GO:2000369 regulation of clathrin-dependent endocytosis: Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:2000369 (regulation of clathrin-dependent endocytosis) is a biological process that modulates the frequency, rate or extent of clathrin-mediated endocytosis, a major route for selective cargo internalization [1,3].
• Clathrin-dependent endocytosis controls the uptake of nutrients, signaling receptors, adhesion molecules, and pathogens, and its dysregulation is linked to cancer, metabolic disorders, and infectious disease [1,6,7].
• Key regulatory nodes include cargo receptors (e.g., integrins, HER2, MRP2, CD63), kinases (PKC, ERK, JNK, p38, PI3K), and the actin cytoskeleton [1,2,6,7,8].
• Phosphorylation of intrinsically disordered proteins and accessory factors tunes actin assembly and endocytic site dynamics.
• Experimental models for studying GO:2000369 include CRISPR knockout, point-mutation, knock-in, and overexpression cell lines, combined with imaging, proteomics, and functional uptake assays [1,6,7].
• The term is distinct from clathrin-independent endocytosis, which represents an increasing degree of complexity and alternative internalization routes.
Description
Regulation of clathrin-dependent endocytosis (GO:2000369) is a biological process that controls the frequency, rate, or extent of clathrin-mediated endocytosis, the principal pathway by which cells internalize specific cargo through clathrin-coated pits [1,3]. This process is essential for nutrient uptake, receptor signaling, cell adhesion, and host-pathogen interactions, and it is subject to tight regulation at multiple levels, including cargo recognition, coat assembly, membrane scission, and cytoskeletal coupling [1,3,8]. Dysregulation of clathrin-dependent endocytosis has been implicated in cancer progression, drug resistance, and metabolic disease, making it a focal point for both basic cell biology and translational research [1,6,7]. Researchers study GO:2000369 to understand how cells selectively internalize receptors such as integrins, HER2, and MRP2, and how signaling pathways (e.g., PKC, ERK/JNK/p38, PI3K) modulate this process [1,6,7]. The pathway is also exploited by pathogens and toxins, and its activity can determine the efficacy of therapeutic antibodies and drug delivery systems [4,5]. Because clathrin-dependent endocytosis is mechanistically distinct from clathrin-independent routes, precise regulation ensures cellular homeostasis and appropriate responses to environmental cues. This article provides a research-grade overview of GO:2000369, covering its definition, molecular machinery, key genes, disease relevance, and state-of-the-art methods including CRISPR-based models and bioinformatics. All statements are grounded in published literature to support reproducibility and further investigation.
regulation of clathrin-dependent endocytosis At A Glance
| GO ID | GO:2000369 |
|---|---|
| GO term | regulation of clathrin-dependent endocytosis |
| Ontology | biological_process |
| Synonym | regulation of clathrin coated pit-dependent endocytosis; regulation of clathrin-mediated endocytosis |
| Major function | Modulates the frequency, rate or extent of clathrin-mediated endocytosis, a key internalization pathway for receptors, nutrients, and pathogens [1,3]. |
| Key regulators | Cargo receptors (integrins, HER2, MRP2, CD63), kinases (PKC, ERK, JNK, p38, PI3K), actin cytoskeleton, and accessory proteins [1,2,6,7,8]. |
| Disease relevance | Cancer, metabolic disorders, infectious disease, and drug resistance [1,6,7]. |
| Research methods | CRISPR knockout/knock-in, live-cell imaging, proteomics, and functional uptake assays [1,6,7]. |
What Is GO:2000369?
GO:2000369, regulation of clathrin-dependent endocytosis, is defined as any process that modulates the frequency, rate or extent of clathrin-mediated endocytosis. It encompasses the molecular events that control the assembly, dynamics, and function of clathrin-coated pits and vesicles, thereby influencing the internalization of specific cargo from the plasma membrane [1,3].
Why Is regulation of clathrin-dependent endocytosis Important in Cell Biology?
Regulation of clathrin-dependent endocytosis is fundamental to cellular physiology because it governs the internalization of signaling receptors, adhesion molecules, and nutrients, thereby controlling signal transduction, cell migration, and metabolic homeostasis [1,3,7]. Its dysregulation contributes to cancer progression, drug resistance, and impaired nutrient absorption, and it is a target for therapeutic intervention [1,6,7].
• Controls the internalization of integrins, affecting cell adhesion and migration.
• Regulates HER2 turnover in non-small cell lung cancer, influencing tumor growth and therapeutic response.
• Mediates the uptake of egg white hydrolysate in the intestine, highlighting a role in nutrient absorption.
• Modulates MRP2 degradation, impacting drug efflux and resistance in hepatic cells.
• Involved in the trafficking of tetraspanin CD63, which affects exosome and lysosomal functions.
• Regulated by phosphorylation of intrinsically disordered proteins that control actin assembly at endocytic sites.
• Distinct from clathrin-independent endocytosis, which provides alternative internalization routes with increasing complexity.
• Exploited by pathogens and toxins for cellular entry, making it a host-target for antiviral and antitoxin strategies.
• Key to G protein-coupled receptor desensitization and signaling.
• Provides a mechanistic basis for drug delivery and targeted therapies [4,6].
What Happens During regulation of clathrin-dependent endocytosis?
Initiation and cargo selection
In simple terms: The cell decides what to bring in by marking specific cargo with tags that recruit the clathrin machinery.
Regulation begins with the selection of cargo, such as integrins, HER2, or MRP2, which are recognized by adaptor proteins and recruited to nascent clathrin-coated pits [1,6,7]. This step is modulated by signaling kinases, including PKC and PI3K, which can alter the phosphorylation state of cargo or adaptors to promote or inhibit internalization. The tetraspanin CD63 also participates in sorting and trafficking decisions within the endosomal system.
Coat assembly and actin coupling
In simple terms: A protein coat builds up around the cargo, and the cytoskeleton helps pull the membrane inward.
Clathrin and adaptor proteins assemble into a lattice that deforms the plasma membrane, a process regulated by phosphorylation of intrinsically disordered proteins that control actin assembly and endocytic site dynamics. Actin polymerization provides force for membrane invagination and scission, and its coupling to the coat is a key regulatory node [1,8].
Membrane scission and vesicle release
In simple terms: The coated pit pinches off to form a vesicle inside the cell.
After coat assembly, dynamin and other scission factors mediate the release of the clathrin-coated vesicle. Regulation of this step determines the frequency and rate of endocytosis and can be influenced by cargo-specific signals and kinases [1,3]. The released vesicle then uncoats and delivers cargo to endosomes.
Cargo-specific regulation and signaling feedback
In simple terms: Different cargoes can have their own on/off switches that fine-tune how much gets inside.
Cargo-specific regulation allows cells to adjust uptake in response to external cues. For example, HER2 mutations enhance clathrin-dependent internalization in lung cancer cells, while rifampicin induces MRP2 endocytosis via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K pathways [6,7]. G protein-coupled receptors also undergo regulated clathrin-dependent endocytosis, which controls their signaling duration.
Distinction from clathrin-independent routes
In simple terms: Not all entry uses clathrin; cells have other ways to bring things in, and regulation keeps them separate.
Regulation of clathrin-dependent endocytosis is distinct from clathrin-independent endocytosis, which encompasses multiple pathways with increasing complexity. Cross-talk between these routes can occur, but the specific molecular players and regulatory inputs differ, underscoring the need for precise definitions when studying GO:2000369.
Key Genes Involved in GO:2000369 regulation of clathrin-dependent endocytosis
The following genes and proteins are central to the regulation of clathrin-dependent endocytosis, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ITGB1 | Integrin beta 1; cargo for clathrin-dependent endocytosis, regulates cell adhesion and migration | Force tuning through regulated integrin endocytosis |
| ERBB2 (HER2) | Receptor tyrosine kinase; internalization via clathrin-dependent endocytosis | HER2 mutations enhance internalization in NSCLC |
| ABCC2 (MRP2) | Drug efflux transporter; undergoes clathrin-dependent endocytosis and degradation | Rifampicin-induced endocytosis via PKC-ERK/JNK/p38 and PI3K |
| CD63 | Tetraspanin; traffics through endosomal compartments, regulates sorting | Trafficking and function in lysosomes and exosomes |
| CLTC | Clathrin heavy chain; structural component of coated pits | Core machinery for clathrin-mediated endocytosis |
| CLTA | Clathrin light chain; regulates coat assembly and disassembly | Accessory role in endocytosis |
| AP2M1 | AP-2 mu subunit; cargo recognition and adaptor function | Cargo selection in clathrin-coated pits |
| DNM2 | Dynamin 2; mediates membrane scission | Vesicle release in clathrin-dependent endocytosis |
| PRKCA | Protein kinase C alpha; phosphorylates substrates to regulate endocytosis | Oxidative stress signaling to MRP2 endocytosis |
| MAPK1 (ERK2) | Mitogen-activated protein kinase; signaling to endocytic machinery | Rifampicin-induced MRP2 endocytosis |
| MAPK8 (JNK1) | c-Jun N-terminal kinase; stress-activated regulation | MRP2 endocytosis via oxidative stress |
| MAPK14 (p38 alpha) | p38 MAP kinase; stress response regulation | MRP2 endocytosis |
| PIK3CA | PI3K catalytic subunit; lipid signaling in endocytosis | Rifampicin-induced MRP2 endocytosis |
| ACTB | Beta-actin; cytoskeletal force for endocytosis | Actin assembly at endocytic sites |
| ACTR2 | ARP2/3 complex subunit; actin nucleation | Phospho-regulation of actin assembly |
| WASL | Wiskott-Aldrich syndrome protein family member; actin nucleation promotion | Regulation by intrinsically disordered proteins |
| SNX9 | Sorting nexin 9; membrane remodeling and scission | Accessory factor in clathrin-mediated endocytosis |
| GAK | Cyclin G associated kinase; regulates coat assembly | Auxiliary factor in endocytosis |
How Is regulation of clathrin-dependent endocytosis Regulated?
Regulation of clathrin-dependent endocytosis is controlled by phosphorylation events, particularly through kinases such as PKC, ERK, JNK, p38, and PI3K, which respond to oxidative stress and other signals. Phosphorylation of intrinsically disordered proteins modulates actin assembly and endocytic site dynamics, providing a mechanism for rapid adaptation. Cargo-specific signals, such as HER2 mutations, can also alter the rate of internalization.
regulation of clathrin-dependent endocytosis and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ERBB2 | Non-small cell lung cancer with HER2 mutations | HER2 mutant knock-in NSCLC cell lines; KO of ERBB2 to assess internalization |
| ABCC2 | Drug-induced cholestasis and altered drug disposition | HepG2 cells with MRP2 knockout or point mutations; rifampicin treatment |
| ITGB1 | Cancer cell migration and mechanotransduction | Integrin beta1 KO or point-mutant cells; force measurements |
| CD63 | Lysosomal storage disorders and exosome biology | CD63 knockout or tagged knock-in cells; trafficking assays |
| CLTC | General endocytic defects and developmental disorders | CLTC knockout or point-mutation cell lines; uptake assays |
Cancer
Dysregulated clathrin-dependent endocytosis affects the turnover of oncogenic receptors such as HER2. In non-small cell lung cancer positive for HER2 mutations, enhanced clathrin-dependent internalization modulates receptor signaling and may influence response to targeted therapies. Integrin endocytosis also contributes to force tuning and migratory behavior of cancer cells.
Metabolic and hepatic disorders
In hepatic cells, rifampicin induces clathrin-dependent endocytosis and ubiquitin-proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways, linking endocytic regulation to drug disposition and cholestasis. Nutrient absorption in the intestine also relies on clathrin-dependent endocytosis, as shown for egg white hydrolysate.
Infectious disease and toxin entry
Many pathogens and toxins exploit clathrin-dependent endocytosis for cellular entry, and the interplay with clathrin-independent routes adds complexity to host-pathogen interactions. Understanding regulation of this process can inform antiviral and antitoxin strategies.
Neurological and lysosomal storage disorders
CD63 trafficking through endosomal and lysosomal compartments is important for cellular homeostasis, and its dysfunction has been linked to lysosomal disorders and neurodegeneration. Regulated endocytosis of signaling receptors in neurons also impacts synaptic function.
From regulation of clathrin-dependent endocytosis-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of a candidate gene alter clathrin-dependent endocytosis rate? | CRISPR knockout cell line (e.g., ITGB1, ERBB2, ABCC2) [1,6,7] |
| Does a specific mutation affect cargo internalization? | Point-mutation knock-in cell line (e.g., HER2 mutations) |
| How does a tag affect protein localization during endocytosis? | Tagged knock-in (e.g., GFP-CD63) |
| Does overexpression of a kinase enhance endocytosis? | Overexpression cell line (e.g., PKC, PI3K) |
| Which genes regulate endocytosis in a genome-wide manner? | CRISPR library screening with uptake readout [1,8] |
| What is the dynamics of actin assembly at endocytic sites? | Live-cell imaging of actin reporters in knockout or knock-in cells |
How to Study the regulation of clathrin-dependent endocytosis Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Live-cell TIRF microscopy | Dynamics of coat assembly and cargo internalization | Visualizing clathrin-coated pit formation [1,8] |
| Phosphoproteomics | Changes in protein phosphorylation | Identifying signaling nodes in endocytosis [7,8] |
| Fluorescent cargo uptake assay | Rate and extent of endocytosis | Comparing wild-type and mutant cells [4,6] |
| CRISPR knockout screening | Genes required for endocytosis | Genome-wide discovery of regulators [1,8] |
| Proximity ligation assay | Protein-protein interactions at endocytic sites | Validating adaptor-cargo interactions |
| Electron microscopy | Ultrastructure of coated pits and vesicles | Morphological analysis of endocytic intermediates |
| Flow cytometry | Cell surface receptor internalization | Quantifying HER2 or integrin endocytosis |
| Western blotting | Protein degradation and trafficking | Assessing MRP2 degradation after endocytosis |
Live-cell imaging and fluorescence microscopy
Live-cell imaging of fluorescently tagged cargo (e.g., integrins, HER2, CD63) and coat proteins allows real-time visualization of endocytic events, including initiation, coat assembly, and scission [1,2,6]. Total internal reflection fluorescence (TIRF) microscopy is particularly useful for studying plasma membrane dynamics.
Proteomics and phosphoproteomics
Mass spectrometry-based proteomics can identify proteins and phosphorylation sites that regulate clathrin-dependent endocytosis, as demonstrated for phospho-regulation of intrinsically disordered proteins in actin assembly. Quantitative phosphoproteomics after kinase activation or inhibition reveals signaling nodes.
Functional uptake assays
Uptake assays using fluorescent or radioactive cargo (e.g., transferrin, egg white hydrolysate) measure the rate and extent of clathrin-dependent endocytosis. These assays can be combined with inhibitors or genetic perturbations to dissect regulatory mechanisms [4,7].
CRISPR-based genetic screens
Genome-wide CRISPR knockout or activation screens coupled with endocytosis readouts (e.g., fluorescent cargo uptake) can identify novel regulators of GO:2000369. Such screens have been used to uncover genes controlling integrin endocytosis and actin dynamics [1,8].
How CRISPR Can Be Used to Study GO:2000369 regulation of clathrin-dependent endocytosis
Knockout
CRISPR knockout of genes such as ITGB1, ERBB2, or ABCC2 can abolish or reduce clathrin-dependent endocytosis of specific cargo, allowing researchers to test causality. For example, MRP2 knockout in HepG2 cells prevents rifampicin-induced endocytosis and degradation. Knockout of clathrin heavy chain (CLTC) disrupts the entire pathway.
Point Mutation
Point mutations can mimic disease-associated variants or phospho-null/phospho-mimetic states. For instance, introducing HER2 mutations found in NSCLC into cell lines enhances clathrin-dependent internalization, providing a model to study altered trafficking. Point mutations in kinase domains can reveal phosphorylation-dependent regulation.
Knock-in
Knock-in of tagged versions of cargo or machinery proteins (e.g., GFP-CD63, HA-tagged integrins) enables real-time tracking and biochemical isolation of endocytic complexes. Knock-in of disease-relevant mutations (e.g., in ABCC2) can model altered drug transport.
Overexpression
Overexpression of regulatory kinases (e.g., PKC, PI3K) or cargo receptors can enhance clathrin-dependent endocytosis and reveal rate-limiting steps. Overexpression of dominant-negative dynamin (DNM2) blocks scission and is a common tool to inhibit the pathway.
How EDITGENE Supports regulation of clathrin-dependent endocytosis Research
Researchers studying regulation of clathrin-dependent endocytosis-related genes often need to determine whether a candidate gene is causally involved in cargo internalization, coat dynamics, or disease-associated trafficking defects. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for regulation of clathrin-dependent endocytosis research.
Frequently Asked Questions About regulation of clathrin-dependent endocytosis
What is GO:2000369 regulation of clathrin-dependent endocytosis?
GO:2000369 is a biological process that modulates the frequency, rate or extent of clathrin-mediated endocytosis, the main pathway for selective cargo internalization [1,3].
What genes are involved in regulation of clathrin-dependent endocytosis?
Key genes include ITGB1, ERBB2, ABCC2, CD63, CLTC, CLTA, AP2M1, DNM2, PRKCA, MAPK1, MAPK8, MAPK14, PIK3CA, ACTB, and others [1,2,3,6,7,8].
How is clathrin-dependent endocytosis regulated?
It is regulated by phosphorylation events via kinases such as PKC, ERK, JNK, p38, and PI3K, as well as by actin cytoskeleton dynamics and cargo-specific signals [7,8].
What diseases are associated with dysregulated clathrin-dependent endocytosis?
Cancer (e.g., HER2-mutant NSCLC), metabolic and hepatic disorders (e.g., MRP2-related drug disposition), and infectious diseases are associated with altered clathrin-dependent endocytosis [5,6,7].
What is the difference between clathrin-dependent and clathrin-independent endocytosis?
Clathrin-dependent endocytosis uses clathrin-coated pits for cargo uptake, while clathrin-independent endocytosis encompasses multiple alternative routes with increasing complexity.
How can I study regulation of clathrin-dependent endocytosis in the lab?
Common methods include live-cell imaging, fluorescent cargo uptake assays, proteomics, and CRISPR-based genetic screens [1,4,8].
What CRISPR models are available for studying clathrin-dependent endocytosis?
Knockout, point mutation, knock-in, and overexpression cell lines can be generated for genes such as ERBB2, ABCC2, and ITGB1 [1,6,7].
Which proteins are cargo for clathrin-dependent endocytosis?
Integrins, HER2, MRP2, CD63, and G protein-coupled receptors are examples of cargo internalized via clathrin-dependent endocytosis [1,2,3,6,7].
How does actin regulate clathrin-dependent endocytosis?
Actin assembly provides force for membrane invagination and scission, and is regulated by phosphorylation of intrinsically disordered proteins.
What is the role of CD63 in endocytosis?
CD63 is a tetraspanin that traffics through endosomal compartments and regulates sorting and lysosomal function.
Conclusion
GO:2000369 regulation of clathrin-dependent endocytosis is a critical biological process that controls the internalization of diverse cargo, including receptors, transporters, and nutrients. Its dysregulation contributes to cancer, metabolic disorders, and infectious disease, making it a prime target for both basic and translational research [1,6,7]. Advances in CRISPR-based models, imaging, and proteomics continue to unravel the complex regulatory networks, offering opportunities for therapeutic intervention.
References
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- 2. Pols MS et al.. 2009. Trafficking and function of the tetraspanin CD63.. Exp Cell Res 315(9):1584-92 PMID: 18930046
- 3. Wolfe BL et al.. 2007. Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis.. Traffic 8(5):462-70 PMID: 17376169
- 4. You H et al.. 2023. Absorption of egg white hydrolysate in the intestine: Clathrin-dependent endocytosis as the main transport route.. Food Res Int 173(Pt 2):113480 PMID: 37803802
- 5. Sandvig K et al.. 2018. Clathrin-independent endocytosis: an increasing degree of complexity.. Histochem Cell Biol 150(2):107-118 PMID: 29774430
- 6. Shimauchi A et al.. 2025. Enhanced HER2 internalization by clathrin-dependent endocytosis in non-small cell lung cancer positive for HER2 mutations.. Br J Cancer 133(7):976-985 PMID: 40721523
- 7. Xu BY et al.. 2020. Rifampicin induces clathrin-dependent endocytosis and ubiquitin-proteasome degradation of MRP2 via oxidative stress-activated PKC-ERK/JNK/p38 and PI3K signaling pathways in HepG2 cells.. Acta Pharmacol Sin 41(1):56-64 PMID: 31316180
- 8. Miao Y et al.. 2018. Phospho-regulation of intrinsically disordered proteins for actin assembly and endocytosis.. FEBS J 285(15):2762-2784 PMID: 29722136
