AP2S1 Antibody Pair

What is AP2S1 and why is it an important target for antibody-based research?

AP2S1 (Adaptor-related protein complex 2, sigma 1 subunit) functions as a critical component of the adaptor protein complex 2 (AP-2), which plays a central role in clathrin-mediated endocytosis. This small adaptin (σ-type subunit) works with other AP2 subunits (alpha, beta2, and mu2) to form a heterotetrameric complex that mediates the sorting of membrane proteins in secretory and endocytic pathways . The AP2 complex contributes to clathrin-coated vesicle (CCV) formation by recruiting the scaffold protein clathrin and facilitates cargo selection by recognizing sorting signals within the cytoplasmic tails of integral membrane proteins . The AP2S1 protein is clinically relevant because mutations affecting the Arg15 residue cause familial hypocalciuric hypercalcemia type 3 (FHH3) , while other variants are associated with neurodevelopmental disorders . This dual relevance to both basic cellular processes and human disease makes AP2S1 an important target for antibody-based research.

What applications are AP2S1 antibodies typically validated for in laboratory settings?

AP2S1 antibodies have been validated for multiple research applications, with Western Blot (WB) and ELISA being the most commonly reported:

For immunoprecipitation studies, researchers have successfully used anti-CaSR antibodies to co-immunoprecipitate AP2S1, demonstrating protein-protein interactions between these molecules . When designing experiments, researchers should note that optimal conditions may be sample-dependent, and titration in each testing system is recommended to obtain optimal results .

What species reactivity considerations are important when selecting an AP2S1 antibody?

Species reactivity is a critical factor when selecting an AP2S1 antibody for research applications. The search results reveal varying reactivity profiles among commercially available antibodies:

When working with less common research models, researchers should verify cross-reactivity or select antibodies specifically validated for their species of interest. For positive controls, mouse and rat brain tissue have been identified as reliable samples for Western blot applications . The high evolutionary conservation of AP2S1 (>99% identity with zebrafish, >96% with fruitfly homologs) may contribute to the broad cross-reactivity observed with some antibodies.

How should AP2S1 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of AP2S1 antibodies is essential for maintaining their activity and specificity. Based on the search results, the following guidelines should be followed:

Adhering to these storage and handling guidelines will help ensure consistent antibody performance across experiments and maximize shelf life.

How can AP2S1 antibodies be utilized to study protein-protein interactions within the AP2 complex?

AP2S1 antibodies can be effectively employed to investigate the complex interactions between AP2S1 and other proteins using several methodologies:

• Co-immunoprecipitation (Co-IP): Multiple studies have utilized this approach to examine interactions between AP2S1 and its binding partners. For example, researchers have:

  • Immunoprecipitated CaSR using anti-CaSR antibodies and then probed with anti-AP2S1 antibodies to confirm interaction

  • Used anti-FLAG and anti-HA antibodies to investigate interactions between tagged CaSR and AP2S1 variants

  • Employed anti-HA antibodies to examine interactions between AP2S1 and other AP2 complex subunits (AP2α, AP2β2, AP2μ2)

• Quantitative analysis protocols: To compare wild-type and mutant interactions, researchers have:

  • Measured >50% decrease in AP2S1 in CaSR immunoprecipitates from Ap2s1+/L15 mouse kidneys compared to wild-type

  • Demonstrated significant reductions (>50%) in the amount of AP2α, AP2β2, and AP2μ2 subunits in immunoprecipitates from cells expressing mutant AP2S1

• Expression systems for studying interactions:

  • HEK293 cells stably overexpressing N-terminal FLAG-tagged CaSR with either HA-tagged wild-type or mutant AP2S1

  • Systems expressing fluorescent protein tags (e.g., RFP) to monitor transfection efficiency

These methods have revealed crucial insights, such as how the FHH3-associated Arg15Leu mutation diminishes protein-protein interactions between CaSR and AP2S1, as well as between AP2S1 and other AP2 complex subunits .

What methodologies are effective for studying the functional consequences of AP2S1 mutations?

Multiple complementary methodologies have proven effective for characterizing the functional impacts of AP2S1 mutations:

• CRISPR/Cas9 genome editing for disease models:

  • Generation of knock-in mouse models with specific mutations (e.g., Ap2s1+/L15)

  • Design of single-guide RNAs (sgRNAs) targeting specific genomic regions: "sgRNAs targeting the genomic region encoding the Arg15 residue of AP2S1 were designed"

  • Homology-directed repair using single-stranded DNA oligo-deoxynucleotide (ssODN) donors containing the desired mutation

• Calcium signaling assays:

  • Flow cytometry-based assays measuring Ca²⁺-induced responses in cells expressing wild-type or mutant AP2S1

  • Concentration-response curves to determine EC₅₀ values for calcium sensitivity

  • Example findings: AP2S1 mutations led to "a rightward shift in the concentration–response curves with significantly higher EC₅₀ values"

• Endocytosis assessment:

  • Transferrin uptake assays to measure clathrin-mediated endocytosis efficiency

  • Cell surface receptor expression measurements before and after stimulation

  • Studies show that AP2S1 mutations reduce CaSR endocytosis, affecting receptor density at the cell surface

• Cell viability and growth analysis:

  • Determination of percentage increase in cell numbers over time (e.g., 24-hour period)

  • Findings indicate that expression of mutant AP2S1 proteins can impair cell growth

• Behavioral assays in model organisms:

  • In zebrafish models with ap2s1 mutations, researchers have measured acoustic startle responses and habituation learning

  • These studies revealed that AP2S1 mutations can "disrupt acoustically evoked habituation learning" and alter "acoustically evoked behavior selection"

How can researchers distinguish between pathogenic and benign variants of AP2S1?

Distinguishing pathogenic from benign AP2S1 variants requires an integrated approach incorporating multiple lines of evidence:

• Functional assays:

  • CaSR signaling assays: Pathogenic variants show rightward shifts in concentration-response curves with significantly increased EC₅₀ values. For example, mutations affecting Arg15 increase EC₅₀ values from approximately 2.5mM to >2.7mM .

  • Transferrin uptake assays: Pathogenic variants reduce clathrin-mediated endocytosis .

  • Cell viability assessments: Some pathogenic variants decrease cell growth by impairing essential cellular processes .

• Protein-protein interaction studies:

  • Co-IP experiments show reduced interaction of pathogenic variants with binding partners .

  • For instance, the Arg15Leu mutation causes ">50% decrease in the amount of AP2S1 in the CaSR immunoprecipitate" .

• Evolutionary conservation and population frequency metrics:

  • AP2S1 is highly conserved across species, with >99% identity with zebrafish homologs .

  • Non-synonymous AP2S1 variants are significantly rarer in population databases than expected: "observed AP2σ variants vs. expected gene variants: missense = 0.009% vs. 0.1%; and nonsense = 0% vs. 0.01%" .

  • Variants affecting conserved residues within functional domains are more likely pathogenic.

• Domain-specific effects:

  • Mutations affecting Arg15 (Arg15Cys, Arg15His, Arg15Leu) consistently cause FHH3 .

  • Variants in the α4-α5 helical region (Thr112Met, Met117Ile, Glu142Lys) affect AP2 complex formation .

  • Other variants (Arg10Trp, Arg10Gln, Lys18Glu, Lys18Asn, Arg61His) associate with neurodevelopmental disorders .

Research has shown that seemingly similar variants can have different effects—for example, while Arg61His was associated with neurodevelopmental disorders in one study , it showed no effect on CaSR signaling in another study, suggesting it might be a benign polymorphism for calcium homeostasis .

How are AP2S1 antibodies utilized in familial hypocalciuric hypercalcemia type 3 (FHH3) research?

AP2S1 antibodies have become essential tools in unraveling the molecular mechanisms underlying FHH3, with several key applications:

• Mutation detection and characterization:

  • Western blot analysis to detect the expression of wild-type and mutant AP2S1 proteins in patient samples and experimental models

  • Identification of specific mutations affecting the Arg15 residue (Arg15Cys, Arg15His, and Arg15Leu)

  • Validation that mutations are present at the protein level rather than just genomic alterations

• Protein-protein interaction assessment:

  • Co-immunoprecipitation experiments to evaluate interactions between AP2S1 and the calcium-sensing receptor (CaSR)

  • Quantitative analysis demonstrating that "the FHH3-associated mutant Leu15 AP2S1 also impairs the interactions between AP2S1 and the other subunits (AP2α, AP2β2 and AP2μ2) of the AP2 heterotetramer"

  • Documentation that these impaired interactions disrupt clathrin-mediated endocytosis of CaSR

• Mouse model characterization:

  • AP2S1 antibodies have been used to study Ap2s1+/L15 knock-in mice, confirming that these animals recapitulate the FHH3 phenotype

  • Measurement of AP2S1 expression levels in different tissues of mutant mice

  • Correlation of biochemical findings with physiological calcium homeostasis parameters

• Cellular signaling analysis:

  • Investigation of how AP2S1 mutations affect downstream CaSR signaling pathways

  • Documentation that AP2S1 mutations "decreased the sensitivity of CaSR-expressing cells to extracellular-calcium"

These applications have collectively established that FHH3 results from impaired interaction between mutant AP2S1 and CaSR, leading to reduced CaSR endocytosis and diminished sensitivity to extracellular calcium .

What role do AP2S1 antibodies play in research on neurodevelopmental disorders?

Recent research has revealed an important connection between AP2S1 variants and neurodevelopmental disorders, with AP2S1 antibodies playing a crucial role in elucidating these associations:

• Characterization of novel disease-associated variants:

  • AP2S1 antibodies have helped identify and characterize five different AP2S1 variants (p.Arg10Trp, p.Arg10Gln, p.Lys18Glu, p.Lys18Asn and p.Arg61His) in 26 patients with neurodevelopmental delay

  • These patients present with distinct clinical features: >70% had epilepsy, 50% had brain abnormalities, and notably, none had hypercalcemia (distinguishing them from FHH3 patients)

• Functional impact assessment:

  • Western blot and other immunodetection methods using AP2S1 antibodies have demonstrated that these variants decrease cell viability

  • Four of the five variants reduced CME transferrin uptake, reflecting impaired endocytosis

  • Four variants disrupted interactions with other AP2 complex subunits, affecting proper complex formation

• Protein interaction network analysis:

  • AP2S1 antibodies have enabled the discovery that the variant AP2σ2 p.Arg10Trp has reduced interactions with 44 human proteins, including intersectin 1

  • This is particularly significant as intersectin 1 is "a component required for clathrin-coated pit formation and synaptic vesicle dynamics in neurones"

• Research in model organisms:

  • Antibodies against AP2S1 have facilitated studies in zebrafish models showing that ap2s1 "regulates acoustically evoked behavior selection and habituation learning"

  • These studies demonstrate that AP2S1's role in neurodevelopment is evolutionarily conserved across species

These findings collectively illustrate how AP2S1 antibodies have been instrumental in establishing that AP2S1 variants can disrupt clathrin-mediated endocytosis leading to neurodevelopmental abnormalities, expanding our understanding beyond the protein's previously established role in calcium homeostasis.

How can AP2S1 antibodies help assess the efficacy of potential therapeutic approaches for AP2S1-related disorders?

AP2S1 antibodies can serve as critical tools for evaluating potential therapeutic strategies for disorders caused by AP2S1 dysfunction:

• Expression system monitoring:

  • AP2S1 antibodies can measure expression levels of wild-type versus mutant AP2S1 in gene therapy approaches

  • Western blot analysis using specific antibodies can confirm successful transfection and expression of therapeutic constructs

  • Fluorescence microscopy combined with immunodetection can assess localization and expression patterns: "Successful transfection was also confirmed by visualising RFP fluorescence using an Eclipse E400 fluorescence microscope"

• Protein-protein interaction restoration assessment:

  • Co-immunoprecipitation studies using AP2S1 antibodies can determine whether therapeutic interventions restore normal interactions between:

    • AP2S1 and calcium-sensing receptor (CaSR) in FHH3

    • AP2S1 and other AP2 complex subunits

    • AP2S1 and neuronal proteins like intersectin 1 in neurodevelopmental disorders

• Functional recovery measurement:

  • Antibody-based detection methods can assess whether treatments normalize:

    • Clathrin-mediated endocytosis efficiency

    • Cell surface receptor density

    • AP2 complex formation and stability

• Target engagement verification:

  • AP2S1 antibodies can confirm that therapeutic compounds or biologics engage with their intended targets

  • Differential detection of wild-type versus mutant AP2S1 could help monitor allele-specific therapeutic approaches

• Biomarker development:

  • AP2S1 antibodies could potentially help develop assays for biomarkers that reflect treatment efficacy

  • Measurement of AP2S1-related protein complexes in accessible samples could provide surrogate endpoints for clinical trials

While the search results do not explicitly describe therapeutic approaches for AP2S1-related disorders, the methodologies employed in basic research using AP2S1 antibodies provide a foundation for therapeutic development and monitoring.

What are common challenges in AP2S1 antibody applications, and how can they be addressed?

Researchers working with AP2S1 antibodies frequently encounter several technical challenges that can be mitigated with appropriate strategies:

• Optimal dilution determination:

  • Challenge: Different AP2S1 antibodies require vastly different dilutions, ranging from 1:500 to 1:16000 for Western blot applications

  • Solution: Perform dilution series experiments using positive control samples (e.g., brain tissue) to determine optimal conditions for your specific detection system

  • As noted in the literature: "It is recommended that this reagent should be titrated in each testing system to obtain optimal results"

• Specificity verification:

  • Challenge: Ensuring the antibody detects AP2S1 with high specificity, especially given its small size (observed molecular weight: 15-17 kDa)

  • Solution: Include appropriate positive controls (e.g., mouse brain tissue, rat brain tissue) and negative controls (tissues or cells with AP2S1 knockdown)

  • Consider validation through multiple detection methods or using antibodies targeting different epitopes

• Detection of endogenous versus overexpressed protein:

  • Challenge: Distinguishing endogenous AP2S1 from transfected constructs in experimental systems

  • Solution: Use epitope tags (HA, FLAG) to differentiate overexpressed constructs , or compare expression levels with vector-only controls

  • Western blot analysis can confirm that "the expression of AP2S1 was shown to be similar in cells transiently transfected with WT or variant proteins and higher than the endogenous expression of AP2S1"

• Co-immunoprecipitation optimization:

  • Challenge: Achieving efficient immunoprecipitation of AP2S1 complexes

  • Solution: Follow established protocols, such as "mixing lysates with antibody for 30 min at 4°C prior to addition to protein G"

  • Consider crosslinking approaches for transient interactions or using specialized IP buffers to maintain complex integrity

• Signal detection sensitivity:

  • Challenge: Obtaining sufficient signal for low-abundance AP2S1

  • Solution: Employ enhanced chemiluminescence or fluorescent secondary antibodies

  • Load adequate protein amounts (may require optimization) and consider signal amplification methods

How should researchers interpret contradictory results when studying AP2S1 variants?

When facing contradictory results in AP2S1 variant studies, researchers should adopt a systematic approach to resolution:

• Integrate multiple functional assays:

  • Different assays may reveal distinct aspects of AP2S1 function

  • For example, search result found that while six non-synonymous AP2S1 variants affected evolutionarily conserved residues, only three (Thr112Met, Met117Ile, Glu142Lys) significantly impaired CaSR function

  • Complete functional characterization requires assessment of multiple parameters: calcium signaling, protein-protein interactions, endocytosis efficiency, and cell viability

• Consider mutation location and domain-specific effects:

  • AP2S1 mutations in different domains may selectively affect specific functions:

    • Arg15 mutations consistently affect CaSR interaction and cause FHH3

    • Variants in the α4-α5 helical region affect AP2 complex formation

    • Different variants associate with distinct clinical phenotypes (hypercalcemia vs. neurodevelopmental disorders)

• Account for model system variables:

  • Contradictions may arise from differences in experimental models

  • Mouse models may show phenotypic differences from cellular systems

  • As noted in search result , some AP2S1 variants (Tyr20Asn, Ile123Asn) showed no effect on CaSR signaling, and mice with a splice variant had normal calcium levels "despite a >50% reduction in AP2S1 protein expression"

  • Cell type-specific effects may also contribute to variable results

• Evaluate dosage effects:

  • Heterozygous versus homozygous mutations may produce different phenotypes

  • Expression levels of mutant proteins relative to wild-type can affect outcomes

  • Some effects may only be apparent with complete loss of function

• Use reference standards and controls:

  • Include known pathogenic mutations (e.g., Arg15His) as positive controls

  • Include validated benign variants (e.g., Arg3His, Ala44Thr) as negative controls

  • Standardize experimental conditions to enable valid comparisons between studies

By systematically addressing these factors, researchers can resolve apparently contradictory results and develop a more comprehensive understanding of AP2S1 variant effects.

What emerging technologies might enhance AP2S1 antibody-based research?

Several cutting-edge technologies hold promise for advancing AP2S1 antibody-based research:

• Proximity labeling techniques:

  • BioID or APEX2-based approaches could reveal the dynamic interactome of AP2S1 in different cellular contexts

  • These methods could identify novel interaction partners beyond the 44 human proteins already known to interact with AP2S1

  • Applications could uncover cell-type specific interactions in neurons versus other tissues

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED microscopy combined with AP2S1 antibodies could visualize AP2S1 localization within clathrin-coated pits at nanometer resolution

  • Multi-color imaging could reveal spatial relationships between AP2S1, CaSR, and other components of the endocytic machinery

  • Live-cell super-resolution could track the dynamics of wild-type versus mutant AP2S1 during endocytosis

• CRISPR-based genome and epigenome editing:

  • Precise knock-in models of disease-associated variants using improved CRISPR systems

  • Base editing or prime editing for introducing specific mutations without double-strand breaks

  • CRISPRi/CRISPRa systems to modulate AP2S1 expression levels in specific tissues

• Cryo-electron microscopy:

  • Structural determination of the entire AP2 complex containing wild-type versus mutant AP2S1

  • Visualization of AP2S1-CaSR interactions at atomic resolution

  • Understanding conformational changes induced by disease-causing mutations

• Single-cell proteomics:

  • Analysis of AP2S1 expression and protein complex formation at the single-cell level

  • Identification of cell populations with differential sensitivity to AP2S1 mutations

  • Correlation of protein expression patterns with cellular phenotypes

These technologies would complement traditional antibody-based approaches and provide deeper insights into AP2S1 function in normal physiology and disease states.

How might AP2S1 antibodies contribute to developing personalized medicine approaches for patients with AP2S1 mutations?

AP2S1 antibodies could play integral roles in advancing personalized medicine for patients with AP2S1-related disorders:

• Variant-specific diagnostic tools:

  • Development of antibodies that specifically recognize disease-causing AP2S1 variants

  • Creation of rapid diagnostic assays to identify patients with AP2S1 mutations

  • Discrimination between FHH3-causing mutations and neurodevelopmental disorder-associated variants

• Functional stratification of patients:

  • AP2S1 antibodies could help classify patients based on the molecular consequences of their specific mutations

  • Functional assays using patient-derived cells could determine:

    • Degree of impairment in calcium sensing for FHH3 patients

    • Extent of endocytosis dysfunction for neurodevelopmental disorder patients

    • Potential for response to targeted therapies

• Biomarker development:

  • Antibody-based assays could monitor disease progression or treatment response

  • Quantification of AP2S1-containing protein complexes in accessible patient samples

  • Correlation of molecular markers with clinical outcomes

• Therapeutic monitoring:

  • Assessment of gene therapy efficacy through detection of functional AP2S1 expression

  • Monitoring restoration of normal protein-protein interactions following treatment

  • Evaluation of drug target engagement in clinical trials

• Pharmacodynamic markers:

  • AP2S1 antibodies could help determine optimal dosing of therapeutic agents

  • Measurement of downstream signaling pathway normalization following treatment

  • Detection of cellular phenotype restoration in patient-derived samples

These applications would contribute to the development of more targeted therapeutic approaches for both FHH3 and AP2S1-associated neurodevelopmental disorders, moving beyond the current symptom-based management toward addressing the underlying molecular pathology.