include appropriate positive controls with known molecular weight

• Consider the effects of sample preparation conditions (reducing vs. non-reducing, denaturation temperature)

• Verify antibody specificity using FLRT2 knockout samples or siRNA-treated cells
  These approaches will help distinguish between true FLRT2 detection and potential artifacts or cross-reactivity with other proteins.

How can FLRT2-Unc5 signaling be targeted for potential therapeutic applications in neurological disorders?

Given the role of FLRT2 and FLRT3 in maintaining proper tangential migration of cortical interneurons , targeting FLRT2-Unc5 signaling may offer therapeutic potential for neurological disorders associated with interneuron migration defects, including aspects of autism and schizophrenia.
Potential therapeutic approaches include:

• Development of small molecule inhibitors or peptide mimetics that can modulate FLRT2-Unc5 interaction with greater specificity than global knockout approaches

• Gene therapy approaches to restore proper FLRT2 expression in developmental disorders

• Cell-based therapies using engineered neural progenitors with optimized FLRT2 expression for transplantation
  Research methodologies should include high-throughput screening for molecules that modify FLRT2-Unc5 interactions, followed by functional assays measuring neuronal migration and circuit formation. In vivo testing using appropriate animal models of neurodevelopmental disorders would be essential for preclinical validation.

What are the challenges in reconciling FLRT2's dual roles in adhesion and repulsion, and how might these be experimentally addressed?

FLRT2 exhibits an intriguing functional duality, mediating both cell adhesion through homotypic interactions and cell repulsion through Unc5 receptor binding . This dual functionality raises the question of how cells integrate these potentially contradictory signals.
To address this challenge, researchers should consider the following experimental approaches:

• Single-cell analysis techniques to identify cell-specific responses to FLRT2 signaling based on receptor expression profiles

• Live imaging of cells expressing fluorescently tagged FLRT2 and Unc5 receptors to visualize the dynamics of adhesion versus repulsion in real time

• Domain-specific mutations that selectively disrupt either adhesive or repulsive functions to dissect their relative contributions in different cellular contexts

• Systems biology approaches incorporating mathematical modeling to predict how the balance between adhesion and repulsion might be regulated under different conditions Understanding the molecular switches that determine whether FLRT2 mediates adhesion or repulsion in a given context could provide valuable insights into both developmental processes and disease mechanisms, potentially opening new avenues for therapeutic intervention.