GRB10 Antibody

What is GRB10 and what are its primary biological functions?

GRB10 (Growth factor receptor-bound protein 10) is an adapter protein that modulates coupling of cell surface receptor kinases with specific signaling pathways . It contains three consensus domains:

• Pleckstrin homology (PH) domain

• SH2/SH3 domain

• Ras-associating domain

GRB10 plays critical roles in:

• Insulin and IGF-1 signaling pathways

• Glucose homeostasis and metabolism

• Tissue-specific growth regulation

• Neuronal development (through paternal expression)

Research in GRB10-knockout mice (Grb10Δ2-4) has demonstrated improved whole-body glucose tolerance, enhanced insulin sensitivity, increased muscle mass, and reduced adiposity, indicating its significant role in metabolic regulation .

What applications are GRB10 antibodies validated for?

GRB10 antibodies have been validated for multiple research applications:

When selecting antibodies, researchers should prioritize those with validation data specifically for their experimental system and application of interest .

How should researchers validate GRB10 antibody specificity?

Methodological approach to validate GRB10 antibody specificity:

• Positive control tissues/cells: Use tissues known to express GRB10, such as HeLa cells, HepG2 cells, mouse/rat brain tissue, or mouse liver tissue

• Knockout/knockdown validation: Compare antibody reactivity between:

  • Wild-type samples

  • GRB10 knockout models (e.g., Grb10Δ2-4 mice)

  • siRNA/shRNA knockdown cells

• Molecular weight verification: Confirm detection at expected molecular weights:

  • Calculated MW: 61-67 kDa (536aa/594aa isoforms)

  • Observed MW: 60-70 kDa in most systems

  • Note that GRB10 antibodies frequently detect at least three major bands (65-85 kDa) representing differently processed forms

• Cross-reactivity assessment: Test reactivity across species of interest (most commercial antibodies react with human, mouse, and rat GRB10)

How can researchers effectively detect GRB10's interaction with receptor tyrosine kinases?

GRB10 interactions with receptor tyrosine kinases can be investigated using these methodological approaches:

• Co-immunoprecipitation studies:

  • Immunoprecipitate the receptor (e.g., insulin receptor, IGF-1R, PDGFR) using specific antibodies

  • Immunoblot with GRB10 antibody to detect association

  • Alternatively, immunoprecipitate with GRB10 antibody and blot for receptor

• Stimulus-dependency experiments:

  • Starve cells to reduce baseline signaling

  • Stimulate with appropriate ligand (insulin, IGF-1, PDGF-BB)

  • Research shows GRB10 associates with PDGFRβ, IGF-IR, and IR strictly in a ligand-dependent manner

• Domain-specific interaction studies:

  • Use GST-fusion proteins of GRB10 domains (SH2 domain, Pro-rich region)

  • Microinjection of dominant-negative GST-GRB10 SH2 domain has been shown to interfere with growth factor-mediated DNA synthesis

  • Cell-permeable fusion peptides of the GRB10 Pro-rich region can specifically interfere with IGF-I and insulin (but not PDGF-BB) signaling

• Phosphorylation-dependent interactions:

  • Investigate how GRB10 phosphorylation affects receptor binding

  • GRB10 is phosphorylated by mTORC1 at Ser150, Ser428, and Ser476 upon insulin stimulation

What methodological approaches should be used to study tissue-specific expression and function of GRB10?

GRB10 exhibits complex tissue-specific expression patterns that require specialized methodological approaches:

• Neuronal expression studies:

  • Challenge: Adult brain is over 70% glial cells that don't express paternal GRB10

  • Solution: Differentiate mouse embryonic stem cells (mESCs) into homogenous populations of postmitotic alpha motor neurons

  • This in vitro model shows repression of the major promoter and activation of the neuron-specific promoter

• Tissue-specific knockout models:

  • Generate conditional knockout mice using tissue-specific Cre recombinase

  • Compare with global knockout models (e.g., Grb10Δ2-4) to identify tissue-specific phenotypes

  • Grb10Δ2-4 mice show improved glucose tolerance, increased muscle mass, and reduced adiposity

• Promoter-specific expression analysis:

  • GRB10 utilizes multiple promoters, including a neuron-specific promoter

  • Track promoter switching during differentiation

  • In neuronal differentiation, expression from the major promoter decreases while neuron-specific promoter activity increases

• Imprinting analysis:

  • Utilize parent-of-origin specific knockout models

  • Employ allele-specific expression assays to distinguish maternal vs. paternal expression

  • GRB10 is maternally expressed in most tissues but paternally expressed in neurons

How should researchers interpret contradictory findings about GRB10's role in insulin signaling?

The literature contains seemingly contradictory findings about GRB10's role in insulin signaling. Researchers should approach this complexity with:

• Experimental context awareness:

  • While most studies suggest GRB10 inhibits insulin signaling , some experiments have shown it to be a positive regulator

  • The consensus view is that "Grb10 may function to inhibit insulin signaling or to redirect the Insr signaling pathway"

• Methodological considerations:

  • Overexpression vs. knockout approaches yield different results

  • Cell type specificity is crucial (hepatocytes vs. muscle cells vs. adipocytes)

  • Acute vs. chronic manipulation may affect outcomes

• Mechanistic models to test:

  • GRB10 prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the insulin receptor kinase activation loop

  • GRB10 physically disrupts IRS association with phosphorylated residues of the insulin receptor

  • GRB10 inhibits insulin-stimulated glycogen synthase via novel pathways

• Context-specific signaling:

  • Design experiments to test how GRB10 functions differently across:

    • Different tissues (muscle vs. adipose vs. liver)

    • Different receptors (insulin receptor vs. IGF1R vs. PDGFR)

    • Different downstream pathways

What are the optimal protocols for detecting phosphorylated forms of GRB10?

GRB10 phosphorylation plays a critical role in its function, particularly in response to growth factors and insulin signaling:

• Phospho-specific antibodies:

  • Use antibodies targeting specific phosphorylation sites, such as Phospho-GRB10 (Ser476)

  • For phospho-Ser476 detection, optimal dilutions are:

    • Western blotting: 1:1000

    • Immunoprecipitation: 1:50

• Stimulation protocols:

  • Serum-starve cells for 16-24 hours

  • Stimulate with appropriate ligand:

    • Insulin (10-100 nM, 5-15 minutes)

    • IGF-1 (50-100 ng/mL, 5-15 minutes)

    • EGF (which induces serine phosphorylation)

  • Immediately lyse cells in buffer containing phosphatase inhibitors

• mTORC1-dependent phosphorylation studies:

  • GRB10 is a direct substrate of mTORC1

  • It is phosphorylated at Ser150, Ser428, and Ser476 upon insulin stimulation

  • Include rapamycin controls to block mTORC1-dependent phosphorylation

• Phosphatase treatment controls:

  • Split samples and treat half with lambda phosphatase

  • Compare migration patterns (phosphorylated forms often migrate slower)

  • Verify phospho-antibody specificity by loss of signal after phosphatase treatment

What techniques should be employed to distinguish between different GRB10 isoforms?

GRB10 exists in multiple isoforms with differing molecular weights and functions. Researchers should use these approaches:

• Isoform-specific antibody selection:

  • Choose antibodies raised against regions unique to specific isoforms

  • Verify calculated molecular weights:

    • 536aa isoform: ~61 kDa

    • 594aa isoform: ~67 kDa

  • Note that GRB10 antibodies typically detect at least three major protein bands ranging from 65-85 kDa

• Molecular techniques for isoform identification:

  • RT-PCR with isoform-specific primers

  • RNA-seq analysis to quantify isoform-specific transcripts

  • Use of minigene constructs to study alternative splicing regulation

• Neuronal vs. non-neuronal isoforms:

  • During neuronal differentiation, GRB10 expression shifts:

    • Major promoter is repressed

    • Neuron-specific promoter is activated

  • When differentiating mESCs into motor neurons, higher relative expression of the neuronal isoform is observed compared to adult spinal cord, likely due to enrichment of neuronal population over glia

• Functional validation approaches:

  • Isoform-specific overexpression studies

  • CRISPR-Cas9 targeting of isoform-specific exons

  • Domain-specific inhibitors (e.g., cell-permeable fusion peptides targeting the Pro-rich region)