Recombinant Human Insulin-like growth factor II (IGF2) (Active)

What is Recombinant Human IGF-II and why is it important for research?

Recombinant Human IGF-II is a synthetic version of the naturally occurring Insulin-like Growth Factor II, produced through genetic engineering techniques. It is structurally homologous to proinsulin and shares approximately 70% sequence identity with IGF-I . Despite being one of the most abundant growth factors in the human body, IGF-II remains relatively understudied compared to insulin and IGF-I. For every research paper published on IGF-II, approximately three are published on IGF-I and thirty on insulin .

IGF-II is particularly important for research because it:

• Functions as a potent mitogenic growth factor during embryonic development

• Is expressed in multiple tissues and cell types with potential autocrine, paracrine, and endocrine functions

• Exhibits highly conserved structure across species (100% identity between human, bovine, and porcine proteins)

• Has complex regulation mechanisms that are dramatically altered during development and disease states

How does IGF-II differ structurally and functionally from IGF-I and insulin?

While IGF-II belongs to the insulin-like family and shares structural similarities with both insulin and IGF-I, it has several distinguishing features:

IGF-II has evolved an incredibly intricate series of checks and balances to control its cellular activity, far more complex than those for IGF-I and insulin, suggesting that stringent control of IGF-II is extremely important for cellular function .

What are the primary receptors for IGF-II and how do they affect its biological activity?

IGF-II interacts with multiple receptors, each mediating different biological responses:

• IGF-I Receptor (IGF1R):

  • IGF-II engages IGF1R to mediate embryonic growth

  • Activates intracellular signaling pathways promoting cell proliferation and survival

  • Binding affinity is lower than that of IGF-I for this receptor

• IGF-II Receptor (IGF2R):

  • Functions primarily as a "sink" receptor leading to IGF-II degradation

  • Does not possess intrinsic tyrosine kinase activity

  • High-affinity binding involves multiple domains, particularly domain 11 with contributions from the fibronectin type II (FNII) region

• Insulin Receptor (IR):

  • IGF-II can bind to IR, particularly the IR-A isoform

  • This interaction contributes to metabolic and mitogenic effects

The balance between these receptor interactions is critical for regulating IGF-II bioavailability and biological effects.

What are the recommended protocols for reconstitution and storage of recombinant IGF-II?

For optimal results when working with recombinant human IGF-II, follow these handling procedures:

Reconstitution Protocol:

• Centrifuge vial before opening to collect material at the bottom

• Gently pipette the recommended solution down the sides of the vial to avoid protein denaturation

• DO NOT VORTEX the solution, as this can damage the protein structure

• Allow several minutes for complete reconstitution before use

Storage Recommendations:

• For prolonged storage, dilute to working aliquots in a 0.1% BSA solution

• Store at -80°C in small, single-use aliquots

• Avoid repeated freeze-thaw cycles, which can lead to protein degradation

• Working solutions may be stored at 4°C for up to 1 week

How can researchers accurately measure IGF-II binding affinities to receptors?

Surface plasmon resonance (SPR) is the gold standard method for measuring IGF-II binding affinities to its receptors. Based on published protocols:

Recommended SPR Protocol:

• Couple IGF2R fragments (e.g., domains 10-13) to a biosensor surface via amine groups to achieve approximately 2000 resonance units

• Conduct binding analysis at 25°C in HBS-EP buffer at a flow rate of 40 μl/min

• Prepare IGF-II and mutants at concentrations ranging from 6.25-100 nM

• Use a two-state conformational change model for curve fitting, as this best describes the 1:1 binding interaction with conformational change upon binding

• Include appropriate regeneration steps (e.g., 10 mM HCl for 1.5 min or 2 M MgCl₂ for 2 min)

Alternative coupling methods include biotinylation of IGF2R constructs and immobilization to streptavidin-coated sensor chips. Note that direct amine coupling typically leads to lower absolute binding affinity measurements compared to biotinylation methods, though relative binding affinities remain consistent .

What challenges are associated with producing and purifying recombinant IGF-II?

Researchers commonly encounter several challenges when working with recombinant IGF-II:

• Proper Folding:

  • IGF-II contains three disulfide bonds that must form correctly for biological activity

  • Expression systems require appropriate oxidizing environments and chaperones

• Aggregation Concerns:

  • IGF-II has hydrophobic regions that can promote aggregation

  • Addition of stabilizers (e.g., BSA) is often necessary for maintaining solubility

• Post-translational Modifications:

  • Different expression systems may yield variations in glycosylation patterns

  • Escherichia coli systems (commonly used) produce non-glycosylated IGF-II

• Purification Challenges:

  • Separation from IGF-binding proteins that co-purify with IGF-II

  • Need for multiple chromatography steps to achieve high purity

• Activity Verification:

  • Essential to confirm biological activity after purification

  • Typically assessed through cell proliferation assays with IGF-responsive cell lines

How can recombinant IGF-II be used to study developmental processes?

IGF-II plays critical roles in embryonic development, making recombinant IGF-II an important tool for developmental studies:

Key Experimental Approaches:

• Ex vivo Organ Culture Systems:

  • Supplement culture media with defined concentrations of recombinant IGF-II

  • Analyze effects on organ growth, differentiation, and morphogenesis

  • Compare with IGF-I supplementation to distinguish specific developmental effects

• Developmental Timing Studies:

  • Administer IGF-II at different developmental stages to identify critical windows of action

  • Monitor E2F3 expression in parallel, as E2F3 appears to drive IGF-II expression during development

• Cell Lineage Specification:

  • Examine effects of IGF-II on stem cell differentiation into specific lineages

  • Use in conjunction with other growth factors to optimize differentiation protocols

• Tissue-Specific Knockdown/Knockout Models:

  • Combine recombinant IGF-II administration with genetic models featuring IGF-II deficiency

  • Assess rescue effects to determine tissue-specific requirements

What are the best cellular assays to evaluate IGF-II biological activity?

Researchers can employ several established assays to evaluate the biological activity of recombinant IGF-II:

• Cell Proliferation Assays:

  • MCF-7 breast cancer cells are highly responsive to IGF-II

  • Measure BrdU incorporation or use MTT/MTS colorimetric assays

  • Compare with known standards to establish relative potency

• Receptor Activation Assays:

  • Western blot analysis of IGF1R phosphorylation

  • Downstream signaling activation (Akt, ERK1/2, p70S6K)

  • Establish dose-response curves (typically 0.1-100 ng/mL range)

• Reporter Gene Assays:

  • Cells transfected with luciferase constructs under control of IGF-responsive elements

  • Allows quantitative measurement of transcriptional activation

• Migration/Invasion Assays:

  • Transwell or wound healing assays to assess motility responses

  • Particularly relevant for studying IGF-II's role in cancer cell behavior

• Specialized Functional Assays:

  • Regulatory T-cell function enhancement evaluation

  • Enzyme delivery assessments for lysosomal storage disorders

How should researchers account for endogenous IGF binding proteins in experimental systems?

IGF binding proteins (IGFBPs) significantly impact IGF-II bioavailability and activity in experimental systems:

Methodological Considerations:

• Serum Considerations:

  • Fetal bovine serum contains variable levels of IGFBPs

  • Consider using defined serum replacement or carefully characterized serum lots

  • Pre-treatment of serum with acid-ethanol extraction can remove endogenous IGFBPs

• Cellular Production of IGFBPs:

  • Many cell lines produce IGFBPs that can interfere with exogenous IGF-II

  • Measure IGFBP levels in conditioned media

  • Consider using IGFBP antibodies or IGF-II analogs with reduced IGFBP binding

• Direct vs. Indirect Effects:

  • Include controls to distinguish direct IGF-II effects from IGFBP-mediated effects

  • Some cellular responses may be due to displacement of endogenous IGFs from IGFBPs

• Quantification Methods:

  • Use free IGF-II-specific assays rather than total IGF-II measurements

  • Consider the impact of IGFBP proteases that may be present in the system

How can researchers investigate the relationship between E2F3 and IGF-II in development and cancer?

Recent evidence suggests E2F3 is a key regulator of IGF-II expression both in development and cancer . Researchers can explore this relationship through:

Investigative Approaches:

• Temporal Expression Analysis:

  • Measure E2F3 and IGF-II expression levels across developmental stages

  • Data indicates E2f3 mRNA expression, protein expression, and binding to the Igf2 promoter all decrease with age postnatally in multiple mouse organs

• Chromatin Immunoprecipitation (ChIP):

  • Confirm direct E2F3 binding to IGF-II promoter regions

  • Focus on the P2 promoter in mouse (equivalent to human P3), which contains consensus E2F-binding sites

• Promoter-Reporter Assays:

  • Construct reporter systems containing the IGF-II promoter

  • Test activation by E2F3 in different cellular contexts

  • Include mutated E2F binding sites as controls

• Cancer Models:

  • Compare E2F3 and IGF-II expression in normal vs. cancer tissues

  • Analyze correlation between E2F3 and IGF-II levels in human cancer datasets

  • Microarray data reveals that E2F3-overexpressing prostate and bladder cancers show increased IGF-II expression with positive correlation between E2F3 and IGF-II mRNA levels

• Mechanistic Manipulation:

  • Use gain- and loss-of-function approaches for E2F3

  • In late juvenile hepatocytes, restoration of high E2f3 expression restored high Igf2 expression, supporting a causal relationship

What techniques are available for studying IGF-II signaling pathway specificity?

Understanding the signaling specificity of IGF-II is crucial for elucidating its biological functions:

Advanced Methodological Approaches:

• Receptor-Specific Mutants and Antagonists:

  • Use IGF-II analogs with altered receptor binding specificities

  • Apply receptor-specific blocking antibodies to isolate signaling through individual receptors

• CRISPR/Cas9 Receptor Modification:

  • Generate cell lines with specific receptor knockouts or mutations

  • Create cells expressing receptor variants with altered downstream signaling capabilities

• Phosphoproteomics:

  • Employ mass spectrometry-based approaches to identify phosphorylation events

  • Compare IGF-II-induced phosphorylation profiles with those of IGF-I and insulin

  • Temporal analysis can reveal signaling dynamics and feedback mechanisms

• Single-Cell Analysis:

  • Investigate heterogeneity in IGF-II responses within seemingly homogeneous populations

  • Correlate receptor expression levels with signaling outputs at single-cell resolution

• Biosensors and Live Imaging:

  • Utilize FRET-based biosensors to monitor signaling events in real-time

  • Track receptor internalization and trafficking using fluorescently tagged IGF-II

How does the IGF-II/IGF2R interaction structure inform the development of targeted therapeutics?

The complex structural relationship between IGF-II and IGF2R provides opportunities for developing targeted therapeutics:

Structure-Based Design Considerations:

• Critical Binding Domains:

  • Domain 11 of IGF2R is primarily responsible for IGF-II binding

  • The fibronectin type II (FNII) domain indirectly enhances binding affinity by interacting with the AB-loop of domain 11

• Key Residue Interactions:

  • The Glu1544Lys mutation in isolated domain 11 increases IGF-II affinity by sixfold

  • This mutation may enhance affinity by introducing an electrostatically favorable interaction with Asp20 of IGF-II

• Binding Affinity Modulation:

  • Deletion of FNII markedly reduces IGF-II binding, causing a 10-fold drop in binding affinity

  • The Glu1544Lys mutation has different effects depending on the context:

    • Negative impact in IGF2R-Dom10-13 (10-fold reduction in affinity)

    • Positive impact in isolated domain 11

    • Increases affinity fourfold compared with ΔFNII alone when FNII is absent

• Therapeutic Design Strategies:

  • Design peptides targeting the IGF-II binding interface of domain 11

  • Develop antibodies that enhance IGF2R-mediated IGF-II clearance

  • Create IGF-II variants with altered receptor specificity profiles for targeted applications

How is IGF-II dysregulation implicated in cancer development and progression?

IGF-II dysregulation plays significant roles in multiple cancer types:

Key Research Findings:

• Overexpression Patterns:

  • IGF-II is frequently overexpressed in childhood malignancies including Wilms tumor, medulloblastoma, and rhabdomyosarcoma

  • Also commonly overexpressed in adult cancers: lung, breast, colorectal, bladder, ovarian, and liver cancers

• Mechanisms of Overexpression:

  • Loss of imprinting (LOI) where the normally silenced maternal IGF-II allele becomes expressed

  • This biallelic expression typically leads to approximately doubled expression

  • Many cancers show much higher increases in IGF-II, suggesting additional mechanisms

  • Aberrant activation of the P3 promoter (human, equivalent to mouse P2) is observed in multiple cancers

  • E2F3 overexpression appears to drive IGF-II overexpression in some cancers, particularly prostate and bladder cancers

• Functional Consequences:

  • Promotes cancer cell proliferation, survival, and invasion

  • Contributes to tumor angiogenesis

  • May facilitate metastatic spread

• Research Approaches:

  • Analyze correlations between E2F3 and IGF-II expression in cancer databases

  • Investigate promoter usage patterns in different cancer types

  • Examine effects of IGF-II neutralization on cancer cell behavior

What experimental models are best suited for studying IGF-II in specific disease contexts?

Researchers investigating IGF-II in disease states can utilize various experimental models:

Model Systems for IGF-II Research:

• Cell Culture Models:

  • Cancer cell lines with variable IGF-II expression

  • Primary cells from disease-relevant tissues

  • 3D organoid cultures to better recapitulate tissue architecture

• Genetic Mouse Models:

  • Igf2 knockout or conditional knockout mice

  • Transgenic mice with tissue-specific IGF-II overexpression

  • Humanized IGF-II mice to better model human-specific regulation

  • E2f3 manipulation models to study regulation of IGF-II expression

• Patient-Derived Xenografts (PDX):

  • Capture tumor heterogeneity and microenvironment interactions

  • Allow testing of IGF-II-targeting approaches in clinically relevant models

• Disease-Specific Models:

  • For cancer: Orthotopic implantation models for tissue-specific microenvironments

  • For metabolic disorders: Diet-induced models with IGF-II pathway modulation

  • For developmental disorders: Ex vivo organ culture systems with recombinant IGF-II supplementation

• Disease Applications Beyond Cancer:

  • Mucopolysaccharidosis type IIIB models to study IGF-II peptide fusion for enzyme delivery

  • Food allergy models to investigate IGF-II's role in regulatory T-cell functions

How can researchers develop IGF-II-based therapeutic approaches?

IGF-II's diverse biological functions offer several avenues for therapeutic development:

Therapeutic Development Strategies:

• IGF-II as a Therapeutic Target:

  • Neutralizing antibodies against IGF-II

  • Antisense oligonucleotides or siRNAs to reduce IGF-II expression

  • Small molecule inhibitors of IGF-II/receptor interactions

  • Targeting E2F3 to modulate IGF-II expression in cancers

• IGF-II as a Therapeutic Agent:

  • Recombinant IGF-II for conditions requiring growth promotion

  • IGF-II fusion proteins for targeted delivery applications

  • IGF-II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts

• Receptor Targeting Approaches:

  • IGF2R-targeted therapies to enhance IGF-II clearance

  • Selective targeting of IGF1R vs. insulin receptor signaling

  • Bispecific antibodies engaging multiple components of the IGF system

• Methodological Considerations:

  • Cell-based screening assays for candidate molecule identification

  • Structure-based design leveraging the IGF-II/IGF2R interaction data

  • Appropriate animal models for efficacy and safety assessment

  • Consideration of compensatory mechanisms in the insulin/IGF system