FZD4 Human

What is the structure and function of FZD4 in human cells?

FZD4, located on human chromosome 11q14.2, contains two exons and encodes a 537-amino acid protein with distinct functional domains, including an N-terminal cysteine-rich domain (CRD), seven transmembrane domains, and a C-terminal S/T-X-V motif . As a member of the Frizzled family of transmembrane receptors, FZD4 plays a crucial role in regulating the Wnt signaling pathway . Recent cryo-EM structural analysis has revealed that FZD4 engages with the DEP domain of Dishevelled 2 (DVL2), a key WNT transducer, through a distinctive binding mode where the DEP finger-loop inserts into the FZD4 cavity to form a hydrophobic interface . Additionally, FZD4's intracellular loop 2 (ICL2) anchors the complex through polar contacts, establishing a molecular basis for signal transduction .

How does FZD4 participate in the Norrin/β-catenin signaling pathway?

FZD4 functions as a receptor in the Norrin/Wnt signaling pathway, which is essential for the development of the retinal vascular system . In canonical WNT signaling, the active WNT-FZD-LRP5/6 complex recruits the key transducer protein, Dishevelled (DVL), to the plasma membrane of cells, which then serves as a hub to recruit Axin and subsequently bring glycogen synthase kinase-3β (GSK3β) and casein kinase 1 (CK1) to the plasma membrane to form the membrane-associated signaling complex . The DEP interface is highly conserved in FZDs, indicating a universal mechanism by which FZDs engage with DVLs to propagate WNT signaling . Studies using Fzd4 knockout models have successfully replicated the observed features of FEVR, mainly attributed to impaired Norrin/β-catenin signals, while restoration of these signals via β-catenin overexpression has been shown to reverse the condition's features .

What pathologies are associated with FZD4 mutations in humans?

FZD4 mutations have been closely linked to Familial Exudative Vitreoretinopathy (FEVR), an inherited disorder characterized by abnormalities in the retinal vasculature . FEVR manifests with typical phenotypes including peripheral avascular areas, retinal folds, and in some cases, retinal detachment . Research has shown that FZD4 mutations account for approximately 15.2-21% of FEVR patients . Additionally, emerging evidence suggests that altered FZD4 expression may play a role in certain cancers, such as cutaneous squamous cell carcinoma (CSCC), where downregulation of FZD4 has been observed . This diverse pathological involvement underscores the critical role of FZD4 in both developmental processes and tissue homeostasis.

What methodological approaches are most effective for identifying novel FZD4 mutations?

For comprehensive mutation detection in FZD4, a multi-faceted approach is recommended. Whole Exome Sequencing (WES) has proven effective for identifying novel point mutations, as demonstrated in studies where researchers discovered previously unreported mutations such as FZD4 c.314T>G (p.M105R) . For targeted analysis, gene panel sequencing can be employed, particularly when investigating a cohort of patients with a specific phenotype like FEVR .

When investigating potential Copy Number Variations (CNVs), which are increasingly recognized as contributors to FZD4-related pathologies, methodologies such as Multiplex Ligation-dependent Probe Amplification (MLPA) or array Comparative Genomic Hybridization (aCGH) should be considered . These techniques can detect larger genomic deletions or duplications that might be missed by traditional sequencing approaches.

For validation of identified variants, Sanger sequencing remains the gold standard, particularly for confirming genotype-phenotype cosegregation within affected families . This comprehensive mutation screening approach ensures detection of the diverse mutation types in FZD4, including nonsense mutations, missense mutations, small deletions/insertions, and gross deletions .

How can researchers effectively assess the functional impact of FZD4 mutations?

To systematically evaluate the functional consequences of FZD4 mutations, a multi-layered experimental strategy is recommended:

These complementary approaches provide a comprehensive understanding of how specific mutations impact different aspects of FZD4 function, from protein expression and localization to downstream signaling events.

What cellular models are optimal for studying FZD4 signaling pathways?

When selecting cellular models for FZD4 research, consider the following options based on specific research objectives:

For basic signaling studies, HEK293T and HEK293STF cell lines have been extensively validated and serve as reliable models . The HEK293STF line is particularly valuable as it contains a stably integrated Super TOP Flash (STF) reporter construct, allowing direct measurement of β-catenin-dependent transcriptional activity.

For studies focusing on subcellular localization and protein trafficking, HeLa cells offer advantages due to their well-defined cellular architecture and compatibility with high-resolution microscopy techniques . These cells are particularly suitable for immunofluorescence studies evaluating membrane localization of wild-type and mutant FZD4 proteins.

For cancer-related FZD4 research, specific cancer cell lines like Colo16 (a CSCC cell line) provide relevant disease context . Studies have demonstrated that manipulating FZD4 expression in these cells affects proliferation and apoptosis, offering insights into potential therapeutic applications.

When investigating retinal vascular development, primary human retinal endothelial cells or established retinal cell lines may provide physiologically relevant contexts, though these models often require specialized culture conditions and expertise.

For complex in vivo questions, Fzd4 knockout mouse models have successfully recapitulated FEVR features and remain valuable for studying developmental aspects and testing potential therapeutic interventions .

How can researchers accurately quantify Norrin-FZD4 binding interactions?

Accurate quantification of Norrin-FZD4 binding interactions requires careful experimental design and standardization. A validated approach involves the following methodology:

First, establish consistent expression of FZD4 proteins across experimental conditions, as variability in expression levels can confound binding assessments. This can be achieved by standardizing transfection protocols and confirming equivalent expression via western blotting or flow cytometry .

For microscopy-based binding assays, standardize FZD4 fluorescence intensity during cell selection to maintain consistency in expression levels across analyzed cells. Since membrane expression can vary considerably between individual cells, select regions with moderate fluorescence intensity for analysis .

Utilize line segment analysis (approximately 1 μm) across the cell membrane to measure both FZD4 and NDP (Norrin) fluorescence intensity at consistent locations. Calculate the relative binding strength by dividing NDP's fluorescence intensity by FZD4's fluorescence intensity .

For immunofluorescence detection, specific antibodies such as rat anti-HA (3F10; Roche), rabbit anti-FLAG (14793S; Cell Signaling Technology), Alexa fluor 488 donkey anti-rat (A-21208; ThermoFisher), and Alexa fluor 647 donkey anti-rabbit (A-31573; ThermoFisher) have been successfully employed .

Advanced quantitative binding studies may employ surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding affinities (Kd values) between purified Norrin and FZD4 CRD domains, providing more precise thermodynamic parameters of the interaction.

What strategies can be employed to investigate the role of FZD4 in tumor biology?

To comprehensively investigate FZD4's role in tumor biology, researchers should implement a multi-dimensional approach:

Gene Expression Analysis: Begin by comparing FZD4 mRNA expression between tumor tissues and corresponding normal tissues or perilesional areas. RT-qPCR provides a quantitative assessment, as demonstrated in CSCC studies where FZD4 was found to be significantly downregulated in tumor tissues compared to perilesional tissues .

Protein Expression Confirmation: Validate mRNA findings at the protein level using western blotting and immunohistochemistry to assess both expression levels and localization patterns within tumor tissues .

Functional Studies Through Genetic Manipulation:

• For loss-of-function studies, utilize siRNA transfection to downregulate FZD4 expression. Validate knockdown efficiency at both mRNA and protein levels before proceeding with functional assays .

• For gain-of-function studies, transfect cells with FZD4-overexpressing plasmids and confirm successful overexpression before functional analysis .

Proliferation and Viability Assays: Employ CCK-8 assays to measure cell proliferation rates following FZD4 manipulation. These assays have revealed that FZD4 overexpression can inhibit proliferation in certain tumor cell lines, such as Colo16 .

Apoptosis Assessment: Use Annexin V and PI double staining analyzed by flow cytometry to evaluate the impact of FZD4 manipulation on apoptosis rates. Studies in CSCC cells demonstrated that FZD4 upregulation significantly increased apoptosis .

Pathway Analysis: Investigate the downstream effects of FZD4 on Wnt signaling components using western blotting and reporter assays to understand the molecular mechanisms underlying observed phenotypic changes.

This integrated approach provides comprehensive insights into both the expression patterns and functional significance of FZD4 in tumor development and progression, potentially identifying novel therapeutic targets.

How can copy number variations (CNVs) in the FZD4 gene be effectively detected and characterized?

Detection of copy number variations (CNVs) in FZD4 requires specialized approaches beyond standard sequencing methods. A systematic methodology includes:

Initial Screening: Implement Multiplex Ligation-dependent Probe Amplification (MLPA) as a first-line technique for detecting exon-level deletions or duplications in FZD4. This method allows for the simultaneous interrogation of multiple exons and can detect whole-gene deletions that might be missed by conventional sequencing .

Confirmation and Refinement: For MLPA-positive cases, proceed with array Comparative Genomic Hybridization (aCGH) or SNP arrays to define the precise boundaries of the CNV. These techniques provide higher resolution mapping of breakpoints and can determine if neighboring genes are affected .

Breakpoint Characterization: For detailed characterization of deletion/duplication breakpoints, design long-range PCR primers flanking the suspected breakpoints, followed by sequencing of the junction fragments. This approach provides nucleotide-level resolution of the CNV boundaries and can inform mechanisms of CNV formation.

Validation in Family Members: Extend testing to family members to establish segregation patterns and penetrance of the identified CNVs. This step is crucial for genetic counseling and establishing genotype-phenotype correlations .

Functional Assessment: For novel CNVs, consider functional studies such as RT-qPCR to assess the impact on transcript levels, or Western blotting to evaluate protein expression. These approaches help establish the pathogenicity of newly identified variants.

This comprehensive strategy ensures accurate detection and characterization of FZD4 CNVs, which have been established as important contributors to conditions such as FEVR but may be overlooked by standard genetic testing approaches.

What are the recommended protocols for analyzing FZD4-DVL2 interactions at the molecular level?

Analysis of FZD4-DVL2 interactions requires specialized techniques spanning structural, biochemical, and cellular approaches:

Cryo-Electron Microscopy (Cryo-EM): This technique has successfully revealed the structure of FZD4 engaged with the DEP domain of DVL2, showing that the DEP finger-loop inserts into the FZD4 cavity to form a hydrophobic interface. Cryo-EM offers the advantage of visualizing the complex in a near-native state without crystallization .

Co-Immunoprecipitation (Co-IP): To assess protein-protein interactions in cellular contexts, implement Co-IP using epitope-tagged versions of FZD4 and DVL2. Following cell lysis, immunoprecipitate one protein using an appropriate antibody and detect the co-precipitated partner by Western blotting. This approach confirms direct interaction and can assess how mutations affect binding .

BRET/FRET Assays: Bioluminescence/Fluorescence Resonance Energy Transfer assays provide quantitative, real-time measurement of protein interactions in living cells. For these assays, tag FZD4 and DVL2 with appropriate donor and acceptor fluorophores/bioluminescent proteins and measure energy transfer as an indicator of molecular proximity.

Domain Mapping through Mutagenesis: Systematically mutate key residues in both FZD4 and DVL2, particularly focusing on the intracellular loops of FZD4 and the DEP domain of DVL2, to pinpoint specific interaction interfaces. Functional experiments have validated that FZD4's intracellular loop 2 (ICL2) anchors the complex through polar contacts .

Cellular Localization Studies: Utilize confocal microscopy with fluorescently tagged proteins to visualize the co-localization of FZD4 and DVL2 in response to pathway activation. This approach provides spatial information about interaction dynamics.

These complementary approaches provide comprehensive insights into the molecular determinants governing FZD4/DVL assembly and contribute to understanding the propagation of WNT signaling.

How can genotype-phenotype correlations in FZD4-related FEVR be systematically established?

Establishing robust genotype-phenotype correlations for FZD4-related FEVR requires a methodical approach combining clinical assessment, genetic analysis, and functional studies:

Standardized Phenotypic Classification: Implement consistent clinical evaluation protocols including detailed fundus photography, fluorescein angiography (FFA), and optical coherence tomography (OCT) to categorize disease severity. The typical FEVR phenotypes include peripheral avascular areas, retinal folds, and in advanced cases, retinal detachment .

Comprehensive Genetic Analysis: Employ a tiered genetic testing strategy:

• Initial screening for point mutations via targeted sequencing or WES

• CNV analysis using MLPA or array-based methods

• Segregation analysis in families to establish variant pathogenicity and penetrance

Functional Categorization of Variants: Classify FZD4 variants based on their impact on protein function using in vitro assays:

• Protein expression analysis

• Membrane localization assessment

• Norrin binding capacity

• DVL2 recruitment ability

• Downstream signal activation via Topflash reporter assays

Longitudinal Follow-up: Track disease progression in mutation carriers over time, as intrafamilial variability has been observed where affected parents often exhibit milder phenotypes than their children, mostly manifesting only peripheral supernumerary branching or avascular areas on FFA .

Statistical Analysis: Apply multivariate analysis to identify correlations between specific mutation types/locations and clinical features, disease onset, progression rate, and treatment response.

This integrated approach facilitates reliable assessment of FEVR recurrence risk and improves genetic counseling by establishing clear connections between specific FZD4 variants and clinical outcomes.

What therapeutic approaches targeting FZD4 signaling are being investigated for retinal vascular disorders?

Research into therapeutic interventions targeting FZD4 signaling pathways for retinal vascular disorders encompasses several promising strategies:

Anti-VEGF Therapy: Intravitreous injection of anti-VEGF agents such as ranibizumab has been employed to treat extraretinal neovascularization in FEVR patients with FZD4 mutations . This approach targets downstream effects rather than the primary signaling defect but has shown efficacy in managing neovascular complications.

Wnt/β-catenin Pathway Modulation: Based on the understanding that FEVR results from impaired Norrin/β-catenin signaling, research is focusing on approaches to restore pathway activity. Studies in Fzd4 knockout models have demonstrated that restoration of β-catenin signals can reverse FEVR features, suggesting potential therapeutic avenues .

Gene Therapy Approaches: Development of vectors for FZD4 gene supplementation in patients with loss-of-function mutations represents a promising direction. Adeno-associated viral (AAV) vectors are being explored for retinal delivery of functional FZD4 genes, potentially addressing the root cause of the disease.

Small Molecule Screening: High-throughput screening efforts are identifying compounds that can enhance residual FZD4 signaling or activate alternative pathways to compensate for FZD4 dysfunction. These studies utilize cell-based assays with the Topflash reporter system to identify potential therapeutic candidates .

Combinatorial Approaches: Emerging research suggests that combining treatments addressing multiple aspects of the disease process (anti-angiogenic therapy plus pathway modulation) may yield superior outcomes compared to monotherapy.

While these approaches show promise in preclinical studies, translation to clinical applications requires careful evaluation of safety, efficacy, and delivery methods specific to ocular tissues. The development of personalized therapeutic strategies based on specific FZD4 mutation types may optimize treatment outcomes.

What are the emerging research frontiers in FZD4 biology?

The future of FZD4 research is expanding across multiple frontiers, driven by technological advances and evolving conceptual frameworks:

Structural Biology Integration: The recent cryo-EM structure of FZD4 engaged with the DEP domain of DVL2 has revealed that DEP mimics G-protein/β-arrestin/GRK to recognize an active conformation of the receptor, expanding current GPCR engagement models . This breakthrough opens opportunities for structure-based drug design targeting specific conformational states of FZD4.

Dimerization Mechanisms: Emerging evidence has identified a distinct FZD4 dimerization interface that may play crucial roles in signal transduction . Further characterization of homo- and hetero-dimerization patterns could reveal new regulatory mechanisms and therapeutic targets.

Cross-talk with Other Signaling Pathways: Investigation of how FZD4 signaling integrates with other pathways beyond canonical Wnt signaling will provide a more comprehensive understanding of its role in development and disease. This includes potential interactions with Notch, Hedgehog, and growth factor signaling networks.

Tissue-Specific Functions: While FZD4's role in retinal vascular development is well-established, its functions in other tissues require further exploration. The finding that FZD4 may play an antioncogenic role in cutaneous squamous cell carcinoma, contrary to its pro-tumorigenic role in other cancers, suggests complex tissue-specific functions .

Single-Cell Analysis: Application of single-cell technologies to study FZD4 expression and signaling at cellular resolution will provide insights into cell-type specific roles and heterogeneous responses to pathway perturbations.