Recombinant Human Protein FAM189A2 (FAM189A2)

What is FAM189A2 and why has it been renamed ENTREP?

FAM189A2 (Family with sequence similarity 189 member A2) has been renamed ENTREP (ENdosomal TRansmembrane binding with EPsin) based on its subcellular localization and function. The protein is a type-I single-pass transmembrane protein containing 450 amino acids, with a structure that includes a signal sequence, a CD20-homologous extracellular region (55 aa), a transmembrane domain, and a downstream cytoplasmic region (343 aa) at the carboxyl-terminus. This renaming reflects its demonstrated role in endosomal trafficking and interaction with proteins like Epsin.

The protein has been definitively identified as an activator for the HECT-type ubiquitin E3 ligase ITCH, providing functional clarity that led to this more descriptive nomenclature. When designing experiments involving this protein, researchers should note both names in their publications to ensure proper cross-referencing with historical literature.

What is the molecular structure and function of FAM189A2/ENTREP?

FAM189A2/ENTREP is a type-I transmembrane protein with a structure that includes:

• Signal sequence (N-terminal)

• CD20-homologous extracellular region (55 amino acids)

• Transmembrane domain

• Cytoplasmic region (343 amino acids) at the C-terminus

• PPxY motifs in the cytoplasmic domain

Functionally, FAM189A2/ENTREP serves as an activator for ITCH, a HECT-type ubiquitin E3 ligase. The PPxY motifs mediate its association with and ubiquitination by ITCH. Additionally, FAM189A2/ENTREP facilitates the association of the chemokine receptor CXCR4 with ITCH, enhancing ITCH-mediated ubiquitination of CXCR4. This interaction plays a crucial role in the regulation of CXCR4 desensitization, which affects various cellular processes, including chemotaxis and cancer cell stemness.

The protein also associates with Epsin and accumulates in early and late endosomes along with ITCH, suggesting its involvement in clathrin-mediated endocytosis pathways. This endosomal localization is critical for its function in regulating receptor trafficking.

What are the known transcript variants of FAM189A2/ENTREP?

Research has identified at least two distinct transcript variants of FAM189A2/ENTREP in the MCF-7 breast cancer cell line:

• Complete transmembrane variant: This transcript is identical to FAM189A2 isoform b (NM_001127608.3, registered under accession number LC496047.1 in the DDBJ/EMBL-EBI/GenBank database). It encodes the full 450 amino acid protein with all functional domains intact.

• Exon 5-skipping variant: This transcript lacks the entire transmembrane domain. When expressed in HEK293T cells, this variant protein is faintly detected under normal conditions but increases to levels comparable to the complete variant in the presence of the protease inhibitor MG132. This suggests significant protein instability of the exon 5-skipping variant.

When designing expression constructs for recombinant production, researchers should carefully consider which variant to use based on their experimental goals. The complete transmembrane variant is recommended for most functional studies due to its stability and complete domain architecture.

How does FAM189A2/ENTREP regulate CXCR4 signaling and what are the implications for cancer research?

FAM189A2/ENTREP regulates CXCR4 signaling through a multi-step mechanism involving ITCH-mediated ubiquitination and subsequent receptor endocytosis:

• FAM189A2/ENTREP serves as an activator for the HECT-type ubiquitin E3 ligase ITCH

• It facilitates the association between CXCR4 and ITCH

• This interaction enhances ITCH-mediated ubiquitination of CXCR4

• The ubiquitination triggers CXCL12-induced endocytosis of CXCR4

• This process leads to CXCR4 desensitization

In FAM189A2-knockout cellular models, CXCL12-induced endocytosis of CXCR4 is prohibited, which enhances the effects of CXCL12 on chemotaxis and mammosphere formation in breast cancer cells. This suggests that FAM189A2/ENTREP functions as a negative regulator of CXCR4 signaling by promoting its desensitization.

For cancer research, these findings have significant implications:

• FAM189A2/ENTREP is downregulated in various cancers, including breast cancer

• Low FAM189A2 expression correlates with reduced relapse-free survival in breast cancer patients

• The loss of FAM189A2 may enhance CXCR4 signaling, promoting cancer cell migration and stemness

This regulatory mechanism represents a potential therapeutic target for cancers where CXCR4 signaling plays a critical role in progression and metastasis.

What is the prognostic significance of FAM189A2/ENTREP expression in different cancer types?

FAM189A2/ENTREP has emerged as a significant prognostic marker in several cancer types, with expression levels correlating with clinical outcomes:

Breast Cancer:

• Low FAM189A2 expression significantly correlates with reduced relapse-free survival (RFS) time across all breast cancer subtypes (HR = 0.62, log-rank P = 1.9e-15)

• Median RFS times: 148 months for low-expression cohort vs. 216.66 months for high-expression cohort

• Particularly significant in luminal A-subtype (HR = 0.59, log-rank P = 4e-09) and HER2-enriched subtype (HR = 0.45, log-rank P = 4.1e-05)

• No significant correlation observed in triple-negative breast cancer

Expression in Various Cancers:
FAM189A2 is downregulated compared to normal tissues in multiple cancer types:

• Bladder cancer

• Breast cancer

• Lung cancer

• Colorectal cancer

• Gastric cancer

• Head and neck cancer

Metastatic Progression:
FAM189A2 expression is lower at metastatic sites compared to primary sites in both prostate and breast cancers, suggesting a potential role in metastatic progression.

These findings indicate that FAM189A2/ENTREP may function as a tumor suppressor, with its downregulation contributing to cancer progression and metastasis. Researchers studying cancer biomarkers should consider including FAM189A2/ENTREP expression analysis in their studies, particularly for breast cancer prognostication.

How does the interaction between FAM189A2/ENTREP and ITCH regulate cellular processes beyond CXCR4 signaling?

While the FAM189A2/ENTREP-ITCH interaction has been primarily characterized in the context of CXCR4 regulation, this relationship likely impacts additional cellular processes:

• General Endosomal Trafficking: FAM189A2/ENTREP associates with Epsin and accumulates in early and late endosomes, suggesting a broader role in endosomal trafficking beyond CXCR4. This may affect the dynamics of multiple membrane receptors and signaling pathways.

• Regulation of Other ITCH Substrates: ITCH is known to ubiquitinate various substrates involved in diverse cellular functions. As an ITCH activator, FAM189A2/ENTREP may influence the ubiquitination and subsequent degradation or trafficking of these additional targets. These might include:

  • Transcription factors

  • Apoptosis regulators

  • Other membrane receptors

• Clathrin-Mediated Endocytosis: The association with Epsin suggests FAM189A2/ENTREP involvement in the cargo selection of clathrin-mediated endocytosis, potentially affecting the internalization of multiple plasma membrane proteins.

Understanding these broader implications requires further research using techniques such as:

• Proximity labeling to identify additional FAM189A2/ENTREP interacting partners

• Global ubiquitinome analysis in FAM189A2/ENTREP knockout versus wildtype cells

• Plasma membrane proteomics to identify additional receptors affected by FAM189A2/ENTREP loss

This expanded understanding would provide insights into the tumor suppressor functions of FAM189A2/ENTREP beyond CXCR4 regulation.

What expression systems are optimal for producing recombinant FAM189A2/ENTREP protein?

Producing functional recombinant FAM189A2/ENTREP requires careful consideration of expression systems due to its transmembrane nature and post-translational modifications. Based on research methodologies used in FAM189A2 studies, optimal approaches include:

Mammalian Expression Systems:

• HEK293T cells: Demonstrated success in expressing both full-length and variant forms of FAM189A2/ENTREP

• Advantages: Proper folding, post-translational modifications including ubiquitination, and appropriate membrane insertion

• Vector considerations: CMV promoter-driven vectors with appropriate epitope tags (e.g., FLAG, HA) for detection and purification

• Transfection method: Lipid-based transfection has shown good results

Expression Optimization Strategies:

• For full-length protein: Standard expression conditions with appropriate membrane protein extraction protocols

• For exon 5-skipping variant: Co-treatment with proteasome inhibitors (e.g., MG132) may be necessary to prevent rapid degradation

• For functional studies: Co-expression with interaction partners like ITCH may stabilize the protein

Purification Considerations:

• Gentle detergent solubilization (e.g., n-dodecyl-β-D-maltoside or digitonin) to maintain structural integrity

• Affinity chromatography using anti-tag antibodies

• Size exclusion chromatography for final purification

Researchers should validate the functionality of recombinant FAM189A2/ENTREP by assessing its ability to interact with known partners such as ITCH and EPN1, and its capacity to localize properly when reintroduced into cells.

What are the most effective knockout and knockdown strategies for studying FAM189A2/ENTREP function?

Multiple genetic manipulation approaches have been successfully employed to study FAM189A2/ENTREP function, each with specific advantages and applications:

CRISPR-Cas9 Knockout:

• Most effective for complete ablation of protein expression

• Target early exons to ensure complete disruption of the reading frame

• Verify knockout by sequencing and Western blotting

• Particularly useful for studying:

  • CXCR4 endocytosis dynamics

  • Migration and mammosphere formation assays

  • Long-term phenotypic consequences

siRNA/shRNA Knockdown:

• Valuable for temporal studies and graded reduction of expression

• Multiple siRNAs targeting different regions should be tested for specificity

• Can be delivered transiently or stably (shRNA)

• Particularly useful for:

  • Studying acute responses to FAM189A2/ENTREP depletion

  • Titrating the level of FAM189A2/ENTREP expression

  • Comparing partial vs. complete loss

Rescue Experiments:

• Essential for confirming specificity of observed phenotypes

• Re-expression of wildtype FAM189A2/ENTREP in knockout cells

• Structure-function analysis using domain mutants:

  • PPxY motif mutants to disrupt ITCH interaction

  • Transmembrane domain mutants

Validation Methods:

• qRT-PCR for transcript levels

• Western blotting for protein levels

• Functional assays (CXCR4 ubiquitination, endocytosis rates)

When studying cancer models, researchers should be aware that many cancer cell lines already have reduced FAM189A2/ENTREP expression. MCF-7 is notably an exception among breast cancer lines, maintaining relatively high expression levels, making it particularly useful for knockout studies.

What methods should be used to investigate the FAM189A2/ENTREP-ITCH interaction and ubiquitination dynamics?

Investigating the FAM189A2/ENTREP-ITCH interaction and subsequent ubiquitination dynamics requires specialized biochemical and cellular approaches:

Protein-Protein Interaction Methods:

• Co-immunoprecipitation (Co-IP):

  • Pull down FAM189A2/ENTREP and blot for ITCH (or vice versa)

  • Include appropriate controls (IgG, unrelated membrane proteins)

• Proximity Ligation Assay (PLA):

  • Visualize interactions in situ within cells

  • Particularly valuable for determining subcellular localization of interactions

• Domain Mapping:

  • Generate constructs with mutations in the PPxY motifs of FAM189A2/ENTREP

  • Evaluate binding to the WW domain of ITCH

  • Use truncation mutants to identify other potential interaction regions

Ubiquitination Assays:

• In vivo ubiquitination assay:

  • Express His-tagged ubiquitin, FAM189A2/ENTREP, and ITCH

  • Purify ubiquitinated proteins under denaturing conditions

  • Detect FAM189A2/ENTREP or CXCR4 by Western blotting

• In vitro ubiquitination assay:

  • Use purified components (E1, E2, ITCH, FAM189A2/ENTREP, CXCR4, ubiquitin)

  • Analyze by SDS-PAGE and Western blotting

  • Quantify ubiquitin chain formation and substrate modification

Real-time Ubiquitination Dynamics:

• FRET-based sensors for monitoring ubiquitination in living cells

• Pulse-chase studies to determine ubiquitination kinetics

• Quantitative mass spectrometry to identify ubiquitination sites and chain types

Functional Validation:

• Endocytosis assays using fluorescently-labeled CXCL12 or antibodies against CXCR4

• Receptor degradation assays following CXCL12 stimulation

• Signaling readouts (calcium flux, ERK phosphorylation) downstream of CXCR4

These approaches will allow researchers to dissect both the biochemical mechanisms and cellular consequences of the FAM189A2/ENTREP-ITCH-CXCR4 regulatory axis.

How can FAM189A2/ENTREP be targeted in cancer therapeutic development?

FAM189A2/ENTREP represents a promising target for cancer therapeutic development, particularly for breast cancers where CXCR4 signaling drives progression and metastasis. Several strategic approaches can be employed:

Restoration of FAM189A2/ENTREP Expression:

• Gene Therapy Approaches: Viral vectors delivering FAM189A2/ENTREP cDNA, particularly to tumors with low expression

• Epigenetic Modulators: If downregulation occurs through epigenetic mechanisms, DNA methyltransferase inhibitors or histone deacetylase inhibitors might restore expression

• Transcriptional Activators: CRISPR-based transcriptional activation systems targeting the FAM189A2/ENTREP promoter

Mimicking FAM189A2/ENTREP Function:

• Peptide Mimetics: Synthetic peptides containing the PPxY motifs that can activate ITCH

• Small Molecule ITCH Activators: Compounds that relieve ITCH autoinhibition

• CXCR4 Ubiquitination Enhancers: Molecules that promote CXCR4 ubiquitination and subsequent degradation

Combinatorial Approaches:

• CXCR4 Antagonists + FAM189A2/ENTREP Restoration: Targeting both the receptor and its regulatory mechanism

• Chemotherapy + FAM189A2/ENTREP Restoration: Sensitizing cancer cells to conventional treatments by altering CXCR4 signaling

Patient Selection Strategies:

• Use FAM189A2/ENTREP expression levels as a biomarker for therapy selection

• Focus on luminal A and HER2-enriched breast cancer subtypes where FAM189A2/ENTREP has demonstrated prognostic value

• Monitor CXCR4 activity as a pharmacodynamic marker

Research efforts should prioritize understanding the mechanisms of FAM189A2/ENTREP downregulation in cancers to inform the most effective therapeutic strategies for expression restoration or functional mimicry.

What biomarker potential does FAM189A2/ENTREP have in cancer diagnostics and prognostics?

FAM189A2/ENTREP demonstrates significant potential as a biomarker in cancer diagnostics and prognostics, particularly in breast cancer. The evidence and applications include:

Prognostic Value:

• In breast cancer patients (all subtypes), low FAM189A2 expression correlates with reduced relapse-free survival (RFS) with a hazard ratio of 0.62 (p = 1.9e-15)

• Median RFS times: 148 months for low-expression vs. 216.66 months for high-expression cohorts

• Strongest prognostic value in:

  • Luminal A subtype: HR = 0.59, p = 4e-09

  • HER2-enriched subtype: HR = 0.45, p = 4.1e-05

Comparative Expression Table Across Cancer Types:

Clinical Application Areas:

• Risk Stratification: Identifying patients with higher risk of relapse based on low FAM189A2 expression

• Treatment Decision Support: Guiding more aggressive therapy for patients with low FAM189A2 expression

• Monitoring Disease Progression: Potential marker for development of metastasis

• Companion Diagnostics: For therapies targeting the CXCR4 signaling pathway or FAM189A2/ENTREP itself

Implementation Considerations:

• RNA-based assays (qRT-PCR, RNA-seq) provide reliable quantification of FAM189A2 expression

• Immunohistochemistry protocols need validation for protein-level detection

• Gene expression panels incorporating FAM189A2 with other prognostic markers may improve predictive power

The consistent downregulation across multiple cancer types suggests FAM189A2/ENTREP could serve as part of a pan-cancer biomarker panel, though its strongest prognostic utility appears to be in specific breast cancer subtypes.

How can differential expression analysis of FAM189A2/ENTREP be integrated into cancer research workflows?

Incorporating FAM189A2/ENTREP differential expression analysis into cancer research workflows can enhance understanding of disease mechanisms and improve patient stratification. Here's a methodological framework for integration:

Bioinformatic Analysis Pipeline:

• RNA-Seq Data Processing:

  • Standard quality control and normalization procedures

  • Use of "edgeR" and "limma" R packages for differential expression analysis with thresholds of log2FC ≥2 and FDR <0.05

  • Generation of volcano plots and hierarchical clustering visualization

• Multi-Omics Integration:

  • Correlation of FAM189A2/ENTREP expression with:

    • DNA methylation status of the FAM189A2 locus

    • Copy number variations

    • Protein expression (if proteomics data available)

    • Associated microRNAs that may regulate FAM189A2

• WGCNA (Weighted Gene Co-expression Network Analysis):

  • Identify co-expressed gene modules containing FAM189A2/ENTREP

  • Characterize the "turquoise module" or other modules containing FAM189A2/ENTREP

  • Perform Gene Ontology and pathway enrichment analysis of co-expressed genes

Experimental Validation Workflow:

• Cell Line Panel Screening:

  • qRT-PCR and Western blot analysis across cancer cell line panels

  • Correlation with cellular phenotypes (migration, invasion, stemness)

• Patient Sample Analysis:

  • Tissue microarray construction and immunohistochemistry

  • Correlation with clinicopathological parameters and outcome data

• Functional Studies:

  • Perturbation of FAM189A2/ENTREP expression in appropriate models

  • Assessment of effects on CXCR4 signaling and cancer hallmarks

Implementation in Clinical Research:

• Incorporation into existing prognostic scores for breast cancer

• Development of subtype-specific risk assessment models including FAM189A2/ENTREP

• Integration into liquid biopsy approaches for monitoring disease progression

This comprehensive approach ensures that FAM189A2/ENTREP expression analysis is meaningfully integrated into both basic research and translational applications, maximizing its utility as both a mechanistic research tool and potential clinical biomarker.

Concluding Insights and Future Research Directions

The characterization of FAM189A2/ENTREP as an ITCH ubiquitin ligase activator with specific roles in CXCR4 regulation represents a significant advance in understanding receptor trafficking in cancer biology. The protein's downregulation across multiple cancer types and its correlation with clinical outcomes, particularly in breast cancer, highlight its potential importance in cancer progression mechanisms.

Future research should focus on:

• Detailed structural studies of FAM189A2/ENTREP to facilitate rational drug design

• Comprehensive identification of additional FAM189A2/ENTREP-regulated receptors beyond CXCR4

• Investigation of mechanisms responsible for FAM189A2/ENTREP downregulation in cancers

• Development of therapeutic strategies to restore or mimic FAM189A2/ENTREP function

• Validation of FAM189A2/ENTREP as a predictive biomarker in prospective clinical trials