Recombinant Dog Protein 4.1 (EPB41), partial

What is Protein 4.1R (EPB41) and what are its primary functions in cells?

Protein 4.1R, encoded by the EPB41 gene, is a membrane-cytoskeleton adaptor protein that serves as a critical component of the erythrocyte membrane cytoskeleton. It functions primarily to control cell surface expression and activity of various transmembrane proteins while also organizing F-actin. The protein plays essential roles in maintaining membrane mechanical stability, cytoskeleton organization, and the cell cycle. As a prototypical member of the FERM domain-containing superfamily of proteins (alongside ezrin, radixin, and moesin), 4.1R links a diverse range of transmembrane proteins—including cell adhesion molecules, ion channels, and receptors—to the cytoskeleton .

What are the major isoforms of Protein 4.1R identified in mammals?

Multiple isoforms of Protein 4.1R have been identified through RT-PCR and western blotting analyses. Research has revealed at least four primary isoforms in mammalian systems: ATG1 4.1R Δexons14,15; ATG1 4.1R Δexons14,15,17B; ATG2 4.1R Δexons14,15; and ATG2 4.1R Δexons14,15,17B. These isoforms exhibit different molecular weights, with western blots showing two highly expressed proteins at approximately 80 kDa and 115 kDa, and two lower abundance proteins at approximately 135 kDa and 170 kDa. The human version of 4.1R has a canonical amino acid length of 864 residues with a protein mass of 97 kilodaltons, although seven isoforms have been identified in total .

How does the subcellular localization of different 4.1R isoforms correlate with their functions?

The subcellular localization of 4.1R isoforms varies significantly, correlating with their diverse functions. Fluorescence microscopy studies utilizing GFP fusion proteins have shown that the ~80 kDa isoform localizes primarily in the cytoplasm and at the leading edge of motile cells, suggesting involvement in cell migration and membrane dynamics. In contrast, the ~115 kDa isoform is predominantly retained in the nucleus, indicating potential roles in nuclear processes. Immunofluorescence staining with anti-4.1R-exon16 antibody confirms this differential localization pattern of endogenous 4.1R isoforms. This distribution pattern explains how 4.1R can simultaneously participate in membrane organization, cytoskeletal arrangement, and potentially nuclear functions such as cell cycle regulation .

What molecular mechanisms underlie the interaction between Protein 4.1R and β1 integrin in cell adhesion processes?

The molecular interaction between Protein 4.1R and β1 integrin represents a critical mechanism regulating cell adhesion. Co-immunoprecipitation experiments have demonstrated that both ATG1 4.1R isoforms can be pulled down with β1 integrin antibody, and conversely, β1 integrin can be pulled down with 4.1R by anti-4.1R antibody. In vitro GST pull-down assays further confirm that both ATG1 4.1R and ATG2 4.1R bind directly to the cytoplasmic domain of β1 integrin, and that β1 integrin specifically binds to the 30 kDa membrane-binding domain of 4.1R. This direct interaction appears to be essential for normal surface expression and activation of β1 integrin, as studies with 4.1R-/- keratinocytes show approximately 43% reduction in the activated form of β1 integrin both in the absence and presence of MnCl₂. The molecular mechanism likely involves 4.1R-mediated stabilization of β1 integrin at the cell surface, potentially through cytoskeletal anchoring that prevents premature internalization or degradation .

How does the knockout or silencing of the EPB41 gene affect cellular morphology and migratory capabilities across different cell types?

Knockout or silencing of the EPB41 gene produces profound effects on cellular morphology and migration across various cell types. In 4.1R-/- keratinocytes, cell adhesion, spreading, migration, and motility are significantly impaired. These cells fail to form proper actin stress fibers and focal adhesions when cultured on fibronectin. Similarly, siRNA-mediated silencing of 4.1R in DC2.4 dendritic cells results in distinct morphological changes, including smaller cell bodies, increased numbers of synapses, and more pronounced protuberances compared to control cells. The migratory capacity of both cell types is significantly reduced following 4.1R depletion, as demonstrated by Transwell migration assays. These consistent effects across different cell types suggest a conserved role for 4.1R in cytoskeletal organization and cell motility, likely through its interaction with adhesion molecules like β1 integrin and its role in F-actin organization .

What is the role of Protein 4.1R in pathological conditions such as myasthenia gravis, and how might recombinant versions be used in therapeutic development?

Protein 4.1R has been implicated in the pathogenesis of myasthenia gravis (MG), with proteomics analysis revealing abnormal overexpression of 4.1R in the thymus tissues of MG patients. The protein appears to play a particularly important role in dendritic cells (DCs), which are critical antigen-presenting cells in the thymus and contribute significantly to thymic immunity and autoimmune diseases. Research shows that silencing 4.1R expression in dendritic cells alters their morphology and reduces their migratory capacity, suggesting that 4.1R overexpression in MG may contribute to abnormal DC function and migration. Given these findings, recombinant 4.1R proteins could be valuable tools for developing targeted therapies for MG. Potential approaches might include using modified recombinant 4.1R to competitively inhibit the abnormal function of overexpressed endogenous 4.1R, or developing antibodies against specific domains of 4.1R to modulate its activity in dendritic cells .

What are the optimal expression systems for producing functional recombinant dog Protein 4.1R, and how do they compare?

The production of functional recombinant dog Protein 4.1R requires careful consideration of expression systems to ensure proper folding, post-translational modifications, and structural integrity. Based on research approaches used for studying 4.1R proteins, several expression systems can be considered:

What gene silencing approaches are most effective for studying the function of endogenous EPB41 in canine cell models?

Several gene silencing approaches can be employed to study endogenous EPB41 function in canine cell models, each with distinct advantages:

• siRNA transfection: Research has demonstrated successful silencing of 4.1R expression using specific siRNAs. For example, in a study using mouse dendritic cells, three different 4.1R-specific siRNAs were designed and transfected, with real-time PCR and Western blot analysis confirming significant downregulation of EPB41 expression. The most effective siRNA reduced expression by approximately 80%. This approach allows for transient knockdown with relatively simple methodology.

• shRNA lentiviral transduction: For more stable, long-term silencing, shRNA delivered via lentiviral vectors can be used. This approach would be particularly valuable for studying the effects of 4.1R depletion in slowly dividing primary canine cells or in extended experimental timelines.

• CRISPR/Cas9 gene editing: For complete knockout studies, CRISPR/Cas9 can be employed to introduce frameshift mutations or large deletions in the canine EPB41 gene. This approach creates stable knockout cell lines that completely lack 4.1R expression.

When designing silencing constructs for canine 4.1R, it's essential to target conserved regions present in all isoforms (like exon 16) if complete silencing is desired, or to target specific exons (like exon 17B) if selective isoform silencing is the goal .

What analytical techniques provide the most comprehensive characterization of recombinant dog Protein 4.1R structure and interactions?

A multi-technique approach provides the most comprehensive characterization of recombinant dog Protein 4.1R:

For studying the key functional interaction between dog 4.1R and β1 integrin, a combination of co-immunoprecipitation and GST pull-down assays has proven particularly informative. Research has successfully used these techniques to demonstrate direct binding between 4.1R and the cytoplasmic domain of β1 integrin, as well as mapping the interaction to the 30 kDa membrane-binding domain of 4.1R .

How can recombinant dog Protein 4.1R be utilized in comparative studies of cytoskeletal regulation across species?

Recombinant dog Protein 4.1R offers valuable opportunities for comparative studies of cytoskeletal regulation across mammalian species. The 4.1R protein family shows evolutionary conservation of core structural domains, but with species-specific variations that may relate to functional specialization. By producing recombinant versions of dog 4.1R alongside human, mouse, and other mammalian 4.1R proteins, researchers can conduct systematic comparisons of binding affinities to conserved partners like β1 integrin.

Such comparative studies require consistent experimental conditions using techniques like:

• Binding affinity assays with common interaction partners

• Cross-species complementation experiments in knockout models

• Structural studies comparing domain organization and flexibility

These approaches can reveal whether functional differences exist in how 4.1R regulates cell adhesion, migration, and membrane stability across species. For example, studies with 4.1R-/- keratinocytes have shown impaired cell adhesion, spreading, and migration, but it remains unclear whether the severity of these phenotypes would be identical across species or whether species-specific adaptations in 4.1R function exist .

What are the most challenging aspects of working with partial recombinant dog Protein 4.1R constructs, and how can researchers overcome them?

Working with partial recombinant dog Protein 4.1R constructs presents several significant challenges:

• Domain interdependence: The functional domains of 4.1R often exhibit interdependence, where the activity of one domain is modulated by other regions of the protein. Studies have shown that 4.1R contains multiple functional domains, including a 30 kDa membrane-binding domain that interacts with β1 integrin. Working with partial constructs may disrupt these interdomain interactions, potentially yielding artificial results.

• Isoform complexity: With multiple isoforms identified (ranging from ~80 kDa to ~170 kDa), determining which partial construct best represents the physiologically relevant form for a specific research question can be difficult. The choice between ATG1 and ATG2 initiation variants significantly affects protein size and potentially function.

• Solubility and stability issues: Partial constructs often exhibit reduced solubility or stability compared to full-length proteins, particularly when expressed in prokaryotic systems.

To overcome these challenges, researchers should:

• Design partial constructs based on known domain boundaries from structural studies

• Include adjacent domains when studying a specific region to maintain contextual regulation

• Validate findings with multiple overlapping constructs

• Compare results with full-length protein whenever possible

• Utilize mammalian expression systems for increased solubility of complex domains

• Employ fusion tags (like MBP or SUMO) that enhance solubility while minimizing functional interference

How does post-translational modification of Protein 4.1R affect its functional properties, and what methodologies best detect these modifications in recombinant preparations?

Post-translational modifications (PTMs) of Protein 4.1R significantly impact its functional properties, influencing subcellular localization, binding partner interactions, and structural conformation. Although the search results don't provide specific information about PTMs in dog 4.1R, research on mammalian 4.1R proteins indicates several important modifications:

To effectively study these modifications in recombinant dog 4.1R preparations, researchers should implement:

• Expression in mammalian systems (preferably canine cells) to ensure proper PTM machinery

• Enrichment strategies prior to analysis (e.g., phosphopeptide enrichment)

• Targeted mass spectrometry approaches like Multiple Reaction Monitoring (MRM)

• Site-directed mutagenesis of potential modification sites to assess functional significance

The differential localization observed between the ~80 kDa cytoplasmic/leading edge isoform and the ~115 kDa nuclear isoform suggests that PTMs may play a crucial role in determining subcellular targeting, making their characterization essential for understanding the complete functional profile of dog 4.1R .

What are the implications of Protein 4.1R dysfunction in canine disease models, and how can recombinant proteins aid in diagnostic development?

Protein 4.1R dysfunction potentially contributes to several canine pathologies, particularly those involving cell adhesion, migration, and membrane stability. While specific canine disease associations are not detailed in the search results, the fundamental roles of 4.1R suggest relevance to:

• Hematological disorders: Given 4.1R's critical role in erythrocyte membrane stability, dysfunction could contribute to canine hemolytic anemias or erythrocyte abnormalities.

• Wound healing deficiencies: Studies in 4.1R-/- mice demonstrated defective epidermal wound healing, suggesting potential involvement in delayed wound healing in dogs, particularly following surgical interventions or traumatic injuries.

• Immune system dysregulation: The association between 4.1R and dendritic cell function indicates possible roles in canine autoimmune conditions or immune deficiencies.

Recombinant dog Protein 4.1R could aid diagnostic development through:

• Generation of specific antibodies for immunohistochemical detection of abnormal 4.1R expression or localization in tissue samples

• Development of binding assays to assess 4.1R-β1 integrin interaction strength in patient samples

• Creation of functional assays to evaluate cytoskeletal integrity in canine cells

These approaches could enable earlier detection of conditions involving cytoskeletal disruption and potentially guide therapeutic interventions targeting 4.1R-dependent pathways .