Recombinant Mouse Protein EFR3 homolog A (Efr3a), partial

What is the structural characterization of EFR3A protein and how does it compare between mouse and human orthologs?

EFR3A belongs to the armadillo-like family (ARMH) of superhelical proteins characterized by a multi-helical motif composed of two curved layers of α-helices arranged in a right-handed superhelix. The protein exhibits an extended superhelical rod-like structure in its first two-thirds, with a typical armadillo repeat motif (ARM) in the N-terminal part and a triple helical motif in its C-terminal region .

Structurally, EFR3A demonstrates remarkable conservation across species, with significant homology between mouse and human orthologs. The N-terminal region (residues 14-111 of human EFR3A) shows substantial sequence identity (23%) and similarity (58%) to the yeast ortholog, indicating evolutionary conservation of this functionally important domain . Modern structural analysis using AlphaFold has provided detailed models of the human EFR3A structure, which can serve as a reference point for studying the mouse ortholog .

What are the key functional domains of mouse EFR3A protein and their roles in cellular processes?

Mouse EFR3A contains several functionally significant domains that mediate its biological activities:

• N-terminal palmitoylation motif: Contains 3-4 cysteine residues that undergo palmitoylation, critical for plasma membrane localization .

• Armadillo repeat motif (ARM): Located in the N-terminal region, this domain is characterized by a superhelical structure that likely facilitates protein-protein interactions .

• Basic patch at N-terminus: This region is responsible for interactions with phosphatidyl inositols, enabling attachment of EFR3A and its protein complexes to the plasma membrane .

• Central basic patch: Located near the middle part of the molecule by loops joining helices 11-18, this region may contribute to membrane interactions .

• C-terminal triple helical region: The conserved middle C-terminal region (residues 724-787 in human) forms a V-shaped structure composed of three α-helices that mediates interactions with partner proteins, particularly in the PI4K complex .

These domains collectively enable EFR3A to function as a scaffold protein anchoring the phosphatidylinositol 4-kinase A complex to the plasma membrane and participating in membrane raft organization through interactions with proteins like flotillin-2 .

How is EFR3A expression altered in neurological disorders and what experimental approaches can detect these changes?

EFR3A has been implicated in several neurological disorders through altered gene expression patterns. In essential tremor (ET), a common neurological disorder, EFR3A is among seven genes showing abnormally changed expression patterns, specifically being upregulated . This finding was established through analysis of both publicly available and author-generated RNA sequencing data, suggesting that RNA-seq is an effective method for detecting EFR3A expression changes in neurological conditions .

In Alzheimer's disease models, the knockout of Efr3a in the CA3 area of the hippocampus leads to Amyloid β-induced depletion of PI(4,5)P2 . Methodology for studying these effects includes selective deletion of Efr3a at presynaptic sites in CA1 pyramidal neurons, which has been shown to improve cognitive function and memory in APP/PS1 mouse models of Alzheimer's disease .

For experimental detection of expression changes, researchers should consider:

• RNA sequencing of affected tissues

• Region-specific knockout or knockdown approaches

• Immunohistochemistry to visualize protein localization

• Western blotting for quantitative protein expression analysis

• Functional assays measuring PI(4,5)P2 levels in affected regions

What are the established experimental models for studying EFR3A function in mice?

Several experimental mouse models have been developed to study EFR3A function:

• Brain-specific Efr3a knockout mice: These models have revealed enhanced hippocampal neurogenesis in adult mice, with newborn neurons characterized by extended survival and decreased apoptosis. The mechanism appears to involve increased expression of brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) genes, which govern signaling pathways controlling survival, particularly the AKT pathway .

• Region-specific Efr3a knockout models: Selective deletion of Efr3a in specific brain regions such as the CA3 area of the hippocampus or at presynaptic sites in CA1 pyramidal neurons provides insights into region-specific functions .

• Efr3a knockdown and overexpression models: These have been used to study the role of EFR3A in cochlear development and function, revealing that loss of Efr3a expression may delay hair cell loss and spiral ganglion degeneration .

• Cell line models: While not mouse-specific, established cell lines can be manipulated to study EFR3A function through knockdown or overexpression approaches .

When selecting an experimental model, researchers should consider the specific aspect of EFR3A function they wish to study (neurogenesis, membrane organization, PI4K complex assembly, etc.) and choose the model system accordingly.

How can brain-specific Efr3a knockout mouse models be effectively used to study neurogenesis?

Brain-specific Efr3a knockout mouse models provide valuable tools for studying neurogenesis, particularly in the hippocampus. For effective use of these models:

• Generation approach: Create conditional knockout mice using a Cre-loxP system with brain-specific promoters driving Cre recombinase expression. This allows temporal and spatial control of Efr3a deletion .

• Confirmation methods: Validate knockout efficiency using RT-PCR, Western blotting, and immunohistochemistry to ensure complete deletion in target regions.

• Neurogenesis assessment: Employ BrdU (bromodeoxyuridine) or EdU (5-ethynyl-2'-deoxyuridine) labeling to track newly formed neurons. Combine with neuronal markers (NeuN, DCX) to identify mature and immature neurons .

• Survival analysis: Conduct pulse-chase experiments with timed BrdU injections followed by analysis at different time points to assess neuronal survival rates, which are extended in Efr3a KO mice .

• Apoptosis measurement: Use TUNEL assay or caspase-3 immunostaining to quantify apoptotic neurons, which are decreased in Efr3a KO mice .

• Molecular pathway analysis: Examine BDNF and TrkB expression levels using qPCR and Western blotting. Assess downstream AKT pathway activation through phospho-AKT levels .

• Behavioral testing: Implement hippocampus-dependent learning and memory tests (Morris water maze, novel object recognition) to correlate enhanced neurogenesis with functional outcomes.

This comprehensive approach can elucidate the mechanistic role of EFR3A in regulating adult neurogenesis and potentially identify therapeutic targets for disorders characterized by impaired neurogenesis .

What methodological approaches are most effective for studying EFR3A's role in membrane raft organization?

Studying EFR3A's role in membrane raft organization requires specialized techniques focusing on membrane dynamics and protein-protein interactions:

• Flotillin interaction analysis: Given EFR3A's interaction with flotillin-2, co-immunoprecipitation assays can be employed to study this interaction under various conditions. Use both endogenous proteins and tagged recombinant proteins for verification .

• Membrane fluidity measurements: Assess changes in membrane fluidity using fluorescence anisotropy with probes like DPH (1,6-diphenyl-1,3,5-hexatriene). Compare wild-type cells with EFR3A knockdown cells to determine EFR3A's influence on membrane properties .

• Giant plasma membrane vesicles (GPMVs): Isolate GPMVs, which are free of membrane skeleton and cytoskeleton proteins, to study intrinsic membrane properties affected by EFR3A presence or absence .

• Cholesterol depletion experiments: Use methyl-β-cyclodextrin (MβCD) to deplete cholesterol and compare effects with EFR3A knockdown to identify shared pathways .

• Signaling pathway analysis: Monitor phosphorylation of EGFR and PLCγ1 upon EGF stimulation in control versus EFR3A-depleted cells to connect membrane organization to downstream signaling .

• Raft isolation: Employ detergent-resistant membrane fraction isolation through sucrose gradient ultracentrifugation to biochemically characterize rafts with and without EFR3A .

• Superresolution microscopy: Techniques like STORM or PALM can visualize nanoscale organization of membrane components in the presence or absence of EFR3A.

• Reconstitution studies: Perform in vitro reconstitution with purified components to confirm direct effects of EFR3A-flotillin complexes on membrane physical properties .

These approaches can collectively build a comprehensive understanding of how EFR3A contributes to membrane raft formation and function.

What are the recommended protocols for analyzing palmitoylation status of mouse EFR3A protein?

Analyzing the palmitoylation status of mouse EFR3A requires specific techniques that can detect this post-translational modification:

• Metabolic labeling: Incubate cells expressing mouse EFR3A with [³H]palmitate to radioactively label palmitoylated proteins. Immunoprecipitate EFR3A and detect incorporation using fluorography .

• Acyl-biotinyl exchange (ABE): This non-radioactive method involves three steps:

  • Blocking free thiols with N-ethylmaleimide

  • Cleaving palmitoyl-thioester bonds with hydroxylamine

  • Biotinylating newly exposed thiols followed by streptavidin pulldown and Western blotting

• Click chemistry approach: Use alkyne-tagged palmitic acid analogs (17-ODYA) in cell culture, followed by copper-catalyzed click reaction with azide-containing reporters for visualization or pulldown.

• Pharmacological inhibition assessment: Attempt to inhibit palmitoylation with 2-bromopalmitate (2-BrP) treatment, although research indicates EFR3A palmitoylation may be unusually stable over extended periods (up to 48 hours) .

• Site-directed mutagenesis: Generate cysteine-to-serine mutations at putative palmitoylation sites (typically in the N-terminal region with 3-4 cysteine residues) to verify specific modification sites .

• Subcellular localization: Compare cellular localization of wild-type versus palmitoylation-deficient mutants using fluorescence microscopy to determine the functional significance of this modification .

• Isoform analysis: Compare palmitoylation status between different EFR3A isoforms, particularly isoform 2 which lacks the N-terminal cysteine motif .

These methods provide complementary approaches to characterize the palmitoylation status of mouse EFR3A and its functional consequences for protein localization and activity.

How can researchers effectively measure changes in PI(4,5)P2 levels in response to EFR3A manipulation in mouse neurons?

Measuring changes in PI(4,5)P2 levels in response to EFR3A manipulation requires specialized techniques for phosphoinositide detection in neuronal systems:

• Genetically encoded biosensors: Express PI(4,5)P2-specific biosensors such as PH-PLCδ1-GFP in neurons with and without EFR3A manipulation. These biosensors bind specifically to PI(4,5)P2 and allow real-time visualization of its dynamics .

• Immunofluorescence approach: Use PI(4,5)P2-specific antibodies for fixed-cell analysis, although this requires careful sample preparation to preserve phosphoinositides.

• Mass spectrometry-based lipidomics: Extract lipids from control and EFR3A-manipulated neurons followed by LC-MS/MS analysis to quantitatively measure all phosphoinositide species simultaneously.

• Biochemical quantification: Use inositol labeling with [³H]myo-inositol followed by lipid extraction, deacylation, and HPLC analysis to quantify changes in PI(4,5)P2 levels.

• Electrophysiological measurements: Since PI(4,5)P2 regulates ion channel function, patch-clamp recordings of KCNQ/M-currents (which are PI(4,5)P2-dependent) can serve as a functional readout of PI(4,5)P2 availability.

• Experimental controls:

  • Compare Efr3a knockout specifically in the CA3 area of the hippocampus, where Aβ-induced depletion of PI(4,5)P2 has been observed

  • Include positive controls such as activation of phospholipase C, which depletes PI(4,5)P2

  • Use rapamycin-inducible depletion systems as an additional comparison

• Temporal dynamics: Measure PI(4,5)P2 levels at different time points following acute manipulation of EFR3A (e.g., using optogenetic or chemogenetic approaches) to distinguish direct versus compensatory effects.

These approaches can elucidate how EFR3A, through its role in anchoring the PI4K complex to the plasma membrane, influences PI(4,5)P2 homeostasis in neuronal systems, particularly in the context of neurodegenerative conditions like Alzheimer's disease .

How do mouse models of EFR3A deficiency compare to human neurological disorders associated with EFR3A mutations?

Mouse models of EFR3A deficiency provide valuable insights into human neurological disorders, though with important comparative considerations:

• Autism Spectrum Disorders (ASD):

  • Human findings: Six somatic, nonsynonymous mutations in EFR3A coding sequence occur twice more frequently in ASD patients than in control subjects

  • Mouse models: While comprehensive ASD-specific Efr3a mouse models are still developing, the expression pattern of EFR3A is shared with ASD-associated genes including synaptic genes and PI(4,5)P2 phosphatase

  • Methodological approach: Researchers should generate mouse models carrying human ASD-associated EFR3A mutations to directly compare phenotypes

• Essential Tremor (ET):

  • Human findings: RNA sequencing data shows EFR3A is upregulated in ET patients

  • Mouse models: Targeted overexpression of Efr3a in regions relevant to tremor generation could be employed to replicate human ET conditions

  • Comparative assessment: Electrophysiological recordings and behavioral tremor analysis should be conducted in both species

• Alzheimer's Disease:

  • Human association: EFR3A is linked to Alzheimer's disease development

  • Mouse models: Knockout of Efr3a in the CA3 hippocampal area leads to Aβ-induced depletion of PI(4,5)P2, while deleting Efr3a at presynaptic sites in CA1 pyramidal neurons improves cognitive function in APP/PS1 mice

  • Translational potential: These findings suggest therapeutic potential for EFR3A modulation in human Alzheimer's patients

• Spiral Ganglion Degeneration and Hearing Loss:

  • Mouse findings: Loss of Efr3a expression delays hair cell loss and spiral ganglion degeneration in drug-induced models

  • Human relevance: This suggests potential protective effects of EFR3A modulation in human sensorineural hearing loss

  • Research approach: Comparative transcriptomics of mouse models and human samples can identify shared pathways

When developing mouse models to study human EFR3A-associated disorders, researchers should:

• Incorporate human mutations when possible

• Focus on brain regions known to be affected in human disorders

• Employ comprehensive behavioral phenotyping relevant to human symptoms

• Analyze molecular pathways (particularly BDNF/TrkB and AKT signaling) implicated in both species

• Consider species differences in expression patterns, isoform usage, and compensatory mechanisms

What are the most promising future research directions for EFR3A?

The multifaceted roles of EFR3A in cellular physiology and pathology suggest several promising research directions:

• Therapeutic targeting: Exploring EFR3A modulation as a therapeutic approach for neurological disorders, particularly Alzheimer's disease where presynaptic Efr3a deletion improves cognitive function in mouse models .

• Membrane organization mechanisms: Further elucidating the molecular details of how EFR3A and flotillin interaction regulates membrane raft formation and organization .

• Isoform-specific functions: Investigating the functional differences between EFR3A isoforms, especially those lacking the palmitoylation motif, to understand their potentially distinct roles .

• Comparative paralogs analysis: Deeper exploration of the functional overlap and distinction between EFR3A and EFR3B, particularly in neuronal contexts where Efr3b depletion in the CA2/CA3 hippocampal area results in excitability and social novelty recognition deficits .

• Post-translational modification: Investigating the unusual stability of EFR3A palmitoylation and its mechanistic significance .

• Signaling pathway integration: Developing comprehensive models of how EFR3A integrates PI4K signaling, membrane organization, and downstream effects on receptors like EGFR .

• Structural biology approaches: Utilizing cryo-EM and other advanced structural techniques to fully characterize the EFR3A protein and its complexes with interaction partners .

These research directions hold significant promise for advancing our understanding of EFR3A biology and potentially developing novel therapeutic approaches for associated disorders.

What methodological challenges remain in studying EFR3A function?

Despite significant progress, several methodological challenges persist in EFR3A research:

• Isoform-specific tools: Developing antibodies and other research tools that can distinguish between highly similar EFR3A isoforms remains challenging but essential for understanding their potentially distinct functions .

• Membrane protein complexes: Studying the interactions of EFR3A with membrane components and other proteins in their native lipid environment requires specialized techniques that preserve these delicate interactions .

• Temporal dynamics: Capturing the dynamic assembly and disassembly of EFR3A-containing complexes in response to cellular stimuli requires sophisticated live-cell imaging approaches.

• Tissue-specific functions: Understanding how EFR3A functions differently across various tissues and cell types necessitates the development of conditional and inducible models with high specificity.

• Palmitoylation analysis: The unusual stability of EFR3A palmitoylation presents challenges for standard inhibition approaches, requiring development of more specialized techniques .

• Compensatory mechanisms: Distinguishing between direct effects of EFR3A manipulation and compensatory responses, particularly between EFR3A and EFR3B paralogs, requires careful experimental design.

• Translation to human disease: Connecting findings from mouse models to human pathology requires innovative comparative approaches and careful consideration of species differences.