Recombinant Mouse Rho-guanine nucleotide exchange factor (Rgnef), partial

What is Rgnef and what are its primary functions?

Rgnef (also known as p190RhoGEF or ARHGEF28) is a ubiquitously expressed 190 kDa guanine nucleotide exchange factor that primarily activates RhoA and RhoC GTPases in cells . It functions as a molecular integrator, translating various intra- and extracellular signals to promote spatiotemporal activity of Rho GTPases. Rgnef plays crucial roles in regulating cell motility, focal adhesion establishment, and cytoskeletal dynamics downstream of integrin signaling . Studies with knockout models have demonstrated that Rgnef is essential for proper RhoA activation following fibronectin-integrin stimulation, directly impacting cellular migration and adhesion formation . The protein contains multiple functional domains, including a DH-PH catalytic core responsible for GEF activity, making it a versatile regulator of cellular processes .

How does Rgnef differ from other RhoGEFs?

While Rgnef belongs to the larger Dbl-family of RhoGEFs characterized by their DH-PH domain architecture, it possesses several distinctive features that set it apart. Rgnef is most closely related to p114 (ARHGEF18), Lbc (ARHGEF13), and GEFH1 (ARHGEF2), but contains unique structural elements . A key distinguishing feature is Rgnef's ability to form a specific interaction with focal adhesion kinase (FAK), which other RhoGEFs typically lack . This Rgnef-FAK interaction enables both canonical GEF-dependent RhoA activation and GEF-independent scaffold functions that enhance FAK signaling . Additionally, Rgnef contains an N-terminal leucine-rich region, a cysteine-rich zinc finger domain, and a C-terminal region with a coiled-coil domain that can interact with microtubules, neurofilament mRNA, and signaling proteins like 14-3-3 and JIP-1 . These structural differences likely contribute to Rgnef's specialized functions in focal adhesion dynamics and cell migration.

What are the known tissue expression patterns of Rgnef in mice?

Rgnef exhibits a ubiquitous expression pattern across mouse tissues, though with varying levels in different cell types . In mouse embryonic fibroblasts (MEFs), Rgnef plays a critical role in cell migration and focal adhesion formation . Studies with transgenic mouse models have enabled the characterization of Rgnef expression and function in vivo. Specifically, researchers have developed Rgnef exon 24 floxed mice (Rgnef flox) crossed with CMV-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development . Analysis of these models revealed that while heterozygous Rgnef WT/flox (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, the number of Rgnef flox/flox (Cre+) mice at 3 weeks of age was significantly lower than expected, suggesting an important developmental role . This expression pattern indicates Rgnef's relevance in multiple tissue contexts and developmental stages, though additional tissue-specific expression data would be valuable for targeted research applications.

What are the key structural domains of Rgnef and their functions?

Rgnef contains several distinct structural domains that contribute to its diverse cellular functions:

• N-terminal leucine-rich region: Mediates protein-protein interactions

• Cysteine-rich zinc finger domain: Involved in protein and potentially DNA interactions

• DH (Dbl-homology) domain: Catalyzes nucleotide exchange on Rho GTPases

• PH (Pleckstrin homology) domain: Critical for membrane targeting and regulation

• C-terminal coiled-coil domain: Facilitates interactions with microtubules, mRNAs, and signaling partners

Recent studies have highlighted the importance of specific regions within the Rgnef PH domain for proper membrane targeting, which is essential for its function . The DH-PH domain tandem constitutes the catalytic core responsible for stimulating GDP release and GTP binding on RhoA and RhoC GTPases . Additionally, structural analyses have revealed that the interaction between Rgnef and focal adhesion kinase (FAK) involves specific binding interfaces that regulate both proteins' activities in cell motility contexts . These domains collectively enable Rgnef to function both as a canonical GEF activating RhoA and as a scaffold protein in signaling complexes.

How does the binding interface between Rgnef and TDP-43 influence their interaction?

Recent structural studies have investigated the binding interface between an RGNEF fragment (residues 1-242) and TDP-43 (residues 96-269), revealing important insights into their molecular interaction . Computational docking analyses using InterEvDock3 and ClusPro platforms, combined with structural data from AlphaFold and available NMR structures, have generated detailed models of this interaction . The stability of the Rgnef-TDP-43 dimer was quantified by measuring conformational spread between output models using RMSD calculations, and the top 50 scoring models were deposited to the ModelArchive database (ma-hepyb) .

Analysis of this binding interface reveals that Rgnef fragments can mitigate the toxic phenotype associated with TDP-43, which has implications for understanding neurodegenerative disease mechanisms . The interaction appears to involve specific residue contacts that stabilize the complex. In experimental validation, transgenic fly models expressing both proteins (elav> RGNEF;TDP-43) showed altered phenotypes compared to TDP-43 expression alone, indicating functional significance of this interaction in vivo . This structural interface represents an important target for understanding how Rgnef may modulate TDP-43-related pathologies and potentially offers insights for therapeutic approaches.

What molecular mechanisms regulate Rgnef activation?

Rgnef activation is regulated through multiple molecular mechanisms:

• Integrin-mediated activation: Fibronectin-integrin stimulation triggers Rgnef activation within 60 minutes, coinciding with RhoA activation . This process is essential for proper focal adhesion establishment and cell motility.

• FAK-dependent regulation: The interaction between Rgnef and focal adhesion kinase (FAK) creates a bidirectional regulatory relationship, where FAK can enhance Rgnef activity while Rgnef acts as both a GEF and scaffold to promote FAK signaling .

• Membrane targeting: Specific regions within the Rgnef PH domain are critical for proper localization to the plasma membrane, which is necessary for GEF activity toward Rho GTPases .

• Protein-protein interactions: Rgnef activity can be modulated through interactions with binding partners including 14-3-3 proteins and c-Jun amino-terminal kinase interacting protein-1 (JIP-1) .

These regulatory mechanisms ensure precise spatiotemporal control of Rgnef activity, allowing for coordinated regulation of downstream Rho GTPase signaling in response to diverse cellular cues. Understanding these activation mechanisms is crucial for designing experiments to modulate Rgnef function in research contexts.

What are the most effective approaches for generating Rgnef knockout models?

Based on published research, several effective approaches have been established for generating Rgnef knockout models:

• Conditional knockout strategy: The creation of Rgnef exon 24 floxed mice (Rgnef flox) that can be crossed with tissue-specific or inducible Cre recombinase lines represents a powerful approach. In particular, researchers have successfully used CMV-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development . This strategy allows verification of knockout through both genotyping and protein expression analysis.

• Verification protocols: It is essential to confirm Rgnef knockout at both genomic and protein levels. Successful studies have isolated Rgnef flox/flox (Cre+) (Rgnef−/−) embryos and primary mouse embryo fibroblasts (MEFs) and verified the absence of Rgnef protein expression through Western blotting or immunohistochemical approaches .

• Phenotypic validation: Functional validation of Rgnef knockout can be performed through multiple assays, including haptotaxis migration, wound closure motility, focal adhesion quantification, and RhoA GTPase activation assays after fibronectin-integrin stimulation . Comparing these parameters between wildtype and knockout cells provides robust confirmation of functional Rgnef depletion.

• Rescue experiments: To confirm knockout specificity, epitope-tagged Rgnef re-expression in knockout cells should rescue the observed phenotypes, as demonstrated in published studies . This control ensures that observed effects are specifically due to Rgnef loss rather than off-target effects.

These approaches have been successfully employed to establish the importance of Rgnef in cell migration, focal adhesion dynamics, and RhoA signaling in research settings.

What assays can be used to measure Rgnef-dependent RhoA activation?

Several established assays can effectively measure Rgnef-dependent RhoA activation:

• RhoA GTPase pulldown assays: This technique uses the Rho-binding domain (RBD) of effector proteins like Rhotekin fused to GST to selectively capture active (GTP-bound) RhoA from cell lysates. Comparing RhoA activation between wildtype and Rgnef-/- cells after fibronectin stimulation reveals Rgnef's contribution to RhoA signaling .

• FRET-based biosensors: Fluorescence resonance energy transfer biosensors can detect RhoA activation in living cells with high spatiotemporal resolution. These biosensors typically incorporate the RBD domain and provide real-time visualization of RhoA activity patterns in different subcellular locations.

• Phosphorylation of downstream targets: Measuring the phosphorylation status of RhoA effector targets such as myosin light chain (MLC) or cofilin can provide indirect measurements of RhoA pathway activation.

• Temporal activation analysis: Studies have shown that Rgnef activation occurs within 60 minutes upon fibronectin plating of cells, associated with RhoA activation . Time-course experiments capturing this window are therefore recommended.

• Rescue experiments: Re-expression of wildtype Rgnef in knockout cells should restore RhoA activation, while expression of GEF-deficient mutants can help dissect which domains are required .

These complementary approaches provide robust assessment of Rgnef's contribution to RhoA signaling and can be adapted for various experimental conditions and cell types.

How can the Rgnef-FAK interaction be effectively studied in cellular systems?

The Rgnef-FAK interaction represents a critical aspect of Rgnef biology that can be studied through several methodological approaches:

• Co-immunoprecipitation (Co-IP): This technique can detect native protein complexes between Rgnef and FAK from cell lysates. Using antibodies against either protein followed by Western blotting for the binding partner can confirm their association .

• Proximity ligation assay (PLA): This technique visualizes protein-protein interactions with high sensitivity in fixed cells, allowing detection of Rgnef-FAK complexes at specific subcellular locations like focal adhesions.

• FRET-based interaction sensors: Fluorescently tagged Rgnef and FAK constructs can be designed to monitor their interaction dynamics in living cells, providing temporal and spatial information about when and where these proteins interact.

• Domain mapping experiments: Expression of truncated versions of Rgnef can help identify the specific domains required for FAK binding. Similarly, FAK mutants can reveal which regions of FAK are necessary for Rgnef interaction .

• Functional consequence analysis: Measuring parameters like focal adhesion formation, cell migration, and RhoA activation in systems where the Rgnef-FAK interaction is specifically disrupted (rather than eliminating either protein entirely) can reveal the biological significance of this interaction .

These approaches, often used in combination, provide comprehensive insights into how the Rgnef-FAK interaction regulates both proteins' activities and influences downstream cellular processes like migration and adhesion formation.

How does Rgnef integrate with integrin signaling networks?

Rgnef serves as a critical node in integrin signaling networks through multiple mechanisms:

• Activation by integrin engagement: Fibronectin-integrin stimulation triggers Rgnef activation within 60 minutes, coinciding with RhoA activation . This timing suggests that Rgnef functions as an intermediate signaling component rather than in immediate-early integrin responses.

• FAK-dependent signaling: Rgnef forms a specific interaction with focal adhesion kinase (FAK), a major integrin-activated tyrosine kinase. This Rgnef-FAK interaction creates bidirectional regulation where FAK can enhance Rgnef activity while Rgnef acts as both a GEF and scaffold to promote FAK signaling .

• Focal adhesion regulation: Studies with Rgnef knockout cells demonstrate significantly reduced focal adhesion numbers following fibronectin stimulation, indicating that Rgnef is essential for proper adhesion formation downstream of integrins .

• Cytoskeletal reorganization: Through RhoA activation, Rgnef mediates integrin-induced cytoskeletal changes necessary for cell spreading and migration. This includes regulation of actomyosin contractility and stress fiber formation .

• Feedback regulation: Rgnef likely participates in feedback loops within integrin signaling networks, as its activation leads to changes in adhesion and contractility that can further modulate integrin engagement and clustering.

Understanding Rgnef's position within these integrin signaling networks provides insights into how cells coordinate adhesion dynamics and migration in response to extracellular matrix engagement.

What is the relationship between Rgnef and NF-κB signaling in oxidative stress responses?

Recent research has revealed a novel relationship between Rgnef and NF-κB signaling in the context of oxidative stress responses, particularly in ovarian cancer:

• Antioxidant gene regulation: RNA-sequencing and bioinformatic analyses have identified a conserved Rgnef-supported anti-oxidant gene signature including GPX4, Nqo1, and Gsta4, which are common targets of the NF-κB transcription factor .

• Functional significance: Experimental evidence demonstrates that Rgnef promotes NF-κB-dependent tumorsphere survival, while antioxidant treatment enhances growth of Rgnef-knockout spheroids . This indicates that Rgnef's role in oxidative stress protection operates through NF-κB signaling pathways.

• Tumor progression link: In ovarian cancer contexts, Rgnef facilitates NF-κB-mediated gene expression protecting cells from oxidative stress, which contributes to tumor progression and resistance to environmental stressors .

• Three-dimensional growth support: Rgnef is selectively required for three-dimensional spheroid growth in vitro and tumor growth in vivo, partly through this NF-κB-mediated antioxidant response .

This relationship between Rgnef and NF-κB represents an important expansion of our understanding of Rgnef's cellular functions beyond its canonical roles in Rho GTPase activation and focal adhesion dynamics. The connection to oxidative stress responses provides new insights into how Rgnef may contribute to tumor progression through multiple signaling mechanisms.

How does Rgnef activity influence cellular mechanotransduction?

Rgnef plays important roles in cellular mechanotransduction through several interconnected mechanisms:

• Focal adhesion dynamics: As a regulator of focal adhesion establishment and turnover, Rgnef mediates the conversion of integrin-based adhesions into mature focal adhesions that serve as critical mechanosensing structures . The significantly reduced focal adhesion formation in Rgnef knockout cells demonstrates its importance in this process.

• RhoA-dependent contractility: Through its GEF activity toward RhoA, Rgnef promotes actomyosin contractility which is essential for cells to generate forces and sense mechanical properties of their environment . This contributes to mechanosensitive cellular behaviors including migration and adaptation to substrate stiffness.

• FAK-mediated signaling: The Rgnef-FAK interaction enhances FAK activation and downstream signaling , which is known to be mechanosensitive and critical for translating physical forces into biochemical signals within cells.

• Cytoskeletal remodeling: By regulating RhoA activity, Rgnef influences stress fiber formation and cytoskeletal tension, key determinants of how cells respond to mechanical cues .

• Spatial regulation: Rgnef's specific targeting to adhesion sites via its PH domain allows for localized control of mechanotransduction signaling at the cell-matrix interface.

These mechanisms position Rgnef as an important regulator of how cells sense and respond to mechanical forces, with potential implications for understanding cellular behavior in both physiological contexts and disease states where mechanotransduction is altered.

How is Rgnef involved in ovarian cancer progression?

Rgnef demonstrates significant involvement in ovarian cancer progression through multiple mechanisms:

These findings establish Rgnef as a significant factor in ovarian cancer progression, particularly through novel mechanisms involving oxidative stress protection, which extends beyond its canonical roles in cell migration and focal adhesion dynamics.

What is known about Rgnef's role in neurodegenerative diseases?

While research on Rgnef's involvement in neurodegenerative diseases is still emerging, several important connections have been established:

• Interaction with TDP-43: Recent structural studies have investigated the binding interface between an RGNEF fragment (residues 1-242) and TDP-43 (residues 96-269) . This interaction is significant because TDP-43 is a major component of pathological inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

• Mitigation of toxic phenotypes: Evidence suggests that RGNEF fragments can mitigate the toxic phenotype associated with TDP-43 in model systems . In transgenic Drosophila models, co-expression of RGNEF with TDP-43 (elav> RGNEF;TDP-43) shows altered phenotypes compared to TDP-43 expression alone .

• RNA binding capabilities: The C-terminal region of Rgnef contains domains that can bind to the 3'-untranslated region of neurofilament mRNA , suggesting potential roles in RNA metabolism that could be relevant to neurodegenerative mechanisms.

• Locomotor effects: Studies in Drosophila models have evaluated negative geotaxis (locomotion) using climbing assays in flies expressing various combinations of RGNEF, TDP-43, and fragments like NF242 . These functional assessments provide insights into how Rgnef-related proteins might influence motor function in neurodegenerative contexts.

These findings suggest that Rgnef may play previously unappreciated roles in neurodegenerative disease processes, particularly through interactions with known disease-associated proteins like TDP-43. This emerging area represents an important direction for future research.

How do Rgnef knockout phenotypes manifest in different disease models?

Rgnef knockout phenotypes manifest distinctly across different disease models, providing insights into its context-dependent functions:

• Developmental consequences: In general knockout models, heterozygous Rgnef WT/flox (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, but Rgnef flox/flox (Cre+) mice numbers at 3 weeks of age were significantly less than expected . This suggests important developmental roles that may impact disease modeling.

• Cellular migration defects: In fibroblast models, loss of Rgnef significantly inhibits haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation . These phenotypes highlight Rgnef's fundamental role in cell motility that could influence multiple disease processes.

• Ovarian cancer implications: In ovarian cancer models, Rgnef knockout inhibits three-dimensional spheroid formation in vitro and tumor growth in vivo . Mechanistically, this involves disruption of NF-κB-mediated antioxidant gene expression that normally protects cells from oxidative stress .

• Comparative analysis table of Rgnef knockout phenotypes:

These diverse phenotypes underscore the multifaceted roles of Rgnef across biological contexts and suggest that therapeutic targeting would need to be carefully tailored to specific disease settings.

How can Rgnef be targeted for potential therapeutic development?

Based on current understanding of Rgnef biology, several potential therapeutic targeting strategies could be considered:

• Small molecule inhibitors of GEF activity: Developing compounds that specifically target the DH domain of Rgnef could inhibit its catalytic GEF activity toward RhoA without affecting other functions. This approach would be particularly relevant for cancer contexts where RhoA hyperactivation promotes tumor progression .

• Peptide disruptors of protein-protein interactions: Designing peptides or peptidomimetics that interfere with specific protein interactions, such as the Rgnef-FAK binding interface, could allow more selective modulation of Rgnef functions . This strategy could potentially reduce side effects compared to complete inhibition of all Rgnef activities.

• RNA-based approaches: siRNA or antisense oligonucleotides targeting Rgnef could be employed for transient knockdown in specific tissues. The association between ARHGEF28 gene loss and better patient survival in ovarian cancer suggests potential benefits of this approach .

• Targeting downstream effectors: In situations where direct Rgnef targeting proves challenging, focusing on key downstream pathways like NF-κB-mediated antioxidant responses might provide alternative intervention points .

• Exploiting oxidative stress vulnerability: Since Rgnef promotes protection from oxidative stress through NF-κB pathways in ovarian cancer, combining Rgnef inhibition with agents that increase oxidative stress could create synthetic lethality in tumor cells .

These strategies should be pursued with careful consideration of context-specific Rgnef functions and potential off-target effects. Validation in appropriate disease models will be essential for advancing any Rgnef-targeted therapeutic approach.

What techniques can be used to visualize Rgnef activity in living cells?

Advanced imaging techniques can provide valuable insights into Rgnef activity dynamics in living cells:

• FRET-based Rgnef biosensors: Constructing intramolecular fluorescence resonance energy transfer biosensors that undergo conformational changes upon Rgnef activation could allow real-time visualization of its activity. This approach has been successful for other GEFs and could be adapted for Rgnef.

• RhoA activity sensors: Since RhoA is a primary Rgnef target, existing FRET-based RhoA biosensors can be used in conjunction with Rgnef manipulation to indirectly monitor its activity. These sensors typically incorporate the Rho-binding domain (RBD) of effector proteins between fluorescent proteins.

• Fluorescently tagged Rgnef: Live cell imaging with fluorescently tagged Rgnef constructs can reveal its dynamic localization to specific subcellular compartments such as focal adhesions, providing spatial information about where Rgnef functions .

• Photoactivatable Rgnef variants: Developing optogenetic tools to precisely control Rgnef activity with light could allow spatiotemporal manipulation while simultaneously monitoring downstream effects.

• Proximity ligation visualization: Adapting proximity ligation assays for live cell applications could enable visualization of specific Rgnef interactions, such as with FAK, in real time .

These visualization approaches, especially when combined with functional readouts like focal adhesion dynamics or cell migration, can provide unprecedented insights into how Rgnef activity is regulated in space and time within living cells.

What are the emerging research frontiers in understanding Rgnef regulation and function?

Several emerging research frontiers are expanding our understanding of Rgnef regulation and function:

• Non-canonical functions beyond GEF activity: Recent discoveries highlight Rgnef's roles as a scaffold in the FAK signaling pathway and in promoting NF-κB-mediated antioxidant gene expression . Further exploration of GEF-independent functions may reveal additional regulatory roles.

• Interaction with disease-associated proteins: The emerging understanding of Rgnef's interaction with TDP-43 , a protein implicated in neurodegenerative diseases, opens new avenues for investigating Rgnef in neurological contexts beyond its established roles in cancer and cell migration.

• Mechanosensitive regulation: Given Rgnef's roles in focal adhesions and cytoskeletal dynamics , investigating how mechanical forces regulate its activity and localization represents an important frontier, particularly in contexts like tissue development and cancer invasion where mechanical cues are critical.

• Isoform-specific functions: Further characterization of potential Rgnef splice variants or post-translationally modified forms could reveal specialized functions in different cellular contexts or disease states.

• Systems-level integration: Understanding how Rgnef functions within larger signaling networks, rather than in isolated pathways, will provide a more comprehensive view of its regulatory roles. Approaches like proteomics and phosphoproteomics combined with computational modeling could advance this frontier.

• Translational applications: Developing Rgnef as a potential therapeutic target or biomarker, particularly in cancers where its expression correlates with patient outcomes , represents an important translational research direction.

These emerging frontiers highlight the expanding significance of Rgnef beyond its classical characterization as a RhoGEF and suggest numerous promising directions for future research.