FCHSD2 Antibody

What is FCHSD2 and what are its structural characteristics?

FCHSD2 (FCH and double SH3 domains 2) is an adaptor protein that plays a role in endocytosis via clathrin-coated pits. The canonical human FCHSD2 protein consists of 740 amino acid residues with a molecular mass of approximately 84.3 kDa. FCHSD2 is a member of the mild curvature-generating F-BAR family of proteins, which are known to function in early stages of clathrin-coated pit initiation and stabilization .

The protein contains multiple domains including an F-BAR domain and SH3 domains that mediate its protein-protein interactions. These structural elements are crucial for its function in regulating actin assembly and membrane dynamics during endocytosis. Up to three different isoforms of FCHSD2 have been identified in humans .

Where is FCHSD2 localized within cells and what are its known functions?

FCHSD2 is primarily localized in the cell membrane and cytoplasm . Its subcellular localization is critical for its function, as it is recruited to the plasma membrane and specifically to clathrin-coated pits (CCPs) where it participates in endocytosis .

The protein plays a crucial role in clathrin-mediated endocytosis (CME), particularly in the initiation of clathrin-coated pits. Research has shown that FCHSD2 affects the rate of CCP initiation without affecting CCP lifetimes, suggesting its importance in the early stages of endocytosis rather than in later maturation steps . Additionally, FCHSD2 regulates the endocytic trafficking and surface expression of epidermal growth factor receptor (EGFR), which has significant implications for cancer cell signaling and behavior .

What are the common synonyms and orthologs of FCHSD2?

Researchers should be aware of several synonyms when searching for literature on FCHSD2:

• NWK1

• SH3MD3

• F-BAR and double SH3 domains protein 2

• FCH and double SH3 domains protein 2

• SH3 multiple domains 3

• NWK

FCHSD2 is evolutionarily conserved, with orthologs reported in multiple species including:

• Mouse

• Rat

• Bovine

• Frog

• Chimpanzee

• Chicken

The conservation across species suggests important fundamental biological roles for this protein, making comparative studies possible across model organisms.

What are the primary applications for FCHSD2 antibodies in research?

FCHSD2 antibodies are used in multiple research applications, with Western Blot (WB) being the most widely utilized method. Common applications include:

When selecting an FCHSD2 antibody, researchers should ensure the antibody has been validated for their specific application of interest .

What types of FCHSD2 antibodies are available and how should they be selected?

Various types of FCHSD2 antibodies are available for different research applications:

• Unconjugated antibodies: These are versatile and can be used for most applications including Western blot, IHC, and ELISA .

• Conjugated antibodies: These include:

  • Biotin-conjugated: Useful for signal amplification strategies

  • HRP-conjugated: Directly applicable for Western blots and ELISA

  • FITC-conjugated: Used for direct fluorescence detection

When selecting an antibody, researchers should consider:

• The specific epitope recognized (N-terminal, C-terminal, or internal regions)

• Species reactivity (human, mouse, rat, etc.)

• Clonality (monoclonal vs. polyclonal)

• Validation data available for specific applications

• Post-translational modifications that might affect epitope recognition

How can researchers validate the specificity of FCHSD2 antibodies?

Validating antibody specificity is crucial for reliable results. For FCHSD2 antibodies, recommended validation approaches include:

• siRNA knockdown validation: Use siRNA to deplete FCHSD2 and confirm the reduction of signal in Western blot or immunofluorescence. This approach has been successfully employed to validate antibodies for Western blotting, although some commercially available antibodies may show nonspecific staining in immunofluorescence applications .

• Recombinant protein expression: Express tagged versions of FCHSD2 (such as FCHSD2-Myc) and confirm detection with both tag-specific and FCHSD2-specific antibodies .

• Multiple antibody approach: Use antibodies recognizing different epitopes of FCHSD2 and confirm consistent results.

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody pulls down the correct protein.

Note that C-terminally GFP-tagged FCHSD2 has been reported to form aggregates in the perinuclear region, which are not seen with Myc-tagged protein, suggesting that the C-terminal tag may affect protein localization and function .

What is the significance of FCHSD2 in cancer progression?

FCHSD2 has emerged as a potentially important factor in cancer progression, particularly in non-small cell lung cancer (NSCLC). Research indicates that FCHSD2 functions as a negative regulator of cancer aggressiveness:

• Survival correlation: NSCLC patients with relatively high FCHSD2 expression demonstrate significantly better survival rates compared to those with low expression .

• Migration and proliferation: FCHSD2 depletion enhances cancer cell migration and proliferation in experimental models. Reconstitution with wild-type FCHSD2 or phosphomimetic mutants (S681E) can reverse these effects .

• EGFR regulation: FCHSD2 regulates the endocytic trafficking and surface expression of EGFR, a predominant oncogenic receptor in NSCLC. Depletion of FCHSD2 increases surface EGFR levels, potentially enhancing oncogenic signaling .

These findings suggest that FCHSD2 may act as a tumor suppressor in certain contexts, making it an important target for cancer research.

How does ERK1/2 signaling regulate FCHSD2 function in cancer cells?

ERK1/2 directly regulates FCHSD2 function through phosphorylation at a specific site:

• Phosphorylation site: ERK1/2 phosphorylates FCHSD2 at serine 681 (S681), which is located within a canonical ERK phosphorylation motif (PXSP) .

• Functional consequences: Phosphorylation of FCHSD2 at S681 is required for:

  • FCHSD2 recruitment to the plasma membrane and clathrin-coated pits

  • ERK1/2-dependent regulation of clathrin-mediated endocytosis

  • Proper control of EGFR trafficking

• Mutational analysis: Research using FCHSD2 mutants has demonstrated:

  • Nonphosphorylatable mutant (S681A): Fails to restore normal endocytic function and ERK1/2 sensitivity

  • Phosphomimetic mutant (S681E): Restores function and renders cells resistant to ERK1/2 inhibition

This regulatory mechanism appears particularly important in cancer cells, where ERK1/2 signaling is often dysregulated.

What experimental models have been used to study FCHSD2 in cancer?

Several experimental models have been employed to study FCHSD2 function in cancer:

Research approaches have included:

• siRNA-mediated knockdown of FCHSD2

• Reconstitution with wild-type or mutant FCHSD2 constructs

• Pharmacological inhibition of ERK1/2 signaling

• Measurement of clathrin-mediated endocytosis using transferrin and EGFR uptake assays

• Live-cell imaging to track clathrin-coated pit dynamics

• Migration assays to assess cancer cell motility

These models have revealed that cancer cells appear particularly dependent on FCHSD2 for proper endocytic function.

How can researchers effectively study FCHSD2's role in clathrin-mediated endocytosis?

Studying FCHSD2's role in clathrin-mediated endocytosis (CME) requires specialized techniques:

• CME Cargo Uptake Assays:

  • Transferrin uptake: Use fluorescently labeled transferrin to measure endocytosis rates

  • EGFR uptake: Measure EGF-stimulated EGFR internalization

  • Surface biotinylation: Quantify internalized vs. surface proteins

• Live-Cell Imaging Approaches:

  • Total internal reflection fluorescence (TIRF) microscopy to visualize events at the plasma membrane

  • Automated detection and analysis of clathrin-coated pit dynamics

  • Quantification of:

    • CCP initiation rates

    • CCP lifetimes

    • CCP maturation events

• Biochemical Analysis:

  • Co-immunoprecipitation to identify FCHSD2 binding partners

  • Phospho-proteomic analysis to identify phosphorylation events (e.g., S681 phosphorylation)

When interpreting results, researchers should consider that FCHSD2 may have different roles in normal versus cancer cells, as demonstrated by its differential effects on CME in these contexts.

What approaches can be used to study FCHSD2 phosphorylation?

Several techniques can be employed to study FCHSD2 phosphorylation:

• Phospho-specific antibodies: While not specifically mentioned in the search results, developing phospho-specific antibodies against the S681 phosphorylation site would be valuable.

• Phospho-proteomic analysis: This approach has been successfully used to confirm EGF-dependent phosphorylation of FCHSD2 at S681 in serum-starved and EGF-treated HCC4017 cells .

• Mutational analysis: Creating phospho-deficient (S681A) and phosphomimetic (S681E) mutants has proven effective for studying the functional significance of S681 phosphorylation .

• Kinase inhibition studies: Using ERK1/2 inhibitors to modulate phosphorylation and assess functional consequences .

• In vitro kinase assays: While not explicitly mentioned in the search results, in vitro kinase assays could confirm direct phosphorylation of FCHSD2 by ERK1/2.

These approaches can be combined to provide comprehensive understanding of how phosphorylation regulates FCHSD2 function.

What are the considerations for localizing FCHSD2 in cells?

Localizing FCHSD2 in cells presents several technical challenges and considerations:

• Antibody selection: Commercial antibodies for immunofluorescence may show nonspecific staining. Validation by siRNA knockdown is essential to confirm specificity .

• Protein tagging considerations:

  • C-terminal GFP tagging can cause aberrant localization, with FCHSD2-GFP forming perinuclear aggregates

  • Myc-tagging appears to preserve proper localization and function

  • Validate tagged constructs functionally before using for localization studies

• Colocalization markers:

  • Use established markers for clathrin-coated pits (e.g., clathrin light chain, AP-2)

  • Consider markers for endosomal compartments to track FCHSD2 throughout the endocytic pathway

• Imaging techniques:

  • Super-resolution microscopy may be necessary to resolve individual clathrin-coated pits

  • TIRF microscopy is valuable for visualizing events at the plasma membrane

  • Confocal microscopy for general subcellular localization

Careful consideration of these factors will help ensure accurate determination of FCHSD2 localization.

How do FCHSD2 interactions with actin cytoskeleton components influence its function?

FCHSD2 belongs to the F-BAR protein family and likely influences actin dynamics during endocytosis:

• WASP activation: Studies of the Drosophila homolog Nwk indicate that FCHSD2 may regulate actin assembly through activation of neural Wiskott–Aldrich syndrome protein (N-WASP) .

• F-BAR domain function: The F-BAR domain of FCHSD2 is associated with membrane curvature generation, which is important for early stages of CCP formation. This mild curvature-generating property distinguishes FCHSD2 from other F-BAR proteins like FCHo1/2 .

• Endosomal sorting: Studies in Drosophila suggest FCHSD2 may regulate sorting in early endosomal compartments, potentially through actin-dependent mechanisms .

• Domain-specific interactions: The domain structure of FCHSD2 and its known protein interactions suggest multiple points of contact with the actin cytoskeleton and endocytic machinery .

Further research is needed to fully characterize the interplay between FCHSD2, actin dynamics, and membrane trafficking in both normal and cancer cells.

How does FCHSD2 function differ between normal and cancer cells?

Notable differences in FCHSD2 function between normal and cancer cells include:

• CME dependency:

  • Cancer cells (H1299, HCC4017): Highly dependent on FCHSD2 for clathrin-mediated endocytosis

  • Normal cells (ARPE-19, HBEC30KT): Less dependent on FCHSD2 for general CME

• EGFR trafficking:

  • In nontumorigenic ARPE-19 cells: EGFR endocytosis is not significantly affected by FCHSD2 knockdown

  • In HBEC30KT cells: EGFR uptake shows moderate sensitivity to FCHSD2 knockdown

  • In cancer cells: EGFR uptake is strongly dependent on FCHSD2

• ERK1/2 sensitivity:

  • Cancer cells show greater sensitivity to ERK1/2 inhibition in terms of FCHSD2-mediated endocytosis

  • This suggests cancer cells may rely more heavily on the ERK1/2-FCHSD2 axis

These differences suggest potential therapeutic opportunities targeting FCHSD2 function specifically in cancer cells while sparing normal tissues.

What is the relationship between FCHSD2 and patient outcomes in different cancer types?

Current evidence suggests FCHSD2 expression positively correlates with better outcomes in lung cancer:

• NSCLC survival data: Non-small cell lung cancer patients with relatively high FCHSD2 expression demonstrate significantly better survival rates than those with low expression .

• Biological basis:

  • FCHSD2 negatively regulates cancer cell migration and proliferation

  • It controls EGFR surface levels and trafficking

  • These mechanisms may explain the correlation with improved survival

• Potential as biomarker:

  • FCHSD2 expression levels might serve as a prognostic biomarker in lung cancer

  • Further validation in larger cohorts and other cancer types is needed

  • Analysis of FCHSD2 phosphorylation status may provide additional prognostic information

Additional research is needed to determine if this relationship extends to other cancer types and to understand the molecular mechanisms underlying the correlation between FCHSD2 expression and patient outcomes.