Recombinant Mouse Furin (Furin)

What is the basic function of furin in cellular processes?

Furin is a cellular endoprotease identified in 1990 that proteolytically activates large numbers of proprotein substrates in the secretory pathway. It belongs to the proprotein convertase family and plays a crucial role in processing precursor proteins into their biologically active forms. Furin typically recognizes and cleaves at consensus sequences containing basic amino acids, such as -Arg-X-Lys/Arg-Arg↓-, although it can also process minimal consensus sites in certain contexts. This proteolytic activity is essential for activating various signaling molecules, growth factors, and other proteins important for cellular function .

How is furin expression regulated during development?

Furin expression is tightly regulated during embryonic development, where it plays essential roles in critical developmental processes. Studies have shown that furin is required both in extra-embryonic tissues and in the cardiogenic mesoderm to promote yolk sac vasculogenesis, ventral closure, heart-looping, and axial rotation. The regulation of furin expression is linked to signaling pathways involving TGF-β family members. For example, TGF-β can bind to its receptor to stimulate furin gene transcription through a convergent pathway involving SMAD2 and mitogen-activated protein kinase (MAPK) . Additionally, in neonatal mouse lungs, furin expression has been observed specifically at the tips of secondary alveolar septa, indicating its role in lung development and alveolarization .

What is the cellular localization pattern of furin in mouse tissues?

Immunohistochemistry studies in mouse tissues have revealed specific localization patterns of furin. In developing mouse lungs, furin-positive cells are concentrated at the tips of secondary alveolar septa. Furin immunoreactivity has been observed in vascular smooth muscle cells but not in airway epithelium. Immunofluorescence studies on primary cultures prepared from mouse peripheral lung cell suspension have confirmed that furin is co-expressed in smooth muscle actin (SMA)-positive cells, suggesting its presence in myofibroblasts . This specific localization pattern is important for understanding furin's functional roles in different tissue contexts.

What are the recommended antibodies and reagents for furin detection?

For reliable detection of furin in research applications, several validated antibodies and reagents are available. The anti-furin antibody from Invitrogen (PA1-062) has been successfully used in immunohistochemistry and immunoblotting studies. For visualization in immunofluorescence applications, secondary antibodies such as Alexa Fluor 594-conjugated anti-rabbit antibody (Invitrogen, A-11012) can be used. When studying furin's role in activating downstream targets like IGF-IR, antibodies specific to both the total receptor (Cell Signaling, 9750) and its phosphorylated form (Cell Signaling, 3024) are essential . For inhibiting furin activity in experimental contexts, researchers can employ specific inhibitors such as hexa-D-arginine (Tocris, 4711) or the membrane-permeable furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKRCMK; Cayman, 14965) .

How can I detect furin expression in specific cell types?

To detect furin expression in specific cell types, immunofluorescence co-localization studies are particularly effective. For example, to identify furin in myofibroblasts, cells can be seeded at a density of 0.1 × 10^6 cells/cm^2 in glass bottom plates, fixed with 4% formalin, permeabilized with methanol, and blocked with 1% goat serum in PBS containing 0.1% Tween 20. The cells can then be incubated with anti-furin primary antibody and Alexa Fluor 488-conjugated anti-alpha smooth muscle actin antibody, followed by appropriate secondary antibodies. DAPI can be used to identify nuclei, and wide-field epifluorescence microscopy can be employed for imaging the cells . This approach allows researchers to visualize furin expression in relation to specific cellular markers.

What methods are effective for quantifying changes in furin expression?

To quantify changes in furin expression, multiple complementary approaches can be used. At the mRNA level, quantitative RT-PCR is effective for measuring changes in furin gene expression. For protein-level analysis, immunoblotting of tissue or cell lysates provides reliable quantification when combined with densitometry. For spatial distribution analysis, immunohistochemistry combined with cell counting can reveal changes in the number of furin-positive cells in different experimental conditions. For example, in hyperoxia exposure studies, researchers observed a significant decrease in furin-positive cells in mouse lungs, which was confirmed by both cell counting in tissue sections and densitometry of immunoblots from whole lung lysates . These methods can be applied to various experimental contexts to accurately measure changes in furin expression.

What is the role of furin in embryonic development?

Furin plays a crucial role in embryonic development, with genetic knockout studies demonstrating that inactivation of the furin gene in mice creates an embryonic lethal phenotype, resulting in death at an early embryonic stage. This essential role appears to be due to furin's involvement in activating key developmental signaling molecules, particularly those in the TGF-β family. Furin is required for activating members of this family, including TGF-β1, bone morphogenetic protein-4 (BMP-4), Nodal, and Lefty-2. The failure to maintain asymmetry in the embryo observed in furin knockout models is likely due to a block in the furin-catalyzed production of Nodal and Lefty-2 . These findings highlight furin's indispensable role in coordinating proper embryonic patterning and development.

How does furin contribute to lung alveolarization?

Furin plays a significant role in lung alveolarization, particularly in neonatal lungs. Studies have shown that furin-positive cells are specifically localized at the tips of secondary alveolar septa in developing mouse lungs. These cells are identified as myofibroblasts based on their co-expression of smooth muscle actin. Importantly, experimental models of impaired alveolarization, such as hyperoxia exposure in neonatal mice, show a significant reduction in furin expression. This reduction correlates with decreased alveolar formation, as evidenced by increased mean linear intercept (Lm) and decreased tissue volume density (TVD) . These findings suggest that furin regulates alveolarization through the activation of alveolarization-driving proteins, potentially including insulin-like growth factor 1 receptor (IGF-1R), which requires furin-mediated processing for activation.

How does furin regulate the activity of morphogens during development?

Furin regulates morphogen activity during development through a sophisticated "measure once, cut twice" processing mechanism. This is particularly well-documented for BMP-4, where furin first cleaves pro-BMP-4 at the consensus furin site that joins the pro- and BMP-4 domains (–Arg–Ser–Lys–Arg ↓–), followed by a second cleavage at a minimal consensus furin site within the propeptide (–Arg–Ile–Ser–Arg ↓–). The context of these two sites ensures the ordered processing of pro-BMP-4 and the correct activity of this morphogen . This sequential cleavage approach appears to be a common mechanism for controlling signaling gradients in development, as similar patterns are observed in other BMP-4-related signaling molecules. The presence of both consensus and minimal furin sites in these proteins suggests that this is an evolutionarily conserved mechanism for fine-tuning morphogen activity during development.

How does furin contribute to cancer progression and metastasis?

Furin contributes to cancer progression and metastasis through several mechanisms. First, furin is upregulated in several cancer types, including non-small-cell lung carcinomas, squamous-cell carcinomas of the head and neck, and glioblastomas. The increased levels of furin in tumors correlate with increased aggressiveness and with elevated levels of one of its substrates, membrane type 1-matrix metalloproteinase (MT1-MMP) . Furin activates MT1-MMP, which in turn activates extracellular pro-MMP2 (pro-gelatinase), inducing rapid tumor growth and neovascularization. Additionally, furin processes insulin-like growth factor-1 (IGF1) and its receptor (IGF1R), both of which are upregulated in colon, breast, prostate, and lung cancers . Inhibition of furin-mediated processing by specific inhibitors like α1-PDX reduces tumor incidence, size, and vascularization in experimental models, highlighting furin's critical role in cancer biology.

What is the role of furin in inflammatory diseases such as rheumatoid arthritis?

In rheumatoid arthritis, furin and TGF-β participate in a positive feedback loop that exacerbates the disease. TGF-β binding to its receptor stimulates furin gene transcription through a SMAD2 and mitogen-activated protein kinase (MAPK) convergent pathway. In synoviocytes (fibroblast- and macrophage-like cells lining joint synovium), the amplified levels of furin and TGF-β together increase the levels of ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs-4), also known as aggrecanase-1. ADAMTS-4 degrades the cartilage protein aggrecan, contributing to joint destruction in rheumatoid arthritis . This example illustrates how furin can participate in pathological positive feedback mechanisms that perpetuate inflammatory diseases.

What are the available inhibitors for furin and how do they differ in mechanism?

Several inhibitors are available for blocking furin activity in experimental settings, each with distinct mechanisms and applications. Peptide-based competitive inhibitors include hexa-D-arginine, which mimics the basic residue-rich recognition sequences of furin substrates. More potent inhibitors include the membrane-permeable furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKRCMK), which covalently modifies the active site of furin . Protein-based inhibitors such as α1-PDX (α1-antitrypsin Portland variant) act as suicide substrates that form stable complexes with furin. These inhibitors have been successfully used to block furin-mediated activation of substrates like MT1-MMP in cancer models and IGF-1R in developmental studies . The choice of inhibitor depends on experimental requirements, including cell permeability needs, specificity, and the biological context being studied.

How can I design experiments to evaluate the effects of furin inhibition on target protein activation?

To evaluate the effects of furin inhibition on target protein activation, cell culture-based approaches provide a controlled experimental system. For example, primary myofibroblast cells can be seeded at 0.3 × 10^6 cells/cm^2 in 6-well plates in complete medium. After allowing cells to attach, they can be subjected to serum starvation (0.2% FBS) with 50 μM of dec-RVKRCMK for 24 hours. Following this initial treatment, cells can be stimulated with 10% FBS, with or without dec-RVKRCMK, for an additional 24 hours . Cell lysates can then be analyzed by immunoblotting to assess the processing status of furin substrates. For targets like IGF-1R, this approach reveals accumulation of the pro-form when furin is inhibited, indicating blocked processing. Additionally, downstream signaling effects can be assessed by examining phosphorylation of the substrate proteins or their signaling partners.

What are the potential off-target effects of furin inhibition to consider in experimental design?

When designing experiments involving furin inhibition, researchers must consider several potential off-target effects. First, furin belongs to a family of proprotein convertases with overlapping substrate specificities, so inhibitors may affect multiple family members depending on their selectivity. Second, furin processes numerous substrates involved in diverse cellular processes, making it challenging to attribute observed effects to a specific substrate pathway. Third, complete inhibition of furin can affect cellular viability, given its essential roles in normal cellular function. To address these concerns, experimental designs should include appropriate controls, such as dose-response studies to identify minimal effective inhibitor concentrations, selective targeting approaches (e.g., siRNA knockdown specific to furin), rescue experiments with processed forms of suspected target substrates, and comparative studies with other proprotein convertase inhibitors to distinguish furin-specific effects .

How can I differentiate between direct and indirect effects of furin on substrate activation?

Differentiating between direct and indirect effects of furin on substrate activation requires a multi-faceted approach. Direct in vitro cleavage assays using purified recombinant furin and substrate proteins can establish whether a protein is a direct furin substrate. For example, recombinant furin from commercial sources (like New England Biolabs, #P8077S) can be used in controlled biochemical assays . Site-directed mutagenesis of putative furin cleavage sites in the substrate protein can confirm direct recognition - if mutation prevents processing both in vitro and in cellular contexts, this strongly suggests direct furin action. Pulse-chase experiments can track the kinetics of substrate processing in the presence and absence of furin inhibitors or in furin-deficient cell lines. Subcellular co-localization studies can determine whether furin and its putative substrate occupy the same cellular compartments, which is necessary for direct interaction. Combined, these approaches can distinguish direct furin substrates from proteins that are affected indirectly through downstream pathways.

What experimental models are most appropriate for studying furin's role in alveolarization?

Several experimental models are appropriate for studying furin's role in alveolarization, each with distinct advantages. In vivo models include neonatal mouse hyperoxia exposure, which has been shown to significantly reduce furin expression and impair alveolarization, as measured by increased mean linear intercept (Lm) and decreased tissue volume density (TVD) . This model allows for the study of furin regulation in a physiologically relevant context. For more mechanistic studies, conditional furin knockout mice targeting specific cell populations (such as myofibroblasts) can elucidate cell type-specific roles. Ex vivo models include precision-cut lung slices, which maintain the complex 3D architecture of the lung while allowing for experimental manipulation. In vitro, primary myofibroblast cultures derived from neonatal mouse lungs enable detailed molecular studies of furin's role in activating alveolarization-driving proteins like IGF-1R . Combining these models provides complementary insights: in vivo studies establish physiological relevance, while in vitro approaches enable detailed molecular mechanism investigations.

How does the "measure once, cut twice" processing mechanism of furin contribute to protein functional diversity?

The "measure once, cut twice" processing mechanism employed by furin represents a sophisticated regulatory approach that contributes to protein functional diversity through several mechanisms. This sequential cleavage approach, exemplified by BMP-4 processing, ensures that proteins are activated in the correct cellular compartment and in the appropriate context. The ordered nature of the cleavages - first at a consensus furin site and then at a minimal consensus site - allows for temporal control over protein activation, potentially creating distinct functional intermediates with unique activities . This mechanism also enables the generation of protein fragments with different diffusion properties, which is particularly important for morphogens like BMP-4 that function in concentration gradients during development. The requirement for specific sequence contexts around both cleavage sites provides an additional layer of regulation, ensuring that only properly folded proteins in the correct cellular environment undergo complete processing. This mechanism has been observed not only in developmental contexts but may extend to viral pathogenesis, as evidenced by sequential cleavage requirements for respiratory syncytial virus (RSV) fusion protein activation .