rasgrf2 Antibody

What is RASGRF2 and what cellular functions does it regulate?

RASGRF2 is a guanosine nucleotide exchange factor for Ras that participates in T-cell signaling and immune responses. Research has shown that RASGRF2 is expressed in T cells, translocates to immune synapses, activates Ras, and stimulates the transcriptional factor NF-AT (nuclear factor of activated T cells) through Ras- and phospholipase C-γ1-dependent routes . Additionally, RASGRF2 has been identified as a dual Ras/Rac-GEF, meaning it can activate both Ras and Rac GTPases, with significant implications for neuronal signaling pathways . In neurons, the Rac-GEF activity of RASGRF2 has been demonstrated to be necessary for NMDA receptor-dependent long-term potentiation (NMDA-LTP) .

What types of RASGRF2 antibodies are available for research applications?

Based on available information, researchers primarily utilize polyclonal antibodies against RASGRF2. For instance, rabbit polyclonal antibodies against RASGRF2 are commercially available and have been validated for applications such as immunohistochemistry (IHC) and immunohistochemistry-paraffin (IHC-P) . Some antibodies cross-react with both RasGRF1 and RasGRF2 isoforms, which researchers should consider when selecting antibodies for experiments requiring isoform specificity .

What are the common applications for RASGRF2 antibodies in research?

RASGRF2 antibodies are primarily used for:

• Immunohistochemistry (IHC) analysis in both fresh and paraffin-embedded tissues

• Detection of RASGRF2 in subcellular locations, such as its translocation to immune synapses in T cells

• Evaluation of RASGRF2 expression levels in various tissues, including comparing expression between normal and diseased states (e.g., tumor vs. adjacent non-tumor tissue)

• Studying the role of RASGRF2 in specific signaling pathways, particularly in T-cell activation and neuronal signaling

How can researchers discriminate between RASGRF1 and RASGRF2 in experimental settings?

Distinguishing between RASGRF1 and RASGRF2 presents a significant challenge in research settings since many antibodies cross-react with both isoforms. As noted in one study, "the RasGRF2 antibody we used cross-reacts with both isoforms of RasGRF" . To overcome this limitation, researchers should:

• Use genetic approaches such as specific knockdown or knockout models targeting RASGRF2

• Employ RNA interference (RNAi) with verified specificity for RASGRF2, such as the shRNA complementary to the target sequence 5′-TGGATTGATGACTATAGTC-3′ used in previous research

• Utilize rescue experiments with mutant constructs to confirm specificity of effects

• Consider complementary techniques like RT-PCR with isoform-specific primers to verify expression patterns

In systems where both isoforms may be present, researchers should explicitly acknowledge potential cross-reactivity limitations in their experimental design and interpretation.

What are the functional implications of RASGRF2's dual Ras/Rac-GEF activity in experimental design?

RASGRF2's dual Ras/Rac-GEF functionality requires careful experimental design to distinguish which activity is responsible for observed phenotypes. Research has demonstrated that these two GEF activities can be separated through specific mutations:

• Isolating Rac-GEF activity: The point mutation R1140A inhibits Ras binding to the catalytic CDC25H domain, preventing Ras activation while preserving Rac-GEF functionality

• Isolating Ras-GEF activity: Deletion of the entire Rac-GEF Dbl homology (DH) domain eliminates Rac-GEF activity, though this can enhance RasGRF2's Ras-GEF activity in the unstimulated state

These mutational approaches can be particularly valuable when coupled with rescue experiments following RASGRF2 knockdown. For example, in neuronal studies, researchers found that RASGRF2 knockdown suppressed NMDA-LTP, but this defect was rescued with the Ras-dead (R1140A) mutant, suggesting that the Rac-GEF activity, rather than Ras-GEF activity, was necessary for this function .

How does RASGRF2 expression correlate with disease progression in cancer research?

Bioinformatic analysis has revealed a significant relationship between RASGRF2 expression and stomach adenocarcinoma (STAD) progression. Research indicates that:

These findings suggest RASGRF2 serves as a negative protective factor in STAD patients, making it a potential biomarker for prognosis and therapeutic targeting. Similar investigations in other cancer types may yield valuable insights into RASGRF2's role in cancer biology more broadly.

What are the optimal protocols for RASGRF2 immunohistochemistry?

Based on validated research methodologies, the following protocol is recommended for RASGRF2 immunohistochemistry:

• Tissue preparation:

  • Fix tissue specimens in neutral formaldehyde

  • Embed in paraffin

  • Section to 4 μm thickness

• Staining procedure:

  • Use the streptavidin-peroxidase immunohistochemical method for enhanced staining intensity

  • Incubate tissue sections at 4°C overnight with anti-RASGRF2 antibody (1:100 dilution)

  • Recommended antibodies include rabbit anti-human monoclonal antibodies (e.g., ab121577, Abcam Inc.)

• Controls and validation:

  • Include both tumor and adjacent non-tumor tissue when available

  • Use positive and negative controls to validate staining specificity

  • Quantify staining intensity using standardized scoring systems

This methodology has been successfully employed to determine differences in RASGRF2 protein expression between tumor and adjacent non-tumor tissues and to correlate expression with clinicopathological characteristics .

What approaches can be used to investigate RASGRF2 activation dynamics?

To study RASGRF2 activation, researchers have developed several effective methodologies:

• GEF activity precipitation assays:

  • Use "bait" proteins consisting of GTPases mutated to tightly bind activated GEFs (e.g., Rac1-G15A or H-Ras-G15A)

  • Compare precipitation of RASGRF2 before and after stimulation (e.g., NMDA stimulation)

  • Increased precipitation indicates enhanced GEF activity

• Subcellular localization studies:

  • Track RASGRF2 translocation using immunocytochemistry or fluorescently tagged constructs

  • In T cells, monitor translocation to immune synapses

  • In neurons, examine spine localization

• Downstream pathway activation:

  • Assess Ras/MAPK pathway activation using phospho-specific antibodies

  • Monitor Rac-dependent cytoskeletal changes using appropriate markers

  • Evaluate transcriptional targets such as NF-AT in immune cells

These approaches allow for comprehensive analysis of both the spatial and temporal aspects of RASGRF2 activation in various cellular contexts.

How can researchers effectively perform RASGRF2 knockdown and rescue experiments?

Based on previously validated methods, the following approach is recommended for RASGRF2 knockdown and rescue experiments:

• RASGRF2 knockdown:

  • Use shRNA complementary to the target sequence 5′-TGGATTGATGACTATAGTC-3′ for rat RASGRF2

  • Validate knockdown efficiency (aim for >80% reduction)

• Rescue construct design:

  • Use species variants resistant to the shRNA (e.g., human RASGRF2 for rescue of rat knockdown)

  • For domain-specific studies, consider the following mutants:

    • R1140A mutation to inhibit Ras-GEF activity while preserving Rac-GEF function

    • DH domain deletion to eliminate Rac-GEF activity

• Functional validation:

  • Confirm rescue construct expression by immunoblotting or immunofluorescence

  • Validate functional rescue through relevant assays (e.g., NMDA-LTP in neurons, NF-AT activation in T cells)

  • Include appropriate controls (e.g., empty vector, wild-type rescue)

This experimental paradigm has been successfully employed to dissect the specific contributions of RASGRF2's Ras-GEF versus Rac-GEF activities in neuronal function .

How is RASGRF2 involved in T-cell signaling and immune responses?

RASGRF2 plays a critical role in T-cell signaling and immune function through several mechanisms:

• Immune synapse localization: RASGRF2 translocates to immune synapses upon T-cell receptor (TCR) engagement

• NF-AT activation: RASGRF2 stimulates the transcriptional factor NF-AT through both:

  • Ras-dependent pathways

  • Phospholipase C-γ1-dependent routes

• Cytokine production: Analysis of RasGRF2-deficient mice shows that this protein is required for the induction of important NF-AT targets, including:

  • Tumor necrosis factor alpha (TNF-α)

  • Interleukin 2 (IL-2)

• Synergistic activation: RASGRF2 pathways synergize with T-cell receptor-, Vav1-, and Ca²⁺-elicited pathways for full NF-AT stimulation

• T-cell proliferation: The RasGRF2 pathway cooperates with the Vav1/RasGRP1 route in the blasting transformation and proliferation of mature T cells

These findings establish RASGRF2 as a key component of the signaling machinery involved in TCR- and NF-AT-mediated immune responses, with potential implications for immunological disorders and therapeutic targeting.

What is the significance of RASGRF2 in neuronal signaling and plasticity?

RASGRF2 plays a crucial role in neuronal signaling and synaptic plasticity through its dual GEF activities:

• NMDA receptor coupling: RASGRF2 couples NMDA receptor calcium flux to Rac activation in neurons

• Spine enlargement: The Rac-GEF activity of RASGRF2 is necessary for:

  • NMDA-induced long-term potentiation (NMDA-LTP)

  • Correlated rapid spine enlargement following NMDA stimulation

• Synapse development: RASGRF2's Rac-GEF activity affects:

  • mEPSC frequency

  • Spine density

  • Synaptic PSD-95 content

• NMDA receptor function: The Ras-GEF activity of RASGRF2 modulates:

  • Peak NMDA current

  • NMDA receptor expression or function

  • Synaptic strength over longer timescales

This functional dichotomy between RASGRF2's Rac-GEF and Ras-GEF activities provides a molecular mechanism for the coordination of different aspects of synaptic development and plasticity, with important implications for learning, memory, and neurological disorders.

How can RASGRF2 expression serve as a biomarker in cancer research?

RASGRF2 shows significant potential as a biomarker in cancer research, particularly in stomach adenocarcinoma (STAD):

These findings suggest that RASGRF2 may serve not only as a prognostic biomarker but also provide insight into the molecular mechanisms underlying cancer progression and potential therapeutic targets.