FGFR2 Human, (22-289)

What is the molecular structure of recombinant FGFR2 Human (22-289)?

FGFR2 Human (22-289) is a single, glycosylated polypeptide chain containing the amino acid sequence from positions 22-289 of the full-length protein. The recombinant protein is produced in Sf9 Baculovirus cells and has a molecular mass of 56.8 kDa. It is expressed with a 239 amino acid hIgG-His-Tag at the C-Terminus to facilitate purification and detection in experimental systems. The protein undergoes post-translational modifications, particularly glycosylation, which may affect its binding properties and interactions with ligands .

What are the optimal storage conditions for FGFR2 Human (22-289)?

For short-term storage (2-4 weeks), store FGFR2 Human (22-289) at 4°C. For longer periods, store the protein frozen at -20°C. To maintain stability during long-term storage, it is recommended to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA). This prevents protein denaturation and loss of activity. Multiple freeze-thaw cycles should be strictly avoided as they can lead to protein degradation and loss of functional activity. When working with the protein, aliquot into single-use volumes before freezing to minimize freeze-thaw cycles .

How does FGFR2 differ structurally and functionally from other FGFR family members?

The FGFR family consists of five genes: FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5 (FGFRL1). While FGFR1-4 share a common structural organization with three extracellular immunoglobulin-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain, FGFR2 has distinct tissue expression patterns. FGFR2 is predominantly expressed in the brain and spinal cord, whereas other family members show different tissue specificities .

What are the primary signaling pathways activated by FGFR2?

FGFR2 activation initiates several major signaling cascades:

• RAS-MAPK Pathway: Upon FGFR2 auto-phosphorylation, FRS2 is recruited and phosphorylated, which then recruits GRB2 and SOS to activate Ras and its downstream MAPK pathway. This pathway is crucial for cell proliferation and differentiation .

• PI3K-AKT Pathway: FGFR2 signaling activates PI3K-AKT through GRB2-associated binding protein 1 (GAB1), which is recruited to phosphorylated FRS2. This pathway regulates cell survival, metabolism, and negatively regulates ERK1/2 signaling .

• PLCγ-PKC Pathway: FGFR2 directly phosphorylates and activates phospholipase C gamma (PLCγ), leading to calcium release and protein kinase C (PKC) activation.

• STAT Signaling: FGFR2 can activate STAT1, STAT3, and STAT5 signaling, which regulate gene expression related to cell growth and immune responses .

• Crosstalk with WNT Signaling: ERK1/2, activated downstream of FGFR2, directly phosphorylates LRP6 at S1490 and T1572, which increases WNT signaling activity .

How do feedback mechanisms regulate FGFR2 signaling?

FGFR2 signaling is tightly regulated by several negative feedback mechanisms to prevent aberrant activation:

• ERK-Mediated Inhibition: Active ERK1/2 directly phosphorylates FGFR at conserved serine residues (analogous to S777 in FGFR1), which dampens FGFR signaling. When this inhibition is blocked, FGFR signaling is enhanced .

• FRS2 Phosphorylation: FGFR-dependent activation of ERK1/2 phosphorylates FRS2 at multiple threonine residues, reducing FGFR-FRS2 signaling. In the absence of ERK1/2, p38 MAPK can mediate this phosphorylation .

• RSK2-Mediated Regulation: Activated RSK2, a downstream target of ERK1/2, binds and phosphorylates FGFR1 at S789 (with similar mechanisms likely for FGFR2), reducing tyrosine phosphorylation, promoting ubiquitination, and regulating endocytosis of the receptor .

• Amplification Mechanisms: Protein kinase C ε (PKCε) phosphorylates S779 of FGFR1 and FGFR2, creating a docking site for the adaptor 14-3-3, which enhances FGFR-RAS-MAPK and FGFR-PI3K-AKT signaling .

What experimental approaches can be used to study FGFR2 phosphorylation status?

To investigate FGFR2 phosphorylation:

• Phospho-specific Western Blotting: Use antibodies that specifically recognize phosphorylated tyrosine residues of FGFR2. This technique can detect auto-phosphorylation following ligand binding or constitutive phosphorylation in mutant proteins.

• Immunoprecipitation followed by Mass Spectrometry: Immunoprecipitate FGFR2 using anti-FGFR2 or anti-tag antibodies (for the His-tagged FGFR2 Human, 22-289), then analyze by mass spectrometry to identify specific phosphorylation sites and their stoichiometry.

• In vitro Kinase Assays: Using purified FGFR2 Human (22-289) protein, perform kinase assays with ATP and substrate proteins to assess kinase activity and patterns of substrate phosphorylation.

• Phospho-flow Cytometry: For cellular studies, use phospho-specific antibodies and flow cytometry to analyze FGFR2 phosphorylation at the single-cell level, allowing correlation with other cellular parameters.

• Proximity Ligation Assay: This technique can detect phosphorylated FGFR2 and its interactions with downstream signaling molecules in situ, providing spatial information about signaling events.

How is FGFR2 implicated in cancer development and progression?

FGFR2 plays complex, context-dependent roles in cancer:

• Gene Fusions and Rearrangements: FGFR2 fusions are the most common FGFR gene alterations in cancer, particularly in intrahepatic cholangiocarcinoma with a prevalence of 9-16%. Common fusion partners include BICC1, KIAA1217, TACC2, CCDC6, and adenosylhomocysteinase like 1 .

• Mechanism of Fusion Activation: The tyrosine kinase domain of FGFR2 is joined with various partners that facilitate dimerization, leading to constitutive activation of FGFR2 kinase activity and downstream signaling pathways. About 32.9% of patients have unique fusion partners, while 15.7% share fusion partners with only one other patient, suggesting diverse biological effects .

• Dual Role as Oncogene or Tumor Suppressor: FGFR2 demonstrates context-specific roles as either a tumor promoter or suppressor. While FGFR2 loss-of-function mutations occur in 10% of melanoma tumors and cell lines and in bladder cancers, overexpression of FGFR2 in gastric cancer, triple-negative breast cancer, and osteosarcoma promotes cancer cell proliferation and survival .

• Resistance Mechanisms: SOX9, stimulated by the Wnt/β-catenin pathway, enhances FGF7 and FGFR2 expression, promoting cholangiocarcinoma cell proliferation and resistance to FGFR inhibitors like pemigatinib. Through AKT/mTOR pathway activation, FGFR2 reduces sensitivity to chemotherapeutic agents like gemcitabine .

What approaches can be used to study FGFR2 function in cancer models?

To investigate FGFR2's role in cancer:

• Gene Expression Analysis: RNA sequencing or qPCR to analyze FGFR2 expression levels and splicing variants in tumor versus normal tissues.

• Fusion Detection: FISH, RNA-seq, or PCR-based methods to identify FGFR2 gene fusions in patient samples or cell lines.

• Functional Assays:

  • Proliferation assays using BrdU incorporation or Ki-67 staining

  • Migration and invasion assays using transwell chambers

  • Apoptosis assays measuring caspase activation or Annexin V binding

  • Colony formation assays to assess anchorage-independent growth

• Signaling Analysis:

  • Western blotting for phosphorylated downstream targets (ERK1/2, AKT, STAT3)

  • Multiplex phosphoprotein assays to simultaneously measure multiple pathway components

  • Reporter gene assays for transcriptional targets

• In vivo Models:

  • Patient-derived xenografts (PDX) maintaining FGFR2 alterations

  • Genetically engineered mouse models expressing FGFR2 fusions

  • Orthotopic transplantation models reflecting the tumor microenvironment

How does FGFR2 interact with the immune system in cancer?

FGFR2 has been shown to influence immune responses in the tumor microenvironment:

• PD-L1 Regulation: FGFR2 mediates immune tolerance in colorectal cancer cells by inducing PD-L1 expression through the JAK/STAT3 pathway. This mechanism may contribute to immune evasion by cancer cells .

• Immune Cell Infiltration: FGFR2 signaling can modulate the recruitment and activation of various immune cell populations within the tumor microenvironment, potentially affecting antitumor immunity.

• Cytokine Production: Activation of FGFR2 pathways can lead to altered cytokine and chemokine production, influencing local inflammatory responses and immune cell function.

• Combination Therapy Potential: The interaction between FGFR2 and immune checkpoints suggests potential synergy between FGFR inhibitors and immunotherapies, particularly in cancers with FGFR2 alterations.

How can FGFR2 Human (22-289) be used to screen for novel therapeutic compounds?

For therapeutic compound screening:

• Biochemical Assays:

  • In vitro kinase assays measuring auto-phosphorylation or substrate phosphorylation

  • Thermal shift assays to assess compound binding by protein stabilization

  • Surface plasmon resonance or isothermal titration calorimetry to determine binding kinetics and affinity

• Cell-Based Screening:

  • Phospho-ERK or phospho-AKT reporter cell lines to measure pathway inhibition

  • Cell viability assays in FGFR2-dependent cancer cell lines

  • High-content imaging to assess multiple parameters simultaneously

• Structure-Based Design:

  • Co-crystallization of FGFR2 Human (22-289) with candidate compounds

  • Molecular docking using FGFR2 crystal structures

  • Fragment-based screening to identify novel binding pockets

• Resistance Mechanism Studies:

  • Long-term culture with FGFR inhibitors to identify resistance mechanisms

  • Combination screens to identify synergistic drug pairs

  • Testing against known resistance mutations in FGFR2

What are the methodological challenges in studying FGFR2 isoforms and splice variants?

Key challenges include:

• Isoform Specificity:

  • FGFR2 has two major splice variants (IIIb and IIIc) with different ligand specificities

  • Design isoform-specific primers for qPCR that span unique exon junctions

  • Use isoform-specific antibodies for Western blotting and immunoprecipitation

• Expression Systems:

  • Maintain proper glycosylation and folding of recombinant FGFR2 variants

  • Establish stable cell lines expressing specific splice variants

  • Control for endogenous FGFR expression that may confound results

• Functional Differentiation:

  • Develop assays that can distinguish biological activities of different isoforms

  • Use domain-specific ligands to activate particular splice variants

  • Employ CRISPR/Cas9 to generate isoform-specific knockout cell lines

• Analytical Techniques:

  • Use RNA-seq with sufficient read depth to accurately quantify splice variants

  • Apply proteomics approaches to identify post-translational modifications specific to each isoform

  • Develop computational methods to predict functional consequences of alternative splicing

How can FGFR2 signaling crosstalk with other pathways be methodically investigated?

To study pathway crosstalk:

• Simultaneous Inhibition Approaches:

  • Combine FGFR2 inhibitors with inhibitors of other pathways (e.g., MEK, PI3K, or WNT inhibitors)

  • Use genetic approaches (siRNA, CRISPR) to simultaneously knock down components of multiple pathways

  • Apply mathematical modeling to predict and test combined pathway perturbations

• Protein-Protein Interaction Analysis:

  • Perform co-immunoprecipitation of FGFR2 with components of other signaling pathways

  • Use proximity ligation assays to visualize interactions in situ

  • Apply BRET or FRET approaches to measure real-time interactions in living cells

• Phosphoproteomic Approaches:

  • Conduct global phosphoproteomic analysis after FGFR2 activation or inhibition

  • Use multiplexed kinase assays to identify substrates shared between FGFR2 and other kinases

  • Apply kinase activity profiling to measure changes across multiple pathways

• Transcriptomic Analysis:

  • Perform RNA-seq after modulating FGFR2 and other pathways

  • Use single-cell approaches to capture heterogeneity in pathway activation

  • Apply network analysis to identify gene expression signatures of pathway crosstalk

What are common issues when working with recombinant FGFR2 in kinase assays?

Common problems and solutions:

• Low Activity:

  • Ensure protein has not undergone multiple freeze-thaw cycles

  • Add carrier protein (0.1% HSA or BSA) to stabilize during storage

  • Verify pH and buffer composition are optimal for kinase activity

  • Include proper cofactors (Mg²⁺ or Mn²⁺) at appropriate concentrations

• High Background:

  • Use ultrapure ATP to minimize contaminating phosphates

  • Include appropriate negative controls (kinase-dead mutants)

  • Pre-clear substrates to remove any pre-existing phosphorylation

  • Optimize antibody dilutions for phospho-specific detection

• Inconsistent Results:

  • Standardize protein concentrations using quantitative methods

  • Establish clear time course parameters to capture linear phase of reaction

  • Control temperature precisely during assay procedures

  • Use internal standards to normalize between experiments

• Inhibitor Testing Problems:

  • Include positive control inhibitors with known IC₅₀ values

  • Test for compound solubility issues in assay buffers

  • Consider compound binding to plastic surfaces

  • Account for ATP concentration when comparing inhibitor potencies

How can researchers optimize transfection of FGFR2 constructs in different cell systems?

Optimization strategies:

• Vector Selection:

  • Choose vectors with appropriate promoters for target cell types

  • Consider using inducible systems for proteins that may affect cell growth

  • Include fluorescent tags for easy visualization, but verify they don't affect function

  • Use codon-optimized sequences for the target cell type

• Transfection Method Optimization:

  • For hard-to-transfect cells, test multiple methods (lipofection, electroporation, nucleofection)

  • Optimize cell density at transfection (typically 70-90% confluence)

  • Adjust DNA:transfection reagent ratios systematically

  • Consider cell cycle synchronization for timing-sensitive experiments

• Expression Verification:

  • Use Western blotting to confirm protein expression at expected molecular weight

  • Perform immunofluorescence to check subcellular localization

  • Verify functional activity through phosphorylation assays

  • Quantify transfection efficiency using flow cytometry

• Stable Cell Line Generation:

  • Choose appropriate selection markers and determine optimal antibiotic concentration

  • Create single-cell clones to ensure homogeneous expression

  • Verify stable integration by genomic PCR

  • Regularly check for expression drift during long-term culture

What controls should be included when studying FGFR2 fusion proteins in cancer models?

Essential controls include:

• Expression Controls:

  • Wild-type FGFR2 to compare with fusion constructs

  • Kinase-dead mutants (e.g., K517M) to confirm kinase-dependent effects

  • Individual fusion partners expressed alone to distinguish their contribution

  • Different breakpoint variants of the same fusion to assess structural requirements

• Signaling Pathway Controls:

  • Positive controls using known FGFR ligands (e.g., FGF7, FGF10)

  • Pathway inhibitors to confirm specificity (e.g., FGFR inhibitors, MEK inhibitors)

  • Constitutively active downstream components as positive controls

  • Phosphatase treatment as negative controls for phosphorylation events

• Functional Assays:

  • Include parental cell lines without FGFR2 alterations

  • Use cells with known FGFR2 fusions as positive controls

  • Compare effects in multiple cell backgrounds to ensure robustness

  • Include time-dependent measurements to capture both acute and chronic effects

• In vivo Models:

  • Compare tumor growth of cells expressing FGFR2 fusions versus wild-type FGFR2

  • Include treatment arms with established FGFR inhibitors

  • Monitor for development of resistance mechanisms

  • Assess biomarkers of pathway activation in tissue samples

How is FGFR2 involved in regulating immune responses in the tumor microenvironment?

Current research has revealed:

• Immune Checkpoint Regulation: FGFR2 can mediate immune tolerance by inducing PD-L1 expression through the JAK/STAT3 pathway in colorectal cancer cells, potentially contributing to immune evasion mechanisms .

• Methodological Approaches:

  • Co-culture systems with cancer cells and immune cells

  • Flow cytometry to assess immune cell activation markers

  • Cytokine profiling in FGFR2-altered versus wild-type tumors

  • In vivo models with intact immune systems versus immunodeficient models

• Combination Therapy Implications:

  • Testing FGFR inhibitors with immune checkpoint inhibitors

  • Monitoring changes in tumor-infiltrating lymphocytes after FGFR inhibition

  • Developing biomarkers to predict response to combination approaches

  • Assessing acquired resistance mechanisms

What is the significance of non-coding RNA regulation of FGFR2 expression in cancer progression?

Emerging evidence indicates:

• MicroRNA Regulation: Several microRNAs directly target FGFR2 to inhibit its expression and cancer progression. These include miR-381-3p, miR-494, miR-5701, and miR-519e-5p, particularly in gastric cancer .

• Long Noncoding RNA Interactions: LncRNA ASNR competitively inhibits miR-519e-5p, thereby indirectly regulating FGFR2 levels .

• Epigenetic Control Mechanisms: Methyl-CpG-binding domain protein 1 and histone deacetylase 3 form a complex that inhibits miR-5701 expression, thus restoring FGFR2 levels .

• Experimental Approaches:

  • RNA immunoprecipitation to identify direct RNA-protein interactions

  • Luciferase reporter assays to validate miRNA binding sites

  • CRISPR-based epigenetic editing to modify regulatory regions

  • Single-cell RNA sequencing to capture heterogeneity in non-coding RNA expression