Recombinant Xenopus laevis Growth factor receptor-bound protein 2-B (grb2-b)

What is the molecular structure of Xenopus laevis GRB2-B and how does it compare to mammalian GRB2?

Xenopus laevis GRB2-B shares the canonical modular architecture of GRB2 proteins, consisting of a single Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains - N-SH3 and C-SH3. This structure is highly conserved across species and represents a classic example of an adaptor protein design optimized for intracellular signal transduction . The crystal structure of mammalian GRB2 has been resolved at 3.1 Å resolution (PDB: 1GR1), revealing the spatial organization of these domains that facilitates its function in recruiting downstream effectors .

The SH2 domain specifically recognizes phosphorylated tyrosine residues, while the SH3 domains bind proline-rich sequences in partner proteins. This modular architecture enables GRB2-B to act as a molecular bridge in signaling cascades, particularly within receptor tyrosine kinase (RTK) pathways that are crucial for embryonic development in Xenopus .

Studies on folding and binding properties indicate that Xenopus GRB2-B, like its mammalian counterpart, exhibits complex domain interactions that contribute to its functional versatility in different cellular contexts .

What are the primary signaling pathways involving GRB2-B in Xenopus development?

GRB2-B in Xenopus laevis functions primarily as a critical adaptor in receptor tyrosine kinase (RTK) signaling cascades, with particular importance in the Ras-MAPK pathway. This pathway is essential for multiple aspects of embryonic development, including mesoderm induction, dorsoventral patterning, and neural development .

The canonical function of GRB2-B involves:

• Recognition of phosphorylated RTKs through its SH2 domain

• Recruitment of Son of Sevenless (SOS) through its SH3 domains

• Facilitation of Ras activation through GDP-GTP exchange

• Subsequent activation of the MAPK cascade

This signaling nexus is particularly important during gastrulation and neurulation in Xenopus embryogenesis . Recent research suggests that GRB2-B may also function in cross-talk between RTK and BMP signaling pathways, potentially connecting to the mechanisms by which R-spondins antagonize BMP signaling during embryonic axis formation in Xenopus .

What expression systems are most effective for producing functional recombinant Xenopus laevis GRB2-B?

Bacterial expression systems, particularly E. coli, remain the most widely used platform for recombinant GRB2-B production due to their efficiency and cost-effectiveness. For optimal expression in E. coli, codon optimization for the Xenopus sequence is recommended as it can significantly improve protein yield.

The following expression parameters have been demonstrated to be effective:

For structural and functional studies requiring higher purity and proper folding, mammalian (HEK293) or insect cell (Sf9) expression systems may be preferable, particularly when post-translational modifications are suspected to play a role in GRB2-B function.

What purification strategy yields the highest purity and activity for recombinant GRB2-B?

A multi-step purification approach is recommended to achieve high purity while preserving the functional activity of recombinant Xenopus GRB2-B:

• Initial capture: Affinity chromatography using a His-tag or GST-tag fusion construct

  • For His-tagged constructs, immobilized metal affinity chromatography (IMAC) with Ni-NTA resin works efficiently

  • Buffer optimization is critical: PBS with 300-500 mM NaCl, 20-50 mM imidazole, pH 7.4-8.0

• Intermediate purification: Ion exchange chromatography

  • GRB2-B typically has a pI of approximately 5.9-6.2

  • Q-Sepharose (anion exchange) at pH 8.0 effectively separates the target from contaminants

• Polishing step: Size exclusion chromatography

  • Superdex 75 or Superdex 200 columns separate monomeric GRB2-B from aggregates and provide buffer exchange

  • Running buffer: 20 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.5

• Tag removal: If necessary, use precision protease or TEV protease cleavage followed by a second affinity step to remove the cleaved tag

Typical yield from optimized bacterial expression systems ranges from 5-15 mg of purified protein per liter of culture. The purified protein should be assessed for proper folding using circular dichroism spectroscopy and functional activity through binding assays with known interaction partners.

How can researcher verify the binding specificity of recombinant GRB2-B to receptor tyrosine kinases in Xenopus systems?

Several complementary approaches can be used to verify binding interactions:

• In vitro binding assays:

  • Surface Plasmon Resonance (SPR) is particularly valuable for determining binding kinetics and affinities

  • For Xenopus GRB2-B, immobilize the protein on a CM5 chip and measure binding to phosphorylated peptides derived from known RTK binding sites

  • Typical binding affinities for GRB2 SH2 domains to phosphotyrosine-containing peptides range from 0.1-10 μM

• Co-immunoprecipitation from Xenopus egg extracts:

  • Xenopus egg extracts provide a physiologically relevant environment for testing interactions

  • GRB2-B can be tagged and used to pull down interacting partners, which can then be identified by Western blotting or mass spectrometry

  • This approach was successfully used for ESCRT-II interactions in Xenopus eggs

• UV cross-linking approach:

  • Particularly useful for detecting direct RNA-protein or protein-protein interactions

  • The methodology described for ESCRT-II in Xenopus egg extracts can be adapted for GRB2-B studies

  • This technique allows identification of specific binding sites and can be used to study sequence specificity

What methods are recommended for investigating GRB2-B role in Ras-MAPK pathway activation during Xenopus development?

The following methodological approaches are particularly effective:

• Embryo microinjection experiments:

  • Microinjection of mRNA encoding wild-type or mutant forms of GRB2-B

  • Typical injection volumes: 5-10 nl at concentrations of 50-200 pg/nl

  • Combine with lineage tracers (e.g., β-galactosidase) to track injected cells

  • Analyze effects on MAPK activation using phospho-ERK immunoblotting or immunostaining

• Dominant negative approaches:

  • Express SH2 or SH3 domain mutants that can bind targets but not propagate signals

  • Common mutations include replacement of critical residues in the SH2 domain that abolish phosphotyrosine binding

• Morpholino knockdown with rescue experiments:

  • Design translation-blocking or splice-blocking morpholinos against GRB2-B

  • Perform rescue experiments with morpholino-resistant mRNA to confirm specificity

  • Analyze embryonic phenotypes and molecular markers of MAPK pathway activation

• Biochemical pathway analysis:

  • Prepare lysates from embryos at different developmental stages

  • Use Western blotting to detect phosphorylated (activated) components of the MAPK pathway

  • Compare pathway activation in control versus GRB2-B manipulated embryos

How do mutations in different domains of GRB2-B affect its function in Xenopus development?

Systematic mutagenesis studies reveal domain-specific effects on GRB2-B function that provide insight into its mechanistic roles:

The folding dynamics of these mutants are particularly informative. As observed in comprehensive mutational analyses of GRB2, the folding mechanism can follow a nucleation-condensation model with a diffused transition state . Importantly, the effects of mutations may not be solely due to direct binding interface disruption, as residues distant from binding pockets can influence ligand recognition through allosteric networks .

When assessing GRB2-B mutations, it's critical to consider both direct effects on binding and potential allosteric consequences that may alter the energetic network governing domain interactions.

What techniques are most informative for studying protein-protein interactions involving GRB2-B in Xenopus systems?

For comprehensive characterization of GRB2-B interactions in Xenopus systems, multiple complementary techniques should be employed:

• UV Cross-linking and Immunoprecipitation (CLIP):

  • Particularly valuable for identifying direct binding partners

  • Successfully applied to study RNA-protein interactions in Xenopus egg extracts

  • Can be adapted to study protein-protein interactions by using protein-specific crosslinkers

• RIP-Seq and CLIP-Seq approaches:

  • Allows genome-wide identification of interaction partners

  • Can reveal sequence or structural motifs recognized by GRB2-B

  • In ESCRT-II studies, these techniques identified GA-rich motifs as preferential binding sites

• Biolayer Interferometry (BLI) and Isothermal Titration Calorimetry (ITC):

  • Provide detailed thermodynamic and kinetic parameters of interactions

  • Particularly useful for comparing binding properties of wild-type and mutant proteins

  • Can distinguish between enthalpy and entropy contributions to binding energy

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps interaction interfaces and conformational changes upon binding

  • Particularly valuable for identifying allosteric effects

  • Can reveal how binding at one domain affects the structure and dynamics of other domains

• In vitro reconstitution assays:

  • Using purified components to reconstruct signaling complexes

  • Allows systematic addition or removal of components to determine minimal requirements for activity

  • Similar approaches with ESCRT-II demonstrated that recombinant proteins can recapitulate selective binding observed in egg extracts

These techniques should be complemented with appropriate controls and validation experiments to ensure specificity and physiological relevance of the observed interactions.

How does GRB2-B contribute to dorsoventral axis formation in Xenopus embryos?

GRB2-B plays a critical role in dorsoventral axis specification through its involvement in multiple signaling pathways:

• RTK-Ras-MAPK signaling:

  • GRB2-B mediates FGF receptor signaling, which contributes to mesoderm induction and patterning

  • The timing and spatial distribution of this signaling helps establish the dorsoventral axis

• Cross-talk with BMP signaling:

  • Evidence suggests GRB2-B may modulate BMP receptor signaling, which is critical for dorsoventral patterning

  • This interaction may be analogous to how R-spondins function as BMP receptor antagonists in Xenopus early embryonic development

  • R-spondins target BMP receptor 1A for degradation, and GRB2-B may influence similar receptor trafficking pathways

• Regulation of organizer formation:

  • The Spemann organizer in Xenopus is essential for axis formation and neural induction

  • GRB2-B likely influences organizer formation and function through its effects on multiple signaling pathways

  • This is similar to how the Spemann organizer integrates BMP signaling to regulate embryonic axis formation

To study these contributions, researchers typically combine microinjection of GRB2-B constructs with molecular markers for dorsal (e.g., chordin, noggin) and ventral (e.g., Vent1, Vent2) tissues, followed by in situ hybridization or RT-qPCR analysis.

What are the best approaches for studying GRB2-B localization during embryonic development?

Several imaging approaches can be combined for comprehensive analysis:

• Fluorescently tagged GRB2-B constructs:

  • mCherry, GFP, or photoconvertible fluorophores (e.g., mEos) fused to GRB2-B

  • Validate that the tag doesn't interfere with protein function through rescue experiments

  • Use microinjection of mRNA encoding the fusion protein at 1-4 cell stage (50-200 pg/embryo)

• Immunohistochemistry with anti-GRB2-B antibodies:

  • Use specific antibodies against Xenopus GRB2-B or cross-reactive antibodies

  • Validate antibody specificity using morpholino knockdown controls

  • Combine with markers for specific cellular compartments or signaling components

• Super-resolution microscopy techniques:

  • Structured Illumination Microscopy (SIM) for whole-embryo imaging with improved resolution

  • Stimulated Emission Depletion (STED) or Single-Molecule Localization Microscopy (SMLM) for nanoscale resolution in specific regions

  • These techniques can reveal co-localization with binding partners at subcellular resolution

• Live imaging approaches:

  • Light sheet microscopy for extended time-lapse imaging with minimal phototoxicity

  • Confocal microscopy for higher resolution imaging of specific regions

  • Combine with photobleaching techniques (FRAP) to assess protein dynamics

When designing these experiments, it's crucial to include appropriate controls and to carefully consider the developmental timing, as GRB2-B localization and function may change throughout development.

How can CRISPR-Cas9 be effectively applied to study GRB2-B function in Xenopus laevis?

CRISPR-Cas9 genome editing in Xenopus laevis requires specific considerations due to its allotetraploid genome:

• Design strategy:

  • Target both homeologs (L and S) of GRB2-B if complete knockout is desired

  • Design sgRNAs that target conserved regions between homeologs when possible

  • Validate sgRNAs using in vitro digestion assays before embryo injection

• Delivery method:

  • Microinject Cas9 protein (1 ng) with sgRNA (500 pg) into fertilized eggs

  • For tissue-specific editing, inject at 8-16 cell stage into blastomeres that will give rise to tissues of interest

  • Use lineage tracers to identify injected cells

• Validation approaches:

  • T7 endonuclease I assay or direct sequencing to confirm editing

  • Western blotting to verify protein reduction

  • RT-PCR to check for altered splicing or nonsense-mediated decay

• Experimental design considerations:

  • Target different domains to create allelic series

  • Generate precise point mutations using homology-directed repair

  • Consider mosaic effects when interpreting phenotypes

• Controls:

  • Include Cas9-only and non-targeting sgRNA controls

  • Perform rescue experiments with wild-type or mutant mRNA to confirm specificity

This approach allows for detailed functional analysis of GRB2-B in its native context, providing insights that complement conventional knockdown or overexpression studies.

What controls should be included in experiments involving GRB2-B knockdown or overexpression in Xenopus laevis?

Rigorous experimental design requires comprehensive controls:

• For morpholino knockdown:

  • Standard control morpholino at equivalent concentration

  • 5-base mismatch control morpholino

  • Rescue with morpholino-resistant GRB2-B mRNA

  • Western blotting to confirm protein reduction

  • Target validation by RT-PCR for splice-blocking morpholinos

• For CRISPR knockout:

  • Non-targeting sgRNA + Cas9 protein

  • Sequencing to confirm on-target editing

  • Rescue with wild-type GRB2-B mRNA

  • Analysis of potential off-target effects

• For overexpression studies:

  • GFP or β-galactosidase mRNA as injection control

  • Dosage series to establish concentration-dependence

  • Functionally inactive mutant versions (e.g., SH2 or SH3 domain mutants)

  • Western blotting to confirm expression levels

• For all approaches:

  • Uninjected embryos as developmental controls

  • Contralateral side comparison in unilateral injections

  • Time-course analysis to distinguish primary from secondary effects

  • Multiple independent biological replicates (minimum n=3 experiments)

  • Appropriate statistical analysis (e.g., ANOVA with post-hoc tests)

These controls ensure that observed phenotypes are specifically attributable to GRB2-B manipulation and not to experimental artifacts or off-target effects.

How should researchers address the allotetraploidy of Xenopus laevis when studying GRB2-B?

Xenopus laevis underwent a whole-genome duplication, resulting in two homeologous subgenomes (L and S). This presents unique challenges and opportunities for GRB2-B research:

• Gene identification and nomenclature:

  • Properly identify both L and S homeologs of GRB2-B

  • Follow established nomenclature conventions for duplicated genes

  • Design primers and probes that can distinguish between homeologs

• Functional redundancy:

  • Assess expression patterns of both homeologs using homeolog-specific primers

  • Consider potential subfunctionalization or neofunctionalization

  • Target both homeologs simultaneously for complete loss-of-function studies

  • Target homeologs individually to assess specific contributions

• Experimental approaches:

  • For morpholinos or CRISPR, design reagents that target conserved regions to affect both homeologs, or design homeolog-specific reagents

  • For overexpression, clone and express each homeolog separately to assess functional differences

  • Use homeolog-specific antibodies or tags for protein detection

• Data interpretation:

  • Consider compensatory mechanisms between homeologs

  • Quantify the relative contribution of each homeolog to the observed phenotypes

  • Compare findings with those in diploid species (e.g., Xenopus tropicalis) when possible

This comprehensive approach to addressing allotetraploidy ensures more accurate and interpretable results when studying GRB2-B function in Xenopus laevis.

What are the most common technical challenges when working with recombinant Xenopus laevis GRB2-B and how can they be overcome?

Researchers commonly encounter several technical challenges that can be addressed with specific strategies:

• Protein solubility issues:

  • Challenge: GRB2-B may form inclusion bodies during bacterial expression

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.3 mM), and include solubility enhancers (10% glycerol, 0.1% Triton X-100)

  • Alternative: Express as fusion with solubility tags such as MBP or SUMO

• Domain stability problems:

  • Challenge: Individual domains may be less stable than the full-length protein

  • Solution: Include short segments of adjacent domains when expressing individual domains

  • Alternative: Use stabilizing mutations identified through directed evolution or rational design

• Binding partner identification:

  • Challenge: Distinguishing specific from non-specific interactions

  • Solution: Implement stringent washing conditions and include appropriate negative controls

  • Alternative: Use crosslinking approaches like those developed for ESCRT-II studies in Xenopus eggs

• Functional assays:

  • Challenge: Establishing robust readouts for GRB2-B activity

  • Solution: Develop in vitro reconstitution assays with purified components

  • Alternative: Use Xenopus egg extracts as a physiologically relevant system for functional studies

• Antibody specificity:

  • Challenge: Generating antibodies that specifically recognize Xenopus GRB2-B

  • Solution: Use conserved epitopes or raise antibodies against unique regions

  • Alternative: Tag endogenous GRB2-B using CRISPR-mediated homology-directed repair

These technical strategies, combined with the methodological approaches discussed in previous sections, provide a comprehensive toolkit for addressing challenges in Xenopus GRB2-B research.