FGFR2 Human, His

What is the basic structure of human FGFR2?

FGFR2 is a receptor tyrosine kinase encoded by the FGFR2 gene located on chromosome 10 in humans. The protein's structure consists of an extracellular region composed of three immunoglobulin domains, a single hydrophobic membrane-spanning segment, and a cytoplasmic tyrosine kinase domain. The extracellular portion interacts with fibroblast growth factors, initiating downstream signaling cascades that influence cellular mitogenesis and differentiation. The amino acid sequence of FGFR2 is highly conserved both between family members and throughout evolutionary history, highlighting its biological significance .

What are the primary physiological functions of FGFR2?

FGFR2 plays critical roles in embryonic development and tissue repair, particularly in bone formation and blood vessel development. On a molecular level, FGFR2 mediates cell division, growth, and differentiation through ligand binding and receptor dimerization, which activates the tyrosine kinase domains to initiate intracellular signaling cascades. These signaling pathways regulate numerous developmental processes and contribute to tissue homeostasis in mature organisms . Disruptions in FGFR2 signaling are associated with several developmental disorders and pathological conditions, including various types of cancer.

What are the main isoforms of FGFR2 and how do they differ?

FGFR2 has two major naturally occurring isoforms, FGFR2IIIb and FGFR2IIIc, which result from alternative splicing of the third immunoglobulin-like domain. FGFR2IIIb is predominantly expressed in ectoderm-derived tissues and endothelial organ linings (such as skin and internal organs), while FGFR2IIIc is primarily found in mesenchymal tissues, including craniofacial bone. The tissue-specific distribution of these isoforms is functionally significant, as mutations in FGFR2IIIc are associated with craniosynostosis, a condition characterized by premature fusion of cranial sutures . The isoforms also differ in their ligand binding preferences, contributing to tissue-specific responses to various fibroblast growth factors.

How does FGFR2 expression vary across cancer types?

FGFR2 expression patterns and genetic alterations vary significantly across different cancer types. Analysis of large comprehensive datasets covering over 10,000 tumor samples across more than 30 cancer types reveals distinct patterns of FGFR2 abnormal expression, methylation, and alterations (mutations, fusions, amplifications, and deletions). For example, cholangiocarcinoma (CHOL) shows the highest frequency of FGFR2 fusions, while uterine corpus endometrial carcinoma (UCEC) exhibits the highest number of FGFR2 mutations, with S252W and N549K being the most frequently mutated positions . These cancer-specific patterns have important implications for diagnosis, prognosis, and therapeutic strategies.

How can researchers optimize protein expression and purification of His-tagged FGFR2?

Optimizing expression and purification of His-tagged FGFR2 requires careful consideration of several factors:

• Expression System Selection:

  • Bacterial systems (E. coli): Suitable for cytoplasmic domains, but may struggle with full-length receptor due to glycosylation requirements

  • Mammalian cell lines: Preferred for full-length FGFR2 with proper post-translational modifications

  • Insect cell systems: Good compromise between yield and post-translational modifications

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

  • Consider adding imidazole gradient elution to reduce non-specific binding

  • Secondary purification steps (ion exchange, size exclusion chromatography) to achieve higher purity

• Buffer Optimization:

  • Include appropriate detergents for membrane-bound regions

  • Add stability enhancers such as glycerol or specific ligands

  • Consider phosphatase inhibitors to preserve phosphorylation states

The inclusion of rigorous quality control steps, including Western blotting and activity assays, is essential to confirm that the purified protein maintains its native conformation and function.

How does FGFR2 regulate cellular interactions with the extracellular matrix?

FGFR2 plays a critical role in regulating epithelial cell interactions with the extracellular matrix (ECM) through mechanisms involving integrin expression and function. Experimental knockdown of FGFR2 in HB2 mammary epithelial cells results in decreased protein levels of specific integrins, including α2, α5, and the mature form of β1, while leaving α1, α3, and α6 unchanged. Additionally, treatment with the FGFR inhibitor AZD4547 significantly decreases integrin β1 protein levels after 48 and 72 hours, coinciding with pronounced inhibition of FGFR signaling . These findings suggest a molecular link between FGFR2 activity and epithelial cell-ECM communication, with FGFR2 loss and concomitant integrin β1 degradation responsible for deregulating these interactions.

What experimental approaches can be used to study FGFR2-mediated cell adhesion and migration?

Researchers investigating FGFR2-mediated cell adhesion and migration can employ multiple complementary approaches:

• Genetic Manipulation:

  • CRISPR/Cas9-mediated FGFR2 knockout or knockdown

  • Overexpression of wild-type or mutant FGFR2 constructs

  • Domain-specific mutations to isolate functional regions

• Functional Assays:

  • Adhesion assays on different ECM components (collagen, fibronectin, laminin)

  • Wound healing assays to assess collective cell migration

  • Single-cell tracking for detailed migration parameters

  • Transwell migration and invasion assays

• Molecular Analysis:

  • Co-immunoprecipitation to identify FGFR2-integrin interactions

  • Western blotting to quantify integrin expression levels and activation states

  • Immunofluorescence to visualize focal adhesion formation and cytoskeletal organization

• Pharmacological Approaches:

  • Specific FGFR2 inhibitors (e.g., AZD4547) to block signaling

  • Integrin-blocking antibodies to dissect downstream effects

  • Pathway-specific inhibitors to determine signaling mechanisms

These approaches, used in combination, can provide comprehensive insights into how FGFR2 regulates cellular interactions with the extracellular environment.

What are the most common FGFR2 alterations in cancer, and how do they affect therapeutic responses?

FGFR2 alterations in cancer include mutations, fusions, and amplifications, with distinct patterns across different cancer types. In cholangiocarcinoma, FGFR2 fusions are the predominant alteration and are highly targetable, with FDA-approved drugs showing efficacy against these alterations . In contrast, uterine corpus endometrial carcinoma exhibits a high frequency of point mutations, particularly S252W and N549K, which have oncogenic effects but show less response to targeted therapies .

The alteration type significantly influences therapeutic outcomes:

Clinical trials targeting FGFR2 rearrangements in cholangiocarcinoma have shown objective remission rates of 14-35% and disease control rates of 75-83%, with progression-free survival of 5.8-6.9 months . The FDA has approved pemigatinib, an FGFR inhibitor, for treating cholangiocarcinomas with FGFR2 rearrangements that have failed prior chemotherapy .

How do clinicopathological features correlate with FGFR2 alterations in cancer?

In genomic analyses, FGFR2 fusions involve various partner genes, with FGFR2-BICC1 being the most common fusion type in cholangiocarcinoma. The most frequent concomitant genetic alterations with FGFR2 rearrangements include BAP1, CDKN2A, and CDKN2B mutations, while KRAS and IDH1 mutations appear to be mutually exclusive with FGFR2 rearrangements . Understanding these molecular and clinical associations is crucial for patient stratification and treatment selection in precision oncology approaches.

What computational approaches can be used for designing novel FGFR2 inhibitors?

Structure-based computational approaches offer powerful tools for developing novel FGFR2 inhibitors. An integrated bioinformatics workflow typically includes:

• Virtual Screening:

  • Structure-based virtual screening of compound libraries (e.g., PubChem database)

  • Application of Tanimoto similarity thresholds (e.g., 80%) to identify structural analogs of known inhibitors

  • Molecular docking to predict binding modes and affinities

• Filtering and Selection Criteria:

  • Drug-likeness properties assessment

  • Binding affinity evaluation

  • Docking score analysis

  • Selectivity profiling

• Advanced Simulation Techniques:

  • All-atom molecular dynamics (MD) simulations (e.g., 200 ns duration)

  • Essential dynamics analysis

  • Principal component analysis (PCA)

  • Free energy landscapes (FELs)

One successful application of this approach identified promising FGFR2 inhibitors from 2336 compounds in the PubChem database, with a compound (PubChem CID:507883) demonstrating particularly favorable characteristics . The docking protocol validation through retrospective procedures, such as redocking co-crystallized ligands, ensures the reliability of the computational predictions.

How can researchers evaluate the specificity of FGFR2 inhibitors against other FGFR family members?

Evaluating the specificity of FGFR2 inhibitors against other FGFR family members requires a multi-faceted approach:

• Computational Methods:

  • Comparative molecular docking against all FGFR family members

  • Binding site analysis to identify unique structural features of FGFR2

  • Molecular dynamics simulations to assess inhibitor stability in different FGFR binding pockets

  • MM-GBSA or FEP calculations to compare binding energies across FGFR family

• Biochemical Assays:

  • In vitro kinase assays with purified recombinant FGFR1-4 proteins

  • Determination of IC50 values for each family member

  • Calculation of selectivity indices (ratio of IC50 values)

• Cellular Systems:

  • Cell lines with differential expression of FGFR family members

  • Phosphorylation assays of downstream targets

  • Cellular phenotypic assays (proliferation, migration, etc.)

• Structural Biology:

  • X-ray crystallography or cryo-EM of inhibitor-bound FGFR2

  • Comparison with structures of other FGFR family members

By combining these approaches, researchers can develop comprehensive selectivity profiles and understand the molecular basis for any observed differences in inhibitor efficacy across the FGFR family, guiding further optimization efforts toward improved specificity.

What is the spectrum of FGFR2 mutations in craniosynostosis, and how do they affect protein function?

FGFR2 mutations are a significant cause of craniosynostosis, a condition characterized by premature fusion of cranial sutures. In a comprehensive genomic screening of 259 patients with craniosynostosis (where mutations in other genes like FGFR1, FGFR3, and TWIST had been excluded), 85 independent FGFR2 mutations were detected . These mutations predominantly affect the FGFR2IIIc isoform, which is expressed in mesenchyme including craniofacial bone .

The mutations typically occur in specific hotspots of the FGFR2 gene and can be categorized based on their effects on protein function:

• Ligand-binding domain mutations: These alter ligand binding specificity or affinity, potentially causing inappropriate activation of the receptor.

• Transmembrane domain mutations: These can lead to constitutive dimerization and activation of the receptor independent of ligand binding.

• Tyrosine kinase domain mutations: These may enhance catalytic activity or alter substrate specificity.

• Splicing mutations: These can affect the balance between different FGFR2 isoforms, disrupting the tissue-specific expression patterns.

The functional consequences of these mutations include increased ligand binding affinity, ligand-independent activation, altered downstream signaling, and disrupted cross-talk with other signaling pathways, ultimately affecting the normal development of craniofacial structures.

What experimental models are most appropriate for studying FGFR2 mutations in developmental contexts?

Studying FGFR2 mutations in developmental contexts requires models that can recapitulate the complex cellular interactions and temporal dynamics of embryonic development. Several complementary approaches include:

• Cell-Based Models:

  • Primary cranial osteoblasts or mesenchymal stem cells

  • Induced pluripotent stem cells (iPSCs) derived from patients with FGFR2 mutations

  • Differentiation protocols to generate relevant cell types (osteoblasts, chondrocytes)

• Organoid Models:

  • Cranial suture organoids

  • Bone developmental organoids

  • Co-culture systems with multiple cell types

• Animal Models:

  • Genetically engineered mouse models carrying specific FGFR2 mutations

  • Conditional knockout or knockin approaches for tissue-specific effects

  • CRISPR/Cas9-mediated introduction of mutations in developing embryos

• Systems for Functional Analysis:

  • Live imaging of suture development in animal models

  • Micro-CT analysis of craniofacial bone development

  • Single-cell RNA sequencing to identify cell-specific effects

  • Phosphoproteomics to map altered signaling pathways

These models provide complementary insights, with cell-based systems offering detailed molecular analyses, organoids capturing 3D tissue architecture, and animal models revealing systemic developmental effects and phenotypic outcomes of FGFR2 mutations.

What are the latest methodologies for studying FGFR2 signaling dynamics in real-time?

Advanced technologies for studying FGFR2 signaling dynamics in real-time include:

• FRET-Based Biosensors:

  • Genetically encoded sensors for monitoring FGFR2 conformational changes

  • Sensors for downstream signaling events (MAPK, PI3K, PLCγ activation)

  • Single-molecule FRET to track individual receptor behavior

• Live-Cell Imaging Techniques:

  • Lattice light-sheet microscopy for high-resolution 3D imaging

  • Total internal reflection fluorescence (TIRF) microscopy for membrane dynamics

  • Fluorescence recovery after photobleaching (FRAP) to measure receptor mobility

• Optogenetic Approaches:

  • Light-controlled activation of FGFR2 signaling

  • Spatiotemporal control of receptor dimerization

  • Reversible modulation of specific signaling branches

• Single-Cell Technologies:

  • Mass cytometry (CyTOF) for high-dimensional analysis of signaling states

  • Microfluidic platforms for single-cell signaling analysis

  • Live-cell RNA imaging to track transcriptional responses

These methodologies enable researchers to dissect the complex spatiotemporal dynamics of FGFR2 signaling with unprecedented resolution, revealing how signal transduction changes over time, varies between individual cells, and responds to different ligands or inhibitors.

How can multi-omics approaches enhance our understanding of FGFR2 biology?

Multi-omics approaches provide a comprehensive view of FGFR2 biology by integrating data from various molecular levels:

• Genomics and Epigenomics:

  • Whole genome/exome sequencing to identify genetic alterations

  • DNA methylation analysis to understand epigenetic regulation

  • ATAC-seq for chromatin accessibility profiling

• Transcriptomics:

  • RNA-seq for global expression changes

  • Single-cell RNA-seq for cell-type-specific responses

  • Spatial transcriptomics to map expression patterns in tissue context

• Proteomics and Post-translational Modifications:

  • Global proteome analysis

  • Phosphoproteomics to map signaling networks

  • Ubiquitylation and SUMOylation profiling

• Interactomics:

  • Affinity purification-mass spectrometry to identify protein interactions

  • Proximity labeling (BioID, APEX) for spatial interaction mapping

  • Cross-linking mass spectrometry for structural insights

• Metabolomics:

  • Targeted and untargeted metabolite profiling

  • Stable isotope tracing for metabolic flux analysis

• Integrative Computational Approaches:

  • Network analysis to connect multi-omics datasets

  • Machine learning for pattern recognition

  • Systems biology modeling of signaling dynamics

The integration of these approaches has revealed that DNA methylation is associated with FGFR2 expression in several cancers, and that FGFR2 alterations correlate with distinct patterns of gene expression and pathway activation . Such multi-omics studies provide a systems-level understanding of FGFR2 biology that cannot be achieved through single-omics approaches alone.

What are the current therapeutic strategies targeting FGFR2 in cancer treatment?

Current therapeutic strategies targeting FGFR2 in cancer treatment include:

• Small Molecule Inhibitors:

  • Selective FGFR inhibitors (e.g., pemigatinib, infigratinib)

  • Multi-kinase inhibitors with FGFR activity (e.g., dovitinib, ponatinib)

  • Irreversible FGFR inhibitors in development

• Clinical Applications:

  • FDA-approved pemigatinib for FGFR2 fusion-positive cholangiocarcinoma that has failed prior chemotherapy

  • Clinical trials showing objective remission rates of 14-35% and disease control rates of 75-83% for advanced cholangiocarcinomas with FGFR2 fusions

  • Progression-free survival of 5.8-6.9 months in targeted therapy trials

• Combination Approaches:

  • FGFR inhibitors with immune checkpoint inhibitors

  • Combinations with cytotoxic chemotherapy

  • Strategies to overcome resistance mechanisms

• Emerging Approaches:

  • Antibody-drug conjugates targeting FGFR2

  • Proteolysis targeting chimeras (PROTACs) for FGFR2 degradation

  • Allosteric modulators of FGFR2 function

These strategies are being evaluated in multiple clinical trials, with particular focus on tumors with specific FGFR2 alterations. The success of pemigatinib in cholangiocarcinoma has established FGFR2-targeted therapy as a viable approach for precision oncology, with ongoing efforts to expand its applications to other cancer types with relevant FGFR2 alterations.

How can researchers optimize patient selection for FGFR2-targeted therapies?

Optimizing patient selection for FGFR2-targeted therapies requires a comprehensive approach:

• Molecular Testing Strategies:

  • Comparison of detection methods shows that FISH and NGS provide concordant results for FGFR2 rearrangements (kappa value=0.696, p<0.01), while IHC shows discordant results

  • NGS offers comprehensive detection of various FGFR2 alterations (mutations, fusions, amplifications)

  • Liquid biopsy approaches for non-invasive monitoring

• Clinicopathological Features:

  • In intrahepatic cholangiocarcinoma, FGFR2-positive tumors tend to correlate with early clinical stage, histologically small duct subtype, and reduced mucus production

  • These features can guide enrichment strategies for molecular testing

• Concomitant Genomic Alterations:

  • FGFR2 rearrangements in cholangiocarcinoma often co-occur with BAP1, CDKN2A, and CDKN2B alterations

  • KRAS and IDH1 mutations appear mutually exclusive with FGFR2 rearrangements

  • These patterns may influence therapeutic responses

• Biomarker Development:

  • Downstream signaling activation markers

  • Expression signatures predictive of response

  • Resistance-associated alterations

By integrating these approaches, researchers can develop evidence-based strategies for identifying patients most likely to benefit from FGFR2-targeted therapies, improving clinical outcomes while minimizing unnecessary treatments in patients unlikely to respond.

What are the emerging areas of investigation in FGFR2 biology?

Emerging areas in FGFR2 research include:

• Structural Biology and Biophysics:

  • Cryo-EM studies of full-length FGFR2 in different activation states

  • Investigation of FGFR2 complexes with co-receptors and modulators

  • Single-molecule studies of receptor dynamics and clustering

• Systems Biology Approaches:

  • Quantitative modeling of FGFR2 signaling networks

  • Integration of multi-omics data to build predictive models

  • Network-based approaches to understand context-dependent signaling

• Developmental and Stem Cell Biology:

  • Role of FGFR2 in tissue-specific stem cell maintenance and differentiation

  • Single-cell analysis of FGFR2 function during embryonic development

  • Organoid models to study FGFR2 in tissue morphogenesis

• Immune System Interactions:

  • Cross-talk between FGFR2 signaling and immune cell function

  • Implications for cancer immunotherapy combinations

  • Role in inflammatory conditions and wound healing

• Novel Therapeutic Approaches:

  • Allosteric modulators of FGFR2 activity

  • Targeted protein degradation strategies

  • RNA-based therapeutics for isoform-specific modulation

These emerging areas promise to deepen our understanding of FGFR2 biology and open new avenues for therapeutic intervention in FGFR2-associated diseases.

How will technological advances shape the future of FGFR2-targeted drug development?

Technological advances are poised to transform FGFR2-targeted drug development in several key areas:

• Artificial Intelligence and Machine Learning:

  • AI-driven drug design targeting FGFR2-specific binding pockets

  • Predictive modeling of drug resistance mechanisms

  • Virtual screening of billions of compounds with improved accuracy

• Advanced Structural Biology:

  • AlphaFold and similar tools for more accurate protein structure prediction

  • Cryo-EM for visualization of FGFR2 in different conformational states

  • Fragment-based drug discovery approaches

• High-throughput Functional Screening:

  • CRISPR-based genetic screens for resistance mechanisms

  • Phenotypic screening in 3D organoid models

  • Patient-derived models for personalized drug testing

• Precision Medicine Implementation:

  • Digital pathology with AI-assisted interpretation

  • Integration of genomic, transcriptomic, and proteomic biomarkers

  • Real-world evidence collection through digital health platforms

• Novel Therapeutic Modalities:

  • Bifunctional degraders (PROTACs) targeting FGFR2

  • mRNA-based therapies for transient FGFR2 modulation

  • Gene editing approaches for permanent correction of pathogenic FGFR2 mutations

These technological advances will enable more rapid development of highly selective FGFR2 inhibitors, better prediction of clinical responses, and more effective strategies to overcome resistance mechanisms, ultimately improving outcomes for patients with FGFR2-driven diseases.