VEGFR2 Human, His

What is the structural composition of human VEGFR2?

Human VEGFR2 (also known as KDR, Flk-1, and CD309) is a type I single-pass membrane receptor that belongs to the class III subfamily of receptor tyrosine kinases (RTKs). The mature receptor contains a 745 amino acid extracellular domain with seven immunoglobulin-like repeats, a 21 amino acid transmembrane domain, and a 571 amino acid cytoplasmic domain . This receptor shares structural homology with VEGFR1 (Flt-1) and VEGFR3 (Flt-4), which together play essential roles in vasculogenesis and angiogenesis .

How does VEGFR2 signaling function at the molecular level?

VEGFR2 signaling begins when dimeric VEGF ligands bind to the receptor, causing monomeric VEGFR2 to dimerize and autophosphorylate tyrosine residues in the cytoplasmic domain . Unlike most receptor tyrosine kinases that primarily activate the Ras pathway or PI3K pathway, VEGFR2 predominantly signals through the PLCγ-PKC-MAPK pathway for endothelial proliferation . The SH2 domain of PLCγ specifically binds to the 1175-PY site of VEGFR2 (1173-PY in mice), activating the PKC pathway, particularly PKCβ . Studies using VEGFR-2 Y1173F knock-in mice have confirmed that this phosphorylation site is critical for angiogenic signaling, as mutation at this position results in embryonic lethality similar to complete VEGFR-2 knockout .

What distinguishes VEGFR2 binding affinities for monomeric versus dimeric states?

Research has revealed significant differences in binding affinities between monomeric and dimeric VEGFR2. Direct measurements using fluorescence-based techniques have demonstrated that the binding affinity of VEGF for dimeric VEGFR2 (KLD = 4.3 × 10^9 M^-1) is approximately 45-fold higher than for monomeric VEGFR2 (KLM = 9.6 × 10^7 M^-1) . These correspond to dissociation constants of 230 pM and 10 nM, respectively . This enhanced affinity for dimeric receptors plays a crucial role in the sensitivity and regulation of VEGFR2 signaling.

What expression systems are optimal for producing functional recombinant human VEGFR2?

For studies requiring fully functional VEGFR2, mammalian expression systems are generally preferred. HEK293T cells have been successfully used to express VEGFR2 ECTM constructs (where the kinase domain is substituted) for binding affinity studies . When designing recombinant constructs, researchers commonly utilize fusion tags such as Fc or Avi-tag for purification and detection purposes. For instance, commercially available recombinant human VEGFR2 may include an Avi-tag for biotinylation at a single site, facilitating various applications including binding studies .

How can researchers accurately quantify VEGFR2 surface density and binding characteristics?

Quantifying VEGFR2 surface density requires specialized techniques. One effective approach involves subjecting cells to reversible osmotic swelling to facilitate the conversion of effective 3D concentrations into 2D membrane protein surface densities . This technique has been used to measure both VEGFR2 surface densities and the concentration of bound VEGF ligands. For example, in studies with VEGFR2 ECTM-YFP constructs, researchers have achieved reproducible measurements across different free VEGF concentrations ranging from 0.21 to 42.4 nM . The bound VEGF surface density can then be plotted against the expressed VEGFR2 surface density to determine binding characteristics.

What methods are effective for studying VEGFR2 dimerization dynamics?

Several approaches can be employed to study VEGFR2 dimerization:

What developmental patterns of VEGFR2 expression have been observed?

Developmental studies have revealed distinct patterns of VEGFR2 expression:

• In early first-trimester embryos (approximately 7 weeks), VEGFR2 immunoreactivity is present in the epidermis, vascular endothelial, and mesothelial cells . Small capillaries with positive endothelia are detected in primitive placenta, truncal, intestinal, and pulmonary mesenchyme .

• In the heart, endothelial staining is more prominent in the atria and peripheral portions of the ventricles, as well as in the orifices of great vessels .

• In the liver, sinusoids show positive VEGFR2 expression, while all parenchymal epithelial, non-endothelial mesenchymal, and primitive neural elements are negative .

• In late first-trimester fetuses, VEGFR2 expression becomes more restricted to capillary endothelia, chondrocytes, and the superficial portion of the epidermis .

• In normal adult tissues, VEGFR2 expression is limited to endothelia and mesothelia .

This developmental pattern provides valuable insights for researchers studying vascular development and angiogenesis-related diseases.

How can binding data be analyzed to understand VEGFR2 signaling dynamics?

Analysis of binding data reveals complex VEGFR2 signaling dynamics. By combining data from multiple experiments with varying free VEGF concentrations, researchers can perform global fits to determine key parameters such as the binding affinities for monomeric and dimeric receptors . This approach enhances the accuracy and precision of the fit compared to analyzing individual experiments separately.

The relationship between bound VEGF and VEGFR2 surface density follows a model that considers the thermodynamic cycle of receptor dimerization and ligand binding . This model predicts the fractions of different receptor states (monomeric VEGFR2, VEGF-bound monomeric VEGFR2, dimeric VEGFR2, and VEGF-bound dimeric VEGFR2) as functions of total VEGFR2 surface density and free VEGF concentration .

An important prediction from these models is the bell-shaped dependence of the active, ligand-bound, dimeric fraction of VEGFR2 on the free VEGF concentration , which has significant implications for understanding optimal ligand concentrations for receptor activation.

How does VEGFR2 serve as a target for anti-angiogenic therapies?

VEGFR2 represents a primary target for anti-angiogenic therapy in cancer treatment due to its central role in promoting angiogenesis . The VEGF-VEGFR2 signaling axis stimulates tumor vascularization, which supports cancer growth and metastasis. Therapeutic approaches targeting VEGFR2 include:

• Tyrosine kinase inhibitors that block the cytoplasmic kinase domain

• Monoclonal antibodies that prevent VEGF binding to VEGFR2

• Decoy receptors that sequester VEGF ligands

Research indicates that targeting the signaling pathways of VEGFR1 and VEGFR2 may be effective for treating inflammation and multiple tumors, including breast, gastric, and lung carcinomas . Additionally, cancer immunotherapies using VEGF and VEGFR2 monoclonal antibodies show promise when combined with PD-1/PD-L1 immune checkpoint blockade .

What considerations are important when developing VEGFR2-targeted therapies?

When developing VEGFR2-targeted therapies, researchers should consider:

• The specificity of different VEGF family members for VEGFR2. For example, VEGF-E (derived from the Orf virus) uniquely binds to and activates VEGFR2 but not other VEGFRs or PDGFR . This specificity provides opportunities for developing highly selective therapeutic approaches.

• The potential for heterodimer formation between VEGFR2 and other VEGFRs (VEGFR1 and VEGFR3) , which may influence the efficacy of receptor-targeted therapies.

• Alternative splicing isoforms of VEGFR2 that lack transmembrane and cytoplasmic domains and function as decoy receptors . These naturally occurring variants might inspire the design of engineered decoy receptors for therapeutic use.

• The potential for combination therapies, as suggested by the synergistic effects observed when combining VEGFR2 inhibitors with immune checkpoint inhibitors .

What are the best practices for measuring VEGFR2 expression in tissue samples?

For accurate measurement of VEGFR2 expression in tissue samples, consider these methodological approaches:

• Immunohistochemistry: Use VEGFR2-specific antibodies such as the rabbit monoclonal antibody 55B11, which has been validated in extensive tissue studies . This approach allows visualization of VEGFR2 distribution and can distinguish between membrane and cytoplasmic staining patterns.

• Sample preparation: Ensure proper fixation and antigen retrieval techniques to preserve epitope accessibility while maintaining tissue morphology.

• Controls: Include appropriate positive controls (such as normal endothelial cells or known VEGFR2-positive tumors) and negative controls (tissues known to lack VEGFR2 expression) in each assay.

• Scoring system: Implement a standardized scoring system to evaluate staining intensity and distribution, which enables quantitative comparison between different samples and studies.

• Complementary techniques: Consider combining immunohistochemistry with other methods such as in situ hybridization or RT-PCR to confirm expression at both protein and mRNA levels.

How can researchers overcome challenges in studying VEGFR2 phosphorylation?

Studying VEGFR2 phosphorylation presents several challenges, including rapid dephosphorylation and technical difficulties in detecting specific phosphorylation sites. To address these issues:

• Use site-specific phospho-antibodies that recognize particular phosphorylated tyrosine residues, such as Y1175 (crucial for PLCγ binding and proliferation signaling) or Y951 (important for migration signals) .

• Implement rapid sample processing with immediate addition of phosphatase inhibitors to prevent dephosphorylation during cell lysis and protein extraction.

• Consider genetic approaches, such as mutagenesis of specific tyrosine residues (e.g., Y1175F mutants), to investigate the functional importance of individual phosphorylation sites .

• For quantitative analysis, use techniques such as ELISA or multiplexed phospho-protein detection platforms that allow simultaneous assessment of multiple phosphorylation sites.

What approaches can improve the specificity of recombinant VEGFR2 in binding assays?

To enhance the specificity of recombinant VEGFR2 in binding assays:

• Utilize Avi-tag biotinylated VEGFR2, which features biotinylation at a single site contained within the Avi-tag (a unique 15 amino acid peptide) . This approach provides uniform orientation when immobilized on streptavidin surfaces.

• Consider using VEGFR2 ECTM constructs where the kinase domain is substituted with a detectable tag (e.g., YFP) via a flexible linker . This modification maintains the extracellular domain's binding properties while facilitating detection.

• When designing binding experiments, account for the dimeric nature of both VEGF ligands and VEGFR2 receptors. Global fitting approaches that incorporate both monomeric and dimeric binding parameters can provide more accurate assessments of binding affinities .

• Vary free VEGF concentrations across multiple orders of magnitude (e.g., 0.21 to 42.4 nM) to capture the full range of binding behaviors and enable robust modeling of receptor-ligand interactions .