GRB7 Antibody

What is GRB7 and what cellular functions does it regulate?

GRB7 is an adaptor molecule that mediates signal transduction from multiple cell surface receptors to various downstream signaling pathways. It belongs to the GRB7 protein family, which also includes GRB10 and GRB14. As a scaffolding protein without intrinsic enzymatic activity, GRB7 connects activated receptor tyrosine kinases to downstream effector proteins, thereby regulating cellular processes including migration, proliferation, and survival. The protein contains several functional domains, most notably an SH2 domain that binds to phosphorylated tyrosine residues on activated receptors like erbB2/HER2 and focal adhesion kinase (FAK) .

What is the relationship between GRB7 and cancer development?

GRB7 overexpression has been linked to enhanced cell migration and metastasis in several cancer types. The GRB7 gene is located within the 17q12 amplicon in close proximity to HER2, and both are frequently co-amplified in cancers. In breast cancer, GRB7 and erbB2 are overexpressed in 20-30% of cases. Importantly, studies in oesophageal adenocarcinoma (OAC) have shown that while high GRB7-expressing tumors are more likely to be HER2-positive (p = 0.03), approximately 70% of high GRB7-expressing tumors are HER2-negative . This suggests that high GRB7 expression is not merely a consequence of co-amplification with HER2 and that alternative mechanisms exist for increasing GRB7 expression.

How does GRB7 structure relate to its function?

GRB7 contains multiple functional domains that facilitate protein-protein interactions. These include:

• SH2 (Src Homology 2) domain: Binds phosphorylated tyrosine residues on activated receptors

• RA (Ras-Associating) domain: Interacts with specific protein partners, including FHL2

• PH (Pleckstrin Homology) domain: Contributes to membrane localization and protein interactions

• GM (GRB and Mig) region: Contains sequences that may interact with SH3 domain-containing proteins

The domain architecture enables GRB7 to function as a molecular scaffold, bringing together multiple signaling proteins. Research has suggested a model of GRB7 autoinhibition, where intramolecular interactions regulate its activity and availability for binding partners .

What techniques are most effective for detecting GRB7 using antibodies?

Multiple antibody-based techniques can effectively detect GRB7 in research applications:

• Immunohistochemistry (IHC): Particularly useful for tissue samples, allowing visualization of GRB7 localization and semi-quantitative scoring. GRB7 expression by IHC is typically scored as:

  • 3+/positive: Strong complete cytoplasmic and basolateral reactivity

  • 2+/equivocal: Weak-to-moderate complete cytoplasmic and basolateral reactivity

  • 1+/low: Faint incomplete cytoplasmic reactivity

  • 0/negative: No cytoplasmic reactivity

• Western blotting: Enables detection of full-length GRB7 protein (approximately 70-72 kDa) and variant forms. This technique is particularly valuable when assessing knockdown efficiency in functional studies .

• Immunofluorescence: Allows for co-localization studies with other proteins of interest, such as FHL2. This technique has revealed that GRB7 and FHL2 show apparent co-localization in HeLa cells, particularly at the inner cell membrane .

• Co-immunoprecipitation: Essential for studying protein-protein interactions involving GRB7. This approach has demonstrated that full-length GRB7 interacts with FHL2 in mammalian cells and that GRB7 must be tyrosine phosphorylated for this interaction to occur .

What are critical considerations for optimizing GRB7 antibody in IHC applications?

When optimizing GRB7 antibody for IHC, researchers should consider:

• Fixation protocol: Standardize formalin fixation time as over-fixation can mask epitopes.

• Antigen retrieval: Compare heat-induced epitope retrieval methods using citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0).

• Antibody selection: Use antibodies validated specifically for IHC applications with demonstrated specificity.

• Controls: Include positive controls (cell lines with known GRB7 expression like OE19 or Eso26) and negative controls.

• Scoring system: Implement a standardized scoring system (0, 1+, 2+, 3+) based on staining intensity and pattern.

• Subcellular localization: GRB7 typically shows cytoplasmic and basolateral staining patterns .

How can I validate GRB7 antibody specificity for my experiments?

Validating GRB7 antibody specificity requires multiple approaches:

• Positive and negative controls: Use cell lines with known GRB7 expression levels (e.g., OE19 and Eso26 for high expression; NES cells for low expression) .

• Knockdown validation: Perform siRNA or shRNA knockdown of GRB7 and confirm reduced signal with the antibody .

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding.

• Cross-reactivity assessment: Test antibody against related family members (GRB10, GRB14) to ensure specificity.

• Multiple antibody comparison: Use antibodies targeting different epitopes of GRB7 and compare staining patterns.

• Correlation with other methods: Correlate antibody-based detection with mRNA expression by qPCR .

How can I effectively study GRB7 phosphorylation and its impact on protein interactions?

GRB7 phosphorylation status significantly impacts its protein interactions and cellular functions. To study this:

• Phosphorylation detection: Use phosphotyrosine-specific antibodies to identify phosphorylated GRB7 in Western blot analysis. Research has shown that GRB7 must be tyrosine phosphorylated to interact with FHL2 .

• Co-immunoprecipitation approach:

  • Immunoprecipitate GRB7 or its binding partners (e.g., FHL2)

  • Probe blots with anti-phosphotyrosine antibodies to detect phosphorylation status

  • Strip and re-probe membranes with GRB7-specific antibodies to confirm identity

• Experimental manipulations:

  • Use tyrosine kinase inhibitors to block phosphorylation

  • Generate phospho-mimetic (Y→E) or phospho-resistant (Y→F) mutations

  • Employ phosphatase treatment to remove phosphate groups

• Functional correlation:

  • Compare binding affinities of phosphorylated versus non-phosphorylated GRB7

  • Assess downstream signaling pathway activation in relation to phosphorylation status

  • Examine subcellular localization changes associated with phosphorylation state

What approaches are most effective for GRB7 knockdown studies?

Multiple approaches can achieve GRB7 knockdown in experimental models, each with specific advantages:

• siRNA-mediated transient knockdown:

  • Implementation: Transfection with siRNAs targeting GRB7 mRNA

  • Optimal protocol: 40 nM siRNA with lipid-based transfection reagents like Lipofectamine RNAiMax

  • Target selection: Use siRNA pools targeting multiple exons (e.g., exons 4, 5, 11, and 14) to ensure knockdown of all variants

  • Advantages: Rapid implementation (results within 24-72 hours); high knockdown efficiency

• shRNA-mediated stable knockdown:

  • Implementation: Lentiviral vectors expressing short hairpin RNAs targeting GRB7

  • System design: Use doxycycline-inducible systems with fluorescent reporters (e.g., mCherry, GFP) for temporal control and visualization

  • Validation: Test multiple shRNA constructs for efficiency (e.g., construct sh2 showed greater knockdown efficiency than sh1 in OE19 cells)

  • Advantages: Long-term knockdown; suitable for in vivo studies

• CRISPR-Cas9 mediated gene knockout:

  • Implementation: CRISPR-Cas9 system targeting GRB7 exons

  • Advantages: Complete gene knockout versus partial knockdown

  • Considerations: Time-consuming establishment; potential for off-target effects

Phenotypic responses to GRB7 knockdown are cell line-dependent. High GRB7-expressing cell lines (OE19, Eso26) show pronounced decreases in proliferation following knockdown, while low GRB7-expressing lines (OE33, OACP4C, NES) do not demonstrate significant changes .

How can I distinguish between the effects of GRB7 and co-amplified genes like HER2?

Distinguishing between GRB7 and HER2 effects requires careful experimental design:

• Cell line selection strategy:

  • Use cell lines with different GRB7/HER2 expression profiles:

    • GRB7+/HER2+ cell lines (e.g., OE19, Eso26)

    • GRB7+/HER2- cell lines (many high GRB7-expressing tumors are HER2-negative)

    • GRB7-/HER2+ cell lines

    • GRB7-/HER2- cell lines as controls

• Selective genetic manipulation:

  • Perform individual knockdown of GRB7 or HER2

  • Compare phenotypic effects and downstream pathway activation

  • Research has shown that GRB7 knockdown has advantages over anti-HER2 therapy in decreasing cellular proliferation in certain contexts

• Pathway analysis:

  • Monitor differential effects on signaling pathways using phospho-specific antibodies

  • Use techniques like Reverse Phase Protein Array (RPPA) to assess pathway activation

  • Look for pathway changes specific to each protein's manipulation

• Clinical correlation:

  • Analyze patient samples with different expression patterns

  • The finding that 70% of high GRB7-expressing tumors are HER2-negative supports independent roles

What downstream signaling pathways are affected by GRB7 modulation?

GRB7 modulation affects several key downstream signaling pathways in cancer cells:

• PI3K/AKT/mTOR Pathway:

  • GRB7 knockdown decreases phosphorylation of:

    • Akt at S473 and T308

    • mTOR at S2448

    • Ribosomal protein S6 (rS6) at S240/S244

    • 4E-BP1 at T37/T46 in some cell contexts (OE19)

  • Functional consequences include reduced cell survival signaling, decreased protein synthesis, and impaired cell cycle progression

• MAPK/ERK Pathway:

  • GRB7 knockdown decreases phosphorylation of ERK1/2 at T202/Y204

  • This results in reduced proliferative signaling and altered gene expression

• Cytoskeletal Regulation:

  • GRB7 interacts with focal adhesion kinase (FAK)

  • Modulation affects cell migration and invasion capabilities

  • Impacts cytoskeletal reorganization and cell-matrix interactions

Reverse Phase Protein Array (RPPA) analysis following GRB7 knockdown has revealed complex changes in signaling networks. In OE19 cells, 6 proteins were upregulated and 19 downregulated, while in Eso26 cells, 22 proteins were upregulated and 4 downregulated . These findings indicate that GRB7 functions as an important signaling hub.

How does GRB7 interact with other proteins in signaling complexes?

GRB7 engages in multiple protein-protein interactions that contribute to its signaling functions:

• Interaction with FHL2:

  • GRB7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with roles in oncogenesis

  • This interaction is mediated by the RA and PH domains of GRB7

  • Full-length GRB7 must be tyrosine phosphorylated for this interaction to occur

  • Immunofluorescence microscopy demonstrates co-localization of GRB7 and FHL2, particularly at the inner cell membrane

• Interaction with receptor tyrosine kinases:

  • GRB7 binds to erbB2/HER2 through its SH2 domain

  • This interaction connects receptor activation to downstream signaling cascades

• Interaction with focal adhesion proteins:

  • GRB7 binds to focal adhesion kinase (FAK) through its SH2 domain

  • GRB7 has been observed localized to focal adhesions

  • These interactions contribute to GRB7's role in cell migration and adhesion

• Complex formation mechanisms:

  • Phosphorylation-dependent interactions: GRB7 must be tyrosine phosphorylated for certain interactions

  • Domain-specific binding: Different domains mediate interactions with different partners

  • Potential conformational regulation: NMR evidence supports a model of GRB7 autoinhibition

How does GRB7 expression correlate with patient outcomes in cancer?

GRB7 expression correlates with patient outcomes in several cancer types:

Importantly, while GRB7 and HER2 expression correlate, GRB7 appears to have independent prognostic value, as demonstrated by the fact that approximately 70% of high GRB7-expressing OAC tumors are HER2-negative .

What cell lines are most appropriate for studying GRB7 function in different cancer contexts?

Selecting appropriate cell lines is crucial for studying GRB7 function:

• Oesophageal Adenocarcinoma (OAC) cell lines:

  • High GRB7/HER2 expression:

    • OE19: High expression at both mRNA and protein levels; responds to GRB7 knockdown

    • Eso26: High expression; shows presence of GRB7 variant protein; responds to knockdown

  • Moderate/Low GRB7 expression:

    • OE33: Moderate mRNA but lower protein expression; no response to knockdown

    • OACP4C: Moderate mRNA but lower protein; responds to GRB7 overexpression

    • FLO1: Lower expression; shows increased migration upon GRB7 overexpression

  • Normal control:

    • NES (normal oesophageal squamous cells): Low GRB7 expression

• Functional response comparison:

  Cell Line	GRB7 Expression	Response to Knockdown	Response to Overexpression
  OE19	High	Decreased proliferation	Not applicable
  Eso26	High	Decreased proliferation	Not applicable
  OE33	Low/Moderate	No effect	No effect
  OACP4C	Low/Moderate	No effect	Increased proliferation
  FLO1	Low	Not reported	Increased migration

• Technical considerations:

  • All cell lines should be authenticated by short tandem repeat (STR) analysis

  • Confirm mycoplasma-free status before experimental use

  • Verify GRB7 expression levels by Western blot and qPCR

For comprehensive studies, researchers should include multiple cell lines with varying GRB7 expression levels, compare lines with and without HER2 co-amplification, and include appropriate positive and negative controls.