Recombinant Mouse Fibroblast growth factor 1 (Fgf1) (Active)

What is the molecular structure and functional role of FGF1?

Recombinant mouse FGF1 is typically produced as a protein encompassing amino acids 16-155, with the sequence: MFNLPLGNYKKPKLLYCSNGGFFLRILPDGTVDGTRDRSDQHIQLQLSAESAGEVYIKGTETGQYLAMDTEGLLYGSQTPNEECLFLERLEENHYNTYTSKKHAEKN WFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD . FGF1 adopts a β-trefoil core structure, with characteristic tryptophan residues that produce fluorescence emission spectra centered at 307 nm when properly folded .

Functionally, FGF1 plays important roles in regulating cell survival, cell division, angiogenesis, cell differentiation, and cell migration . It acts as a potent mitogen in vitro and functions as a ligand for FGFR1 and integrins . When bound to FGFR1 in the presence of heparin, FGF1 induces receptor dimerization and activation through sequential autophosphorylation on tyrosine residues . This creates docking sites for interacting proteins and activates several signaling cascades including MAPK3/ERK1, MAPK1/ERK2, and AKT1 pathways .

How does FGF1 signaling operate at the cellular level?

FGF1 operates through both extracellular and intracellular mechanisms:

Extracellular signaling:

• Binds to FGFR1 in the presence of heparin, leading to receptor dimerization

• Forms a ternary complex with integrin ITGAV:ITGB3 and FGFR1

• Recruits PTPN11 (SHP-2) to this complex, which is essential for signaling

• Activates downstream pathways including MAPK and AKT cascades

Intracellular actions:

• Can cross the cellular membrane and translocate to the cytosol and nucleus

• Interacts with at least 20 identified intracellular binding partners

• Many of these binding partners are involved in apoptosis, cell cycle, and proliferation, suggesting a role in cell survival

What experimental systems best demonstrate FGF1 activity?

Researchers should consider these experimental approaches when studying FGF1:

In vitro systems:

• Cell proliferation assays to measure mitogenic activity

• Receptor phosphorylation detection via Western blotting (FGFR1, FRS2, MAPK, AKT)

• Angiogenesis assays such as endothelial tube formation

• Neuronal electrophysiology to measure membrane potential changes in responsive neurons

In vivo models:

• Diabetic mouse models (db/db, ob/ob) for studying metabolic effects

• Cardiomyopathy models for cardiac protection studies

• Central nervous system administration to study neuronal activation

How should researchers validate recombinant mouse FGF1 activity?

A comprehensive validation approach includes:

Structural validation:

• SDS-PAGE to confirm molecular weight and purity (>95%)

• Circular dichroism to verify proper protein folding

• Fluorescence spectroscopy to confirm characteristic emission patterns (307 nm)

Functional validation:

• Cell-based assays measuring:

  • Proliferation (mitogenic activity)

  • Receptor phosphorylation (FGFR1, FRS2, MAPK, AKT)

  • Angiogenic response

• Binding assays (SPR or pull-down) to confirm interactions with FGFR1, integrins, or known binding partners

• Electrophysiological recordings to confirm neuronal activation patterns

Quality control parameters:

• Endotoxin levels should be <1 EU/μg for in vivo applications

• Confirmation of absence of microbial contamination

• Batch-to-batch consistency verification

What experimental design considerations are critical when studying FGF1's role in metabolism?

When investigating FGF1's metabolic effects, researchers should consider:

Model selection:

• Diabetic models (db/db, ob/ob mice or Zucker diabetic fatty rats) are appropriate for studying glucose regulation

• Diet-induced obesity models can reveal effects in metabolic syndrome contexts

Administration routes:

• Intracerebroventricular (i.c.v.) injection into the lateral ventricle can induce long-term reduction in hyperglycemia (up to 18 weeks)

• Direct injection into specific brain nuclei like the arcuate nucleus (ARH) can be sufficient to reduce hyperglycemia

Neuronal targeting:

• Focus on proopiomelanocortin (POMC) neurons in the ARH, which are activated by FGF1

• In contrast, neuropeptide Y (NPY) neurons in the ARH do not respond to FGF1, but NPY neurons in the nucleus tractus solitarius (NTS) do respond

Readouts:

• Blood glucose measurements over extended time periods

• Food intake monitoring

• Expression of metabolic genes

• Mitochondrial function parameters (fragmentation, ROS generation, respiration rate, β-oxidation)

How can researchers effectively study the heparin-dependency of FGF1 functions?

The interaction between FGF1 and heparin is critical for many of its functions:

Experimental approaches:

• Compare activities with and without heparin supplementation

• Use FGF1 variants with modified heparin-binding sites (e.g., FGF1 ΔHBS) to distinguish heparin-dependent and independent functions

• Study differential effects on proliferation versus metabolic activities (FGF1 ΔHBS retains metabolic effects while showing reduced proliferative potency)

Relevant assays:

• Receptor binding and activation with/without heparin

• Cell proliferation and metabolic assays in parallel

• In vivo studies comparing wild-type FGF1 and FGF1 ΔHBS

Key findings to consider:

• FGF1 ΔHBS prevents diabetes-induced cardiac injury and remodeling through AMPK/Nur77-dependent mechanisms

• The favorable metabolic activity combined with reduced proliferative properties makes FGF1 ΔHBS a promising candidate for treating metabolic disorders

How can FGF1 be engineered for enhanced therapeutic properties?

Based on current research, several strategies have proven successful:

Chimeric protein design:

• Structure-based chimeras combining FGF1 with other FGF family members can enhance stability and function

• For example, replacing the core of FGF21 with a thermally stable paracrine FGF1 (sFGF1) creates a chimera with increased stability and enhanced antidiabetic activities

Functional domain modification:

• Creating variants with modified heparin-binding sites (FGF1 ΔHBS) reduces proliferative potential while maintaining beneficial metabolic effects

Validation approaches:

• Differential scanning calorimetry to confirm increased thermal stability

• Fluorescence spectroscopy and circular dichroism to verify proper protein folding

• Functional assays comparing wild-type and engineered variants

• In vivo testing in appropriate disease models

What approaches should be used to study FGF1's neuronal activation mechanisms?

FGF1 activates specific neuronal populations through mechanisms that require careful experimental design:

Neuronal subtype targeting:

• Use transgenic reporter mice (NPY-GFP, POMC-EGFP) to identify specific neuronal populations

• Focus on POMC neurons in the arcuate nucleus, which are depolarized by FGF1 (100 nM)

• Note that NPY neurons in the arcuate nucleus do not respond to FGF1, but NPY neurons in the NTS do respond

Electrophysiological approaches:

• Whole-cell patch-clamp recordings to measure membrane potential changes

• Test the effects of tetrodotoxin (TTX) to determine if effects require voltage-gated sodium channels

• Compare responses in healthy versus metabolically compromised models (e.g., diet-induced obesity)

Activation mapping:

• c-Fos immunohistochemistry to map neuronal activation patterns following FGF1 administration

• Focus on key brain regions including arcuate nucleus, area postrema (AP), and nucleus tractus solitarius (NTS)

How should researchers investigate intracellular FGF1 binding partners?

For studying FGF1's intracellular interactions, use these methodological approaches:

Protein interaction discovery methods:

• Yeast two-hybrid screening (use FGF1 aa 22-155 as bait)

• Tandem Affinity Purification (TAP) followed by mass spectrometry analysis

• Co-precipitation from cell lysates using recombinant FGF1 as bait

Interaction validation:

• Pull-down assays with recombinant proteins

• Surface Plasmon Resonance (SPR) measurements to confirm direct interactions

• Co-immunoprecipitation from cells expressing tagged versions of the proteins

Known binding partners to consider:

• Previously identified: FGF-BP1, protein kinase CK2, FGF1 intracellular binding protein (FIBP), mortalin (GRP75/hthsp70/PB74), and p34

• Twenty additional novel intracellular proteins have been identified, many involved in apoptosis, cell cycle regulation, and proliferation

What are the critical factors affecting FGF1 stability and activity in experimental settings?

Storage and handling considerations:

• Store at -80°C for long-term storage

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Use low-protein binding tubes to prevent loss through adsorption

• Include stabilizers in storage buffer (specific formulations may vary)

Activity-preserving factors:

• Heparin supplementation for receptor-dependent activities

• Temperature control during experiments (particularly important as FGF1 has thermal stability considerations)

• Proper pH maintenance (typically physiological range)

• Use of carrier proteins for dilute solutions may prevent loss of activity

Validation methods:

• Periodically test activity in established assays (cell proliferation, receptor activation)

• Verify protein integrity by SDS-PAGE

• Use positive controls from previously validated batches

How can researchers distinguish between direct and indirect effects of FGF1?

Experimental strategies:

• Time-course studies (direct effects typically occur rapidly, within minutes to hours)

• Pathway inhibition (use specific inhibitors of known FGF1 signaling pathways)

• Receptor blocking (FGFR inhibitors or neutralizing antibodies)

• Use of FGF1 variants with altered receptor binding properties (e.g., FGF1 ΔHBS)

• Transcription/translation inhibitors to determine if effects require new gene expression

Neuronal studies specific approaches:

• Use tetrodotoxin (TTX) to block action potentials and determine if effects are direct or require neuronal network activity

• Compare responses of different neuronal populations (e.g., POMC vs NPY neurons)

• Use cell-type specific receptor knockouts to confirm direct targeting

What are the key considerations when investigating FGF1's role in diabetic cardiomyopathy?

Based on research with FGF1 ΔHBS , critical methodological considerations include:

Model selection:

• Use established diabetic models (db/db mice, ob/ob mice, Zucker diabetic fatty rats)

• Consider using AMPK null mice as controls to verify pathway involvement

Cardiac function assessment:

• Echocardiography to measure fraction shortening and other functional parameters

• Hemodynamic measurements to assess cardiac performance

Molecular analyses:

• RNA-Seq to identify gene expression changes in cardiac tissues

• Focus on anti-oxidative genes and Nur77 expression

• Western blotting for AMPK activation and downstream targets

Mitochondrial function assessment:

• Measure mitochondrial fragmentation

• Quantify reactive oxygen species (ROS) generation

• Assess cytochrome c leakage

• Determine mitochondrial respiration rate and β-oxidation capacity

Clinical relevance:

• Consider correlations with human data (serum FGF1 levels have been found to positively correlate with fraction shortening in diabetic cardiomyopathy patients)

What emerging applications of FGF1 warrant further investigation?

Metabolic disease therapeutics:

• Long-lasting glycemic control following central administration

• Potential applications in type 2 diabetes treatment using FGF1 variants with reduced proliferative potency

• Further exploration of brain-periphery connections in FGF1-mediated metabolic effects

Cardiac protection:

• Mechanisms of FGF1 ΔHBS in preventing diabetes-induced cardiac injury and remodeling

• Application to other cardiomyopathies beyond diabetic etiology

Neuronal regulation:

• Further characterization of FGF1's effects on specific neuronal populations

• Investigation of potential applications in appetite regulation and energy balance

Engineered FGF1 variants:

• Development of additional chimeric proteins with enhanced stability and function

• Creation of tissue-specific targeting variants to reduce off-target effects

What technologies will advance FGF1 research in the coming years?

Emerging methodologies:

• Single-cell transcriptomics to identify cell-specific responses to FGF1

• CRISPR-based approaches for receptor and pathway component manipulation

• Advanced imaging techniques for tracking FGF1 signaling in real-time

• In silico protein engineering to design optimized FGF1 variants

Data integration approaches:

• Multi-omics analysis combining transcriptomics, proteomics, and metabolomics

• Systems biology modeling of FGF1 signaling networks

• Machine learning for prediction of FGF1 binding partners and pathway interactions