GO:1990175 EH domain binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:1990175 (EH domain binding) is a molecular function describing the binding of a protein to an EH (Eps15 homology) domain, a motif originally identified in three copies at the NH2-termini of Eps15 and Eps15R.
• EH domains are small protein-protein interaction modules that recognize short peptide motifs, most notably asparagine-proline-phenylalanine (NPF) sequences, to coordinate endocytosis, vesicle trafficking and signal transduction.
• EH domain-containing proteins such as EHD1 and EHBP1/EHBP1L1 act as scaffolds that link membrane remodeling to cargo selection and receptor recycling.
• Dysregulation of EH domain binding contributes to cancer immune evasion, metabolic liver disease and lipid disorders, making these interactions attractive therapeutic targets.
• CRISPR knockout, point-mutation, knock-in and overexpression models are essential to dissect which EH domain interactions are causal in disease.
• EH domain binding is studied using affinity proteomics, NMR structural biology, live-cell imaging and CRISPR library screening to map interaction networks and functional consequences.
Description
EH domain binding (GO:1990175) is a molecular function that describes the selective interaction of a protein with an EH (Eps15 homology) domain. The EH domain was originally identified as a motif present in three copies at the NH2-termini of Eps15 and the related molecule Eps15R, and it has since emerged as a versatile protein-protein interaction module in endocytic and trafficking machinery. Proteins that bind EH domains typically do so through short linear motifs, allowing them to assemble dynamic complexes that control membrane traffic, receptor signaling and cytoskeletal remodeling. For researchers, GO:1990175 provides a precise annotation for experiments that map EH-domain interactomes and test how these contacts influence cellular physiology. Because EH domain binding sits at the interface of endocytosis, signal transduction and organelle dynamics, it is relevant to cancer biology, metabolic disease and neurodegeneration. Understanding which proteins bind EH domains, and under what conditions, is therefore a prerequisite for targeting these interactions therapeutically.
EH domain binding At A Glance
| GO ID | GO:1990175 |
|---|---|
| GO term | EH domain binding |
| Ontology | molecular_function |
| Synonym | None listed in QuickGO |
| Major function | Binding to an EH (Eps15 homology) domain of a protein, typically through short peptide motifs such as NPF, to mediate protein-protein interactions in endocytosis and trafficking |
| Definition source | QuickGO definition: Binding to an EH domain of a protein. The EH stand for Eps15 homology. This was originally identified as a motif present in three copies at the NH2-termini of Eps15 and of the related molecule Eps15R |
| Example domain-containing proteins | Eps15, Eps15R, EHD1, EHBP1, EHBP1L1 |
| Related cellular processes | Endocytosis, vesicle trafficking, receptor recycling, signal transduction |
| Disease relevance | Cancer immune evasion, metabolic dysfunction-associated steatohepatitis, liver fibrosis, familial hypercholesterolemia |
What Is GO:1990175?
GO:1990175 EH domain binding is defined as binding to an EH domain of a protein. The EH stands for Eps15 homology, a motif originally identified in three copies at the NH2-termini of Eps15 and of the related molecule Eps15R. In practice, this term is used when an experimental assay demonstrates a physical interaction between a protein and an EH domain, such as co-immunoprecipitation, pull-down with an EH domain bait, or structural analysis of an EH domain-peptide complex.
Why Is EH domain binding Important in Cell Biology?
EH domain binding is important because it governs how cells internalize and sort receptors, lipids and signaling molecules. EH domain-containing proteins such as EHD1 and EHBP1/EHBP1L1 use these interactions to coordinate membrane remodeling with cargo selection, and disruption of these contacts has been linked to cancer immune evasion, metabolic liver disease and lipid disorders. Because EH domains are compact and modular, they are tractable targets for chemical biology and for CRISPR-based functional genomics, making GO:1990175 a high-value annotation for both basic and translational research.
• EH domain binding controls endocytic sorting and receptor recycling, which determine how cells respond to growth factors and nutrients.
• EHBP1L1 binding to JAK1 stabilizes the kinase and drives immune evasion in renal cell carcinoma, linking EH domain interactions to immunotherapy resistance.
• EHBP1 suppresses liver fibrosis in metabolic dysfunction-associated steatohepatitis, showing that EH domain-associated proteins can protect against chronic liver disease.
• Autophagy, lipid droplets and liver disease are interconnected processes in which EH domain proteins participate through membrane trafficking.
• Mutations in genes related to lipid handling, such as those causing familial hypercholesterolemia, highlight the clinical importance of trafficking pathways that EH domain proteins regulate.
• EH domain binding is a druggable protein-protein interaction class, and structural knowledge of EH domains supports rational inhibitor design.
• CRISPR screens can identify which EH domain interactions are essential in specific cell types, accelerating target validation.
• EH domain binding is conserved from yeast to humans, enabling cross-species model systems for mechanistic studies.
• Dysregulated EH domain interactions contribute to neurodegeneration through impaired endosomal trafficking, a hallmark of several neurological disorders.
• Mapping EH domain interactomes provides biomarkers and therapeutic hypotheses for cancer and metabolic disease.
Molecular Mechanism of EH domain binding
EH domain structure and target recognition
In simple terms: The EH domain is a small protein module that grabs specific short sequences on partner proteins.
EH domains are compact protein-protein interaction modules originally identified in three copies at the NH2-termini of Eps15 and Eps15R. They recognize short linear motifs, most commonly asparagine-proline-phenylalanine (NPF) sequences, in target proteins. Structural studies of the EHD1 EH domain have provided high-resolution insights into its fold and ligand-binding surface, revealing how it engages peptide partners with moderate affinity and high specificity. This modular recognition allows EH domain-containing proteins to act as scaffolds that bring together membrane lipids, cargo receptors and cytoskeletal regulators during endocytosis and vesicle trafficking.
Binding to EHBP1 and EHBP1L1
In simple terms: EHBP1 and EHBP1L1 are proteins that interact with EH domains to control membrane trafficking and signaling.
EHBP1 and its paralog EHBP1L1 are key EH domain-associated proteins that function in endocytic recycling and signal transduction. EHBP1L1 binds and stabilizes JAK1, thereby promoting immune evasion in renal cell carcinoma, demonstrating that EH domain binding can directly modulate oncogenic signaling. EHBP1 suppresses liver fibrosis in metabolic dysfunction-associated steatohepatitis, indicating that EH domain-dependent trafficking protects against chronic liver injury. These findings establish EHBP1/EHBP1L1 as central nodes where EH domain binding intersects with disease pathways.
EHD1 and the endocytic machinery
In simple terms: EHD1 is an EH domain-containing protein that helps pinch off vesicles and recycle receptors.
EHD1 is a well-characterized EH domain-containing ATPase that participates in endocytic recycling and membrane remodeling. Its EH domain mediates interactions with NPF-motif-containing partners, coupling cargo recognition to vesicle formation. The EHD1 EH domain has been structurally characterized by NMR, providing a template for understanding how EH domain binding is regulated at the atomic level. Through these interactions, EHD1 coordinates the return of receptors to the plasma membrane, a process essential for nutrient uptake and signal transduction.
Coordination with autophagy and lipid metabolism
In simple terms: EH domain binding helps cells manage fat and recycle damaged components.
EH domain-associated trafficking intersects with autophagy and lipid droplet biology, processes that are dysregulated in liver disease. The ménage à trois of autophagy, lipid droplets and liver disease highlights how membrane trafficking pathways, including those dependent on EH domain interactions, influence hepatic lipid storage and injury. EHBP1 suppression of liver fibrosis further supports a role for EH domain binding in metabolic liver pathology. These connections position GO:1990175 as a molecular function relevant to both vesicle trafficking and metabolic homeostasis.
Regulation by interaction partners and post-translational modifications
In simple terms: EH domain binding is tuned by other proteins and chemical modifications.
EH domain binding is not constitutive; it is regulated by the availability of NPF-motif partners, by phosphorylation of target proteins, and by the local lipid environment. Transcription factors can also interact with RNA to regulate gene expression programs that include trafficking components, indirectly shaping EH domain interaction networks. Selective targeting of BET proteins has been shown to modulate inflammatory and cancer gene expression programs, illustrating how transcriptional regulators can influence the expression of EH domain-containing proteins. Together, these layers of regulation determine when and where EH domain binding occurs.
Key Genes Involved in GO:1990175 EH domain binding
The following genes encode proteins that bind EH domains or contain EH domains, and they are central to research on GO:1990175.
| Gene | Major Role | Research Relevance |
|---|---|---|
| EPS15 | Contains three EH domains at its NH2-terminus; scaffold in endocytosis | Foundational gene for EH domain discovery and endocytic trafficking studies |
| EPS15R | EH domain-containing paralog of Eps15 | Defines the EH domain family and its interaction repertoire |
| EHD1 | EH domain-containing ATPase involved in endocytic recycling | Structurally characterized EH domain; model for EH domain binding |
| EHBP1 | EH domain-binding protein regulating membrane trafficking | Suppresses liver fibrosis in metabolic dysfunction-associated steatohepatitis |
| EHBP1L1 | EH domain-binding protein that stabilizes JAK1 | Drives immune evasion in renal cell carcinoma |
| JAK1 | Kinase stabilized by EHBP1L1 binding | Links EH domain binding to cytokine signaling and cancer immunity |
| EHD2 | EH domain-containing protein in caveolae and membrane remodeling | Candidate for EH domain interaction studies |
| EHD3 | EH domain-containing protein in endosomal sorting | Potential regulator of receptor recycling |
| EHD4 | EH domain-containing protein in endocytosis | Model for tissue-specific EH domain functions |
| REPS1 | EH domain-containing adaptor in endocytosis | Expands the EH domain interactome |
| REPS2 | EH domain-containing protein linked to receptor trafficking | Candidate for cancer trafficking studies |
| ITSN1 | EH domain-containing scaffold in endocytosis | Relevant to synaptic vesicle recycling |
| ITSN2 | EH domain-containing scaffold | Potential role in membrane dynamics |
| LDLR | Receptor whose trafficking intersects with EH domain pathways | Familial hypercholesterolemia mutations affect LDLR trafficking |
| BET proteins | Transcriptional regulators influencing expression of trafficking genes | Selective BD1/BD2 targeting modulates gene expression programs |
| Transcription factors | Interact with RNA to regulate genes including trafficking components | Provide upstream control of EH domain protein expression |
How Is EH domain binding Regulated?
EH domain binding is regulated at multiple levels. The availability of NPF-motif-containing partners determines which interactions occur, and phosphorylation of target proteins can create or abolish binding sites. Transcriptional programs controlled by factors that interact with RNA regulate the expression of EH domain-containing proteins and their partners. Selective targeting of BET proteins modulates inflammatory and cancer gene expression, indirectly affecting trafficking networks that depend on EH domain interactions. In metabolic contexts, autophagy and lipid droplet dynamics influence membrane composition and the recruitment of EH domain proteins to specific organelles. Finally, disease-associated mutations, such as those in familial hypercholesterolemia, can alter receptor trafficking pathways that intersect with EH domain-dependent sorting.
EH domain binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| EHBP1L1 | Renal cell carcinoma immune evasion via JAK1 stabilization | KO and knock-in in renal carcinoma cell lines; JAK1 reporter assays |
| EHBP1 | Metabolic dysfunction-associated steatohepatitis and liver fibrosis | KO and overexpression in hepatocyte cell lines and liver organoids |
| LDLR | Familial hypercholesterolemia | Point-mutation knock-in in hepatocyte-like cells; LDL uptake assays |
| EHD1 | Endocytic recycling defects linked to neurodegeneration | KO and tagged knock-in in neuronal cell lines; live-cell imaging |
| EPS15 | Endocytic trafficking and receptor sorting | KO and overexpression in HeLa and HEK293 cells; affinity proteomics |
EH domain binding in cancer immune evasion
EHBP1L1 binds and stabilizes JAK1, promoting immune evasion in renal cell carcinoma. This demonstrates that EH domain binding can directly enhance oncogenic signaling and suppress anti-tumor immunity. Targeting the EHBP1L1-JAK1 interaction may restore immune surveillance and improve immunotherapy responses. The involvement of JAK1 links EH domain biology to cytokine signaling pathways that are frequently dysregulated in cancer.
EH domain binding in metabolic liver disease
EHBP1 suppresses liver fibrosis in metabolic dysfunction-associated steatohepatitis, indicating that EH domain-associated trafficking protects against chronic liver injury. Autophagy, lipid droplets and liver disease are interconnected, and EH domain proteins participate in the membrane trafficking events that govern hepatic lipid storage. These findings suggest that modulating EH domain interactions could be therapeutically beneficial in steatohepatitis and fibrosis.
EH domain binding and lipid disorders
Familial hypercholesterolemia is caused by mutations that impair LDL receptor function and trafficking. Because EH domain proteins regulate endocytic recycling, they may influence the surface availability of LDLR and other lipoprotein receptors. Studying EH domain binding in patient-derived cells could reveal modifier pathways that affect disease severity.
EH domain binding in neurodegeneration
Endosomal trafficking defects are a hallmark of several neurodegenerative diseases, and EH domain-containing proteins such as Eps15 and EHD1 are core components of this machinery. Disruption of EH domain interactions could contribute to impaired receptor recycling and neuronal dysfunction. Model systems that manipulate EH domain binding are needed to test causality in neurodegeneration.
From EH domain binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is EHBP1L1 required for JAK1 stability and immune evasion? | EHBP1L1 knockout in renal cell carcinoma cells with JAK1 western blot and immune assays |
| Does EHBP1 protect against liver fibrosis? | EHBP1 knockout and overexpression in hepatocytes and hepatic stellate cells |
| Which residues in the EH domain mediate NPF-motif binding? | Point mutations in the EH domain of EHD1 or Eps15 followed by binding assays |
| Can a tagged EH domain be used to map interactomes? | Knock-in of an epitope-tagged EH domain in cell lines followed by affinity purification |
| Does overexpression of EHBP1L1 enhance tumor immune evasion? | Overexpression of EHBP1L1 in cancer cells followed by co-culture with T cells |
| Which genes modulate EH domain-dependent trafficking? | CRISPR library screening in cells expressing an EH domain-dependent reporter |
How to Study the EH domain binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Affinity purification-mass spectrometry | Proteins that bind an EH domain bait | Mapping the EH domain interactome |
| NMR spectroscopy | Atomic structure and dynamics of EH domain-ligand complexes | Structural characterization of EHD1 EH domain |
| Live-cell fluorescence imaging | Localization and dynamics of EH domain proteins | Tracking endocytic recycling |
| CRISPR knockout | Loss-of-function effects on EH domain-dependent processes | Testing causality of candidate genes |
| CRISPR library screening | Genome-wide modifiers of EH domain-dependent phenotypes | Identifying synthetic lethal partners |
| Co-immunoprecipitation | Physical interaction between EH domain proteins and partners | Validating binding in endogenous contexts |
| Western blotting | Protein stability and expression changes | Assessing JAK1 stabilization by EHBP1L1 |
| Reporter assays | Transcriptional or signaling output | Measuring immune evasion or inflammatory programs |
Affinity proteomics and interactome mapping
Affinity purification using EH domains as bait, followed by mass spectrometry, identifies proteins that bind EH domains and defines the interactome for GO:1990175. This approach can be combined with stable isotope labeling to quantify changes in binding under different conditions. Tagged knock-in cell lines expressing epitope-tagged EH domain proteins enable endogenous interactome capture.
Structural biology of EH domain complexes
NMR and crystallography have been used to solve the structure of the EHD1 EH domain, revealing its ligand-binding surface and providing a template for understanding EH domain binding at atomic resolution. Structural studies guide mutagenesis experiments that test which residues are required for interaction. These methods are essential for rational design of inhibitors targeting EH domain interactions.
Live-cell imaging of trafficking
Fluorescently tagged EH domain proteins and their cargoes can be imaged in live cells to track endocytic recycling and vesicle dynamics. This approach reveals where and when EH domain binding occurs in real time. Imaging combined with CRISPR knockout of candidate genes tests the functional requirement for specific interactions.
CRISPR screening and functional genomics
CRISPR library screening can identify genes that modulate EH domain-dependent processes, such as receptor recycling or immune evasion. Screens in cancer cell lines with EHBP1L1 or JAK1 reporters can uncover synthetic lethal interactions. Bioinformatics analysis of screen data prioritizes candidate EH domain binding partners for validation.
How CRISPR Can Be Used to Study GO:1990175 EH domain binding
Knockout
CRISPR knockout of EH domain-containing genes such as EHBP1 or EHBP1L1 is used to test their requirement in disease models. For example, EHBP1 knockout can assess its protective role in liver fibrosis, while EHBP1L1 knockout can determine whether JAK1 stabilization and immune evasion are lost. Knockout cell lines provide clean backgrounds for rescue experiments with wild-type or mutant EH domain proteins.
Point Mutation
Point mutations in the EH domain or in NPF motifs can dissect which residues are required for binding. CRISPR-mediated point mutation of the EHD1 EH domain can test structural predictions from NMR studies. Similarly, point mutations in JAK1 or EHBP1L1 can identify the interface required for stabilization and immune evasion.
Knock-in
Knock-in of epitope tags or fluorescent proteins into endogenous EH domain genes enables interactome mapping and live-cell imaging under native expression levels. Tagged knock-in models avoid artifacts from overexpression and allow precise tracking of EH domain binding dynamics. Knock-in of disease-associated mutations, such as those in LDLR, can model familial hypercholesterolemia.
Overexpression
Overexpression of EH domain proteins or their binding partners is used to test gain-of-function effects, such as enhanced immune evasion by EHBP1L1. Overexpression combined with reporter assays can quantify signaling output and identify downstream pathways. These models are complementary to knockout and knock-in approaches for establishing causality.
How EDITGENE Supports EH domain binding Research
Researchers studying EH domain binding-related genes often need to determine whether a candidate gene is causally involved in a specific trafficking or disease phenotype. EDITGENE provides end-to-end CRISPR services to generate precisely engineered cell models that answer these questions.
Contact EDITGENE today to design your custom CRISPR model for EH domain binding research.
Frequently Asked Questions About EH domain binding
What is GO:1990175 EH domain binding?
GO:1990175 is a molecular function term describing binding to an EH (Eps15 homology) domain of a protein, a motif originally identified in three copies at the NH2-termini of Eps15 and Eps15R.
What genes are involved in EH domain binding?
Key genes include EPS15, EPS15R, EHD1, EHBP1 and EHBP1L1, which encode EH domain-containing or EH domain-binding proteins.
What is the EH domain?
The EH domain is a small protein-protein interaction module that recognizes short peptide motifs, most commonly NPF sequences, and was first identified in Eps15 and Eps15R.
How does EH domain binding regulate endocytosis?
EH domain binding recruits cargo and membrane remodeling machinery to endocytic sites, controlling receptor internalization and recycling.
Is EH domain binding involved in cancer?
Yes, EHBP1L1 binding to JAK1 stabilizes the kinase and drives immune evasion in renal cell carcinoma.
What diseases are linked to EH domain binding?
EH domain binding has been linked to renal cell carcinoma, metabolic dysfunction-associated steatohepatitis, liver fibrosis and familial hypercholesterolemia.
How can I study EH domain binding in the lab?
Common methods include affinity proteomics, NMR structural biology, live-cell imaging and CRISPR knockout or knock-in models.
What is the structure of the EHD1 EH domain?
The EHD1 EH domain has been characterized by NMR, revealing its fold and ligand-binding surface.
Can CRISPR be used to study EH domain binding?
Yes, CRISPR knockout, point mutation, knock-in and overexpression models are widely used to test the function of EH domain proteins and their partners.
Why is EH domain binding important for drug discovery?
EH domains are compact and modular, making them tractable targets for inhibitors that disrupt disease-relevant protein-protein interactions.
Conclusion
GO:1990175 EH domain binding defines a fundamental protein-protein interaction function that coordinates endocytosis, vesicle trafficking and signal transduction. Its relevance spans cancer immune evasion, metabolic liver disease and lipid disorders, making it a high-priority area for functional genomics and therapeutic development. By combining CRISPR models with structural and proteomic approaches, researchers can map EH domain interaction networks and identify causal nodes for intervention.
References
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- 3. Oksuz O et al.. 2023. Transcription factors interact with RNA to regulate genes.. Mol Cell 83(14):2449-2463.e13 PMID: 37402367
- 4. Gilan O et al.. 2020. Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.. Science 368(6489):387-394 PMID: 32193360
- 5. Confalonieri S et al.. 2002. The Eps15 homology (EH) domain.. FEBS Lett 513(1):24-9 PMID: 11911876
- 6. Pan Y et al.. 2023. EHBP1L1 Drives Immune Evasion in Renal Cell Carcinoma through Binding and Stabilizing JAK1.. Adv Sci (Weinh) 10(11):e2206792 PMID: 36775874
- 7. Kieken F et al.. 2007. EH domain of EHD1.. J Biomol NMR 39(4):323-9 PMID: 17899392
- 8. Chiou KR et al.. 2012. Common mutations of familial hypercholesterolemia patients in Taiwan: characteristics and implications of migrations from southeast China.. Gene 498(1):100-6 PMID: 22353362
