FGFR3 Human, His
Description
Fundamental Characteristics of FGFR3 Human, His
FGFR3 Human, His refers to a recombinant form of the human fibroblast growth factor receptor 3 protein that incorporates a histidine tag for purification and experimental purposes. The FGFR3 gene, located on chromosome 4 at position p16.3, encodes this transmembrane receptor protein that belongs to the tyrosine kinase receptor family . The histidine tag, typically consisting of six consecutive histidine residues (6×His), is engineered at either the N-terminus or C-terminus of the protein to facilitate purification through metal affinity chromatography techniques.
The native FGFR3 protein is expressed in various tissues throughout the human body, including cartilage, brain, intestine, and kidneys . This widespread expression pattern underscores the protein's multifunctional role in developmental processes. The FGFR3 gene has been extensively studied due to its association with multiple skeletal dysplasias, most notably achondroplasia, the most common form of human dwarfism.
Production and Characterization of FGFR3 Human, His
Commercial recombinant human FGFR3 with histidine tags typically includes specific portions of the protein essential for research applications. For instance, one formulation comprises human FGFR3 (IIIb) (Glu23-Gly377) with a C-terminal 6-His tag . This segment represents the extracellular domain responsible for ligand binding without the transmembrane or intracellular regions.
Alternative Formulations
Various commercial formulations exist with distinctive features to serve different research needs. For example, some preparations incorporate an Avi-tag in addition to the His-tag, allowing for site-specific biotinylation and oriented immobilization on streptavidin-coated surfaces . This dual-tagged variant offers advantages for binding studies and protein interaction assays by ensuring uniform orientation when immobilized.
Functional Mechanisms of FGFR3
FGFR3 Human, His preserves the fundamental biological properties of the native receptor, particularly those associated with the extracellular domain. Understanding these functions provides context for the applications of the recombinant protein in research.
Signal Transduction Pathways
The FGFR3 protein spans the cell membrane, allowing it to interact with growth factors outside the cell while transmitting signals internally. When fibroblast growth factors attach to the extracellular portion of FGFR3, they trigger activation of the receptor . This initiates a complex cascade of chemical reactions inside the cell that direct cellular processes such as:
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Regulation of cell growth and division (proliferation)
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Determination of cell type (differentiation)
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Formation of blood vessels (angiogenesis)
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Wound healing
FGFR3 primarily transduces signals through three dominant pathways: RAS/MAPK, PI3K/AKT, and PLC gamma . These signaling cascades mediate the diverse cellular responses to FGFR3 activation.
Tissue-Specific Functions
The FGFR3 gene produces multiple isoforms through alternative splicing, with tissue-specific expression patterns. The FGFR3(IIIb) isoform, which is the basis for many His-tagged recombinant preparations, is predominantly expressed in epithelial cells . In contrast, the FGFR3(IIIc) isoform is expressed in mesenchymal tissues. This selective expression contributes to tissue-specific responses to FGF ligands.
In bone development, FGFR3 plays a critical regulatory role by limiting the formation of bone from cartilage (ossification) . This function is particularly important during early skeletal development, where FGFR3 helps to control the rate of endochondral ossification.
Clinical Significance and Disease Associations
FGFR3 Human, His has become an essential tool for studying the pathogenic mechanisms underlying multiple human diseases associated with FGFR3 dysfunction.
Skeletal Dysplasias
Mutations in the FGFR3 gene cause several skeletal disorders that vary in severity. The most well-characterized conditions include:
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Achondroplasia: The most common form of short-limbed dwarfism, caused primarily by a specific mutation (p.Gly380Arg) that results in constitutive activation of FGFR3 . This gain-of-function mutation leads to inhibition of chondrocyte proliferation and restricted long bone growth .
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Hypochondroplasia: A milder skeletal dysplasia often caused by the p.Asn540Lys mutation in FGFR3 . This condition presents with short stature and disproportionately short limbs, but with less severe manifestations than achondroplasia.
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Thanatophoric Dysplasia: A severe and typically lethal skeletal disorder characterized by extremely short limbs and a narrow chest . Type I is caused by various FGFR3 mutations that change a single amino acid or create an elongated protein, while Type II is specifically associated with the p.Lys650Glu mutation .
The frequency of these mutations has been extensively documented. For instance, the International Skeletal Dysplasia Registry analyzed 324 cases and found that the p.Gly380Arg mutation accounts for virtually all cases of achondroplasia, while p.Asn540Lys is responsible for approximately 76% of hypochondroplasia cases .
Cancer Associations
FGFR3 mutations and aberrant signaling have been implicated in several types of cancer:
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Bladder Cancer: Somatic mutations in FGFR3 are frequently associated with bladder cancer, and some mutations correlate with better prognosis . FGFR3-TACC3 and FGFR3-BAIAP2L1 fusion proteins have also been identified in bladder malignancies .
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Glioblastoma: FGFR3-TACC3 fusions serve as primary mitogenic drivers in approximately 4% of glioblastomas and other gliomas . These molecular alterations represent potential therapeutic targets.
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Other Malignancies: Dysregulation of FGFR3 has been observed in various other cancers, with FGFR3(IIIb) specifically upregulated in hepatocellular carcinoma but downregulated in colorectal cancer .
Applications in Research and Therapeutic Development
FGFR3 Human, His has diverse applications in basic research, disease modeling, and drug discovery efforts.
Binding Studies and Interaction Analysis
The recombinant protein enables detailed analysis of binding interactions between FGFR3 and potential ligands or inhibitors. Functional ELISA assays have demonstrated that recombinant human FGFR3(IIIb) His-tag protein binds to FGF1 with measurable affinity . These binding studies help elucidate the molecular mechanisms of FGFR3 activation and inhibition.
Animal Models and Therapeutic Testing
Mouse models expressing human FGFR3 with the G380R mutation have been developed to recapitulate achondroplasia . These knock-in models express the human mutant FGFR3 cDNA in place of the endogenous mouse Fgfr3 gene, resulting in phenotypes that closely resemble those observed in human patients with achondroplasia. The severity of disease manifestations corresponds to the copy number of the activated FGFR3, with homozygous mice (FGFR3^ACH/ACH) showing more pronounced phenotypes than heterozygous mice (FGFR3^ACH/+) .
These animal models offer invaluable tools for testing potential therapeutic approaches for skeletal dysplasias related to FGFR3 overactivation. Studies using these models have demonstrated that:
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The phenotypes become more pronounced during postnatal skeletal development
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The models exhibit growth retardation, disproportionate shortening of limbs, round head, mid-face hypoplasia, and progressive kyphosis
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Premature fusion of cranial sutures and low bone density occur in newborn FGFR3^G380R mice
Therapeutic Development
FGFR3 represents a promising therapeutic target for multiple conditions. In bladder cancer, post-translational modifications of FGFR3 that do not occur in normal cells can be targeted by immunotherapeutic antibodies . For skeletal dysplasias, several approaches are under investigation:
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Tyrosine kinase inhibitors that block FGFR3 signaling
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Neutralizing antibodies against FGFR3
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Antisense oligonucleotides to reduce FGFR3 expression
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C-type natriuretic peptide analogs that counteract the effects of FGFR3 overactivation
Technical Considerations for Laboratory Applications
When working with FGFR3 Human, His in laboratory settings, several technical aspects warrant consideration.
Experimental Applications
FGFR3 Human, His finds utility in diverse experimental setups:
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Binding Assays: Measuring interactions with natural ligands, inhibitors, or antibodies
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Structural Studies: Crystallography or cryo-electron microscopy to determine three-dimensional structure
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Cell-Based Assays: Examining effects of exogenous FGFR3 on cellular processes
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High-Throughput Screening: Identifying potential therapeutic compounds that modulate FGFR3 activity
Future Directions and Emerging Research
Recent advances in FGFR3 research point to several promising directions:
Novel Therapeutic Approaches
The development of targeted therapies for FGFR3-related disorders represents an active area of investigation. For achondroplasia, vosoritide (a C-type natriuretic peptide analog) has shown promising results in clinical trials, demonstrating improved growth velocity in children . Other approaches include:
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RNA-based therapies to modulate FGFR3 expression
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Small molecule inhibitors with improved specificity for mutant FGFR3
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Combination therapies targeting multiple components of the FGFR3 signaling pathway
Product Specs
The fibroblast growth factor (FGF) family consists of at least 18 structurally related proteins with diverse roles in physiological and pathological processes, such as cell growth, differentiation, angiogenesis, wound healing, and tumorigenesis. FGFs exert their biological effects by binding to and activating a family of type I transmembrane tyrosine kinase receptors known as fibroblast growth factor receptors (FGFRs). Upon ligand binding, FGFRs undergo dimerization and autophosphorylation. Four distinct genes (FGFR-1 to -4) encode these closely related receptors. Alternative splicing of mRNAs gives rise to multiple isoforms of FGFR-1 to -3. A common splicing event in FGFR-1 and -2 produces receptors containing all three immunoglobulin-like domains (Ig domains), known as the alpha isoform, or only IgII and IgIII, known as the beta isoform. Notably, only the alpha isoform has been identified for FGFR-3 and FGFR-4. Further splicing events in FGFR-1 to -3, specifically in the C-terminal half of the IgIII domain encoded by two mutually exclusive exons, generate receptors with alternative IgIII domains (IIIb and IIIc). Additionally, a secreted FGF-binding protein called the IIIa isoform has been reported for FGFR-1. This isoform comprises the N-terminal half of the IgIII domain and some intron sequences. Mutations in FGFR-1 to -3 have been implicated in birth defects involving craniosynostosis.
Recombinant human FGFR3, produced in Sf9 insect cells using a baculovirus expression system, is a single glycosylated polypeptide chain. This protein consists of 592 amino acids (residues 23-375a.a.) and has a molecular weight of 65.1 kDa. Note that its apparent molecular size on SDS-PAGE may appear between 70-100 kDa.
This FGFR3 protein is expressed with a C-terminal 239 amino acid hIgG-His tag and purified using proprietary chromatographic methods.
The FGFR3 protein solution is provided at a concentration of 1 mg/ml and is formulated in phosphate-buffered saline (PBS) at pH 7.4 with 10% glycerol.
For short-term storage (up to 2-4 weeks), the product can be stored at 4°C. For extended storage, it is recommended to freeze the product at -20°C. To ensure long-term stability during frozen storage, consider adding a carrier protein (0.1% HSA or BSA). It's important to avoid repeated freeze-thaw cycles to maintain protein integrity.
The purity of this protein is determined to be greater than 95.0% as assessed by SDS-PAGE analysis.
CD333 Antigen, HSFGFR3EX, EC 2.7.10, CD333, CEK2, ACH, Fibroblast Growth Factor Receptor 3, EC 2.7.10.1, FGFR-3, JTK4, Fibroblast Growth Factor Receptor 3 Variant 4, Achondroplasia, Thanatophoric Dwarfism, Hydroxyaryl-Protein Kinase, Tyrosine Kinase JTK4.
ESLGTEQRVV GRAAEVPGPE PGQQEQLVFG SGDAVELSCP PPGGGPMGPT VWVKDGTGLV PSERVLVGPQ RLQVLNASHE DSGAYSCRQR LTQRVLCHFS VRVTDAPSSG DDEDGEDEAE DTGVDTGAPY WTRPERMDKK LLAVPAANTV RFRCPAAGNP TPSISWLKNG REFRGEHRIG GIKLRHQQWS LVMESVVPSD RGNYTCVVEN KFGSIRQTYT LDVLERSPHR PILQAGLPAN QTAVLGSDVE FHCKVYSDAQ PHIQWLKHVE VNGSKVGPDG TPYVTVLKTA GANTTDKELE VLSLHNVTFE DAGEYTCLAG NSIGFSHHSA WLVVLPAEEE LVEADEAGSV YAGLEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKHHHH HH.
Q&A
What is the basic structure of human FGFR3 protein?
Human FGFR3 belongs to a family of type I transmembrane tyrosine kinases that mediate the biological functions of fibroblast growth factors (FGFs). The protein possesses an extracellular domain (ECD) with three immunoglobulin (Ig)-like domains, an acid-box region containing acidic residues between the IgI and IgII domains, a transmembrane domain, and a split tyrosine-kinase domain . His-tagged recombinant variants typically include amino acids Glu23-Gly377 with a C-terminal 6-His tag, focusing on the extracellular portion of the receptor .
What are the different isoforms of FGFR3 and how do they differ?
Alternative splicing generates multiple isoforms of FGFR3, each with unique signaling characteristics. Specifically for FGFR3, alternative splicing of the extracellular domain, particularly the IgIII domain, results in IIIb or IIIc isoforms . These splice variants exhibit distinct and varying binding affinities for different FGF ligands . The FGFR3A(IIIb) splice variant is predominantly expressed in epithelial cells, while other isoforms show different tissue distribution patterns .
What are the primary signaling pathways associated with FGFR3?
FGFR3 mediates the FGF signaling cascade through three dominant pathways: RAS/MAPK, PI3K/AKT, and PLCγ . These pathways regulate critical developmental processes including cellular proliferation, differentiation, migration, morphogenesis, and patterning . The specific pathway activation pattern may depend on the isoform, tissue context, and ligand binding characteristics.
What is the normal tissue expression pattern of FGFR3?
FGFR3 is normally expressed in tissues of the central nervous system, brain, kidney, and testis . Recent studies have revealed that FGFR3 is also expressed by human primordial germ cells (PGCs) during the first and second trimester of development, becoming repressed as PGCs differentiate into primordial oocytes . This expression pattern suggests important developmental roles beyond those previously characterized.
How does FGFR3 function in embryonic development?
FGFR3 plays a critical role in embryonic and fetal development, particularly in skeletal development and bone growth regulation . In human germ cell development, FGFR3 is expressed by PGCs during early developmental stages, with expression predominantly in PGCs and retinoic acid (RA)-responsive meiotic germ cells . This expression pattern becomes repressed as ovarian meiotic germ cells upregulate genes like SCP3 and SPO11 in prophase I of meiosis I .
How can FGFR3 expression be used to identify specific cell populations?
FGFR3 expression can be utilized as a cell surface marker to identify and isolate specific cell populations, particularly human PGCs. Fluorescence-activated cell sorting (FACS) with antibodies recognizing FGFR3 has been demonstrated to effectively enrich for PGCs from prenatal ovarian tissue . Single-cell RNA sequencing confirms that isolating FGFR3-positive cells significantly enriches for human PGCs, making this approach valuable for identifying maturing PGCs in vitro .
What are the optimal conditions for reconstituting recombinant FGFR3-His proteins?
Reconstitution conditions for recombinant FGFR3-His proteins should be carefully controlled to maintain structural integrity and functionality. While specific conditions may vary between commercial preparations, most FGFR3 recombinant proteins should be reconstituted in sterile, buffered solutions with stabilizing agents, avoiding repeated freeze-thaw cycles . Researchers should consider using reconstitution calculators provided by manufacturers to ensure appropriate concentration .
What methods can be used to verify the binding functionality of recombinant FGFR3?
Functional ELISA is a reliable method to verify the binding ability of recombinant FGFR3. For example, recombinant Human FGFR3 alpha (IIIb) His-tag protein can be tested by measuring its binding to recombinant Human FGF acidic/FGF1 protein, with expected ED50 values in the range of 0.700-7.00 μg/mL . Additionally, SDS-PAGE under reducing and non-reducing conditions can be used to visualize the protein, which typically appears as bands at 60-66 kDa .
How can single-cell RNA sequencing be utilized to study FGFR3 expression patterns?
Single-cell RNA sequencing (scRNA-seq) provides valuable insights into FGFR3 expression at the cellular level. This approach has been used to identify FGFR3 mRNA enrichment in germ cell populations of human embryonic and fetal ovaries and testes . By defining cell populations based on marker genes (such as NANOG, POU5F1, BLIMP1 for PGCs; STRA8, ZGLP1, SPO11 for meiotic cells), researchers can precisely map FGFR3 expression throughout development and differentiation .
What is the relationship between FGFR3 and heat shock protein 90 (Hsp90)?
FGFR3 has been established as a strong Hsp90 client protein, in contrast to other FGFR family members . This interaction affects the stability and signaling capacity of the receptor. Both immature (120 kDa) and mature (130 kDa) forms of wild-type, constitutively active, and kinase-dead FGFR3 interact with Hsp90, suggesting that Hsp90 can engage with the receptor throughout its maturation and trafficking in the secretory pathway .
How do Hsp90 inhibitors affect FGFR3 function and stability?
Hsp90 inhibitors like 17-AAG and radicicol interfere with Hsp90-FGFR3 interactions in a concentration-dependent manner, which correlates with their affinities for Hsp90 . Inhibition of Hsp90 function alters the chaperone complexes associated with FGFR3 and reduces the stability and signaling capacity of the receptor . This suggests that modulating Hsp90 chaperone complexes may beneficially influence the stability and function of FGFR3 in disease contexts .
How does FGFR3 interaction with Cdc37 differ from other FGFR family members?
Co-chaperone Cdc37 interacts more strongly with FGFR3 compared to other FGFR family members . Specifically, Cdc37 pulls down more strongly with FGFR3 and is only faintly observed for FGFR1 and FGFR4, but not for FGFR2 . This differential interaction may contribute to the unique stability and signaling characteristics of FGFR3 compared to other family members.
What are the key FGFR3 mutations associated with human disease?
The G380R mutation in FGFR3 is the most common mutation causing achondroplasia (ACH), the most prevalent form of genetic dwarfism in humans . This gain-of-function mutation leads to over-activation of FGFR3 signaling. Other FGFR3 mutations are associated with various skeletal dysplasias, and FGFR3A(IIIb) is specifically upregulated in hepatocellular carcinoma but downregulated in colorectal cancer .
How has the human FGFR3 G380R mutation been modeled in mice?
A mouse model expressing human FGFR3 G380R has been developed by replacing the endogenous mouse Fgfr3 gene with human FGFR3 G380R cDNA . Both heterozygous (FGFR3^ACH/+) and homozygous (FGFR3^ACH/ACH) mice recapitulate phenotypes observed in human ACH patients, including growth retardation, disproportionate shortening of limbs, round head, mid-face hypoplasia at birth, and kyphosis progression during postnatal development . Additional features include premature fusion of cranial sutures and low bone density in newborn mutant mice .
What methodological approaches can be used to study FGFR3 in disease contexts?
Multiple methodological approaches can be employed to study FGFR3 in disease contexts:
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Mouse models expressing human FGFR3 mutations provide valuable tools for assessing potential therapeutic approaches for skeletal dysplasias related to over-activation of human FGFR3 .
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Immunoprecipitation combined with Western blotting can reveal interactions between FGFR3 and chaperone proteins like Hsp90 and Cdc37 .
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Site-directed mutagenesis and creation of chimeric receptors allow for studying specific domains and their roles in FGFR3 function .
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FACS with FGFR3 antibodies can isolate specific cell populations expressing the receptor for further analysis .
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Single-cell RNA sequencing provides high-resolution data on FGFR3 expression patterns in normal and disease contexts .
How might Hsp90 inhibitors be utilized in FGFR3-mediated diseases?
Hsp90 inhibitors may offer therapeutic potential for FGFR3-mediated diseases since FGFR3 is a strong Hsp90 client protein . Geldanamycin derivatives such as 17-AAG and other Hsp90 inhibitors have progressed to phase II clinical trials for cancer therapy . By disrupting the Hsp90-FGFR3 interaction, these inhibitors reduce the stability and signaling capacity of FGFR3, which could be beneficial in conditions characterized by FGFR3 overactivation, such as skeletal dysplasias and certain cancers .
What preclinical models are most appropriate for testing FGFR3-targeted therapeutics?
Mouse models expressing human FGFR3 mutations, particularly the G380R mutation, provide valuable preclinical platforms for testing FGFR3-targeted therapeutics . These models closely recapitulate human disease phenotypes, including both developmental and postnatal features of achondroplasia . The severity of disease phenotypes corresponds to the copy number of activated FGFR3, allowing for dose-response studies . Cell-based systems expressing wild-type or mutant FGFR3 can also be useful for initial screening of compounds targeting FGFR3 signaling or stability .
What methods can be used to assess the efficacy of potential treatments for FGFR3-related disorders?
Assessment of treatment efficacy for FGFR3-related disorders should include multiple parameters:
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Morphological measurements to evaluate skeletal phenotypes, including limb length, skull shape, and spine curvature .
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Histological analysis of growth plates and other affected tissues .
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Molecular assays to assess FGFR3 signaling pathway activation (RAS/MAPK, PI3K/AKT, PLCγ) .
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Protein interaction studies to examine changes in FGFR3 associations with chaperones like Hsp90 .
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Developmental progression evaluations to determine if treatments can prevent or reverse disease features that emerge postnatally, such as kyphosis .
What are the key unanswered questions regarding FGFR3 biology?
Despite significant advances, several aspects of FGFR3 biology remain poorly understood:
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The precise mechanisms by which different FGFR3 isoforms regulate tissue-specific development and function.
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The role of FGFR3 in human primordial germ cell development and how its expression is regulated during differentiation.
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The complete interaction network of FGFR3 with chaperones and co-chaperones beyond Hsp90 and Cdc37.
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The factors determining why FGFR3 is a stronger Hsp90 client than other FGFR family members.
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The potential non-canonical signaling pathways activated by FGFR3 in different cellular contexts.
How might emerging technologies advance FGFR3 research?
Emerging technologies poised to advance FGFR3 research include:
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CRISPR/Cas9 gene editing to create more precise disease models and to study endogenous FGFR3 regulation.
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Advanced proteomics approaches to comprehensively map FGFR3 interactions and post-translational modifications.
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Organoid systems to study FGFR3 function in complex, three-dimensional tissue environments.
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Single-cell multi-omics to correlate FGFR3 expression with epigenetic states, protein levels, and cellular phenotypes.
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Structural biology techniques like cryo-electron microscopy to visualize FGFR3-chaperone complexes and conformational changes associated with receptor activation or inhibition.
What opportunities exist for developing novel FGFR3-targeted therapeutics?
Several opportunities exist for developing novel FGFR3-targeted therapeutics:
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Development of isoform-specific FGFR3 inhibitors to minimize off-target effects.
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Exploration of targeted protein degradation approaches (PROTACs) specific for FGFR3.
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Modulation of FGFR3-chaperone interactions as an alternative to direct receptor inhibition.
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Investigation of combination therapies targeting multiple nodes in FGFR3 signaling networks.
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Development of allele-specific therapies for FGFR3 mutations like G380R that cause achondroplasia.
Product Science Overview
FGFR3 consists of:
- Extracellular Region: Composed of three immunoglobulin-like domains.
- Membrane-Spanning Segment: A single hydrophobic segment.
- Cytoplasmic Tyrosine Kinase Domain: Responsible for signal transduction .
The extracellular portion of FGFR3 interacts with fibroblast growth factors, setting in motion a cascade of downstream signals that ultimately influence mitogenesis and differentiation . This receptor binds both acidic and basic fibroblast growth factors and plays a crucial role in bone development and maintenance .
Mutations in the FGFR3 gene are associated with several disorders, including:
- Craniosynostosis: A condition where the bones in a baby’s skull join together too early.
- Skeletal Dysplasia: A group of disorders characterized by abnormalities in bone and cartilage growth .
FGFR3 has also been implicated in various cancers, including lung and breast cancers. The receptor’s activation can lead to the activation of pathways such as the RAS-MAPK and PI3K-AKT pathways, which are known to play significant roles in cancer development .
