NECAP2 Human
Description
Functional Role in Endocytosis
NECAP2 regulates clathrin adaptor complexes through two primary mechanisms:
AP2 Complex Inactivation
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Binds phosphorylated µ2 subunit (pT156) of AP2 via its PHear domain, clamping AP2 into a closed, inactive conformation .
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Requires simultaneous engagement of its Ex domain with the β subunit of AP2 for full inactivation .
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Counterbalances muniscin proteins (e.g., FCHO-1) that activate AP2, ensuring cyclical AP2 activity .
AP1 Recruitment to Early Endosomes
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Facilitates AP1-clathrin coat assembly on early endosomes for fast recycling of receptors (e.g., EGFR, transferrin) .
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NECAP2 knockdown delays transferrin recycling by 40–50% within 5 minutes, indicating its role in rapid endocytic cycling .
Pathway-Specific Regulation
Disease Associations
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Cancer: Overexpression correlates with poor prognosis in gliomas (HR = 1.93, p < 0.001) and immune infiltration .
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Toxicology: Altered expression under bisphenol A, arsenic, and ivermectin exposure .
Experimental Tools and Applications
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Recombinant NECAP2 Protein: Used in pulldown assays to study AP2/AP1 interactions (Source: abcam ab185838) .
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Genetic Models: C. elegans screens identified NECAP2 residues critical for AP2 binding (e.g., PHear domain R35A, K37A) .
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Protease Sensitivity Assays: Demonstrate NECAP2-induced conformational changes in AP2 .
Clinical and Therapeutic Implications
Product Specs
Q&A
What is the primary functional role of NECAP2 in endocytic pathways?
NECAP2 regulates clathrin coat recruitment to early endosomes, facilitating fast recycling of receptors like EGFR and transferrin receptor (TfR) to the plasma membrane . Unlike slow Rab11-dependent recycling, NECAP2-mediated recycling bypasses perinuclear compartments, directly returning cargo via AP-1/clathrin-coated vesicles. To validate this:
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Knockdown experiments: Use siRNA or CRISPR-Cas9 to deplete NECAP2 and quantify recycling kinetics via pulse-chase assays with fluorophore-conjugated transferrin or EGFR ligands .
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Morphometric analysis: Image early endosomes (EEA1-positive) in NECAP2-depleted cells; enlarged endosomes indicate disrupted AP-1 recruitment .
Which protein interaction interfaces are essential for NECAP2 function?
NECAP2 binds AP-1 through two conserved domains:
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Ex domain: Engages β-appendage of AP-1 in a conformation-dependent manner .
Experimental validation:
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Co-immunoprecipitation (Co-IP): Truncate NECAP2 domains (e.g., ∆PHear or ∆Ex) and test AP-1 binding in HEK293T lysates .
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Structural mapping: Cryo-EM of NECAP2-AP-1 complexes (3.5 Å resolution) reveals clamping of AP-1 into closed, inactive states .
How does NECAP2 coordinate phosphorylation-dependent inactivation of AP-2 complexes?
NECAP2 inactivates AP-2 via a three-step mechanism:
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Recognition: NECAP Ex binds open, membrane-activated AP-2 (induced by PIP2 or heparin) .
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Clamping: PHear domain engages phosphorylated µ-subunit, stabilizing AP-2 in a closed conformation .
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Functional consequence: Occludes cargo-binding sites and blocks clathrin recruitment .
Methodological approaches:
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Protease sensitivity assays: Treat AP-2 ± NECAP2 with trypsin; closed complexes resist cleavage at Thr156 (µ-subunit) .
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Genetic screens: Use C. elegans mutants (e.g., ncap-1∆) to identify suppressors restoring AP-2 activity .
How to resolve contradictory findings on NECAP2’s role in endocytosis vs. recycling?
Some studies report NECAP2 as an endocytosis promoter , while others emphasize recycling . Resolve this via:
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Pathway-specific inhibitors: Compare TfR recycling (bafilomycin A1-sensitive) vs. EGFR degradation (MG132-sensitive) .
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Live-cell imaging: Track pHrodo-labeled EGFR in NECAP2-KO cells; delayed acidification indicates recycling defects .
What experimental models best recapitulate NECAP2 dysregulation?
How to characterize NECAP2-AP-1/AP-2 interactions structurally?
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Cryo-EM: Resolve NECAP2-AP complexes in presence of PIP2 mimetics (e.g., inositol hexakisphosphate) .
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Mutagenesis: Introduce phosphomimetic (S/T→D) or phosphorylation-resistant (S/T→A) mutations in AP-1/AP-2 subunits .
Why do some studies report NECAP2 as an AP-2 activator vs. inactivator?
Discrepancies arise from conformational states:
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AP-2 activation: NECAP2 binds open, unphosphorylated AP-2 to facilitate cargo loading .
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AP-2 inactivation: Phosphorylation triggers NECAP2-mediated closure, terminating activity .
Validation: Use FRET-based sensors to monitor AP-2 conformation in live cells ± NECAP2 inhibitors.
Could NECAP2 be targeted to modulate receptor signaling in disease?
Therapeutic rationale:
Product Science Overview
The recombinant form of NECAP2 is typically expressed in Escherichia coli and is purified to a high degree of purity, often exceeding 85% as validated by SDS-PAGE . The full-length protein consists of 263 amino acids and has a predicted molecular weight of approximately 31 kDa . The protein is tagged with a His tag at the N-terminus to facilitate purification and detection .
NECAP2 contains two characteristic WXXF motifs that are crucial for its function. These motifs mediate the binding of accessory proteins to the ear-domain of adapter protein complexes such as AP-1 and AP-2 . The selective binding to these domains is influenced by the acidic context surrounding the motif and the properties of the second residue of the motif itself .
NECAP2 is primarily localized to the cytoplasmic vesicles, particularly the clathrin-coated vesicle membrane . It colocalizes with AP-2 at the plasma membrane, where it plays a significant role in the formation and regulation of clathrin-coated vesicles . These vesicles are essential for the internalization of various molecules, including nutrients, receptors, and other macromolecules, into the cell.
The proper functioning of NECAP2 is vital for efficient endocytosis. Disruptions in its function can lead to defects in vesicle formation and trafficking, which can have downstream effects on cellular processes and signaling pathways. NECAP2 is also associated with various cellular pathways, including vesicle-mediated transport and clathrin-mediated endocytosis .
Recombinant NECAP2 is used in various research applications to study its role in endocytosis and vesicle trafficking. It is also utilized in mass spectrometry and SDS-PAGE for protein analysis . The availability of recombinant NECAP2 allows researchers to investigate its interactions with other proteins and its involvement in cellular processes.
