GFRA3 Human, Sf9

What is the molecular structure of GFRA3 when expressed in Sf9 cells?

GFRA3 Human Recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 585 amino acids (residues 32-374) with a molecular mass of 65.5kDa. Due to glycosylation, the protein appears at approximately 70-100kDa when analyzed by SDS-PAGE. The protein is typically fused to a 239 amino acid hIgG-His-Tag at the C-terminus to facilitate purification through proprietary chromatographic techniques .

For structural studies, researchers should consider that GFRA3 belongs to the GDNF receptor family and forms a signaling receptor complex with RET tyrosine kinase receptor to bind its ligand artemin (ARTN). The full amino acid sequence includes specific domains essential for ligand binding and receptor interaction, which can be experimentally verified through structural analysis techniques like cryo-EM .

How does GFRA3 compare structurally to other GDNF family receptors?

GFRA3 displays approximately 33% amino acid identity with GFRα1 and 36% identity with GFRα2, suggesting evolutionary conservation of key functional domains while maintaining specificity . When studying receptor-ligand interactions, it's important to note that:

Unlike other family members, GFRA3 is unable to activate RET in conjunction with GDNF, highlighting its binding specificity . Cryo-EM studies reveal that the D1 domain of GFRA3 is invisible in 3D reconstructions of ARTN/GFRα3/RET complexes, suggesting it doesn't participate in binding to either RET or ligands, contrasting with GFRα2 where the D1 domain packs closely with the D3 domain .

To experimentally distinguish between these receptors, researchers should design binding assays that account for these structural differences and specificity of ligand interactions.

What experimental approaches effectively characterize GFRA3's binding specificity?

To characterize GFRA3's binding specificity, implement a multi-faceted approach:

• Binding assays: Utilize pull-down binding assays with a double strep-tagged version of GFRA3 to assess interactions with Artemin and RET .

• Structural analysis: Employ cryo-EM to visualize complex formation. GFRA3's interface with RET (buried surface areas ~730–1010 Ų) is more extensive than that in GFRAL/RET (~680 Ų), potentially compensating for weaker interfaces elsewhere in the complex .

• Domain mapping: Generate constructs with specific domain deletions to identify critical binding regions. For instance, the D1 domain's role can be assessed since it doesn't appear to participate in binding .

• Receptor competition assays: Test whether GFRA3 competes with other GFRα receptors for RET binding, which can reveal binding kinetics and preference.

These methodological approaches provide complementary data about binding specificity beyond simple affinity measurements.

What are optimal conditions for maintaining GFRA3 stability during purification and storage?

Maintaining GFRA3 stability requires specific buffer conditions and handling protocols:

Purification protocol:

• Express GFRA3 (residues 32-374) in Sf9 Baculovirus cells

• Capture using Ni²⁺-Sepharose 6 Fast Flow resin

• Further purify via gel filtration chromatography with Superdex S200 column

• Verify purity (>85%) by SDS-PAGE

Storage conditions:

• Store in solution containing Phosphate Buffered Saline (pH 7.4) with 10% glycerol

• For long-term storage, add a carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles to preserve protein integrity

Researchers should validate protein stability by periodic functional assays and SDS-PAGE analysis to ensure the protein maintains its native conformation throughout experimental use.

What expression systems are most effective for studying GFRA3 interactions?

Based on published research, several expression systems have proven effective for GFRA3 studies:

For optimal results when studying GFRA3-RET-Artemin interactions, express GFRA3 (residues 32-363) with an alkaline phosphatase signal peptide at the N-terminus and a C-terminal His₈-tag in FreeStyle 293F cells or HEK293S-GnTI⁻ cells using the BacMam system . Enhance expression by supplementing with 3 mM sodium butyrate 12 hours after infection .

This approach ensures proper protein folding and post-translational modifications needed for studying physiologically relevant interactions.

How can researchers effectively verify functional activity of recombinant GFRA3?

Verification of GFRA3 functional activity requires multiple complementary approaches:

• Binding assays: Implement pull-down assays using strep-tagged GFRA3 to confirm binding to Artemin and RET . Quantify binding affinities using surface plasmon resonance or bio-layer interferometry.

• Complex formation: Verify proper complex formation with RET and Artemin through size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS).

• Structural integrity: Use circular dichroism spectroscopy to confirm proper folding of the recombinant protein.

• Signaling assays: In cellular systems, measure RET phosphorylation and downstream signaling activation (MAPK, PI3K/AKT pathways) upon Artemin binding to GFRA3.

Active GFRA3 should demonstrate specific binding to Artemin, complex formation with RET, and trigger appropriate downstream signaling events when tested in cellular systems.

What is GFRA3's expression pattern during embryonic development?

GFRA3 exhibits a distinct developmental expression pattern:

GFRA3 is highly expressed by embryonic day 11 but not appreciably expressed in adult mice . In situ hybridization analyses demonstrate that GFRA3 is specifically located in dorsal root ganglia and the superior cervical sympathetic ganglion during development .

To study this expression pattern, researchers should employ techniques such as in situ hybridization, immunofluorescence on cryosections, and qRT-PCR analysis at different developmental timepoints. Gene expression profiling reveals that GFRA3 mRNA is strongly enriched in endocrine progenitors (FC=33.25; FDR=0.02) .

How does GFRA3 function in pancreatic development?

GFRA3's role in pancreatic development presents an intriguing research area:

GFRA3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors and embryonic α- and β-cells . Gene expression profiling in sorted Neurog3-positive cells from Ngn3-EYFP/+ E15.5 embryonic pancreas confirmed strong enrichment of GFRA3 in islet lineage cells .

To investigate GFRA3's function in pancreatic development:

• Use GFRα3-deficient mice to study loss-of-function effects

• Generate transgenic mice overexpressing Artemin in the embryonic pancreas to study gain-of-function effects

• Perform lineage tracing experiments to follow the fate of GFRA3-expressing progenitors

Interestingly, despite this expression pattern, analysis of GFRα3 knockout mice and transgenic mice overexpressing Artemin revealed that the Artemin/GFRα3 signaling pathway is not essential for islet formation, innervation, and function . This suggests potential redundancy with other signaling pathways or more subtle roles in pancreatic development that require sensitive assays to detect.

What is the relationship between GFRA3 and RET expression in the developing nervous system?

The relationship between GFRA3 and RET expression provides insight into potential functional interactions:

Comparison of GFRA3 and RET expression patterns suggests they could form a receptor pair and interact with GDNF family members to play unique roles in development . GFRA3 is specifically located in dorsal root ganglia and the superior cervical sympathetic ganglion, regions where RET is also expressed .

To study this relationship experimentally:

• Perform double-labeling immunohistochemistry or in situ hybridization to map co-expression patterns

• Use proximity ligation assays to detect physical interactions between endogenous GFRA3 and RET

• Implement conditional knockout approaches targeting either receptor in specific tissues

• Analyze phenotypes of single and double knockouts to determine functional redundancy or synergy

These approaches can elucidate whether GFRA3 and RET function together in specific developmental contexts or have independent roles.

How does the ARTN/GFRA3/RET complex assemble?

The assembly of ARTN/GFRA3/RET complex has been characterized through cryo-EM analyses:

The model building of ARTN/GFRA3/RET complex involves docking the crystal structure of ARTN/GFRα3 complexes (PDB ID: 2GH0) into the one-wing map . The interfaces between GFRA3 and RET (buried surface areas ~730–1010 Ų) are more extensive than those in other similar complexes, which may compensate for potentially weaker interactions elsewhere .

To experimentally study this complex assembly:

• Employ multi-angle light scattering to determine complex stoichiometry

• Use hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Generate domain-specific mutations to disrupt specific interfaces

• Apply single-particle electron microscopy to visualize different assembly states

The D1 domain of GFRA3 is invisible in 3D reconstructions, suggesting it doesn't participate in binding to either RET or ligands . This contrasts with GFRα2, where the D1 domain packs closely with the D3 domain and makes contacts with RET-CLD1 .

What methodological approaches are most effective for studying GFRA3 structural biology?

Structural biology studies of GFRA3 benefit from several complementary approaches:

• Cryo-electron microscopy: This has been successfully used to determine the structure of ARTN/GFRα3/RET complexes. Use the following protocol:

  • Express proteins in HEK293S-GnTI⁻ cells for optimal glycosylation control

  • Apply symmetry expansion and focused refinement approaches

  • Resolution estimation using gold-standard Fourier shell correlation (FSC) = 0.143 criterion

• X-ray crystallography: This can provide higher resolution for specific domains:

  • The crystal structure of ARTN/GFRα3 complex (PDB ID: 2GH0) has been determined

  • Focus on stable subdomains if full-length protein proves challenging

• Molecular dynamics simulations: To study conformational changes and dynamic interactions:

  • Use existing structures as starting points

  • Simulate ligand binding and complex formation events

  • Identify potential allosteric sites and conformational changes

• Hydrogen-deuterium exchange mass spectrometry: For mapping flexible regions and binding interfaces that may not be resolved in static structures

These methodologies provide complementary information about GFRA3 structure from different perspectives, enabling a more complete understanding of its function.

What are the key differences in binding interfaces between GFRA3/RET and other GFRα/RET complexes?

Comparative analysis of binding interfaces reveals important structural distinctions:

Interfaces II of GFRα1, GFRα2, and GFRα3 with RET (buried surface areas ~730–1010 Ų) are more extensive than that in GFRAL/RET (buried surface areas ~680 Ų), which may partially compensate for potentially weaker interfaces elsewhere .

The D1 domain of GFRA3 is invisible in 3D reconstructions of ARTN/GFRα3/RET complexes, suggesting it doesn't participate in binding. This contrasts with GFRα2, where the D1 domain is resolved in the cryo-EM map of the NRTN/GFRα2/RET complex and packs closely with the D3 domain .

To experimentally investigate these differences, researchers should design mutagenesis studies targeting specific interface residues and assess their impact on complex formation and signaling.

How can structural insights into GFRA3 complexes inform drug discovery efforts?

Structural insights into GFRA3 complexes offer several avenues for drug discovery:

• Structure-based drug design: The high-resolution structures of ARTN/GFRα3/RET complexes provide templates for in silico screening of compounds that could modulate this interaction. Focus on the extensive interface II (buried surface areas ~730–1010 Ų) as a potential target .

• Peptide mimetics: Design peptides that mimic key binding regions of either GFRA3 or Artemin to develop agonists or antagonists of the signaling pathway.

• Allosteric modulators: Identify potential binding pockets away from the primary interaction sites that could alter the conformation and function of GFRA3.

• Bispecific antibodies: Develop antibodies that can simultaneously target GFRA3 and RET to either enhance or inhibit complex formation based on therapeutic needs.

These approaches could lead to therapeutics for neurological disorders involving GFRA3 signaling, particularly in sensory and sympathetic neurons where GFRA3 is predominantly expressed .

What are the implications of GFRA3's expression in neuronal populations for neurodegenerative disease research?

GFRA3's expression in specific neuronal populations has important implications for neurodegenerative disease research:

GFRA3 is located in dorsal root ganglia and the superior cervical sympathetic ganglion , suggesting a role in sensory and sympathetic neuron development and maintenance. The GDNF family of ligands, including Artemin, are known to promote neuronal survival and regeneration .

To investigate GFRA3's potential in neurodegenerative disease research:

• Examine GFRA3 expression in animal models of peripheral neuropathy and sensory neuron degeneration

• Test whether Artemin administration can protect GFRA3-expressing neurons in disease models

• Develop conditional knockout or overexpression models to assess GFRA3's role in neuronal survival under stress conditions

• Investigate potential genetic variants of GFRA3 in patient populations with sensory neuropathies

Since GFRA3 is unable to activate RET in conjunction with GDNF , therapeutic approaches should specifically target Artemin/GFRA3 signaling rather than general GDNF family signaling.

What are unresolved questions regarding GFRA3 signaling that warrant further investigation?

Several important questions about GFRA3 signaling remain unresolved and merit further investigation:

• Ligand specificity: Despite structural similarity to other GFRα receptors, GFRA3 is unable to activate RET in conjunction with GDNF . What structural features determine this specificity, and are there undiscovered ligands for GFRA3?

• Developmental redundancy: Analysis of GFRα3 knockout mice revealed that the Artemin/GFRα3 signaling pathway is not essential for islet formation, innervation, and function . What compensatory mechanisms exist, and in what contexts is GFRA3 signaling truly indispensable?

• GPI-linkage significance: GFRA3 is anchored to the cell membrane by a phosphatidylinositol-specific phospholipase C-resistant glycosyl-phosphatidylinositol linkage . How does this anchoring mechanism regulate signaling dynamics and receptor localization?

• Signaling beyond RET: Are there RET-independent signaling mechanisms for GFRA3, similar to what has been described for other GFRα receptors?

• Temporal regulation: What mechanisms regulate the high embryonic expression of GFRA3 that diminishes in adulthood , and can this developmental expression pattern be manipulated for therapeutic purposes?

Addressing these questions will require innovative experimental approaches combining genetic, biochemical, and advanced imaging techniques to fully elucidate GFRA3's role in development and disease.