Recombinant Mouse Leucine-rich repeat-containing protein 24 (Lrrc24)

What is the structural composition of mouse LRRC24?

Mouse LRRC24 is a single-pass transmembrane protein belonging to the leucine-rich repeat (LRR) superfamily. The protein structure consists of several distinct domains: a signal peptide (residues 1-23), an N-terminal leucine-rich repeat domain (LRRNT), six leucine-rich repeats (LRRs), a C-terminal leucine-rich repeat domain (LRRCT), an immunoglobulin-like domain, a transmembrane domain, and an arginine-rich motif . The mature protein is primarily composed of alpha helices and has a molecular weight of approximately 53 kDa. Mouse LRRC24 (UniProt ID: Q8BHA1) shares significant sequence homology with human LRRC24, with the extracellular domains exhibiting 86% amino acid sequence identity .

How is the LRRC24 gene organized and expressed?

The mouse LRRC24 gene (Gene ID: 378937) is structurally similar to its human ortholog, which is located on chromosome 8 (8q24.3) in humans . The gene contains five exons, and current evidence indicates a single gene isoform. Expression analysis reveals that LRRC24 has limited tissue distribution, with most notable expression in retinal bipolar neurons and retinal rod cells according to scRNA-seq data . The gene appears to be conserved among Euteleostomi (bony vertebrates) with the exception of Aves (birds), suggesting evolutionary conservation of its function in specific lineages .

What are the primary biological functions of LRRC24 in neuronal development?

LRRC24 has been implicated in several neuronal development processes. Most significantly, recombinant LRRC24 has demonstrated the ability to enhance neurite outgrowth when immobilized at 2.5 μg/mL concentration on experimental surfaces . Mechanistically, this function appears to be related to LRRC24's interaction with Robo2, a receptor known to play crucial roles in axon guidance and neuronal cell migration . The positive regulation of synapse assembly (GO:0051965) is among the biological processes associated with LRRC24, further supporting its neuronal function . These findings suggest that LRRC24 may function as an extracellular cue that guides neurite extension and potentially influences synaptic development during neural circuit formation.

How does LRRC24 contribute to cellular signaling pathways?

Current research suggests that LRRC24 may function as a negative regulator of the ErbB family of receptor tyrosine kinases . Through this activity, LRRC24 appears to suppress ErbB-driven tumor cell proliferation and motility, suggesting potential tumor-suppressive properties. The protein's localization to the extracellular space (GO:0005615) and the external side of the plasma membrane (GO:0009897) positions it as a potential ligand or co-receptor in cellular signaling cascades . The presence of multiple leucine-rich repeats, which are known protein-protein interaction motifs, further supports LRRC24's role in signaling networks. The arginine-rich motif in the cytoplasmic portion may mediate interactions with intracellular signaling components or influence protein trafficking.

What are the optimal conditions for detecting and quantifying mouse LRRC24 in experimental samples?

For quantitative detection of mouse LRRC24, ELISA-based methods offer reliable results with a detection range of 0.156-10 ng/mL . When working with tissue homogenates, cell lysates, or biological fluids, samples should be diluted to concentrations within the mid-range of available detection kits to ensure accurate quantification. Consistency in experimental conditions is critical, as the activity loss rate should be maintained below 5% throughout the experimental procedures . For stability considerations, LRRC24 samples should be stored according to manufacturer recommendations, typically at 4°C during shipping and at specified temperatures for long-term storage. To minimize performance fluctuations, it is advisable that the same researcher perform the entire assay and that lab conditions remain strictly controlled throughout the experimental process .

What are the methodological considerations for producing recombinant mouse LRRC24?

Production of recombinant mouse LRRC24 typically involves expression in mammalian cell systems, such as Chinese Hamster Ovary (CHO) cells, to ensure proper post-translational modifications and protein folding . The mature protein (post-signal peptide cleavage) is the form typically used in recombinant protein production. For purification purposes, affinity tags such as histidine tags can be incorporated, typically at the C-terminus to minimize interference with the N-terminal domains that may be critical for protein function . The purified protein is often formulated in phosphate-buffered saline (PBS) and lyophilized for stability. Upon reconstitution, a concentration of approximately 500 μg/mL in PBS is recommended for most experimental applications . To verify production quality, SDS-PAGE analysis under both reducing and non-reducing conditions should be performed, with authentic recombinant mouse LRRC24 displaying bands at approximately 45-55 kDa .

How can researchers effectively study LRRC24's role in neurite outgrowth and neural development?

To investigate LRRC24's role in neurite outgrowth, researchers should consider substrate immobilization assays where recombinant LRRC24 is applied at concentrations of approximately 2.5 μg/mL on appropriate cell culture surfaces . Primary neuronal cultures or neuronal cell lines can then be seeded on these surfaces, and neurite extension can be quantified using neurite tracing software. For more sophisticated analysis, time-lapse microscopy combined with fluorescent labeling of cytoskeletal elements can provide insights into the dynamics of LRRC24-mediated neurite extension.

To explore the molecular mechanism, co-immunoprecipitation experiments targeting LRRC24 and Robo2 can confirm their physical interaction in neural tissues or cell models . Additionally, knockdown or knockout models using siRNA or CRISPR-Cas9 approaches, respectively, followed by rescue experiments with recombinant protein, can help establish the necessity and sufficiency of LRRC24 in neural development processes. Tissue-specific conditional knockout models focusing on retinal neurons may be particularly informative given the documented expression in retinal bipolar neurons and rod cells .

What approaches are recommended for investigating LRRC24's potential tumor-suppressive properties?

To study LRRC24's reported negative regulation of ErbB signaling and potential tumor-suppressive function, researchers should employ both gain- and loss-of-function experimental paradigms in appropriate cancer cell models . Overexpression of LRRC24 in ErbB-positive cancer cell lines followed by assessment of proliferation rates, migration assays, and invasion assays can provide insights into its tumor-suppressive capacity. Complementary loss-of-function studies using CRISPR-Cas9 or shRNA approaches in cells with endogenous LRRC24 expression can confirm the necessity of this protein in regulating ErbB-driven oncogenic processes.

Mechanistic studies should include phosphorylation analysis of ErbB receptors and downstream signaling components (such as MAPK and PI3K/Akt pathways) following LRRC24 modulation. Co-immunoprecipitation and proximity ligation assays can determine whether LRRC24 directly interacts with ErbB receptors or influences their dimerization. Additionally, xenograft models with LRRC24-modulated cancer cells can assess the in vivo relevance of these findings to tumor growth and metastasis.

How can researchers address specificity concerns when working with anti-LRRC24 antibodies?

Antibody specificity is a critical concern in LRRC24 research due to its structural similarity with other LRR-containing proteins. To ensure specific detection, researchers should validate antibodies using multiple approaches. Positive controls should include recombinant LRRC24 protein at known concentrations, while negative controls should include samples from LRRC24 knockout models or cell lines known not to express LRRC24. Western blot analysis should demonstrate a specific band at the expected molecular weight (45-55 kDa), and additional validation can be performed using immunoprecipitation followed by mass spectrometry .

For immunohistochemistry or immunocytochemistry applications, pre-absorption controls using recombinant LRRC24 can help confirm specificity. Cross-reactivity testing against closely related LRR proteins is also advisable. When possible, use of multiple antibodies targeting different epitopes of LRRC24 can provide added confidence in experimental results. For quantitative applications, standard curves using recombinant LRRC24 should be established to ensure accurate quantification within the assay's linear range .

What are the key considerations for designing LRRC24 knockout or knockdown experiments?

When designing genetic manipulation experiments targeting LRRC24, researchers should carefully consider several factors to ensure interpretable results. For CRISPR-Cas9 knockout approaches, guide RNA design should target early exons to ensure complete loss of protein function, with multiple guide RNAs tested for efficiency and specificity. Off-target effects should be assessed using whole-genome sequencing or targeted sequencing of predicted off-target sites.

For conditional knockout models, the choice of promoter driving Cre recombinase expression should reflect the tissues of interest, particularly focusing on neural tissues given LRRC24's expression pattern and function . For knockdown approaches using siRNA or shRNA, researchers should design multiple sequences targeting different regions of the LRRC24 transcript and validate knockdown efficiency at both mRNA (qRT-PCR) and protein (Western blot) levels.

Phenotypic analysis should include careful examination of neural development, particularly focusing on neurite outgrowth, axon guidance, and synapse formation given LRRC24's association with these processes . Additionally, assessment of ErbB signaling components should be included to evaluate the proposed tumor-suppressive function of LRRC24.

How might LRRC24's role in retinal neurons inform therapeutic approaches for visual system disorders?

The significant expression of LRRC24 in retinal bipolar neurons and retinal rod cells suggests a specialized function in visual system development or maintenance . This expression pattern warrants investigation into LRRC24's potential roles in retinal circuit formation, synaptic connectivity between photoreceptors and bipolar cells, and possibly in retinal regeneration following injury.

To explore these functions, researchers should consider retina-specific knockout models of LRRC24, followed by detailed electrophysiological and morphological analysis of retinal circuits. The ability of recombinant LRRC24 to enhance neurite outgrowth suggests potential therapeutic applications in promoting regeneration or rewiring in degenerative retinal conditions . Ex vivo retinal explant cultures treated with recombinant LRRC24 could help assess its capacity to promote neurite extension and synaptic integration in a complex neural tissue environment.

Given LRRC24's interaction with Robo2, which has established roles in neural circuit formation, investigation into how this interaction specifically influences retinal development could provide insights into fundamental visual system wiring mechanisms . These findings could potentially inform regenerative approaches for conditions such as retinitis pigmentosa or age-related macular degeneration.

What are the implications of LRRC24's potential tumor-suppressive properties for cancer research?

The proposed function of LRRC24 as a negative regulator of ErbB family signaling suggests potential applications in cancer research, particularly for ErbB-driven malignancies . Researchers should investigate whether LRRC24 expression correlates with cancer progression or patient outcomes in tumors with known ErbB involvement, such as certain breast, lung, and glioblastoma cancers.

Mechanistic studies should determine whether recombinant LRRC24 could be developed as a biological therapeutic to suppress aberrant ErbB signaling in cancer cells. Structure-function analyses can identify the specific domains of LRRC24 responsible for ErbB inhibition, potentially leading to the development of peptide mimetics or small molecules that mimic this activity. Additionally, researchers should investigate whether genetic or epigenetic alterations in LRRC24 contribute to ErbB dysregulation in cancer, which could establish LRRC24 as a biomarker for certain cancer subtypes.

Combination studies examining how LRRC24 modulation might enhance the efficacy of existing ErbB-targeting therapies (such as trastuzumab, erlotinib, or afatinib) could reveal synergistic approaches to overcome resistance mechanisms in ErbB-dependent cancers.

How does mouse LRRC24 compare structurally and functionally with human LRRC24 and other orthologs?

Comparative analysis reveals that mouse LRRC24 shares 86% amino acid sequence identity with human LRRC24 in the extracellular domain, suggesting substantial functional conservation . This high degree of conservation indicates that findings from mouse models may have translational relevance to human biology and potential therapeutic applications. The mature form of mouse LRRC24, like its human counterpart, contains six leucine-rich repeats, an immunoglobulin-like domain, and a transmembrane domain .

LRRC24 is conserved across Euteleostomi (bony vertebrates) but notably absent in Aves (birds), suggesting possible lineage-specific loss or significant divergence in this branch of vertebrate evolution . This evolutionary pattern raises interesting questions about the functional redundancy or compensation in avian species, and comparative studies examining neural development in organisms with and without LRRC24 could provide insights into its evolutionary significance.

Research focused on structural and functional comparisons between species could identify conserved domains that are essential for LRRC24's core functions, versus more variable regions that might contribute to species-specific adaptations or functions.

What are the most significant knowledge gaps and future research priorities for LRRC24?

Despite growing understanding of LRRC24's structure and potential functions, significant knowledge gaps remain. The precise signaling mechanisms through which LRRC24 enhances neurite outgrowth are not fully characterized, nor is the complete spectrum of its binding partners beyond Robo2 . The physiological relevance of LRRC24's proposed tumor-suppressive properties requires validation in diverse cancer models and primary tumor samples.

Future research priorities should include:

• Development of comprehensive knockout models to assess LRRC24's function in neural development and potential redundancy with other LRR family members

• Detailed characterization of LRRC24's expression pattern throughout development and in adult tissues beyond the retinal neurons currently identified

• Structural studies to determine the three-dimensional configuration of LRRC24 and how it interacts with Robo2 and potentially ErbB receptors

• Investigation of LRRC24's role in retinal development and function, given its expression in retinal neurons

• Exploration of potential therapeutic applications in neurological disorders and cancer, based on its neurite outgrowth-promoting and potential tumor-suppressive functions