Recombinant Human Amphiregulin protein (AREG), partial (Active)

What is the molecular structure of recombinant human AREG?

Recombinant human AREG (partial, active) is typically expressed as a 98-amino acid protein corresponding to positions 101-198 of the full-length sequence. The protein sequence is: SVRVEQVVKP PQNKTESENT SDKPKRKKKG GKNGKNRRNR KKKNPCNAEF QNFCIHGECK YIEHLEAVTC KCQQEYFGER CGEKSMKTHS MIDSSLSK, with a molecular weight of approximately 11.3 kDa as determined by SDS-PAGE analysis. The protein contains critical EGF-like domains including cysteine-rich regions essential for receptor binding and biological activity . Unlike its membrane-bound native form, recombinant AREG is produced as a soluble protein, typically expressed in E. coli expression systems and purified to >95% purity for research applications .

How does AREG differ from other EGF family members?

AREG belongs to the EGF family but exhibits distinct functional characteristics compared to other family members. While sharing the conserved EGF domain structure, AREG demonstrates unique receptor binding properties and activation dynamics. Notably, AREG requires heparin sulfate expression on target cells to efficiently signal via the EGFR, a requirement not shared by all EGF family ligands . Additionally, AREG shows specific tissue distribution patterns and is uniquely associated with type 2 immune responses, distinguishing it from other EGF family members . The 98-amino acid form of recombinant AREG demonstrates approximately 5-10 fold higher biological activity compared to the shorter 78-amino acid variant in proliferation assays using Balb/c 3T3 fibroblasts .

What are the optimal storage and handling conditions for recombinant AREG?

Recombinant AREG is typically supplied as a lyophilized powder that should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For optimal stability and activity retention, the addition of 5-50% glycerol (final concentration) is recommended for long-term storage. Following reconstitution, the protein should be aliquoted to avoid repeated freeze-thaw cycles, which can significantly reduce biological activity . The shelf life of the lyophilized form is approximately 12 months at -20°C/-80°C, while the reconstituted form maintains stability for approximately 6 months when stored at -20°C/-80°C . For short-term research applications, working aliquots can be stored at 4°C for up to one week with minimal loss of activity .

What are the primary signaling pathways activated by AREG?

AREG primarily signals through binding and activation of the Epidermal Growth Factor Receptor (EGFR). Upon binding, AREG induces EGFR tyrosine kinase transphosphorylation, particularly at tyrosine 845, initiating downstream signaling cascades . These pathways include MAPK/ERK, PI3K/Akt, and JAK/STAT signaling, which collectively regulate cellular proliferation, survival, and differentiation. Experimental evidence demonstrates that AREG-induced cellular responses can be effectively blocked by EGFR inhibitors such as AG1478, confirming the central role of EGFR in AREG signaling . In certain cell types, the biological activity of AREG appears to be modulated by cell-specific factors, suggesting that AREG may activate different signaling pathways depending on the cellular context .

How does AREG contribute to immune system regulation?

AREG plays multifaceted roles in immune regulation, particularly in type 2 inflammatory responses. Research indicates that AREG expression is elicited by diverse stimuli yet is primarily associated with immune cell populations activated in type 2 immune responses, wound repair, and inflammation resolution . Notably, multiple leukocyte populations express AREG, including mast cells, basophils, group 2 innate lymphoid cells (ILC2), and a subset of tissue-resident regulatory CD4+ T cells . AREG has been shown to reduce T-cell proliferation following polyclonal T-cell stimulation with OKT3, both in the presence and absence of monocytes . Additionally, AREG regulates phagocytosis-induced cell death of monocytes in peripheral blood through mechanisms involving both intrinsic and extrinsic apoptotic pathways, including factors such as BCL-2, BCL-XL, and death ligand/receptor CD95/CD95L .

What is the role of AREG in tissue repair and homeostasis?

AREG serves as a critical mediator in tissue repair and homeostasis by promoting epithelial cell proliferation and migration. It functions primarily as a key factor that induces tolerance by facilitating the restoration of tissue integrity following damage associated with acute or chronic inflammation . In airway tissues, AREG has been shown to induce expression of factors essential for repair and remodeling, including VEGF, which plays a critical role in both airway remodeling through angiogenesis and inflammatory processes . The ability of hematopoietic cells to migrate to sites of inflammation and locally up-regulate AREG expression can substantially influence local concentrations of this growth factor, contributing to tissue-specific repair mechanisms . Importantly, AREG expression patterns differ between neonates and adults, suggesting age-dependent roles in tissue homeostasis particularly during bacterial infections .

How is AREG biological activity measured in vitro?

The biological activity of recombinant human AREG is typically assessed through cell proliferation assays, with the murine Balb/c 3T3 fibroblast assay being the gold standard. In this assay, the effective dose (ED50) for AREG typically ranges between 5-10 ng/mL . Researchers should establish dose-response curves using serial dilutions of recombinant AREG (0.1-100 ng/mL) and measure proliferation after 48-72 hours using standard methods such as MTT/XTT assays or BrdU incorporation. For more specific applications, EGFR phosphorylation assays can be employed to measure AREG activity, focusing particularly on tyrosine 845 phosphorylation using phospho-specific antibodies in western blot or ELISA formats . Additionally, downstream signaling can be monitored through expression analysis of AREG-induced genes such as CXCL8, VEGF, and COX-2 using qRT-PCR and ELISA methods .

What cell culture models are optimal for studying AREG functions?

Several cell models have proven valuable for investigating AREG functions. Human airway smooth muscle cells (HASMC) and human bronchial epithelial cells (HBEC) represent excellent models for studying AREG's role in airway inflammation and remodeling . These systems have successfully demonstrated both autocrine and paracrine effects of AREG through conditioned medium experiments. For immune regulation studies, monocyte and T-cell co-culture systems are recommended, as they can effectively demonstrate AREG's impact on T-cell proliferation and monocyte survival . When establishing these models, researchers should consider cell-specific expression of heparin sulfate proteoglycans, which are required for efficient AREG-EGFR signaling . Control experiments should include EGFR inhibitors (e.g., AG1478) and neutralizing antibodies against AREG to confirm specificity of observed effects .

How can AREG-specific responses be distinguished from other EGFR ligands?

Distinguishing AREG-specific responses from those induced by other EGFR ligands requires careful experimental design. One approach involves using neutralizing antibodies specifically against AREG in combination with recombinant AREG stimulation . Another strategy employs RNA interference (siRNA) targeting AREG while maintaining expression of other EGFR ligands . For mechanistic studies, researchers should consider the unique requirement of AREG for heparin sulfate proteoglycans; therefore, modulating heparin sulfate expression can help discriminate between AREG and other EGFR ligands . Comparative studies using multiple EGFR ligands at equimolar concentrations can identify differential activation patterns of downstream signaling pathways. Temporal analysis is also valuable, as AREG may induce distinct kinetics of EGFR activation and internalization compared to other family members, potentially leading to different biological outcomes despite activating the same receptor.

How does AREG contribute to inflammatory disease pathogenesis?

AREG plays complex roles in inflammatory disease pathogenesis, particularly in conditions characterized by type 2 inflammation. In asthma, increased AREG expression contributes to airway remodeling processes through induction of VEGF, which promotes angiogenesis . AREG also enhances the production of inflammatory mediators such as CXCL8 and increases expression of cyclooxygenase-2 (COX-2) in airway epithelial cells, amplifying inflammatory cascades . Experimental approaches to study these mechanisms include airway cell co-culture systems, where bradykinin-stimulated airway smooth muscle cells increase AREG secretion through COX-2/PGE2/EP2-EP4 receptor pathways, creating a paracrine signaling loop with epithelial cells . The dynamic interaction between different cell types through AREG signaling establishes amplification circuits that may maintain chronic inflammation. Targeting these pathways with EGFR inhibitors or anti-AREG antibodies in disease models can provide insight into therapeutic potential.

How does post-translational modification affect AREG function?

Post-translational modifications significantly impact AREG functionality, particularly in the context of its proteolytic processing and release from the cell membrane. Native AREG is expressed as a membrane-bound protein whose activation is regulated largely at the level of release from the cell membrane . This release is mediated by membrane metalloproteinases, primarily ADAM17 (TACE), whose activity can be modulated by inflammatory stimuli. Additional modifications including glycosylation patterns may influence AREG-EGFR binding affinity and specificity. To study these effects, researchers can employ site-directed mutagenesis of key residues to generate modified recombinant AREG variants, comparing their biological activities in standard assays. Protease inhibitor studies can help delineate the contribution of specific proteolytic processing to AREG function. Mass spectrometry-based approaches are valuable for characterizing post-translational modifications in both recombinant and naturally produced AREG under different physiological and pathological conditions.

What are common challenges in maintaining AREG activity during experiments?

Several challenges can affect AREG activity in experimental settings. Protein aggregation during reconstitution or storage represents a common issue that can significantly reduce biological activity. To minimize this problem, researchers should reconstitute lyophilized AREG slowly at room temperature, avoid vortexing, and filter through a 0.2 μm filter if necessary . Another challenge involves protein adsorption to laboratory plasticware, which can substantially reduce effective concentration. This can be mitigated by using low-binding tubes and adding carrier proteins (0.1-0.5% BSA) to dilution buffers. Repeated freeze-thaw cycles significantly diminish activity, so single-use aliquots are strongly recommended . For cell culture applications, AREG stability can be compromised by proteases present in serum or produced by cells; therefore, optimization of treatment duration and consideration of protease inhibitors may be necessary for consistent results.

How can researchers optimize AREG stimulation protocols for different cell types?

Optimizing AREG stimulation protocols requires consideration of cell type-specific factors. First, researchers should determine the EGFR expression level and activation status in their target cells through flow cytometry or western blotting, as this will influence response magnitude. The expression of heparin sulfate proteoglycans is critical for efficient AREG-EGFR signaling , so characterization of these components in the target cells is recommended. Dose-response experiments (typically 0.1-100 ng/mL) should be performed to identify the optimal concentration for specific readouts, recognizing that different outcomes (proliferation, gene expression, etc.) may have distinct dose requirements. Temporal optimization is equally important, with kinetic studies (30 minutes to 72 hours) helping to identify peak response times for various endpoints. For complex cell systems like co-cultures or primary cells, serum starvation conditions and cell density should be carefully optimized to minimize background signaling while maintaining cell viability.

What experimental controls are essential for AREG research?

Essential controls for AREG research include both positive and negative controls to ensure experimental validity. Positive controls should include a well-characterized EGFR ligand such as EGF at a concentration known to elicit responses in the system under study. Negative controls should incorporate heat-inactivated AREG (95°C for 10 minutes) to demonstrate specificity of the observed effects to the active protein. For mechanistic studies, EGFR inhibitor controls (e.g., AG1478) are critical to confirm that observed effects are mediated through EGFR signaling pathways . When studying AREG in conditioned media, neutralizing antibody controls should be employed to specifically deplete AREG and demonstrate its requirement for the observed effects . For gene expression studies, time-matched vehicle controls are essential, as many AREG-regulated genes can be affected by experimental manipulation independent of AREG stimulation. When using siRNA approaches to study endogenous AREG, appropriate non-targeting siRNA controls and rescue experiments with recombinant protein should be included to confirm specificity.