Recombinant Human Sprouty-related, EVH1 domain-containing protein 1 (SPRED1)

What is SPRED1 and what is its fundamental role in cellular signaling?

SPRED1 (Sprouty-related, EVH1 domain-containing protein 1) is a 50-kDa protein that functions as a negative regulator of the Ras/MAPK signaling pathway. It specifically inhibits this pathway in response to growth factor-, cytokine-, and chemokine-induced ERK activation . SPRED1 acts as a tumor suppressor in multiple contexts, including pediatric acute myeloblastic leukemia . Its primary function involves binding to neurofibromin (the protein product of the NF1 gene) via its N-terminal EVH1 domain and mediating the translocation of neurofibromin to the plasma membrane, where it can inactivate Ras through its GTPase activating protein (GAP) activity .

The significance of SPRED1 in cellular signaling is underscored by the fact that loss-of-function mutations in the SPRED1 gene cause Legius syndrome, a disorder with partially overlapping symptoms with Neurofibromatosis type 1 (NF1) . This clinical connection provides strong evidence for SPRED1's critical role in regulating the same signaling pathway affected in NF1.

How is the structure of SPRED1 organized and how does it relate to function?

SPRED1 comprises three distinct functional domains:

• N-terminal Ena/VASP Homology 1 (EVH1) domain: This domain functions as a protein-protein interaction module that specifically binds to the GAP-related domain (GRD) of neurofibromin .

• Central c-Kit binding domain: Located between the EVH1 and SPR domains .

• C-terminal Sprouty-related domain (SPR): This domain undergoes palmitoylation, which is crucial for SPRED1's membrane localization .

This domain organization is directly related to SPRED1's function as a molecular chaperone for neurofibromin. The EVH1 domain binds specifically to neurofibromin's GRD domain, while the SPR domain facilitates membrane targeting of the SPRED1-neurofibromin complex . This dual functionality enables SPRED1 to recruit neurofibromin to the plasma membrane where it can access and inactivate membrane-bound Ras proteins .

What molecular interactions mediate SPRED1's function in Ras/MAPK pathway inhibition?

SPRED1 forms a ternary complex with neurofibromin and active KRAS, as revealed by structural studies . The EVH1 domain of SPRED1 binds specifically to the extra domain (GAPex) of the GAP-related domain (GRD) of neurofibromin with nanomolar affinity . This binding does not interfere with neurofibromin's interaction with Ras or its GAP activity .

The molecular mechanism works as follows:

• The EVH1 domain of SPRED1 binds to the GAPex portion of neurofibromin's GRD

• The SPR domain of SPRED1 undergoes palmitoylation, which targets the SPRED1-neurofibromin complex to the plasma membrane

• At the membrane, neurofibromin can then access and inactivate Ras through its GAP activity

This model is supported by the finding that SPRED1 and KRAS interact with neurofibromin via two separate interfaces, allowing the formation of a functional ternary complex . The binding is highly specific, as demonstrated by the fact that pathogenic mutations that cause Legius syndrome disrupt the SPRED1-neurofibromin interaction .

What are the optimal methodologies for producing and purifying recombinant human SPRED1 protein?

For successful production of recombinant human SPRED1 protein, researchers have employed several complementary approaches:

Expression Systems:

• E. coli: Suitable for producing individual domains (particularly the EVH1 domain) with high yields .

• Insect cells: Preferred for full-length SPRED1 or multi-domain constructs that require eukaryotic post-translational modifications .

Purification Strategy:

• Affinity chromatography using immobilized recombinant SPRED1(EVH1) has proven effective for capturing GRD-containing fragments of neurofibromin .

• For analyzing interactions, in vitro affinity purification approaches with immobilized recombinant proteins have been successful in defining binding specificities .

When studying the interaction between SPRED1 and neurofibromin, it is advisable to prepare various soluble segments of these proteins. The EVH1 domain of SPRED1 can be successfully expressed and purified as an isolated domain (residues 1-137), while the GRD domain of neurofibromin can be expressed either as the minimal catalytic domain (GRDmin) or including the extra domain (GAPex) .

For structural studies, purification to homogeneity is essential, which typically requires a combination of affinity chromatography followed by size-exclusion chromatography to achieve the purity required for crystallization or other structural techniques .

How can researchers effectively study SPRED1-NF1-KRAS interactions in experimental settings?

Several complementary approaches have proven effective for studying the SPRED1-NF1-KRAS interactions:

In Vitro Binding Assays:

• Immobilized protein binding: Purified recombinant SPRED1(EVH1) can be immobilized and used to capture neurofibromin fragments containing the GRD domain .

• Surface Plasmon Resonance (SPR): Useful for determining binding affinities between SPRED1 and neurofibromin domains.

Cellular Approaches:

• Co-immunoprecipitation: Flag-tagged neurofibromin constructs can be co-expressed with SPRED1 in human embryonic kidney cells to analyze their interaction .

• Biochemical fractionation: This technique can be used to determine whether SPRED1 expression increases the membrane localization of neurofibromin fragments .

Structural Analysis:
The structure of the ternary complex formed by NF1(GRD), SPRED1(EVH1), and active KRAS has been determined, showing that SPRED1 and KRAS interact with NF1 via two separate interfaces . This provides a powerful framework for designing experiments to test specific aspects of the interaction.

Functional Assays:
Researchers can assess the functional consequences of the SPRED1-NF1 interaction by measuring:

• RAS activation status (RAS-GTP loading) in cells expressing wildtype or mutant SPRED1/NF1

• ERK phosphorylation as a downstream readout of RAS/MAPK pathway activity

By combining these approaches, researchers can gain comprehensive insights into both the structural details and functional significance of the SPRED1-NF1-KRAS interaction.

What are the molecular determinants of pathogenic SPRED1 mutations in Legius syndrome?

Legius syndrome is caused by loss-of-function mutations in the SPRED1 gene. Analysis of these pathogenic mutations provides valuable insights into the critical functional regions of the SPRED1 protein:

Types of Pathogenic Mutations:

• Truncating mutations: The majority of pathogenic SPRED1 mutations result in premature stop codons, producing truncated translation products .

• Missense mutations: Non-truncating missense mutations are predominantly located within the EVH1 domain .

Functional Consequences:
EVH1 domain mutations (such as T102→R and W31→C) prevent interaction with the GRD-containing fragments of neurofibromin . These mutations disrupt the SPRED1-neurofibromin binding interface, preventing proper recruitment of neurofibromin to the plasma membrane.

Structural Basis:
The location of these mutations corresponds to the surface of the EVH1 domain that interacts with the GAPex region of neurofibromin's GRD . Understanding the structural basis of these interactions helps explain why these specific mutations cause disease.

Experimental Validation:
To study the effects of Legius syndrome mutations, researchers have:

• Introduced patient-derived mutations into recombinant SPRED1 constructs

• Assessed binding to neurofibromin fragments using in vitro pull-down assays

• Examined membrane localization of neurofibromin in cells expressing mutant SPRED1

These approaches have confirmed that pathogenic mutations disrupt the SPRED1-neurofibromin interaction, providing a molecular explanation for how these mutations lead to dysregulated Ras/MAPK signaling in Legius syndrome.

How can SPRED1 expression be accurately quantified in patient samples for diagnostic and prognostic purposes?

SPRED1 expression analysis in patient samples can be performed using multiple complementary techniques:

mRNA Quantification:
Real-time quantitative PCR (RT-qPCR) on bone marrow mononuclear cells has been successfully used to measure SPRED1 mRNA levels in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) . This approach allows for sensitive detection of differences in expression levels between different patient groups.

Protein Detection:

• Immunocytochemistry: This technique can confirm mRNA findings at the protein level in patient samples .

• ELISA: Measurement of serum SPRED1 levels using enzyme-linked immunosorbent assay provides a less invasive method for monitoring SPRED1 expression .

Data Analysis and Interpretation:
When analyzing SPRED1 expression in patient samples, researchers should:

• Include appropriate control groups (e.g., healthy donors, other disease types)

• Consider disease subtypes (e.g., AML subtypes such as M2 and M3 show significant downregulation of SPRED1)

• Correlate expression data with clinical outcomes to assess prognostic value

Prognostic Value:
Studies have shown that SPRED1 expression has prognostic significance in AML. Non-APL (acute promyelocytic leukemia) patients with decreased SPRED1 expression had significantly lower 2-year progression-free survival and event-free survival rates . This finding highlights the potential utility of SPRED1 as a biomarker in certain cancer types.

What experimental approaches are recommended for studying SPRED1 membrane localization?

SPRED1's function depends critically on its ability to localize to the plasma membrane and recruit neurofibromin. Several experimental approaches can be used to study this process:

Biochemical Fractionation:
This technique separates cellular components into membrane and cytosolic fractions, allowing quantification of the proportion of SPRED1 and/or neurofibromin in each fraction . It has been successfully used to demonstrate that:

• Spred1 expression increases the membrane localization of endogenous neurofibromin

• Only GRD-containing neurofibromin fragments are recruited to the membrane by Spred1

Live Cell Imaging:
Fluorescently tagged SPRED1 constructs can be used to visualize its localization in living cells. This approach allows researchers to:

• Track dynamic changes in SPRED1 localization in response to stimuli (e.g., EGF stimulation)

• Visualize the co-localization of SPRED1 with neurofibromin and membrane markers

• Assess the effects of mutations on SPRED1 localization patterns

Mechanistic Studies:
To understand the molecular basis of SPRED1 membrane localization, researchers can:

• Study the role of palmitoylation of the SPR domain using palmitoylation inhibitors or site-directed mutagenesis

• Investigate the effects of EGF stimulation or galectin-1-mediated induction of RAF dimers, which have been shown to translocate SPRED1 to the plasma membrane

• Examine how pathogenic mutations in the SPR domain prevent membrane localization of the SPRED-NF1 complex

Functional Consequences:
The biological significance of SPRED1 membrane localization can be assessed by measuring:

• RAS activation status in cells expressing wildtype vs. membrane-localization-deficient SPRED1 mutants

• Downstream signaling events such as ERK phosphorylation

• Cellular phenotypes relevant to RAS/MAPK pathway activity (e.g., proliferation, differentiation)

What is the role of SPRED1 in cancer development and how can it be targeted therapeutically?

SPRED1 functions as a tumor suppressor in multiple contexts, with particularly strong evidence in hematological malignancies:

Expression in Cancer:
SPRED1 is significantly downregulated in acute myeloid leukemia (AML) compared to both acute lymphoblastic leukemia (ALL) patients and healthy controls . This downregulation has been confirmed at both the mRNA and protein levels through multiple detection methods .

Clinical Significance:
Expression analysis reveals that:

• SPRED1 expression is significantly higher in AML patients who achieve complete remission after induction treatment compared to expression levels at diagnosis

• SPRED1 is particularly downregulated in specific AML subtypes (M2 and M3)

• Non-APL patients with decreased SPRED1 expression have significantly lower 2-year progression-free and event-free survival rates

This data supports SPRED1's role as a prognostic biomarker in AML and suggests that restoring SPRED1 function could have therapeutic potential.

Therapeutic Implications:
Understanding the SPRED1-NF1-KRAS interaction provides potential opportunities for therapeutic intervention:

• Compounds that enhance SPRED1-NF1 binding could potentially restore normal regulation of RAS signaling

• Small molecules that mimic SPRED1's action in recruiting NF1 to the membrane might bypass the need for SPRED1 itself

• Gene therapy approaches to restore SPRED1 expression in cancers where it is downregulated

Future therapeutic development will require deeper understanding of the structural basis of the SPRED1-NF1 interaction and the mechanisms regulating SPRED1 expression in cancer cells.

How do mutations in SPRED1 contribute to the pathophysiology of Legius syndrome?

Legius syndrome is caused by loss-of-function mutations in the SPRED1 gene and shares clinical features with Neurofibromatosis type 1 (NF1):

Mutation Spectrum:
The majority of Legius syndrome-associated mutations in the SPRED1 gene result in premature stop codons, likely producing truncated proteins . Most identified non-truncating missense mutations are located within the EVH1 domain of SPRED1 .

Molecular Mechanism:
These mutations disrupt the normal function of SPRED1 by:

• Preventing binding between the EVH1 domain of SPRED1 and the GRD of neurofibromin

• Disrupting membrane localization of the SPRED1-NF1 complex

This results in impaired recruitment of neurofibromin to the plasma membrane, leading to insufficient suppression of RAS activity and hyperactivation of the RAS/MAPK pathway.

What sequence analysis methods should be used to identify and characterize SPRED1 mutations?

For comprehensive identification and characterization of SPRED1 mutations, researchers should employ the following methodological approach:

PCR Amplification:
The exons and flanking areas of the SPRED1 gene should be amplified by PCR. The optimal PCR mixture (25 μl) should contain:

• 50 ng of template DNA

• 1X PCR buffer

• 0.2 mmol/L dNTP mix

• 50 ng of each primer

• 2.5 mmol/L MgCl2

• 1 unit of AmpliTaq Gold (Applied Biosystems)

Sequencing:
The ddNTP terminator reaction should be carried out with the ABI BigDye Terminator v3.1 Cycle Sequencing kit, with sequencing products loaded and data collected on an ABI 3130 xl genetic analyzer .

Mutation Analysis:
When analyzing sequencing data, researchers should:

• Compare sequences to reference databases to identify variants

• Distinguish between pathogenic mutations and polymorphisms

• Predict the functional consequences of identified mutations using in silico tools

• Validate novel mutations through functional studies

Functional Validation:
For novel or uncharacterized mutations, functional validation is essential to determine pathogenicity:

• In vitro binding assays to assess the effect on interaction with neurofibromin

• Cell-based assays to evaluate effects on membrane localization

• Signaling assays to measure impact on RAS/MAPK pathway regulation

This comprehensive approach ensures accurate identification and characterization of SPRED1 mutations for both research and diagnostic purposes.

What are the latest structural insights into the SPRED1-Neurofibromin-KRAS complex?

Recent structural studies have provided detailed insights into the SPRED1-Neurofibromin-KRAS complex:

Ternary Complex Structure:
The structure of a ternary complex formed by the NF1(GRD) with the SPRED1(EVH1 domain) and active KRAS (GMPPNP bound) has been determined . This structure reveals that:

• SPRED1 and KRAS interact with NF1 via two separate interfaces

• The EVH1 domain of SPRED1 binds to the extra domain (GAPex) of neurofibromin's GRD

• This binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity

Binding Interface:
The EVH1 domain of SPRED1 specifically recognizes the extra domain (GAPex) of the GRD of neurofibromin . This interaction is critical for the function of both proteins in regulating RAS signaling.

Functional Implications:
The structural data provides a molecular explanation for how:

• SPRED1 recruits neurofibromin to RAS through the EVH1-GRD interaction

• Pathogenic mutations in both proteins can disrupt this interaction

• The complex functions cooperatively to regulate RAS activity

Pathogenic Mutations:
The structure also provides a structural explanation for pathogenic mutations in SPRED1 and NF1. For instance, a patient-derived single residue deletion in neurofibromin (ΔM1215) that fails to interact with SPRED1 is located in the binding interface identified in the structure .

These structural insights provide a solid foundation for understanding the molecular basis of SPRED1 and NF1 function in normal physiology and disease.