FLRT3 Human, HEK

What is the molecular structure of human FLRT3 protein?

Human FLRT3 is an 85-95 kDa type I transmembrane glycoprotein synthesized as a 649 amino acid precursor. Its structure includes 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 areas, and a juxtamembrane fibronectin type III domain. This complex structure facilitates FLRT3's diverse protein-protein interactions and signaling capabilities .

What are the primary functions of FLRT3 in neural systems?

FLRT3 serves as a multifunctional protein in neural development and function. It participates in cell-cell adhesion, cell migration, and axon guidance, exerting either attractive or repulsive roles depending on its interaction partners. It plays a crucial role in the spatial organization of brain neurons and promotes neurite outgrowth. Significantly, FLRT3 functions as an endogenous postsynaptic ligand for latrophilins (including LPHN3), with this interaction being essential for glutamatergic synapse development and maintenance .

How can recombinant human FLRT3 be produced in HEK293 cells?

Recombinant human FLRT3 protein can be expressed in HEK293 cells as a fragment spanning amino acids 1-528, typically with a C-terminal 6-His tag for purification purposes. When properly expressed, the protein achieves >98% purity with endotoxin levels below 1 EU/μg. The recombinant protein corresponds specifically to the extracellular portion (Lys29-Pro528) of FLRT3, making it suitable for investigating extracellular interactions without membrane constraints .

What sequence homology exists between human FLRT3 and other species?

The human FLRT3 extracellular domain exhibits high evolutionary conservation, sharing 96%, 96%, 97%, 97%, 98%, and 81% amino acid sequence identity with mouse, rat, canine, bovine, equine, and Xenopus FLRT3 ECDs, respectively. Within the human FLRT family, the FLRT3 ECD shares 61% amino acid identity with FLRT2 and 48% with FLRT1, indicating functional specialization within this protein family while maintaining core structural elements .

What methodologies can be used to study FLRT3-LPHN3 interactions?

Several complementary approaches can be employed to investigate FLRT3-LPHN3 interactions:

• Affinity chromatography and mass spectrometry: For identifying binding partners and interaction domains.

• Direct binding assays: Using purified proteins (FLRT3-His and ecto-LPHN3-Fc) in cell-free systems to confirm direct interactions and eliminate potential co-receptor involvement.

• Surface plasmon resonance (SPR): For quantitative characterization of binding affinity between FLRT3 and LPHN3 ectodomains.

• Cellular binding assays: Expressing FLRT3-myc in HEK293 cells and applying ecto-LPHN3-Fc to assess binding specificity compared to control proteins.

• Co-immunoprecipitation: To verify protein-protein interactions in cellular contexts.

• Co-culture systems: LPHN3-expressing neurons cultured with FLRT3-expressing HEK293 cells to observe protein clustering at contact sites .

How can researchers manipulate FLRT3-LPHN3 signaling in neuronal systems?

Three distinct experimental approaches have proven effective for perturbing FLRT3-LPHN3 complexes:

• Competition with soluble ectodomains: Application of excess soluble ecto-LPHN3-Fc protein to competitively disrupt endogenous LPHN3 complexes.

• shRNA knockdown of FLRT3: Electroporation of neurons with FLRT3-targeting shRNA (shFlrt3), with validation through rescue experiments using shRNA-resistant FLRT3 constructs.

• shRNA knockdown of LPHN3: Targeted reduction of presynaptic LPHN3 expression.

All three approaches result in reduced glutamatergic synapse density, supporting the critical role of FLRT3-LPHN3 interaction in synapse formation and maintenance .

What functional assays can evaluate the impact of FLRT3 manipulation in neurons?

Following genetic or pharmacological manipulation of FLRT3, several functional assays can assess the resulting phenotypes:

• Immunofluorescence analysis: Quantifying synaptic puncta density and size using markers like PSD95 and synapsin.

• Electrophysiology: Recording miniature excitatory postsynaptic currents (mEPSCs) to measure both frequency (reflecting synapse number) and amplitude (reflecting synaptic strength).

• In vivo manipulations: Reducing FLRT3 levels with shRNA in vivo to evaluate effects on afferent input strength and dendritic spine numbers in specific neuronal populations like dentate granule cells.

• Heterologous cell assays: Testing whether FLRT3 or LPHN3 expression in HEK293 cells can induce pre- and postsynaptic differentiation in contacting neurons .

How does FLRT3 contribute to glutamatergic synapse development?

FLRT3 plays a critical role in glutamatergic synapse development through multiple mechanisms:

• FLRT3 functions as a postsynaptic adhesion molecule that interacts with presynaptic latrophilins (including LPHN3) across the synaptic cleft.

• Knockdown of FLRT3 with shRNA significantly reduces excitatory synapse density in cultured hippocampal neurons.

• This reduction in synapse density correlates with decreased miniature excitatory postsynaptic current (mEPSC) frequency.

• In vivo reduction of FLRT3 levels decreases both afferent input strength and dendritic spine numbers in dentate granule cells.

• The FLRT3-LPHN3 interaction appears to positively regulate synapse number, as disruption of this interaction through multiple approaches consistently leads to reduced glutamatergic synapse density .

What is the dual role of FLRT3 in adhesion and repulsion signaling?

FLRT3 exhibits remarkable functional duality through distinct molecular surfaces that mediate opposing cellular responses:

• Homophilic adhesion: FLRT3 can interact with FLRT3 molecules on adjacent cells to promote cell-cell adhesion.

• Heterophilic repulsion: Simultaneously, FLRT3 can interact with Unc5 receptors to mediate repulsive guidance.

• Integrative signaling: Neurons expressing both Unc5 and FLRT integrate these adhesive and repulsive signals from FLRT3, resulting in a balanced response that directs precise neuronal positioning and connectivity.

• Structural basis: Crystal structures of FLRT proteins and their complexes with Unc5 receptors have revealed that these opposing functions are mediated by structurally distinct binding interfaces .

What domain-specific functions have been identified in FLRT3?

FLRT3's diverse functions are mediated by its distinct structural domains:

• Leucine-rich repeat (LRR) domain:

  • Mediates homophilic FLRT-FLRT interactions

  • Responsible for localization in areas of cell contact

  • Facilitates homotypic cell-cell association

  • Involved in binding to Unc5 receptors

• Fibronectin type III domain:

  • Responsible for binding to FGF receptors

  • Regulates FGF signaling during development

• Juxtamembrane linker region:

  • Contains a metalloprotease cleavage site

  • Allows proteolytic shedding of the FLRT ectodomain, which can modulate neuronal migration

These domain-specific functions allow FLRT3 to participate in multiple cellular processes through different molecular interactions .

How does FLRT3 contribute to cortical development?

FLRT3 plays multiple critical roles in cortical development:

• Migration regulation: FLRT3 directs both radial migration and tangential spread of cortical neurons.

• Adhesion-repulsion balance: FLRT proteins fine-tune adhesion and repulsion between cells migrating through the neocortex.

• Integrative signaling: FLRT3 can trigger both adhesive and repulsive signals in the same receiving cell, leading to an integrative response that precisely positions neurons.

• Proteolytic regulation: Similar to FLRT2, proteolytic shedding of FLRT3 may modulate migration of cortical neurons expressing Unc5 receptors .

What is the role of FLRT3 in vascular development?

FLRT3 is actively involved in vascular system development:

• Retinal vascularization: FLRT3 specifically controls the formation of blood vessels in the retina.

• Guidance mechanisms: Vascular cells are guided by FLRT using structurally conserved mechanisms similar to those employed in neuronal guidance.

• Developmental regulation: As part of the FLRT family (FLRT1-3), FLRT3 functions as a regulator of early embryonic and vascular development.

These findings highlight the parallel mechanisms employed in neuronal and vascular patterning, with FLRT3 serving as a key signaling molecule in both systems .

How is FLRT3 expression regulated during development and after injury?

FLRT3 expression exhibits spatiotemporal regulation throughout development and in response to injury:

• Developmental expression:

  • Located in somitic regions on dermatomyotomal muscle precursors and myotomal cells before their migration

  • Expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge

  • Genetic deletion in mouse embryos leads to defective headfold fusion and endoderm migration

• Postnatal expression:

  • FLRT3 mRNA is widely expressed in multiple tissues

  • Shows significant upregulation following experimental peripheral nerve injury in rats

  • The upregulation after nerve injury correlates with enhanced neurite outgrowth, suggesting a role in neural repair mechanisms .

How should recombinant FLRT3 protein be prepared and stored for experimental use?

For optimal experimental results with recombinant FLRT3:

• Formulation: Recombinant human FLRT3 protein is typically supplied lyophilized from a 0.2 μm filtered solution in PBS.

• Reconstitution: It should be reconstituted at 200 μg/mL in sterile PBS.

• Shipping and receipt: The product is shipped at ambient temperature but should be stored immediately upon receipt at the recommended temperature.

• Storage conditions: Use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain protein integrity.

• Carrier considerations: For applications where the presence of BSA could interfere, carrier-free (CF) versions are available. Otherwise, BSA-containing formulations provide enhanced stability and longer shelf-life .

What control experiments are essential when studying FLRT3-LPHN3 interactions?

Rigorous control experiments are crucial for validating FLRT3-LPHN3 interaction studies:

• Binding specificity controls:

  • Test binding of control Fc proteins alongside ecto-LPHN3-Fc

  • Verify that ecto-LPHN3-Fc does not bind to cells expressing unrelated proteins (e.g., LRRTM2)

  • Confirm binding of ecto-LPHN3-Fc to all FLRT isoforms (FLRT1-3)

• Cell-free binding assays:

  • Perform precipitation experiments with purified proteins to eliminate the possibility that unknown co-receptors mediate the interaction

• Rescue experiments:

  • When using shRNA knockdown, include rescue experiments with shRNA-resistant FLRT3 constructs to confirm specificity

• Multiple interference approaches:

  • Validate findings using different approaches (competition with ecto-domains, knockdown of FLRT3, knockdown of LPHN3) to ensure consistency of results .

What quantitative methods can measure FLRT3-mediated effects on synapse formation?

Several quantitative approaches can precisely measure FLRT3's impact on synapse formation:

• Synaptic puncta analysis:

  • Immunofluorescence quantification of synaptic marker density (number of puncta per unit length of dendrite)

  • Measurement of synaptic puncta size (area)

  • Co-localization analysis with pre- and postsynaptic markers

• Electrophysiological measurements:

  • mEPSC frequency analysis to assess functional synapse numbers

  • mEPSC amplitude measurements to evaluate synaptic strength

• Structural analysis:

  • Quantification of dendritic spine numbers in vivo after FLRT3 manipulation

  • Assessment of afferent input strength to determine functional connectivity

These complementary approaches provide a comprehensive assessment of both structural and functional aspects of synapse formation regulated by FLRT3 .