EGF Mouse, His Active

What is mouse EGF and what are its key structural features?

Mouse Epidermal Growth Factor (EGF) is a 54-amino acid polypeptide growth factor that stimulates proliferation, differentiation, and survival of epithelial and epidermal cells. It contains three intramolecular disulfide bonds that are critical for its biological activity and has a molecular weight of approximately 6.2 kDa as a monomer. The amino acid sequence (MNSYPGCPSS YDGYCLNGGV CMHIESLDSY TCNCVIGYSG DRCQTRDLRW WELR) enables high-affinity binding to the epidermal growth factor receptor (EGFR) . Researchers should note that mouse EGF binds to its receptor with an affinity constant of approximately 5.44 nM as determined by Surface Plasmon Resonance (SPR) assays .

What methodologies are most effective for verifying mouse EGF activity?

Mouse EGF activity can be verified through several complementary approaches:

• Cell proliferation assay: The dose-dependent proliferation of BALB/c 3T3 cells is a standard method, with typical ED50 values less than 250 pg/ml, corresponding to a specific activity of approximately 4 × 10^6 units/mg .

• Binding assays: Immobilized Mouse EGF, Fc Tag (2 μg/mL) can bind Mouse EGFR with a linear range of 0.02-0.313 μg/mL .

• Surface Plasmon Resonance: SPR assays can determine binding kinetics and affinity constants between EGF and EGFR (typically around 5.44 nM) .

• Phosphorylation analysis: Western blotting with phospho-specific antibodies can detect EGF-induced autophosphorylation of EGFR at specific tyrosine residues (Tyr845, Tyr992, and Tyr1068) and activation of downstream signaling molecules .

How is EGF gene expression regulated during mouse development?

EGF gene expression shows tissue-specific and developmental regulation in mice. In testicular tissue, EGF mRNA content increases progressively with sexual maturation, with expression levels following the pattern: day 15 < day 30 < day 45 postnatal . This expression pattern correlates with testis maturation and proliferation of Leydig cells, which are the primary producers of EGF in the testis. Using RT-PCR with specific oligonucleotide primers is an effective method to quantify these developmental changes in EGF expression . The developmental regulation suggests that EGF plays important roles in organ maturation and cell differentiation during specific developmental windows.

How does Growth Hormone (GH) modulate EGFR expression and signaling in mice?

GH exerts complex regulatory effects on EGFR expression and signaling in mice:

• In GH Receptor Knockout (GHR-KO) mice, EGFR protein levels are significantly lower compared to normal mice, resulting in decreased EGF-induced phosphorylation of the receptor at tyrosine residues (Tyr845, Tyr992, and Tyr1068) .

• Downstream signaling through AKT, ERK1/2, STAT3, and STAT5 pathways is significantly diminished in GHR-KO mice following EGF stimulation, despite normal basal phosphorylation levels .

• Conversely, transgenic mice overexpressing GH display increased EGFR content and elevated basal tyrosine phosphorylation .

• Interestingly, while AKT and ERK1/2 pathways remain responsive to EGF in GH-overexpressing mice, STAT3 and STAT5 activation is heterologously desensitized, demonstrating pathway-specific regulation .

This complex relationship between GH and EGFR signaling indicates that experimental interpretations must consider the hormonal context and specific signaling pathways being investigated.

What are the primary signaling pathways activated by mouse EGF and how do they interact?

Mouse EGF activates several interconnected signaling pathways through EGFR:

• MAPK/ERK pathway: EGF stimulation induces phosphorylation of ERK1/2 at residues Thr202 and Tyr204, driving cell proliferation and differentiation responses .

• PI3K/AKT pathway: EGF induces AKT phosphorylation at Ser473, promoting cell survival, metabolism, and protein synthesis .

• STAT pathway: EGF activates STAT3 and STAT5 proteins, which translocate to the nucleus and regulate gene expression .

• Receptor autophosphorylation: EGF binding induces EGFR phosphorylation at specific tyrosine residues (Tyr845, Tyr992, and Tyr1068), creating docking sites for various adaptor proteins and initiating downstream signaling cascades .

These pathways do not operate in isolation but form an integrated network with extensive cross-talk. For example, in GH-overexpressing mice, AKT and ERK1/2 pathways remain responsive to EGF while STAT pathways become desensitized, suggesting differential regulatory mechanisms .

How do different phosphorylation sites on mouse EGFR impact downstream signaling events?

The phosphorylation of specific tyrosine residues on mouse EGFR creates unique binding sites for different signaling proteins:

• Tyr845: Located in the activation loop of the kinase domain, this site is phosphorylated by Src kinase and contributes to maximal receptor activation .

• Tyr992: When phosphorylated, this residue creates a binding site for phospholipase C-γ (PLC-γ), initiating calcium signaling and protein kinase C activation .

• Tyr1068: This is a major binding site for the adaptor protein Grb2, which links EGFR activation to the Ras-RAF-MEK-ERK pathway .

What are the optimal storage and handling protocols for maintaining mouse EGF and EGFR stability?

To maintain optimal stability and activity of mouse EGF and EGFR preparations:

• Long-term storage: Store lyophilized proteins at -20°C or lower to preserve activity .

• Reconstitution: For mouse EGFR, reconstitute in PBS, pH 7.4 with trehalose as a protectant . For mouse EGF, use 10 mM sodium phosphate, pH 7.5 buffer .

• Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and activity loss .

• Follow reconstitution protocols provided in the Certificate of Analysis for optimal performance .

• For working solutions, prepare fresh dilutions or store small aliquots to minimize freeze-thaw cycles.

• Quality verification: Confirm protein purity and activity using SDS-PAGE, SEC-MALS, and functional assays before experimental use .

How can researchers effectively study EGF-EGFR interactions in different mouse experimental models?

Effective strategies for studying EGF-EGFR interactions in mouse models include:

• Genetic models: Compare wild-type, knockout (e.g., GHR-KO), and transgenic mice (e.g., GH-overexpressing) to understand regulatory mechanisms .

• Binding studies: Surface Plasmon Resonance (SPR) or ELISA-based binding assays can quantify interactions between EGF and EGFR with high sensitivity .

• Phosphorylation analysis: Western blotting with phospho-specific antibodies targeting EGFR (Tyr845, Tyr992, Tyr1068) and downstream effectors (AKT, ERK1/2, STAT3/5) provides insights into pathway activation .

• Functional readouts: Cell proliferation assays using BALB/c 3T3 cells can determine biological activity with ED50 values typically below 250 pg/ml .

• Tissue-specific studies: RT-PCR analysis can track developmental expression patterns in specific tissues, such as testis during sexual maturation .

• Protein-protein interactions: Co-immunoprecipitation studies can reveal associations between EGFR and downstream signaling molecules or regulatory proteins like phosphatases .

What controls and validation steps are critical when investigating EGFR signaling in transgenic mouse models?

When investigating EGFR signaling in transgenic mouse models, several critical controls and validation steps should be implemented:

How do developmental changes affect EGF-EGFR signaling in mouse models?

Developmental effects on EGF-EGFR signaling in mice are tissue-specific and temporally regulated:

• Testicular expression: EGF mRNA levels in mouse testis increase progressively during sexual maturation (day 15 < day 30 < day 45 postnatal), correlating with Leydig cell proliferation .

• Neural development: In the hippocampus, HB-EGF (a related ligand) activates EGFR to induce reactive neural stem cells, suggesting developmental roles in neural plasticity and response to seizures .

• Receptor expression patterns: EGFR expression and glycosylation patterns change during development, affecting receptor functionality and ligand sensitivity .

• Pathway sensitivity: The responsiveness of different signaling pathways to EGF stimulation may shift during development, as observed in the differential effects on AKT/ERK versus STAT pathways in mature GH-overexpressing mice .

• Cross-talk with other growth factors: Developmental changes in the expression of other growth factors and their receptors can modulate EGFR signaling through heterologous interactions .

Researchers should consider these developmental variables when designing experiments and interpreting results, particularly in studies comparing animals of different ages or developmental stages.

What are the key considerations when interpreting contradictory EGF-EGFR signaling data in different experimental contexts?

When faced with contradictory EGF-EGFR signaling data across different experimental contexts, consider the following factors:

• Receptor expression levels: Differences in EGFR abundance can dramatically impact signaling outcomes, as seen in GHR-KO (decreased EGFR) versus GH-overexpressing (increased EGFR) mice .

• Pathway-specific regulation: Different downstream pathways may be differentially regulated, as observed with the selective desensitization of STAT pathways despite normal AKT/ERK activation in GH-overexpressing mice .

• Developmental context: EGF signaling changes during development (e.g., increasing EGF expression in maturing testis), so age differences between experimental models may explain divergent results .

• Post-translational modifications: Variations in receptor glycosylation can affect molecular weight (86-110 kDa observed vs. 71.2 kDa calculated) and potentially signaling properties .

• Compensatory mechanisms: Chronic alterations in EGFR signaling may trigger compensatory changes in other pathways or feedback mechanisms, especially in genetic models .

• Technical variables: Differences in experimental techniques, protein sources (e.g., commercially sourced vs. lab-prepared), and assay conditions can contribute to apparent contradictions .

• Biological heterogeneity: Primary cells or tissues may display greater variability in EGFR responses compared to established cell lines .

Careful consideration of these factors can help reconcile seemingly contradictory findings and develop more nuanced models of EGF-EGFR signaling regulation.