EFNA5 Human

What is EFNA5 and what is its structural classification?

EFNA5 (ephrin A5) is a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the ephrin-A subclass of ephrin ligands. Unlike ephrin-B ligands, EFNA5 lacks a transmembrane domain and cytoplasmic region, instead being tethered to the cell membrane solely through its GPI anchor . The human EFNA5 gene is located on chromosome 5 and encodes this membrane-bound ligand that primarily interacts with EphA receptors .

The protein's structure is characterized by an extracellular ephrin domain that mediates binding to Eph receptors. Despite lacking an obvious intracellular signaling domain, EFNA5 participates in bidirectional signaling cascades through mechanisms that are still being elucidated by researchers.

What are the primary binding partners for EFNA5?

• EphA class receptors as primary binding partners

• EphB2 as a demonstrated cross-class binding partner

• Potential concentration-dependent binding specificity

• The influence of local cellular context on binding preferences

The binding affinity to different receptors may vary significantly and should be experimentally validated in the specific cellular context under investigation.

How does EFNA5 participate in bidirectional signaling?

Despite lacking an obvious intracellular domain, EFNA5 participates in bidirectional signaling through both forward signaling (from ligand to receptor-expressing cell) and reverse signaling (from receptor to ligand-expressing cell):

• Forward signaling: When EFNA5 binds to EphA receptors, it triggers tyrosine kinase activation in the receptor-expressing cell, leading to downstream signaling cascades.

• Reverse signaling: EFNA5 can transmit signals into its own cell upon binding to Eph receptors, despite lacking a conventional cytoplasmic domain. This GPI-dependent mechanism has been demonstrated in motor neuron growth cones, where EFNA5 reverse signaling promotes growth cone spreading .

The mechanism of EFNA5 reverse signaling depends on its GPI anchor, as evidenced by experiments showing that elimination of GPI linkages through phosphatidlyinositol-specific phospholipase C application abolishes the positive effects of EFNA5 on growth cone spreading . This finding provides important methodological considerations for researchers investigating EFNA5 signaling.

How do EFNA5 and EphA7 establish region-specific neural connections?

EFNA5 and EphA7 exhibit mutually exclusive expression patterns in the cortex and basilar pons, creating a molecular basis for establishing region-specific neural connections. Research has revealed that:

• EphA7 and EFNA5 are expressed in region-specific and mutually exclusive patterns in the cortex and basilar pons .

• Their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts .

• EphA7-positive frontal and occipital cortical areas extend axon collaterals into EFNA5-negative regions of the basilar pons .

• EFNA5-positive parietal cortical areas extend collaterals into EphA7-negative regions of the basilar pons .

This molecular mechanism differs from the gradient-based topographic mapping seen in other neural systems. Instead, it represents a binary, region-to-region connection pattern based on repulsive signaling between mutually exclusive molecular territories. When investigating these patterns, researchers should consider using region-specific markers alongside EFNA5/EphA7 to accurately map expression domains.

What role does EFNA5 play in retinotopic map formation?

EFNA5 is instrumental in establishing the retinotopic map, a precise representation of the visual field in the superior colliculus (SC). The process functions through repulsive signaling:

• EFNA5 is highly expressed in the posterior region of the superior colliculus.

• Retinal ganglion cells (RGCs) from the temporal retina express EphA receptors.

• When temporal RGC axons encounter high EFNA5 expression, EphA activation induces growth cone collapse.

• This repulsion guides temporal RGCs away from the posterior SC toward the anterior SC, where EFNA5 expression is lower .

Methodologically, this process can be studied through:

• In situ hybridization to map EFNA5 expression gradients

• Explant cultures to observe growth cone behaviors

• DiI tracing to visualize RGC projections

• Genetic manipulation to alter EFNA5 expression levels or patterns

Understanding this mechanism provides insight into how molecular repulsion can generate precise topographic maps during neural development.

What experimental approaches can verify EFNA5 expression patterns?

Researchers investigating EFNA5 expression patterns can employ several methodological approaches:

• RNA analysis:

  • RT-PCR with EFNA5-specific primers (e.g., 5′-CTTTTGGCAATCCTACTGTTCC-3′ and 5′-TGCTCACTTCCACACTCCTAGA-3′)

  • In situ hybridization to visualize spatial expression patterns in tissue sections

  • RNA-seq for genome-wide expression analysis

• Protein detection:

  • Immunohistochemistry using EFNA5-specific antibodies

  • Western blotting for protein quantification

  • Flow cytometry for cell-specific expression analysis

• Cloning and expression:

  • EFNA5 CDS can be amplified using specific primers and cloned into expression vectors

  • Expression constructs can be created for functional studies (e.g., pCAGGS-5MCS or pCAGGS-5MCS-FLAG vector)

When designing primers for EFNA5 detection, researchers should reference validated sequences such as NCBI sequence ID: NM_207654.2 for mouse EFNA5 CDS .

How does EFNA5 reverse signaling influence neuronal development?

EFNA5 reverse signaling exerts effects on neuronal development that are often opposite to those of EphA forward signaling. Experimental evidence demonstrates:

• EFNA5 stimulates the spreading of growth cones in cultured mouse spinal motor neurons .

• This effect is GPI-dependent, as elimination of GPI linkages abolishes growth cone spreading .

• In contrast, EphA receptor activation reduces growth cone size .

The opposing actions of EFNA5 reverse signaling and EphA forward signaling create a mechanism by which:

• Migrating axons expressing EphAs preferentially avoid EFNA5-expressing cells

• Axons may be guided toward regions with lower EFNA5 expression

Methods to study this phenomenon include:

• Growth cone collapse assays with purified proteins

• Time-lapse microscopy of neuronal cultures

• Quantitative morphometric analysis of growth cone dynamics

• Pharmacological manipulation of GPI anchors to block reverse signaling

These findings have significant implications for understanding axon guidance mechanisms that establish specific neural circuits during development.

What is known about EFNA5's role in neurological disorders?

Research indicates potential connections between EFNA5 and several neurological conditions:

• Alzheimer's disease: The Eph/ephrin signaling system, including EFNA5, has been implicated in Alzheimer's disease pathology. The EphA1 receptor gene has been associated with Alzheimer's disease in genome-wide association studies .

• Sleep and circadian rhythm disorders: Eph/ephrin signaling affects memory, circadian rhythms, and sleep patterns, suggesting EFNA5 may contribute to related neurological disorders .

• Neuroinflammatory conditions: Eph/ephrin signaling influences microglial function, with implications for neuroinflammatory processes in various disorders .

Methodologically, researchers investigating these connections should consider:

• Genetic association studies in patient populations

• Animal models with altered EFNA5 expression or function

• Electrophysiological measurements to assess neuronal excitability

• Behavioral assays to evaluate memory, sleep, and circadian rhythms

The multifaceted effects of EFNA5 on neural development, function, and pathology make it a compelling target for neurological disease research.

How does EFNA5 interact with viral infection mechanisms?

Ephrin-Eph signaling, including EFNA5-mediated pathways, has been implicated in the infection processes of various viruses:

• Viral entry: Some viruses utilize Eph receptors as entry factors, suggesting EFNA5 may influence viral tropism and cellular susceptibility .

• Viral pathogenesis: The relationship between ephrin signaling and highly pathogenic viruses such as Hendra and Nipah has been documented, indicating potential roles for EFNA5 in viral disease mechanisms .

• Host-pathogen interactions: Viral proteins may co-opt or disrupt normal EFNA5 signaling pathways to facilitate viral replication or spread .

Research methodologies for investigating EFNA5-viral interactions include:

• Viral infection assays with EFNA5-overexpressing or knockdown cells

• Co-immunoprecipitation to identify viral-EFNA5 protein interactions

• Live-cell imaging to visualize viral entry in relation to EFNA5 expression

• CRISPR-based screening to identify EFNA5-dependent viral infection mechanisms

This emerging area of research offers insights into both viral pathogenesis and the fundamental biology of EFNA5 signaling.

What techniques are most effective for manipulating EFNA5 expression in experimental models?

Researchers have several options for modulating EFNA5 expression:

• Genetic approaches:

  • CRISPR/Cas9 genome editing for knockout or knockin models

  • RNA interference (siRNA, shRNA) for transient knockdown

  • Overexpression using viral vectors or plasmid transfection

• Expression constructs:

  • Full-length EFNA5 CDS can be amplified using specific primers (e.g., 5′-atacagatctgccaccATGTTGCACGTGGAGATGTTGACGC-3′ and 5′-atacgatatcTAATGTCAAAAGCATCGCCAGGAGGAAC-3′ for mouse EFNA5)

  • Cloning into expression vectors such as pCAGGS-5MCS or pCAGGS-5MCS-FLAG

  • Creation of tagged constructs for protein visualization and interaction studies

• Protein-level manipulation:

  • Application of recombinant EFNA5-Fc fusion proteins

  • Phosphatidlyinositol-specific phospholipase C to cleave GPI anchors and eliminate EFNA5 surface expression

  • Function-blocking antibodies to inhibit EFNA5-receptor interactions

When designing experiments, researchers should consider the temporal aspects of EFNA5 function, particularly in developmental contexts where precise timing of expression may be critical.

How can researchers effectively study EFNA5-receptor interactions?

Several methodologies can be employed to investigate EFNA5 interactions with its receptors:

• Biochemical approaches:

  • Co-immunoprecipitation to identify protein complexes

  • Pull-down assays using tagged EFNA5 constructs

  • Surface plasmon resonance to measure binding kinetics

  • Proximity ligation assays for visualizing protein interactions in situ

• Imaging techniques:

  • FRET or BRET for real-time interaction analysis

  • Super-resolution microscopy for nanoscale visualization

  • Live-cell imaging to track receptor-ligand dynamics

• Functional assays:

  • Growth cone collapse assays to measure repulsive signaling

  • Cell migration assays to assess chemorepulsion or attraction

  • Stripe assays to evaluate axon guidance preferences

  • Calcium imaging to detect immediate signaling responses

When designing these experiments, researchers should consider the multimeric nature of Eph-ephrin complexes and the potential for both cis and trans interactions between receptors and ligands.

What bioinformatic resources are available for EFNA5 research?

Researchers studying EFNA5 can utilize various bioinformatic resources:

• Sequence and variant data:

  • NCBI Gene database (Gene ID: 1946) for sequence information

  • Genomic sequence: NC_000005.10 (Chromosome 5 Reference GRCh38.p14)

  • ClinVar for variant interpretation

  • dbVar for structural variation data

• Protein structure and function:

  • UniProtKB (P52803) for protein information

  • STRING database for protein-protein interaction networks

  • PheGenI for eQTL and phenotype association data

• Expression databases:

  • GTEx for tissue-specific expression patterns

  • Allen Brain Atlas for spatial expression in neural tissues

  • Single-cell RNA-seq databases for cell-type specific expression

These resources provide valuable information for experimental design, hypothesis generation, and contextualizing research findings within the broader scientific literature.