GPR126 Antibody

What is GPR126 and why is it significant for research?

GPR126 (G protein-coupled receptor 126, also known as ADGRG6) is a member of the adhesion GPCR family that plays crucial roles in multiple physiological processes. It is essential for normal differentiation of promyelinating Schwann cells and myelination of axons in the peripheral nervous system . Research has demonstrated its importance in neural, cardiac, and ear development through G-protein- and/or N-terminus-dependent signaling mechanisms .

More recently, GPR126 has been identified as a progesterone receptor that promotes breast cancer progression through Gi-SRC signaling, particularly in triple-negative breast cancer models . Its expression is elevated in breast cancer tissues and associated with poor prognosis, making it a potential therapeutic target . Additionally, GPR126 stimulates VEGF signaling and angiogenesis by modulating VEGF receptor 2 expression through STAT5 and GATA2 in endothelial cells .

What types of GPR126 antibodies are available and how do they differ?

Several types of GPR126 antibodies are available for research applications:

The key differences lie in the immunogen used for antibody generation and the validated applications. Polyclonal antibodies recognize multiple epitopes on the antigen, while recombinant antibodies offer greater batch-to-batch consistency. When selecting an antibody, researchers should consider the specific domain of GPR126 they wish to target, as the receptor undergoes proteolytic processing at the GPS site, resulting in a membrane-bound C-terminal fragment (CTF) and a soluble N-terminal fragment (NTF) .

How is GPR126 protein structurally organized and what domains should I target with antibodies?

GPR126 possesses a complex domain architecture with distinct functional regions:

• A signal peptide at the N-terminus

• An extended N-terminus containing:

  • CUB (Complement, Uegf, Bmp1) domain

  • PTX (Pentraxin) domain

  • Hormone binding domain

  • 27 putative N-glycosylation sites

• A GPS (GPCR proteolysis site) motif where cleavage occurs

• A 7-transmembrane domain homologous to secretin-like GPCRs

Experimental evidence indicates that GPR126 is cleaved at the GPS motif into a CTF that localizes to the plasma membrane and an NTF. The NTF may be further cleaved by furin at an additional site (S2 site) between the GPS motif and the PTX domain .

For studying full-length GPR126, antibodies targeting conserved regions are preferable. For investigating specific signaling mechanisms, antibodies targeting the N-terminal domains or the 7TM region may be more appropriate based on your research question .

What are the optimal conditions for using GPR126 antibodies in immunohistochemistry?

For optimal immunohistochemistry results with GPR126 antibodies, consider the following protocol parameters:

For Proteintech 17774-1-AP antibody:

• Dilution range: 1:50-1:500 for IHC applications

• Antigen retrieval: TE buffer pH 9.0 is suggested; alternatively, citrate buffer pH 6.0

• Positive control tissues: Mouse kidney tissue and mouse lung tissue

General considerations for all GPR126 antibodies:

• Optimize antibody concentration through titration in your specific testing system

• Include appropriate positive controls (tissues with known GPR126 expression)

• Include negative controls (either no primary antibody or isotype controls)

• For paraffin-embedded sections, effective antigen retrieval is crucial

• Sample-dependent optimization may be necessary

The detection of GPR126 in tissue sections is particularly valuable for studying its expression patterns in developmental contexts, disease states like breast cancer, and in the peripheral nervous system .

How can I effectively use GPR126 antibodies in Western blot applications?

For optimal Western blot results when detecting GPR126:

• Sample preparation:

  • Use appropriate lysis buffer (e.g., Cell Signaling buffer plus protease inhibitor mix and 1 mM PMSF)

  • Sonication (six pulses of 5 seconds each) can improve protein extraction

  • Centrifuge lysates (17,000 × g, 10 min, 4°C) and collect supernatant

• Gel electrophoresis and transfer:

  • Resolve samples on NuPAGE Novex Bis-Tris Gels or similar

  • Transfer to appropriate membrane following standard protocols

• Antibody incubation:

  • For Affinity Biosciences DF4945: Recommended for Western blot of human samples

  • For Proteintech 17774-1-AP: Validated for Western blot with human, mouse, and rat samples

  • Expected molecular weight: 137-145 kDa (calculated molecular weight is 137 kDa)

• Important considerations:

  • GPR126 is highly glycosylated, which may affect its apparent molecular weight

  • The receptor undergoes proteolytic processing, resulting in fragments of different sizes (CTF: ~35 kDa; NTF: ~70 kDa containing CUB and PTX domains)

  • When studying GPR126 cleavage or processing, select antibodies targeting appropriate domains

Due to post-translational modifications and processing, researchers should be prepared to observe multiple bands representing different forms of GPR126 .

How can I validate the specificity of GPR126 antibodies in my experimental system?

Validating antibody specificity is critical for reliable research outcomes. For GPR126 antibodies, consider these approaches:

• Genetic validation:

  • Use GPR126 knockdown (shRNA) or knockout (CRISPR-Cas9) cell lines or tissues as negative controls

  • Several studies have used shRNA against GPR126 in cell lines like MDA-MB-231

  • Mouse models with GPR126 deletion (Gpr126Δ7/Δ7) can serve as negative controls for mouse-reactive antibodies

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide before application

  • Signal reduction confirms specificity to the target epitope

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of GPR126

  • Concordant results increase confidence in specificity

• Expression system validation:

  • Compare endogenous expression with overexpression systems

  • Transfection of tagged GPR126 (e.g., HA-tagged or GFP-tagged) allows validation with anti-tag antibodies

• Cross-reference with mRNA expression:

  • Validate protein detection with RNA expression data

  • In situ hybridization probes for GPR126 have been described (e.g., 692-bp fragment amplified using primers 5′-CTCCGATAACCTGGGGAAAT-3′ and 5′-TTCTTGGGGTTCTCCTCTCA-3′)

Research has shown that tunicamycin treatment, which inhibits N-glycosylation, can affect the detection of GPR126, suggesting the importance of glycosylation for antibody recognition in some cases .

How can antibodies be used to study the mechanical activation of GPR126?

GPR126 functions as a mechano-sensor that translates binding of extracellular matrix (ECM) molecules to its N-terminus into metabotropic intracellular signals. Antibodies can be powerful tools to study this mechanical activation:

• Antibody-mediated receptor activation:

  • Antibodies targeting the N-terminus of GPR126 can act as agonists

  • Studies have used antibodies recognizing N-terminal HA epitopes on GPR126 to induce receptor signaling

  • The activation effect can be enhanced through secondary antibodies, which may cross-link receptors

• Force application methods:

  • Single-cell atomic force microscopy (AFM) combined with fluorescent cAMP sensors allows real-time monitoring of GPR126 activation

  • Force-clamp setup can apply controlled pulling forces (0.25-0.75 nN) to study mechanical activation

  • Different mechanical forces (pushing vs. pulling) can be applied using antibody-coated AFM cantilevers

• Comparison with natural ligands:

  • Antibody-mediated activation can mimic the activation induced by endogenous ligands like collagen IV and laminin 211

  • Collagen IV mediates GPR126 activation through pushing forces, while laminin 211 activates through pulling forces

  • Antibodies can achieve activation through combined pushing and pulling forces

This approach allows researchers to investigate the structure-function prerequisites for mechanical activation of GPR126 and potentially other adhesion GPCRs, providing insights into their activation mechanisms .

How does GPR126 signaling differ across tissue types, and how can antibodies help investigate this?

GPR126 exhibits tissue-specific functions and signaling mechanisms that can be investigated using antibodies:

• Peripheral nervous system:

  • Essential for Schwann cell myelination

  • Signals primarily through G(s)-proteins and cAMP production

  • Antibodies can detect GPR126 expression in Schwann cells during development and myelination

• Cardiac development:

  • The N-terminal fragment (NTF) of GPR126 can act independently as a ligand or coreceptor

  • Domain-specific antibodies can help distinguish between CTF and NTF functions

  • Multiplex in situ hybridization has been used to examine cardiomyocyte-specific expression using probe sets against Gpr126 (1,226 bp to 2,226 bp of NM_001002268)

• Breast cancer progression:

  • GPR126 is activated by progesterone to promote cell growth through Gi-SRC signaling

  • Highly expressed in breast cancer tissues compared to adjacent normal tissues

  • Immunohistochemistry with GPR126 antibodies can assess expression levels and correlation with prognosis

• Placental development:

  • Essential for placentation

  • Antibodies against endomucin, α-SMA, and pan-cytokeratin can be used alongside GPR126 antibodies to study placental architecture

• Experimental approaches:

  • Tissue-specific conditional knockout models can isolate GPR126 functions

  • Immunohistochemistry with GPR126 antibodies can reveal expression patterns across tissues

  • Co-immunoprecipitation using GPR126 antibodies can identify tissue-specific binding partners

Research indicates that tissue-specific functions may involve different domains of GPR126, with the NTF potentially functioning independently from the CTF in certain contexts .

What are the best approaches for studying GPR126 interactions with its ligands using antibodies?

Investigating GPR126 interactions with its ligands requires specialized techniques where antibodies play crucial roles:

• In situ protein binding assays:

  • Generate fusion proteins containing GPR126 N-terminal fragments

  • For example, a 2,310-bp fragment of mouse Gpr126-NTF without GPS motif can be fused with Fc fragments of murine IgG2b subclass (mFc)

  • Purify using Protein A-Sepharose and apply to tissue sections or cells

• Co-immunoprecipitation:

  • Use anti-GPR126 antibodies to pull down receptor complexes

  • Western blot for known ligands (collagen IV, laminin 211, progesterone) or potential binding partners

  • For membrane proteins, crosslinking before lysis may preserve transient interactions

• Proximity ligation assays:

  • Detect protein-protein interactions in situ with high sensitivity

  • Requires antibodies against both GPR126 and its ligand from different species

  • Signal is generated only when proteins are in close proximity (<40 nm)

• Competitive binding studies:

  • Use antibodies to block specific domains of GPR126

  • Assess how domain-specific blocking affects ligand binding and signaling

  • Helps map interaction interfaces between GPR126 and its ligands

• Signaling pathway analysis:

  • Monitor downstream effects of ligand binding (cAMP for Gs signaling, SRC activation for Gi signaling)

  • Compare antibody-mediated activation with ligand-mediated activation

  • Studies show that progesterone activates GPR126-dependent Gi-SRC signaling, while other ligands activate Gs-cAMP pathways

Different ligands activate distinct signaling pathways through GPR126, suggesting biased signaling properties that constitute a fine-tuning signaling network for these receptors with diverse functions .

How can I address weak or non-specific signals when using GPR126 antibodies?

Researchers frequently encounter challenges with signal strength or specificity when using GPR126 antibodies. Here are methodological solutions:

• For weak signals:

  • Optimize antibody concentration (recommended range for IHC: 1:50-1:500)

  • Improve antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase incubation time or temperature

  • Use signal amplification systems (e.g., tyramide signal amplification)

  • Consider protein glycosylation status, as GPR126 is heavily glycosylated with 27 putative N-glycosylation sites

• For non-specific signals:

  • Include proper blocking steps (BSA, serum, or commercial blocking reagents)

  • Increase washing duration and frequency

  • Reduce primary and secondary antibody concentrations

  • Pre-adsorb antibody with tissue lysates from knockout models

  • Use monoclonal antibodies instead of polyclonal when possible

• For Western blot applications:

  • Account for protein processing (GPR126 undergoes proteolytic cleavage at the GPS site)

  • Expected full-length molecular weight: 137 kDa (1222 amino acids)

  • C-terminal fragment (CTF): approximately 35 kDa

  • N-terminal fragment (NTF): approximately 70-100 kDa depending on glycosylation

  • Additional furin cleavage at S2 site may produce smaller fragments

• For fixed tissues:

  • Fixation conditions affect epitope accessibility

  • For formalin-fixed tissues, extend antigen retrieval time

  • Consider alternative fixatives if standard protocols fail

  • Fresh frozen sections may preserve some epitopes better than paraffin embedding

Remember that antibody performance is sample-dependent, requiring optimization for each experimental system .

How do post-translational modifications of GPR126 affect antibody recognition, and how can I account for this?

Post-translational modifications (PTMs) of GPR126 can significantly impact antibody recognition:

• N-glycosylation effects:

  • GPR126 contains 27 putative N-glycosylation sites

  • Glycosylation may mask epitopes or alter protein conformation

  • Experimental approach: Compare detection in samples treated with/without deglycosylation enzymes (PNGase F or Endo H)

  • Studies using tunicamycin (N-glycosylation inhibitor) showed that glycosylation is not essential for GPR126 cellular localization but may affect antibody recognition

• Proteolytic processing:

  • GPR126 undergoes autoproteolytic cleavage at the GPS motif

  • Results in a membrane-bound CTF and a soluble NTF that remains non-covalently associated

  • Further processing by furin at the S2 site releases additional fragments

  • Solution: Use antibodies targeting different domains to detect specific fragments

• Methodological approaches:

  • For Western blot: Include samples processed to retain or remove specific PTMs

  • For glycosylation: Compare native samples with those treated with PNGase F

  • For analyzing cleavage products: Include reducing and non-reducing conditions

  • For detecting secreted N-terminal fragments: Analyze both cell lysates and culture media

• Experimental controls:

  • Express tagged versions (HA-tag, GFP) of GPR126 with mutations at glycosylation sites

  • Generate constructs with modified cleavage sites to prevent processing

  • Use domain-specific antibodies to track different fragments simultaneously

Understanding the impact of PTMs on GPR126 is critical for accurate interpretation of experimental results, particularly when studying the receptor's activation mechanisms and trafficking .

How can I design experiments to differentiate between the functions of cleaved GPR126 fragments?

GPR126 undergoes proteolytic processing, generating fragments with potentially distinct functions. Here's how to design experiments to differentiate their roles:

• Domain-specific antibody approach:

  • Use antibodies targeting the N-terminal domains (CUB, PTX) to detect NTF

  • Use antibodies targeting the 7TM domain to detect CTF

  • Compare localization patterns in immunofluorescence studies

  • Perform co-immunoprecipitation with domain-specific antibodies to identify fragment-specific binding partners

• Expression of truncated constructs:

  • Generate constructs expressing only the NTF or CTF

  • Create fusion proteins with GPR126-NTF ΔGPS-mFc for secretion studies

  • Express these constructs in knockout backgrounds to assess rescue of phenotypes

  • Example: A 2,310-bp fragment of mouse Gpr126-NTF without GPS motif has been used to study N-terminal functions

• Cleavage-resistant mutants:

  • Introduce mutations at the GPS site to prevent autoproteolysis

  • Compare signaling outcomes between wild-type and cleavage-resistant receptors

  • Assess downstream effects on cAMP production (Gs signaling) or SRC activation (Gi signaling)

• Tissue-specific approaches:

  • Compare the roles of GPR126 fragments across tissues (PNS, heart, placenta)

  • Evidence suggests the NTF may function independently in cardiac development

  • Use conditional expression systems to express specific fragments in appropriate contexts

• Mechanistic investigations:

  • For N-terminal function: Apply recombinant NTF to cells and measure responses

  • For CTF function: Use synthetic peptide agonists (e.g., Stachel peptides) that activate the receptor independently of the NTF

  • For mechanical activation: Compare antibody-mediated forces on full-length versus truncated receptors using AFM techniques

Research has demonstrated that the membrane-bound CTF can act as an independent receptor while the soluble NTF can function as a ligand or coreceptor for unknown receptors, highlighting the importance of distinguishing their separate functions .

What are the most effective approaches for studying GPR126 in disease models using antibodies?

Investigating GPR126 in disease models requires careful experimental design and appropriate antibody selection:

GPR126 represents a promising target for various diseases, and antibody-based approaches are crucial for understanding its role in pathological processes and developing potential therapeutics .