FGFR4 Human, His

What is the basic structure of human FGFR4?

Human Fibroblast Growth Factor Receptor 4 (FGFR4) is a transmembrane receptor tyrosine kinase consisting of an extracellular domain with three immunoglobulin (Ig)-like domains, an acid-box region between IgI and IgII domains, a transmembrane domain, and a split tyrosine-kinase domain. Unlike other FGFR family members (FGFR1-3), FGFR4 does not undergo alternative splicing in the IgIII domain, resulting in a single isoform . The genomic organization of FGFR4 encompasses 18 exons rather than the 19 or 20 found in other family members .

How does FGFR4 differ from other FGFR family members?

FGFR4 distinguishes itself from other FGFR family members (FGFR1-3) in several key aspects:

• Lacks alternative splicing variants in the C-terminal half of the IgIII domain

• Contains 18 exons compared to 19-20 in other family members

• Has distinct ligand binding preferences, particularly for acidic fibroblast growth factor (FGF1)

• Demonstrates unique tissue expression patterns during development

• Exhibits a distinct role in metabolic regulation not shared by other FGFRs

What are the primary physiological functions of FGFR4?

FGFR4 functions as a cell-surface receptor for fibroblast growth factors and plays critical roles in:

• Regulation of cell proliferation, differentiation, and migration

• Lipid metabolism and bile acid biosynthesis

• Glucose uptake and energy homeostasis

• Vitamin D metabolism

• Phosphate homeostasis

• Normal down-regulation of CYP7A1 (rate-limiting enzyme in bile acid synthesis) in response to FGF19

What is the expression pattern of FGFR4 during human development?

FGFR4 exhibits a distinct expression pattern during development:

• High expression in mouse pre-implantation blastocytes (along with FGFR3)

• Present in primitive ectoderm at days E5-E6

• Expression increases until days E14-E15 and then declines

• At days E8-E9, FGFR4 is expressed in gut endoderm and myotome of somites

• By day E14.5, strong expression is observed in muscles throughout the embryo (but not heart muscle)

• Widespread expression in cartilage, gut, pancreatic ducts, liver, and lung
  This expression pattern differs significantly from the other three FGFRs, suggesting specialized developmental functions .

How is FGFR4 gene expression regulated at the transcriptional level?

The human FGFR4 core promoter region:

• Spans from position -198 to -9

• Is CG-rich with multiple transcription start points (TSPs)

• Lacks TATA- or CCAAT-like elements, a feature common to many housekeeping genes, oncogenes, growth factors, and transcription factors

• Contains several binding motifs for Sp1, AP2, and GCF transcription factors located 40-80 bp upstream of the TSPs

• Shares characteristics with the promoters of FGFR1-3, which display similar features

What is the significance of the FGFR4 Gly388Arg polymorphism in cancer research?

The FGFR4 Gly388Arg polymorphism (rs351855) is a significant genetic variant present in approximately 55% of the human population . Research findings include:

How does FGFR4 expression differ between cancer subtypes, and what are the functional implications?

Studies of FGFR4 in rhabdomyosarcoma (RMS) subtypes revealed:

• FGFR4 is expressed in both embryonal RMS (eRMS) and alveolar RMS (aRMS), but with distinct patterns and functions

• Higher expression levels are observed in aRMS compared to eRMS

• In eRMS, FGFR4 appears to stimulate cell proliferation

• In aRMS, FGFR4 primarily supports cell survival

• FGFR4 protein is increased in all RMS cell lines compared to non-transformed primary HSMM cells

• In aRMS, FGFR4 expression is induced by PAX3-FOXO1 fusion protein, suggesting FGFR4 is downstream from this oncogenic driver
  These dichotomous roles suggest that FGFR4-targeted therapies may need to be tailored to specific cancer subtypes .

What signaling pathways are activated by FGFR4 in cancer cells?

FGFR4 activates several key signaling cascades in cancer cells:

• PLCG1 pathway:

  • FGFR4 phosphorylates PLCG1

  • Leads to production of diacylglycerol and inositol 1,4,5-trisphosphate

  • Activates downstream calcium signaling and protein kinase C

• FRS2/MAPK pathway:

  • Phosphorylation of FRS2 triggers recruitment of GRB2, GAB1, PIK3R1, and SOS1

  • Mediates activation of RAS, MAPK1/ERK2, MAPK3/ERK1

  • Activates the MAP kinase signaling pathway

• AKT1 signaling pathway:

  • Promotes cell survival and anti-apoptotic responses

• MMP14 regulation:

  • Promotes SRC-dependent phosphorylation of matrix protease MMP14

  • Affects MMP14 lysosomal degradation

  • MMP14 promotes internalization and degradation of FGFR4, creating a feedback loop
    Mutations leading to constitutive kinase activation or impaired receptor inactivation result in aberrant signaling, contributing to oncogenesis .

What are the optimal methods for detecting FGFR4 expression in human tissue samples?

For detection of FGFR4 in human tissue samples, researchers can employ several complementary techniques:

• Immunohistochemistry (IHC):

  • Suitable for FFPE (formalin-fixed paraffin-embedded) tissue sections

  • Requires specific anti-FGFR4 monoclonal antibodies (e.g., MAB6852)

  • Protocol typically involves overnight incubation at 4°C followed by HRP-DAB staining

  • Can localize FGFR4 expression to specific cellular compartments (primarily cytoplasmic)

• Western Blot Analysis:

  • Optimal for cell lysates from cancer cell lines or tissue extracts

  • Detects FGFR4 protein as multiple discrete bands (approximately 110 kDa)

  • Multiple bands often represent differential phosphorylation or glycosylation states

  • PVDF membrane with specific antibody concentration (e.g., 0.2 μg/mL) yields optimal results

• Tissue Microarrays:

  • Effective for analyzing FGFR4 expression across multiple patient samples simultaneously

  • Can distinguish expression patterns between cancer subtypes (e.g., aRMS vs. eRMS)

How can researchers effectively analyze FGFR4 polymorphisms in clinical samples?

For analysis of FGFR4 polymorphisms, particularly the Gly388Arg variant (rs351855):

• Genotyping Approaches:

  • PCR-RFLP (Restriction Fragment Length Polymorphism)

  • Allele-specific PCR

  • TaqMan SNP genotyping assays

  • Next-generation sequencing

• Statistical Analysis for Clinical Correlation:

  • Odds ratios (OR) for associations with clinical parameters

  • Hazard ratios (HR) for survival analyses

  • Multivariable analysis adjusting for confounding factors

• Meta-analysis Considerations:

  • Comprehensive database search (PubMed, Medline, Ovid)

  • Rigorous inclusion criteria for studies

  • Assessment of study heterogeneity using I² statistics

  • Random or fixed-effects models based on heterogeneity

What expression systems yield optimal results for producing functional His-tagged human FGFR4?

For producing functional His-tagged human FGFR4:

• HEK293 Expression System:

  • Preferred for maintaining proper mammalian post-translational modifications

  • Yields recombinant protein with ≥80% purity

  • Suitable for applications requiring native-like protein conformation

• Bacterial Expression Systems:

  • More economical but may lack essential post-translational modifications

  • Better suited for structural studies of individual domains

  • May require refolding protocols to obtain functional protein

• Quality Control Parameters:

  • SDS-PAGE verification of protein purity and molecular weight

  • Western blotting confirmation of His-tag presence

  • Functional assays to verify ligand binding capability

How should researchers design experiments using His-tagged FGFR4 for functional studies?

When designing experiments with His-tagged FGFR4:

• Experimental Applications:

  • SDS-PAGE analysis for purity assessment

  • Surface Plasmon Resonance for binding kinetics

  • Sandwich ELISA for quantification

  • Functional assays for downstream signaling activation

• Critical Considerations:

  • The His-tag location (N- or C-terminal) may affect protein functionality

  • Optimal buffer conditions must be established to maintain protein stability

  • For cell-based assays, receptor clustering and oligomerization properties may differ from native FGFR4

  • Careful validation against untagged controls is essential to ensure the tag doesn't interfere with function

• Storage and Handling:

  • Optimal storage typically at -80°C in small aliquots

  • Avoid repeated freeze-thaw cycles

  • Addition of carrier proteins or glycerol may improve stability

  • Monitor for potential aggregation or degradation before use

What advanced applications are possible with His-tagged FGFR4 in structural biology research?

His-tagged FGFR4 enables several advanced structural biology applications:

• Protein-Ligand Interaction Studies:

  • Immobilized metal affinity chromatography (IMAC) for pull-down assays

  • Surface plasmon resonance (SPR) for binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Crystallography Applications:

  • The His-tag provides a purification method that preserves protein structure

  • Can be used for co-crystallization with potential therapeutic compounds

  • May require tag removal via precision proteases before crystallization

• Cryo-EM Analysis:

  • His-tagged FGFR4 can be used to study receptor oligomerization

  • Analysis of conformational changes upon ligand binding

  • Investigation of receptor-ligand complexes in near-native conditions

What are the current approaches for targeting FGFR4 in cancer therapy research?

Current approaches for FGFR4-targeted cancer therapy include:

• Small Molecule Inhibitors:

  • Tyrosine kinase inhibitors targeting the ATP-binding site

  • Selective FGFR4 inhibitors vs. pan-FGFR inhibitors

  • Development of inhibitors specific to oncogenic FGFR4 variants

• Therapeutic Antibodies:

  • Antibodies targeting the extracellular domain of FGFR4

  • May block ligand binding or induce receptor internalization

  • Potential for antibody-drug conjugates for targeted delivery

• Considerations for Different Cancer Types:

  • Different approaches may be needed for different cancer subtypes

  • In RMS, FGFR4 blockade exerts distinct anti-tumorigenic effects in embryonal versus alveolar subtypes

  • Need to consider the dual role of FGFR4 in proliferation versus survival depending on cancer context

How can FGFR4 Gly388Arg polymorphism status inform personalized treatment approaches?

The FGFR4 Gly388Arg polymorphism has significant implications for personalized medicine: