FLT1 D5 Human
Description
Biological Activity and Mechanism
FLT1 D5 Human functions as a VEGF decoy receptor, binding VEGF-A with high affinity but lacking intracellular signaling domains. This prevents VEGF-A from interacting with its primary signaling receptor, VEGFR-2 (Flk-1), thereby modulating angiogenesis .
Key Functional Data:
Comparative Analysis with Other FLT1 Variants
FLT1 D5 Human differs structurally and functionally from other FLT1 isoforms:
| Variant | Domains | Amino Acids | Mass | Expression Host | Function |
|---|---|---|---|---|---|
| FLT1 D6 | D1–D6 | 687 | 96 kDa | Baculovirus | Full extracellular domain binding |
| FLT1 His-Tag | D1–D3 | 298 | 43 kDa | E. coli | Non-glycosylated, truncated fragment |
| FLT1 D5 | D1–D5 | 562 | 70 kDa | Baculovirus | Intermediate decoy activity |
Research Applications
FLT1 D5 Human is employed in:
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ELISA/Western Blot: Detection of VEGF binding or receptor interaction .
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Functional Assays: Studying angiogenesis inhibition in models like the Chick Chorioallantoic Membrane (CAM) assay .
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Mechanistic Studies: Exploring VEGF-A/Flt-1 signaling pathways in endothelial cell migration, differentiation, and vessel anastomosis .
Stability and Handling Guidelines
| Parameter | Recommendation |
|---|---|
| Storage | Lyophilized: -18°C; Reconstituted: 4°C (2–7 days) |
| Freeze-Thaw Cycles | Avoid repeated cycles to prevent protein degradation |
| Solubility | Reconstitute in sterile water (≥100 µg/mL); avoid sodium azide or preservatives |
Role in Angiogenesis
FLT1 D5 Human mimics soluble Flt-1 (sFlt-1), a natural regulator that sequesters VEGF-A, reducing its availability for VEGFR-2. This mechanism is critical in balancing pro-angiogenic (VEGFR-2) and anti-angiogenic (Flt-1) signaling .
Clinical Relevance
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Sickle Cell Disease (SCD): Genetic variants in FLT1 (e.g., rs115695442) correlate with fetal hemoglobin (HbF) levels, suggesting a role in hypoxia-driven erythropoiesis .
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Placental Angiogenesis: FLT1 knockdown in human placental endothelial cells disrupts VEGF-A/PI3K-Akt signaling, impairing tube formation and wound healing .
Product Specs
Q&A
What is FLT1 and what role does Domain 5 play in its function?
FLT1 (Fms-like tyrosine kinase 1), also known as VEGFR1, is a receptor tyrosine kinase that functions primarily as a decoy receptor during blood vessel formation. It binds to Vascular Endothelial Growth Factor A (VEGFA) with approximately 10-fold higher affinity than VEGFR2 (Flk1/Kdr) .
Methodological approach: To study D5's contribution to FLT1 function, researchers typically employ site-directed mutagenesis of specific residues within this domain, followed by protein expression in mammalian cell systems. Binding assays using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can then quantify how D5 mutations affect VEGF binding kinetics.
How do the membrane-bound and soluble isoforms of FLT1 differ in research applications?
FLT1 exists in two primary isoforms:
| Isoform | Structure | Function | Research Applications |
|---|---|---|---|
| mFLT1 (membrane) | Contains transmembrane and tyrosine kinase domains | Weak kinase activity; primarily functions as a decoy receptor | Study of membrane-localized VEGF sequestration; cell signaling |
| sFLT1 (soluble) | Lacks transmembrane and tyrosine kinase domains | Acts exclusively as a VEGF ligand sink | Investigation of extracellular VEGF gradient formation |
Both isoforms contain Domain 5 and maintain high binding affinity for VEGFA . The soluble form (sFlt1) results from alternative splicing and plays a crucial role in establishing VEGF gradients during development.
Methodological approach: To distinguish between isoform-specific effects, researchers can use isoform-selective antibodies for immunohistochemistry or western blotting. For functional studies, expression constructs encoding either full-length or truncated (soluble) FLT1 can be transfected into cells to observe differential effects on angiogenic responses.
What experimental models are most effective for studying human FLT1 D5 function?
Several experimental models offer unique advantages for investigating human FLT1 D5:
| Model System | Advantages | Limitations | Key Applications |
|---|---|---|---|
| Cell Culture Systems | Controlled environment; easy genetic manipulation | Lacks physiological context | Binding kinetics; signaling studies |
| Mouse Models | Similar vascular development to humans | Species-specific differences in FLT1 structure | In vivo angiogenesis studies |
| Zebrafish | Transparent embryos allow real-time visualization | Greater evolutionary distance from humans | Dynamic vessel formation analysis |
| Retina Explants | Maintains tissue architecture | Short-term viability | Sprout anastomosis studies |
Methodological approach: For domain-specific studies, researchers often generate knock-in models expressing human FLT1 with mutations in D5. The mouse retinal angiogenesis model provides an excellent system for visualizing effects on vessel branching and anastomosis, as demonstrated in studies showing that reduced Flt1 levels promote sprout interactions .
How does Domain 5 of human FLT1 contribute to the spatial regulation of vessel anastomosis?
Analysis of mouse retinas with reduced Flt1 levels showed increased sprout interactions likely to lead to new branches, suggesting that FLT1 (including its D5 domain) plays a crucial role in regulating vessel connection patterns .
Methodological approach: To investigate D5's specific contribution to vessel anastomosis, researchers can employ:
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CRISPR/Cas9-mediated mutation of D5-specific residues
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Live cell imaging of fluorescently labeled endothelial cells expressing wild-type or D5-mutated FLT1
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Quantitative analysis of sprout interaction frequency, duration, and stability
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Computational modeling of VEGF gradient formation and its disruption by D5 mutations
What molecular techniques are optimal for analyzing structural modifications in FLT1 D5?
| Technique | Application | Resolution | Data Output |
|---|---|---|---|
| X-ray Crystallography | 3D structure determination | Atomic resolution | Electron density maps |
| Cryo-EM | Structure of FLT1-VEGF complexes | Near-atomic resolution | 3D molecular models |
| Hydrogen-Deuterium Exchange MS | Protein dynamics and binding interfaces | Peptide-level | Protection factors |
| FRET Analysis | Conformational changes upon binding | Nanometer range | Energy transfer efficiency |
| Molecular Dynamics Simulations | Dynamic behavior prediction | Atomic movement | Trajectory analysis |
Methodological approach: For comprehensive structural analysis of D5, researchers should combine experimental techniques with computational approaches. For example, after solving the crystal structure of D5 alone or in complex with VEGF, molecular dynamics simulations can predict how specific mutations might alter the domain's flexibility or binding interface. These predictions can then be validated using site-directed mutagenesis and binding assays.
How do FLT1 knockout/knockdown models reflect the specific contribution of Domain 5?
Complete genetic deletion of Flt1 is embryonic lethal in mice, highlighting its critical developmental role . Since D5 is integral to FLT1's structure and function, domain-specific approaches are necessary to isolate its contribution.
Methodological approach: Researchers can:
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Generate domain-specific knockouts using CRISPR/Cas9 to delete or modify only D5
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Create knock-in models where D5 is replaced with a homologous domain from another receptor
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Use inducible systems to control the timing of D5 modification
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Employ endothelial cell-specific promoters to restrict modifications to vascular cells
Studies in mouse retinas have shown that reduced Flt1 levels in endothelial cells promote sprout interactions leading to new branches , suggesting that domain-specific modifications would likely produce intermediate phenotypes depending on how they affect VEGF binding.
What are the methodological challenges in distinguishing between FLT1 D5 effects on VEGF gradient formation versus direct signaling?
FLT1's dual role as both a decoy receptor (affecting VEGF gradients) and a signaling receptor creates challenges in isolating domain-specific contributions.
Methodological approach:
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Use fluorescently tagged VEGF to visualize gradient formation in the presence of wild-type versus D5-modified FLT1
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Employ phospho-specific antibodies to detect downstream signaling events
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Utilize FRET-based biosensors to monitor real-time signaling in living cells
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Implement microfluidic devices to establish controlled VEGF gradients while monitoring cell responses
Research indicates that while FLT1 has weak kinase activity that is not required for developmental angiogenesis, it functions critically as an endothelial cell-intrinsic decoy receptor to modulate VEGF signaling amplitude . Understanding D5's specific contribution to these processes requires sophisticated experimental designs that can separate gradient effects from direct signaling.
How can high-throughput screening approaches be applied to identify modulators of FLT1 D5 function?
Methodological approach:
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Develop cell-based assays using reporter systems (e.g., luciferase) downstream of FLT1 activation
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Utilize automated microscopy to analyze endothelial cell behaviors in response to D5 modulators
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Implement AlphaScreen or FRET-based binding assays amenable to 384 or 1536-well formats
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Apply computational docking studies to virtually screen compound libraries against the D5 structure
When developing screening assays, it's crucial to include controls that can distinguish between compounds affecting D5 specifically versus other domains or general FLT1 function.
What are the sex-specific differences in FLT1 expression and function that might influence D5-focused research?
Interestingly, search results indicate potential sex-specific differences in FLT1 biology. In a model of reduced nephron number, ACE2 activity was higher in males compared to females, potentially reflecting a compensatory mechanism for lower expression of neprilysin and greater hypertension and renal injury . While this finding is not directly related to D5, it suggests that sex differences should be considered in FLT1 research.
Methodological approach:
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Include both male and female samples in all studies
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Analyze sex-specific expression patterns of FLT1 and its isoforms
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Investigate hormonal influences on FLT1 expression and function
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Conduct stratified analyses of experimental data by sex
How can advanced imaging techniques be optimized for studying FLT1 D5 dynamics in vessel anastomosis?
Methodological approach:
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Implement super-resolution microscopy (STORM, PALM) to visualize D5-tagged FLT1 distribution
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Use light sheet microscopy for rapid 3D imaging of vessel formation in developing tissues
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Apply intravital microscopy to monitor vessel anastomosis in living organisms
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Develop FLT1 D5-specific biosensors for visualizing domain activity in real-time
Research has shown that vessel anastomosis is a regulated rather than stochastic process, with VEGFA signaling (modulated by FLT1) playing a key role . Advanced imaging techniques are essential for capturing the dynamic nature of this process and understanding D5's contribution.
Product Science Overview
Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1), also known as Flt-1, is a receptor tyrosine kinase that plays a crucial role in angiogenesis and vasculogenesis. It is one of the receptors for Vascular Endothelial Growth Factor (VEGF), which is a signal protein that stimulates the formation of blood vessels. The D5 domain of VEGFR-1 is particularly significant as it is involved in the binding of VEGF.
VEGFR-1 is composed of several domains, including seven immunoglobulin-like domains in its extracellular region, a single transmembrane helix, and an intracellular tyrosine kinase domain. The D5 domain is one of these extracellular immunoglobulin-like domains and is critical for the receptor’s ability to bind VEGF.
VEGFR-1 acts as a decoy receptor, sequestering VEGF and preventing it from binding to VEGFR-2, which is more directly involved in promoting angiogenesis. This regulatory mechanism is essential for maintaining the balance of angiogenic signals in the body.
Recombinant human VEGFR-1 D5 is produced using recombinant DNA technology, which involves cloning the gene encoding the D5 domain into an expression vector, introducing this vector into a host cell (such as E. coli or CHO cells), and then purifying the expressed protein. This recombinant protein can be used in various research and therapeutic applications.
- Research: Recombinant VEGFR-1 D5 is widely used in research to study the mechanisms of angiogenesis and to develop anti-angiogenic therapies. It can be used in binding assays to investigate the interaction between VEGF and its receptors.
- Therapeutics: Due to its ability to bind VEGF, recombinant VEGFR-1 D5 has potential therapeutic applications in diseases characterized by excessive angiogenesis, such as cancer and age-related macular degeneration. By sequestering VEGF, it can inhibit the growth of new blood vessels that supply nutrients to tumors or diseased tissues.
The production of recombinant VEGFR-1 D5 typically involves the following steps:
- Cloning: The gene encoding the D5 domain is cloned into an expression vector.
- Expression: The vector is introduced into a host cell line, such as E. coli or CHO cells, which then express the recombinant protein.
- Purification: The expressed protein is purified using techniques such as affinity chromatography. For example, a hexahistidine tag can be added to the protein to facilitate purification using nickel affinity chromatography.
