Recombinant Mouse Reticulon-4 receptor-like 2 (Rtn4rl2)

What is the basic structure of RTN4RL2 and how does it anchor to the cell membrane?

RTN4RL2 is a leucine-rich repeat (LRR) protein that contains eight leucine-rich repeats flanked by cysteine-rich sequences at both N- and C-termini. It anchors to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage. The protein can be solubilized through phospholipase action or by an unidentified MTMMP to generate a 46 kDa soluble receptor from the membrane-anchored 60 kDa form . The LRR domain is critical for its interactions with binding partners, as demonstrated in crystal structure studies of the related RTN4R in complex with BAI1 .

What are the expression patterns of RTN4RL2 in the mouse cochlea?

RTN4RL2 is expressed in both inner hair cells (IHCs) and spiral ganglion neurons (SGNs) of the mouse cochlea. RNAscope in situ hybridization confirms RTN4RL2 mRNA expression in both outer and inner hair cells, as well as in SGNs. Immunostaining studies show that RTN4RL2 protein colocalizes with βIII-tubulin-positive neurons in spiral ganglia . This expression pattern suggests a potential role in auditory signal transmission at IHC-SGN synapses.

How can I detect RTN4RL2 expression in tissue samples?

For detecting RTN4RL2 expression, several complementary approaches are recommended:

• mRNA detection: RNAscope in situ hybridization using probes specific for RTN4RL2 mRNA, combined with cell-type markers (e.g., Myo7a for hair cells, βIII-tubulin for neurons) .

• Protein detection:

  • Immunostaining with anti-RTN4RL2 antibodies (ensure specificity using knockout controls)

  • Western blot analysis using verified antibodies that recognize RTN4RL2 at approximately 68 kDa .

  • ELISA for quantitative analysis

Always include appropriate controls, including RTN4RL2 knockout tissues and negative controls (omitting primary antibody) .

What is known about RTN4RL2's molecular interactions?

RTN4RL2 has been implicated in interactions with several molecules:

Studies with the related RTN4R show that its interactions with BAI1 involve specific glycosylation patterns. The RTN4R LRR domain binds to the C-terminal part of the BAI1 TSR3 domain, and this interaction requires O-fucosylation mediated by POFUT2 . Similar mechanisms may apply to RTN4RL2 interactions.

How does RTN4RL2 deletion affect auditory synapses?

Deletion of RTN4RL2 in mice results in multiple alterations to auditory synapses:

Presynaptic changes:

• Enlarged synaptic ribbons (increased volume)

• Depolarized shift in Ca2+ channel activation (~+5 mV)

• Tendency toward higher Ca2+ influx at single active zones

Postsynaptic changes:

• Significantly smaller Homer1 patches juxtaposing presynaptic ribbons

• Decreased percentage of presynaptic ribbons juxtaposing Homer1

• Severe reduction of GluA2/3 positive puncta despite maintained Gria2 mRNA expression

• Presence of "orphan" PSDs located away from IHCs

These structural and functional alterations collectively contribute to impaired hearing, as evidenced by increased auditory brainstem response thresholds in RTN4RL2 knockout mice .

What physiological deficits are observed in RTN4RL2 knockout mice?

RTN4RL2 knockout mice (RTN4RL2-/-) exhibit significant hearing impairment characterized by:

• Increased auditory brainstem response (ABR) thresholds by approximately 30-45 dB across all tested frequencies (4, 8, 16, 32 kHz) and in response to click stimulations

• Heterozygous mice (RTN4RL2+/-) show an intermediate phenotype with ABR threshold increases of approximately 10-15 dB at 4, 16, and 32 kHz frequencies

• Impaired synaptic connectivity between SGNs and IHCs, with some SGN peripheral neurites failing to contact IHCs

These findings indicate that RTN4RL2 is essential for normal hearing function, likely through its role in establishing and maintaining proper synaptic connections in the auditory system.

How does RTN4RL2 contribute to calcium channel regulation in inner hair cells?

RTN4RL2 appears to modulate Ca2+ channel function in inner hair cells. In RTN4RL2-/- mice, Ca2+ imaging studies using the low-affinity Ca2+ indicator Fluo4-FF (kD: 10 μM) revealed that:

• Voltage-gated Ca2+ channels at active zones show a significant depolarizing shift in activation voltage (V half) of approximately +5 mV compared to wild-type controls .

• Maximal Ca2+ influx at single active zones tends to be higher in IHCs of RTN4RL2-/- mice, though this difference did not reach statistical significance .

These findings suggest that RTN4RL2 may regulate Ca V1.3 Ca2+ channel properties, potentially through direct interactions or by modulating the local environment of the active zone. For studying this phenomenon, researchers should consider using patch-clamp electrophysiology combined with high-resolution Ca2+ imaging near ribbon synapses, visualized with fluorescently labeled ribbon-binding peptides .

What is the relationship between RTN4RL2 and AMPA receptor expression/localization?

RTN4RL2 deletion leads to complex changes in AMPA receptor expression and localization:

• Immunolabeling studies show a severe reduction of GluA2/3 positive puncta in RTN4RL2-/- mice, suggesting decreased expression or altered localization of these AMPA receptor subunits at postsynaptic sites .

• Interestingly, Gria2 mRNA (which encodes GluA2) remains expressed in SGNs of RTN4RL2-/- mice, indicating that the reduction occurs at a post-transcriptional level .

• This discrepancy suggests RTN4RL2 may regulate AMPA receptor trafficking, anchoring, or stability at the synapse rather than gene expression.

To investigate this relationship, researchers should employ a combination of:

• Subcellular fractionation to track receptor localization

• Super-resolution microscopy to visualize receptor distribution

• Co-immunoprecipitation to identify potential interactions between RTN4RL2 and AMPA receptor trafficking machinery

• Live-cell imaging with tagged receptors to monitor trafficking dynamics in RTN4RL2-deficient versus wild-type cells

How do post-translational modifications affect RTN4RL2 function?

Based on studies of the related protein RTN4R, post-translational modifications likely play crucial roles in RTN4RL2 function. In RTN4R:

• O-fucosylation catalyzed by protein fucosyltransferase POFUT2 is critical for complex formation with BAI1 .

• The Thr424Val mutation that blocks O-fucosylation of BAI1 abolishes binding to RTN4R .

• Knockout of POFUT2 completely abolishes the interaction between RTN4R and BAI1 .

• Mannosyl modifications of tryptophan residues contribute to intramolecular stability .

For RTN4RL2, researchers should investigate:

• Whether similar glycosylation patterns exist

• The enzymes responsible for these modifications

• How these modifications affect protein-protein interactions

• The impact of these modifications on RTN4RL2 stability and turnover

Mass spectrometry, site-directed mutagenesis, and enzymatic deglycosylation experiments would be valuable approaches for these investigations.

What environmental factors influence RTN4RL2 expression?

Multiple environmental factors have been reported to affect RTN4RL2 expression or epigenetic regulation:

These findings suggest RTN4RL2 expression may be susceptible to toxicological regulation. Researchers investigating environmental influences should consider:

• Dose-response relationships

• Tissue-specific effects

• Mechanisms of regulation (transcriptional, post-transcriptional, epigenetic)

• Temporal dynamics of expression changes

• Functional consequences of expression alterations

What are the best approaches for generating and validating RTN4RL2 knockout models?

Based on the research with existing RTN4RL2 knockout mice, researchers should consider:

• Generation approaches:

  • CRISPR/Cas9-mediated gene editing targeting exons encoding functional domains

  • Conditional knockout strategies using Cre-loxP system for tissue-specific deletion

  • Inducible systems for temporal control of gene deletion

• Validation methods:

  • Genotyping using PCR to confirm genetic modification

  • RNAscope in situ hybridization to verify absence of RTN4RL2 mRNA in targeted tissues

  • Immunostaining with anti-RTN4RL2 antibodies to confirm protein absence

  • Western blot analysis to verify complete protein knockout

  • Functional assays (e.g., ABR for auditory phenotypes) to confirm physiological effects

• Control considerations:

  • Include both wild-type (RTN4RL2+/+) and heterozygous (RTN4RL2+/-) controls

  • Use littermates as controls to minimize genetic background effects

  • Consider possible compensatory mechanisms by related proteins (e.g., other Nogo receptors)

How can I design experiments to investigate RTN4RL2 interactions with putative binding partners?

To investigate RTN4RL2 interactions with potential binding partners:

• In vitro binding assays:

  • Surface plasmon resonance (SPR) with purified recombinant proteins

  • Pull-down assays using tagged RTN4RL2

  • Co-immunoprecipitation from native tissues or transfected cells

  • ELISA-based binding assays

• Structural studies:

  • X-ray crystallography of RTN4RL2 in complex with binding partners (as done for RTN4R-BAI1)

  • Cryo-EM for larger complexes

  • NMR for dynamic interaction studies

• Cell-based assays:

  • Surface staining with biotinylated potential binding partners

  • FRET or BiFC to detect interactions in living cells

  • Cell adhesion assays if RTN4RL2 mediates intercellular interactions

• Mutational analysis:

  • Generate point mutations in key residues predicted to be involved in interactions

  • Focus on LRR domain residues, as this domain mediates interactions in related proteins

  • Consider the role of post-translational modifications in mediating interactions

What are the best approaches for investigating RTN4RL2 function in synaptic development and plasticity?

Based on current research, optimal approaches include:

How might RTN4RL2 contribute to neurodevelopmental or neurodegenerative disorders?

Given RTN4RL2's role in synaptic development and function, potential contributions to neurological disorders warrant investigation:

• Neurodevelopmental disorders:

  • RTN4RL2 is involved in corpus callosum development and negative regulation of neuron projection development , suggesting potential roles in disorders characterized by altered connectivity

  • The severe auditory phenotype in knockout mice indicates potential involvement in hearing disorders

  • Research should examine whether RTN4RL2 variants are associated with developmental disorders affecting synaptic function

• Neurodegenerative disorders:

  • RTN4RL2 may play a role in regulating axonal regeneration and plasticity in the adult central nervous system

  • Investigation into whether RTN4RL2 modulation could promote neural repair after injury

  • Examine age-dependent changes in RTN4RL2 expression and function

• Research approaches:

  • Human genetic studies to identify disease-associated variants

  • Creation of disease-relevant models carrying human mutations

  • Therapeutic strategies targeting RTN4RL2 or its signaling pathways

What is the relationship between RTN4RL2 and other Nogo receptor family members?

Understanding the functional redundancy and distinct roles of Nogo receptor family members:

• Comparative analysis:

  • Examine expression patterns of RTN4R (NgR1), RTN4RL1 (NgR3), and RTN4RL2 (NgR2) in various neural tissues

  • Determine whether these receptors compensate for each other in single knockout models

  • Generate and characterize double or triple knockout models

• Binding specificity:

  • Compare binding partners and affinities between different family members

  • Identify unique versus shared downstream signaling pathways

  • Examine structural differences that confer binding specificity

• Evolutionary conservation:

  • Analyze evolutionary relationships between family members

  • Identify conserved domains and motifs that may indicate core functions

  • Compare functions across species to identify evolutionarily conserved roles

This systematic approach would help elucidate the specific contributions of RTN4RL2 within the broader context of Nogo receptor signaling.

Could modulation of RTN4RL2 function represent a therapeutic approach for hearing disorders?

Given the critical role of RTN4RL2 in auditory function, therapeutic possibilities include:

• Potential applications:

  • Hearing loss disorders with synaptic pathology

  • Age-related hearing loss involving synapse degeneration

  • Noise-induced hearing damage

• Therapeutic strategies:

  • Gene therapy approaches to restore or enhance RTN4RL2 expression

  • Small molecules targeting RTN4RL2 signaling pathways

  • Protection of synaptic structures during acoustic trauma

• Research priorities:

  • Determine if RTN4RL2 enhancement could promote synaptic recovery after damage

  • Investigate critical periods during which RTN4RL2 modulation would be most effective

  • Develop cell-type specific targeting strategies for the auditory system

What experimental readouts best measure the efficacy of RTN4RL2-targeted interventions?

To evaluate interventions targeting RTN4RL2:

• Structural readouts:

  • Quantification of synaptic ribbons and postsynaptic densities

  • Analysis of synaptic protein composition and organization

  • Assessment of cochlear innervation patterns

• Functional readouts:

  • Auditory brainstem responses to measure hearing thresholds

  • Otoacoustic emissions to assess outer hair cell function

  • Single-unit recordings to evaluate SGN firing properties

  • Ca2+ imaging to assess presynaptic function

• Molecular readouts:

  • Expression and localization of synaptic proteins

  • AMPA receptor composition and trafficking

  • Signaling pathway activation downstream of RTN4RL2