ADGRL2 Antibody

What is ADGRL2 and why is it significant in neurological research?

ADGRL2 (Adhesion G Protein-Coupled Receptor L2) encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. This receptor plays a crucial role in brain development, as evidenced by its expression in the telencephalon, mesencephalon, and rhombencephalon in embryonic mice and chickens. In human brain embryos and fetuses, ADGRL2 immunoreactivity has been observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons, and cerebellar cortex . The significance of ADGRL2 in neurodevelopment has been highlighted by the discovery that de novo variants in this gene can lead to extreme microcephaly with rhombencephalosynapsis, making it an important target for neurological research . Studies involving knockout models have demonstrated that constitutive Adgrl2−/− mice exhibit embryonic lethality, while Adgrl2+/− mice show microcephaly and vermis hypoplasia, further emphasizing its critical role in brain development .

How do ADGRL2 antibodies differ from other adhesion GPCR antibodies?

ADGRL2 antibodies are specifically designed to target the latrophilin 2 protein, unlike other antibodies targeting the broader adhesion GPCR family. While antibodies against different adhesion GPCRs share some common design principles, they differ in their epitope specificity, binding affinity, and functional effects. For example, synthetic antibody fragments developed against ADGRL3 ECR (extracellular region) can bind to both ADGRL3 and ADGRL1 with similar affinities but act as agonists only for ADGRL3, demonstrating ligand-specific modulation . This distinction is crucial when selecting antibodies for experimental purposes, as cross-reactivity between ADGRL family members must be carefully evaluated. Unlike antibodies targeting other adhesion GPCRs such as ADGRG3, which has been shown to modulate immune cell function, ADGRL2 antibodies primarily find applications in neurodevelopmental and synaptic function studies .

What are the recommended applications for ADGRL2 antibodies in neurological research?

ADGRL2 antibodies are valuable tools for multiple experimental applications in neurological research. Primary applications include immunohistochemistry for tissue localization, Western blotting for protein expression analysis, flow cytometry for cell surface expression studies, and functional assays to evaluate G protein signaling pathways. For immunohistochemistry, ADGRL2 antibodies (typically used at 1:200 dilution) enable visualization of receptor distribution in brain tissue sections, providing critical insights into expression patterns during development and in disease states . For Western blotting, anti-ADGRL2 polyclonal antibodies (used at 1:500 dilution) allow quantification of protein expression levels relative to housekeeping proteins like GAPDH . Additionally, ADGRL2 antibodies can be employed in calcium mobilization assays to evaluate receptor functionality, as demonstrated in studies measuring intracellular calcium release in response to α-latrotoxin binding .

What are the optimal conditions for immunohistochemistry using ADGRL2 antibodies?

For optimal immunohistochemistry results with ADGRL2 antibodies, tissue preparation and antibody incubation conditions are critical. Based on established protocols, fixed tissue sections should be processed using the following approach: incubation with primary ADGRL2 antibody (diluted 1:200) for 1 hour at room temperature, preferably using an automated system such as the Benschmark Ultra system . The primary antibody should be diluted in an antibody diluent reagent solution to minimize background staining. After incubation, slides should be processed with an appropriate detection kit (such as Ultraview), with visualization using an alkaline phosphatase detection system . For negative controls, either omit the primary antibody or use antibodies of known reactivity patterns. Following detection, slides should be rinsed in tap water, counterstained with hematoxylin for nuclear visualization, and mounted with appropriate mounting medium . This methodology ensures specific labeling of ADGRL2 in brain tissues, particularly in developmental studies examining expression patterns in germinal zones and cortical regions.

How can researchers validate the specificity of ADGRL2 antibodies?

Validating ADGRL2 antibody specificity requires a multi-pronged approach to ensure experimental results are reliable and reproducible. First, researchers should perform Western blot analysis using both positive controls (tissues known to express ADGRL2) and negative controls (tissues or cell lines with confirmed absence of ADGRL2 expression). The antibody should detect a protein of the expected molecular weight (~150-180 kDa for full-length ADGRL2, with potential variations due to glycosylation) . Second, immunohistochemistry patterns should be compared with in situ hybridization results to confirm correlation between protein localization and mRNA expression . Third, knockout/knockdown validation is essential - comparing antibody staining in wild-type versus ADGRL2-deficient samples can definitively demonstrate specificity. For heterologous expression systems, researchers can transfect cells with ADGRL2 constructs and confirm antibody detection by flow cytometry or immunofluorescence . Additionally, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, can further validate specificity by demonstrating signal reduction or elimination.

What are the recommended protocols for Western blotting with ADGRL2 antibodies?

For effective Western blotting using ADGRL2 antibodies, the following optimized protocol is recommended: After protein extraction and SDS-PAGE separation, transfer proteins to a membrane and block for 1 hour with 5% skimmed milk in PBS. Incubate the membrane with anti-ADGRL2 polyclonal antibody (diluted 1:500) in 0.05% Tween-PBS (PBST) overnight at 4°C under gentle agitation . For loading control, use anti-GAPDH polyclonal antibody (1:1000). After washing with PBST, detect the primary antibody using peroxidase-labeled anti-rabbit or anti-goat antibodies (1:10,000) . Visualize signals with chemiluminescence reagents and acquire images using appropriate imaging systems. For quantification, calculate the ratio of ADGRL2 signal versus GAPDH to normalize for loading variations . Special attention should be paid to sample preparation, as ADGRL2 is a membrane protein that may require specific lysis conditions to ensure complete solubilization. Additionally, researchers should be aware that ADGRL2 undergoes autoproteolytic cleavage, resulting in multiple bands that represent different fragments of the protein, which may complicate interpretation if not properly accounted for .

How can ADGRL2 antibodies be used to study G protein coupling and signaling pathways?

ADGRL2 antibodies can serve as valuable tools for investigating G protein coupling and downstream signaling pathways through various functional assays. To study ADGRL2 signaling, researchers can utilize luminescence-based gene expression assays for serum response element (SRE), which produces a robust response when G protein coupling is activated . This approach involves measuring the transcription and translation of firefly luciferase normalized to a control reporter (Renilla luciferase) expressed from the same plasmid under a constitutive promoter . For more direct measurement of G protein activation, bioluminescence resonance energy transfer (BRET) assays can be employed to monitor energy transfer between membrane-anchored luminescent donors and fluorescent acceptors coupled to G protein subunits . By applying ADGRL2 antibodies as potential agonists or antagonists in these systems, researchers can assess their effects on receptor activation and signaling. Studies have shown that ADGRL2 primarily signals through Gα₁₂/₁₃, which can be verified using selective G protein inhibitors such as YM-254890 for Gαq . These approaches allow researchers to dissect the specific G protein coupling preferences of ADGRL2 and how antibody binding might modulate these interactions.

What challenges exist in developing function-modulating ADGRL2 antibodies?

Developing function-modulating antibodies against ADGRL2 presents several significant challenges that researchers must address. First, ADGRL2, like other adhesion GPCRs, has a complex structure with multiple domains in its extracellular region, making it difficult to target specific functional epitopes. The receptor undergoes autoproteolytic cleavage at the GPS domain, resulting in a bipartite receptor structure that remains non-covalently associated at the cell surface . This structural complexity means that antibodies might bind without affecting function or might disrupt the natural association between fragments. Second, ADGRL2 interacts with multiple ligands through different domains, so antibodies that block one interaction might not affect others. For example, studies with ADGRL3 have shown that antibodies can selectively block interaction with one ligand (TEN2) while maintaining interaction with another (FLRT3) . Third, the activation mechanism of ADGRL2 involves a tethered agonist segment that becomes exposed upon receptor cleavage or ligand binding . Developing antibodies that specifically modulate this activation mechanism requires precise epitope targeting. Fourth, ensuring specificity within the ADGRL family is challenging, as demonstrated by antibodies that bind to both ADGRL3 and ADGRL1 with similar affinities but only activate ADGRL3 .

What role does ADGRL2 play in neurodevelopmental disorders?

Recent research has revealed that ADGRL2 plays a crucial role in neurodevelopmental disorders, particularly those involving brain formation and connectivity. A groundbreaking study identified a de novo missense variant in the ADGRL2 gene in a fetus with extreme microcephaly and rhombencephalosynapsis, making ADGRL2 the first gene identified as being responsible for this specific combination of brain malformations . Functional characterization of this variant through microfluorimetry experiments revealed significantly reduced cytosolic calcium release in response to α-latrotoxin binding in the fetus's amniocytes compared to age-matched controls . Similar results were observed in HeLa cells transfected with mutant ADGRL2 cDNA versus wild-type constructs. Additionally, studies in ADGRL2 heterozygous mice (Adgrl2+/-) using MRI revealed microcephaly and vermis hypoplasia, further supporting the role of ADGRL2 in brain development . Cellular studies demonstrated that the pathogenic variation increased cell adhesion properties and reduced cell motility, while also altering cytoskeletal dynamics as evidenced by a highly developed cytoplasmic F-actin network in cells overexpressing mutant ADGRL2 . These findings suggest that ADGRL2 mutations may disrupt normal neuronal migration and positioning during brain development, providing new insights into the molecular mechanisms underlying certain neurodevelopmental disorders.

How can synthetic antibody fragments be used to study ADGRL receptor specificity?

Synthetic antibody fragments (sABs) represent an innovative approach for studying ADGRL receptor specificity with unprecedented precision. Recent work has demonstrated the successful generation of domain-specific sABs against the extracellular region (ECR) of ADGRL3 using phage display technology . These sABs were selected through four rounds of screening with decreasing concentrations of target in each round to increase specificity and affinity . The resulting antibodies were the first to target the ADGRL subfamily of adhesion GPCRs. Importantly, one sAB (LK30) showed high specificity for a single domain (Lec domain) of ADGRL3, as confirmed by size exclusion chromatography and SDS-PAGE analysis . Flow cytometry experiments verified that these sABs could also bind to full-length ADGRLs expressed on cell surfaces . Remarkably, functional assays revealed that LK30 acted as an agonist for ADGRL3 but not for ADGRL1, despite binding to both with similar affinities, demonstrating isoform-specific modulation . Structural studies showed that the sAB overlapped with the teneurin-2 (TEN2) binding epitope on ADGRL3, suggesting a mechanism whereby the antibody could selectively disrupt interactions with specific ligands while maintaining others . This approach of developing domain-specific antibodies could be applied to ADGRL2 to dissect its specific interaction partners and signaling mechanisms.

What are the latest advances in using ADGRL2 antibodies for cellular localization studies?

Recent advances in using ADGRL2 antibodies for cellular localization studies have significantly enhanced our understanding of this receptor's distribution and function in the developing nervous system. Immunohistochemical studies using ADGRL2-specific antibodies have revealed detailed expression patterns in human brain embryos and fetuses, with notable immunoreactivity in hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons, and cerebellar cortex . These studies have been complemented by in situ hybridization approaches to correlate protein localization with mRNA expression patterns . Technical improvements in antibody specificity and detection sensitivity have enabled researchers to identify cell type-specific expression patterns and subcellular localization of ADGRL2, which is crucial for understanding its role in neural circuit formation. Advanced imaging techniques, including super-resolution microscopy, are now being employed with ADGRL2 antibodies to precisely map receptor distribution at synapses and growth cones. Additionally, novel approaches combining ADGRL2 antibody labeling with electrophysiological recordings allow correlation between receptor localization and functional properties of neurons . These methodological advances provide powerful tools for investigating how ADGRL2 contributes to circuit formation and synaptic function in both normal development and pathological conditions.

How should researchers interpret ADGRL2 antibody staining patterns in relation to receptor activation states?

Interpreting ADGRL2 antibody staining patterns requires careful consideration of the receptor's unique activation mechanisms and structural dynamics. Adhesion GPCRs like ADGRL2 exist in multiple conformational states depending on their autoproteolytic cleavage status and interaction with ligands . When analyzing immunostaining results, researchers should consider that antibodies may preferentially recognize specific conformational states of the receptor. For instance, some antibodies might preferentially bind to the uncleaved receptor, while others might recognize either the N-terminal fragment (NTF) or C-terminal fragment (CTF) after autoproteolysis . The distribution pattern of ADGRL2 may also reflect different activation states across brain regions or developmental stages. Dense staining in germinal zones might indicate roles in neurogenesis, while synaptic localization suggests functions in synaptogenesis or synaptic transmission . Changes in staining patterns following experimental manipulations (such as ligand application or genetic modifications) can provide insights into receptor trafficking and internalization in response to activation. When comparing wild-type and mutant ADGRL2 staining patterns, differences may reflect altered processing, trafficking, or stability of the receptor rather than just expression levels . For comprehensive interpretation, researchers should combine antibody staining with functional assays that directly measure receptor activation, such as calcium mobilization or G protein coupling assays .

What are the common pitfalls in quantifying ADGRL2 expression using antibody-based methods?

Quantifying ADGRL2 expression using antibody-based methods presents several challenges that researchers must address to obtain reliable results. First, ADGRL2 undergoes autoproteolytic cleavage, resulting in a bipartite receptor with NTF and CTF fragments that remain non-covalently associated . This processing complicates Western blot analysis, as antibodies targeting different regions will detect different fragments, potentially leading to misinterpretation of expression levels. Second, as a membrane protein, ADGRL2 extraction efficiency can vary significantly depending on the lysis conditions, potentially underestimating actual expression levels if solubilization is incomplete. Third, ADGRL2 is subject to post-translational modifications, particularly N-glycosylation, which can affect antibody binding and create heterogeneous banding patterns on Western blots . Fourth, in immunohistochemistry or immunofluorescence, epitope masking can occur if ADGRL2 is engaged with its ligands or if its conformation changes upon activation, leading to underestimation of expression levels. To address these issues, researchers should use multiple antibodies targeting different epitopes, incorporate appropriate positive and negative controls, and validate findings with complementary techniques such as qPCR or in situ hybridization . For Western blotting, specific attention to sample preparation, including proper denaturation and the use of appropriate detergents for membrane protein solubilization, is essential for accurate quantification.

How can researchers reconcile contradictory results obtained with different ADGRL2 antibodies?

Reconciling contradictory results obtained with different ADGRL2 antibodies requires a systematic approach to identify the sources of discrepancy. First, researchers should comprehensively characterize each antibody's properties, including the exact epitope recognized, species reactivity, and whether it targets the N-terminal or C-terminal regions of ADGRL2 . Since ADGRL2 undergoes autoproteolytic cleavage, antibodies directed against different regions may give apparently contradictory results that actually reflect different aspects of receptor processing or trafficking . Second, validation with knockout or knockdown controls is essential for each antibody to confirm specificity. Third, researchers should consider the possibility that different antibodies might preferentially recognize specific conformational states or post-translationally modified forms of ADGRL2 . Fourth, experimental conditions such as fixation methods for immunohistochemistry or lysis conditions for Western blotting can significantly impact epitope accessibility and detection sensitivity . To systematically address contradictory results, researchers should perform side-by-side comparisons of different antibodies under identical experimental conditions, complemented by non-antibody-based methods such as mRNA detection or tagged recombinant constructs . Additionally, functional assays that measure receptor activity, such as calcium signaling or G protein coupling, can provide important context for interpreting expression data . By integrating multiple approaches and understanding the limitations of each antibody, researchers can develop a more comprehensive understanding of ADGRL2 biology.

How might new antibody engineering techniques improve ADGRL2 research tools?

Emerging antibody engineering techniques hold tremendous promise for developing next-generation ADGRL2 research tools with enhanced specificity, functionality, and versatility. One promising approach involves the generation of domain-specific single-chain variable fragments (scFvs) or nanobodies that can target discrete regions of the ADGRL2 extracellular domain with high precision . These smaller antibody formats offer advantages including better tissue penetration, reduced immunogenicity, and the potential for intracellular expression as "intrabodies" to track or modulate ADGRL2 in living cells. Advanced phage display technologies with high-diversity synthetic libraries, similar to those used for generating ADGRL3 antibodies, can be applied to develop highly specific ADGRL2 binders . Bispecific antibody designs that simultaneously target ADGRL2 and one of its ligands could provide unique tools for studying receptor-ligand interactions and their downstream effects. Additionally, antibody fragments conjugated to fluorophores, quantum dots, or enzyme reporters could enable multiplexed imaging of ADGRL2 alongside other proteins of interest. The development of conformation-specific antibodies that selectively recognize active versus inactive states of ADGRL2 would be particularly valuable for studying receptor dynamics and activation mechanisms . Furthermore, conditionally activated antibodies that respond to specific cellular conditions (pH, protease activity, etc.) could provide spatiotemporal control over ADGRL2 targeting. These innovative approaches would significantly enhance our ability to dissect ADGRL2 functions in complex biological systems and potentially lead to new therapeutic strategies for ADGRL2-associated disorders.