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

What is Insulin-like Growth Factor II (IGF2) and what are its primary biological functions?

IGF2 (Insulin-like growth factor II) is a member of the insulin family of polypeptide growth factors, also known historically as multiplication-stimulating polypeptide (MSP) and somatomedin-A. It functions within a complex system of growth and metabolic-regulating proteins that is particularly critical during development. IGF2 has been associated with multiple developmental processes including nervous system proliferation and differentiation, myelination, adrenal cortical proliferation, and skeletal growth and differentiation .

In humans, IGF2 is primarily synthesized by the liver and circulates at high levels in both fetus and adults. Interestingly, in rodents, IGF2 levels drop significantly after the perinatal period, representing an important species difference researchers should consider when designing experimental models .

Which cell types produce IGF2 and what receptors does it interact with?

IGF2 is produced by multiple cell types including astrocytes, hepatocytes, osteoblasts, embryonic striated muscle cells, Kupffer cells, and Ito cells. This diverse expression pattern reflects its wide-ranging biological functions .

The protein exerts its effects by binding to several receptors and binding proteins:

• IGF1R (type-1 insulin-like growth factor receptor)

• Insulin R (IR)-type A

• IGF1R:Insulin R-A hybrids

• IGF2R

• IGF binding proteins 1-6 (IGFBPs)

These interactions activate downstream signaling cascades, particularly the PI3K/AKT and MAPK/ERK pathways, which promote cell survival, growth, and metabolism .

What expression systems are most effective for producing recombinant human IGF2?

Yeast expression systems have been successfully employed to produce recombinant human IGF2 and related binding proteins. Specifically, researchers have expressed IGFBPs as ubiquitin (Ub)-IGFBP fusion proteins in yeast systems. The resulting fusion proteins undergo processing to yield functional recombinant proteins with appropriate binding characteristics .

When characterizing recombinant IGF2 or IGFBPs, techniques such as Western ligand blotting with 125I-IGF II under nonreducing conditions have been effective for confirming specific binding and molecular weight characteristics. High-performance liquid chromatography (HPLC) purification has been used to obtain pure proteins with virtually the same amino acid composition, amino acid number, and NH2-terminal sequences as their native counterparts .

How should researchers validate the structural and functional integrity of recombinant IGF2?

Validation of recombinant IGF2 should include:

• Structural analysis: Compare amino acid composition and NH2-terminal sequencing with native protein

• Molecular weight confirmation: Use Western blotting to verify appropriate molecular mass

• Binding affinity determination: Measure affinity constants for IGF1R and other cognate receptors

• Functional bioassays: Assess the ability to stimulate DNA and glycogen synthesis in appropriate cell types, such as human osteoblastic cells

The recombinant protein should demonstrate binding affinity consistent with native IGF2, which typically shows affinity constants between 1.7 and 3.3 × 10^10 M^-1 for its receptors—approximately 25-100 times higher than the IGF I and II affinities for the type I IGF receptor .

What are the recommended concentrations and protocols for using recombinant IGF2 in cell culture experiments?

When designing experiments with recombinant IGF2, researchers should consider the high binding affinity of IGFBPs present in culture media or serum supplements. Since IGFBPs can sequester IGF2 and inhibit its activity, experiments should be designed to account for this interaction.

In functional assays measuring DNA and glycogen synthesis in human osteoblastic cells, IGFBPs have been observed to inhibit IGF-stimulated activity when present in excess. Therefore, researchers should titrate recombinant IGF2 concentrations to overcome potential inhibitory effects of binding proteins in the experimental system .

Additionally, when studying receptor-mediated effects, it's important to note that different downstream pathways may be activated depending on which receptor IGF2 binds to. For instance, IGF2 binding to IGF1R activates mitogenic signals and antiapoptotic/pro-survival activities through PI3K/AKT and MAPK/ERK pathways .

How can researchers effectively measure IGF2 expression and activity in experimental models?

Several methodological approaches are effective for measuring IGF2 expression and activity:

• qPCR analysis: Quantitative PCR can be used to measure IGF2 mRNA expression levels. The ΔΔCt method with appropriate housekeeping genes is commonly employed for analysis, with results typically presented as 2^(-ΔΔCt) values to show relative expression .

• Protein detection: Western blotting with specific anti-IGF2 antibodies or Western ligand blotting with labeled IGF receptors can detect IGF2 protein levels.

• Functional assays:

  • DNA synthesis assays (e.g., thymidine incorporation)

  • Glycogen synthesis measurements

  • Downstream signaling detection (phosphorylation of AKT, ERK)

• Bioinformatic analysis: Tools such as STRING can identify protein-protein interaction networks involving IGF2, while pathway analysis using DAVID (Database for Annotation, Visualization and Integrated Discovery) can elucidate the role of IGF2 in various metabolic pathways .

What are the most significant genetic variations in IGF2 and their functional consequences?

The IGF2 gene exhibits considerable genetic diversity. Analysis of large-scale genomic data reveals that the 236-codon human IGF2 protein has 85 different documented genetic changes, including:

• 78 missense mutations and in-frame insertions/deletions

• 4 frameshift mutations or stop codons

• 2 splicing site changes

• 1 loss of start codon

This translates to approximately 0.36 variants per codon, with variant alleles present in about 2.5% of the population . These variations may have significant functional consequences, including altered binding affinities, expression levels, or signaling capabilities.

How does IGF2 expression change in pathological conditions and what are the molecular mechanisms involved?

IGF2 expression undergoes significant changes in various pathological conditions:

How can IGF2 and its signaling pathways be targeted for therapeutic applications?

Research into therapeutic applications targeting IGF2 and its signaling pathways is progressing in several areas:

• Cancer therapy: Since IGF2 is frequently overexpressed in cancers and promotes cell survival and proliferation, inhibitors of IGF1R (which mediates many of IGF2's effects) are being investigated as potential anti-cancer drugs. These inhibitors have shown promise in reducing proliferation in various cancer models .

• Neurodevelopmental disorders: Evidence suggests that IGF2 plays important roles in brain function, particularly in plasticity, memory, and cognition. Administration of recombinant IGF2 has been shown to enhance memories in healthy animals and may reverse symptoms in laboratory models of neurodevelopmental disorders .

• Metabolic diseases: Given IGF2's role in the development and maintenance of adipocytes, pancreatic β islet cells, and skeletal muscle mass, it represents a potential target for treating metabolic disorders, including diabetes .

When designing therapeutic strategies, researchers should consider the complexity of the IGF system, including the various receptors and binding proteins that modulate IGF2 activity. The high affinity of IGFBPs for IGF2 (25-100 times higher than receptor affinities) means these proteins significantly influence IGF2 bioavailability and function .

What are the most promising biomarkers associated with IGF2 expression and signaling?

Several promising biomarkers associated with IGF2 have been identified:

• miRNA biomarkers: miR-93-5p and other microRNAs that target IGF2 have been investigated as potential non-invasive biomarkers for prostate cancer. These miRNAs may modulate IGF2 expression and could serve as indicators of disease progression or treatment response .

• SNP markers: Specific single nucleotide polymorphisms in the IGF2 gene, such as rs1004446 and rs3741211, have been evaluated for clinical associations with cancer aggressiveness and treatment response .

• Expression patterns: Changes in IGF2 expression levels themselves may serve as biomarkers for disease progression, particularly in cancers. For example, in prostate cancer, IGF2 expression increases with higher Gleason scores (indicating more aggressive disease) .

• Protein complexes: The expression patterns of IGF2 binding proteins (IGFBPs) and their complexes with IGF2 may provide additional biomarker opportunities, as these proteins regulate IGF2 bioavailability and have been shown to attenuate IGF activity in various tissues .

When developing biomarker assays, researchers should employ multiple analytical techniques including qPCR for mRNA expression, protein detection methods, and potentially microRNA profiling to capture the complex regulatory networks surrounding IGF2 signaling .

How can researchers overcome common challenges when working with recombinant IGF2 in experimental systems?

Researchers frequently encounter several challenges when working with recombinant IGF2:

• Binding protein interference: IGFBPs in culture media or biological samples can sequester IGF2, reducing its bioavailability. To overcome this, researchers can:

  • Use IGF analogs with reduced binding to IGFBPs

  • Pre-treat samples to dissociate IGF2-IGFBP complexes

  • Account for IGFBP effects in experimental design and data interpretation

• Receptor cross-reactivity: IGF2 binds to multiple receptors (IGF1R, insulin receptor-A, IGF2R) with different affinities, complicating the interpretation of functional studies. To address this:

  • Use receptor-specific blocking antibodies to isolate effects through specific pathways

  • Employ receptor-knockout or knockdown models

  • Perform parallel experiments with receptor-specific ligands

• Species differences: As noted, IGF2 levels drop significantly after the perinatal period in rodents but remain high in humans, representing an important consideration when translating findings between species .

What controls and validation steps are critical when measuring IGF2 expression and function?

When measuring IGF2 expression and function, several controls and validation steps are essential:

• For qPCR analysis:

  • Use multiple housekeeping genes for normalization

  • Perform technical replicates (at least three per sample)

  • Apply statistical methods such as Tukey's range test to detect anomalous values

  • Consider analyzing both patient averages and individual replicates to increase statistical power

• For functional assays:

  • Include both positive controls (known IGF2 responders) and negative controls (receptor-deficient cells)

  • Test multiple concentrations of recombinant IGF2 to establish dose-response relationships

  • Confirm specificity by comparing effects with related growth factors (IGF1, insulin)

  • Validate key findings using multiple methodological approaches

• For protein detection:

  • Confirm antibody specificity using recombinant standards

  • Include appropriate positive and negative controls

  • Consider using both Western blotting and ligand blotting techniques for comprehensive analysis

Proper experimental design should also account for potential confounding factors such as treatment timing, cell density, and medium composition, which can significantly impact IGF2 signaling and downstream effects .

What are the most promising emerging areas of IGF2 research?

Several emerging research areas hold particular promise for advancing our understanding of IGF2:

• Epigenetic regulation: Further investigation into the mechanisms of IGF2 imprinting and its dysregulation in disease states, particularly cancer, could reveal new therapeutic targets. The role of E2F transcription factors in regulating IGF2 expression postnatally represents an important area for exploration .

• Neuroscience applications: The role of IGF2 in brain function, particularly in memory formation and cognitive processes, is an exciting frontier. Evidence suggests that IGF2 enhances memories in healthy animals and may ameliorate symptoms in models of neurodevelopmental disorders .

• Metabolic disease connections: The observation that IGF2 variants may decrease risk of type 2 diabetes points to important roles in metabolism regulation that warrant deeper investigation. Understanding how IGF2 regulates the development and function of adipocytes, pancreatic β islet cells, and skeletal muscle could yield insights into metabolic disorders .

• Non-canonical signaling pathways: While the canonical IGF2 signaling through IGF1R and insulin receptor is well-studied, non-canonical pathways and interactions with other signaling networks may reveal new biological functions and therapeutic opportunities .

What technological advances are needed to better understand IGF2 biology and applications?

Advancing our understanding of IGF2 biology will require several technological innovations:

• Improved protein engineering: Developing IGF2 variants with modified receptor specificities could help dissect the contributions of different receptors to IGF2 function and potentially yield more targeted therapeutic agents.

• Advanced imaging techniques: Real-time visualization of IGF2 binding and signaling in living cells and tissues would provide valuable insights into its spatial and temporal dynamics.

• Single-cell analysis: Understanding cell-type-specific responses to IGF2 through single-cell transcriptomic and proteomic approaches could reveal previously unrecognized heterogeneity in IGF2 function.

• Systems biology approaches: Integrating multi-omics data (genomics, transcriptomics, proteomics, metabolomics) with computational modeling could help unravel the complex regulatory networks governing IGF2 expression and function in different physiological and pathological contexts .

• Improved animal models: Developing more sophisticated conditional knockout and knockin models that better recapitulate human IGF2 expression patterns would facilitate more translatable research, particularly given the species differences in postnatal IGF2 expression .