PTH1R Antibody, HRP conjugated

What is PTH1R and why is it important in biological research?

PTH1R (parathyroid hormone 1 receptor) is a G protein-coupled receptor with seven transmembrane domains that mediates the biological actions of both parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). It plays critical roles in bone and mineral metabolism, skeletal development, and calcium homeostasis. The receptor has an extracellular N-terminus region and an intracellular C-terminus region, with its activity primarily mediated by G proteins that activate adenylyl cyclase . PTH1R is associated with the differentiation of bone and cartilage during development, making it a valuable target for researching bone disorders and calcium metabolism .

What are the molecular characteristics of the PTH1R protein?

PTH1R is a glycoprotein with a calculated molecular weight of approximately 66 kDa, although in immunoblotting it typically appears at 70-80 kDa due to post-translational modifications . The human protein consists of 593 amino acids and has several isoforms. PTH1R contains an extracellular domain (ECD) that's critical for ligand binding and signal transduction. The receptor's gene is identified by GenBank accession number BC110388 and NCBI gene ID 5745 . PTH1R structure includes the seven transmembrane helices characteristic of G protein-coupled receptors, with the first transmembrane helix (TM1) containing important residues involved in both ligand binding and receptor activation processes .

How do HRP-conjugated PTH1R antibodies function in experimental assays?

HRP-conjugated PTH1R antibodies combine the specific binding capabilities of anti-PTH1R antibodies with the enzymatic activity of horseradish peroxidase. This conjugation enables sensitive detection through various peroxidase substrate systems. When these antibodies bind to PTH1R, the HRP enzyme catalyzes reactions with substrates to produce colorimetric, chemiluminescent, or fluorescent signals, allowing visualization and quantification of the receptor . The functional principle is similar to fusion proteins like PTH-HRP, where the enzymatic activity allows for sensitive detection of receptor binding events at the cell surface. HRP-conjugated antibodies can be used in various applications including western blotting, immunohistochemistry, and ELISA, with detection possible through multiple substrate systems such as TrueBlue™, tetramethylbenzidine (TMB), luminol, or biotin-phenol with streptavidin-Qdots .

What are the optimal applications for PTH1R antibodies in research?

PTH1R antibodies are optimal for Western Blot (WB), Immunohistochemistry (IHC), and ELISA applications. For Western blotting, a dilution range of 1:500-1:1000 is typically recommended, while for IHC applications, dilutions between 1:50-1:500 are suggested depending on the specific tissue being examined . PTH1R antibodies have been successfully used to detect the receptor in various human tissues including pancreatic cancer and kidney tissues, as well as in multiple cell lines such as HeLa and HepG2 cells. Additionally, these antibodies show reactivity with mouse and rat tissue samples including kidney and liver . For specialized applications detecting endogenous PTH1R, such as in osteoblastic cell lines like HOS, peroxidase-based detection systems with various co-substrates (TMB, luminol, biotin-phenol) can be used .

How can I optimize detection protocols when using HRP-conjugated PTH1R antibodies?

Optimizing detection protocols for HRP-conjugated PTH1R antibodies requires careful consideration of several parameters:

• Substrate selection: Different substrates offer varying sensitivity levels. For instance, PTH-HRP constructions have shown excellent sensitivity with multiple co-substrates including TrueBlue™, tetramethylbenzidine (TMB), luminol, and biotin-phenol with streptavidin-Qdots .

• Antigen retrieval: For IHC applications, TE buffer at pH 9.0 is recommended for optimal antigen retrieval, though citrate buffer at pH 6.0 may be used as an alternative .

• Antibody titration: Each experimental system requires specific antibody dilution optimization. Starting with the recommended dilutions (1:500-1:1000 for WB; 1:50-1:500 for IHC), perform serial dilutions to determine the optimal concentration for your specific application .

• Signal amplification: For detecting low-abundance receptors, signal amplification systems like tyramide signal amplification can enhance sensitivity.

• Blocking optimization: Use appropriate blocking buffers (typically containing BSA or serum) to minimize background signal while preserving specific binding.

• Incubation conditions: Optimize both primary and secondary antibody incubation times and temperatures based on your specific experimental requirements and sample type.

What methodologies can be used to verify the specificity of PTH1R antibody binding?

To verify PTH1R antibody specificity, several methodological approaches are recommended:

• Competition assays: Specificity can be determined by competition with an excess of PTH 1-34, which should displace specific antibody binding. This approach was used to confirm the specificity of PTH-HRP fusion protein binding to PTHR1 .

• Controls with known PTH1R expression: Compare antibody binding patterns in cell lines or tissues with established PTH1R expression levels (such as HOS cell line for endogenous PTHR1) against negative controls .

• Receptor knockout/knockdown validation: Use PTH1R knockout/knockdown models to confirm absence of signal when the receptor is not expressed.

• Flow cytometry validation: Flow cytometry can confirm binding to cell surface expressed PTH1R, as demonstrated with purified antibodies in transfected cells .

• Western blot analysis: Verify antibody specificity by confirming the molecular weight of detected proteins matches the expected size for PTH1R (observed at 70-80 kDa) .

• Cross-reactivity testing: Test the antibody against related receptors to ensure specificity for PTH1R over other similar proteins.

How can PTH1R antibodies be used to investigate receptor trafficking and internalization?

PTH1R antibodies can be valuable tools for studying receptor trafficking and internalization through several methodological approaches:

• Live-cell imaging: HRP-conjugated or fluorescently labeled PTH1R antibodies can track receptor movement in real-time, particularly when the antibody recognizes an extracellular epitope.

• Pulse-chase experiments: Label cell surface receptors with antibodies, then monitor their internalization and subsequent trafficking over time using fixed time-point analysis.

• Co-localization studies: Combine PTH1R antibodies with markers for different cellular compartments (endosomes, lysosomes, etc.) to track the receptor's intracellular fate after internalization.

• RAMP co-expression analysis: Studies have shown that RAMP3 co-expression resulted in reduced cell surface expression of PTH1R, suggesting a role in receptor trafficking or internalization. Similar experimental designs can be employed to investigate other potential modulators of PTH1R trafficking .

• Biotinylation assays: Surface biotinylation followed by PTH1R immunoprecipitation can quantitatively assess internalization rates in response to various stimuli.

• Flow cytometry: Quantitative analysis of surface vs. internalized receptor populations can be performed using non-permeabilized vs. permeabilized cell preparations.

What experimental considerations are important when studying PTH1R signaling pathways using antibody-based approaches?

When investigating PTH1R signaling pathways using antibody-based approaches, several important experimental considerations should be addressed:

• Ligand selection: Different PTH and PTHrP-derived ligands (PTH(1-34), PTH(1-84), PTHrP(1-34), PTHrP(1-108), PTHrP(1-141), and analogs like ZP2307) can induce distinct signaling responses and should be carefully selected based on the pathway of interest .

• Signaling readouts: Multiple downstream pathways should be monitored, including cAMP accumulation, β-arrestin recruitment, and calcium signaling, as PTH1R activates various signaling cascades that can be differentially affected by experimental conditions .

• RAMP interactions: Consider the influence of Receptor Activity-Modifying Proteins (RAMPs) on PTH1R signaling. Research has shown that RAMP2 enhances PTH1R-mediated signaling while RAMP3 attenuates responses .

• G-protein coupling analysis: Different ligands can induce distinct patterns of G protein subtype activation through PTH1R. Techniques like antibody-capture scintillation proximity assays can be employed to investigate these patterns .

• Cell system selection: The choice between overexpression systems and endogenous receptor-expressing cells is critical, as signaling dynamics may differ significantly between these models.

• Temporal considerations: Both the kinetics of signaling responses and the duration of receptor activation should be carefully monitored, as they may reveal important aspects of signaling bias.

How can I design experiments to investigate PTH1R autoantibodies in disease models?

To investigate PTH1R autoantibodies in disease models, consider the following experimental design approaches:

• Detection assays: Implement Luciferase Immunoprecipitation Systems (LIPS) technology using PTH1R-luciferase fusion proteins to detect autoantibodies in patient serum samples, as demonstrated in previous research .

• Domain mapping: Design truncation mutants of PTH1R encompassing specific domains (extracellular domain, transmembrane domain) to map the binding regions of autoantibodies. Previous studies identified that certain patient autoantibodies primarily targeted the extracellular domain (ECD) of PTH1R .

• Functional assessment: Develop cell-based assays measuring cAMP responses to PTH 1-34 in PTH1R-expressing cells to assess whether purified IgG from patient samples can block PTH signaling through PTH1R .

• IgG purification: Purify IgG free of endogenous PTH from serum samples to avoid interference in functional assays .

• Control selection: Include appropriate control groups such as healthy controls and patients with other autoimmune or kidney disorders to establish specificity of PTH1R autoantibodies .

• Correlation with clinical parameters: Design experiments to correlate autoantibody levels with clinical manifestations such as hypocalcemia, hyperphosphatemia, or resistance to PTH therapy.

What are common challenges when working with PTH1R antibodies and how can they be overcome?

When working with PTH1R antibodies, researchers frequently encounter these challenges and solutions:

How should I analyze data from experiments involving PTH1R mutants?

When analyzing data from experiments involving PTH1R mutants, consider the following methodological approaches:

• Functional comparison to wild-type: Compare parameters like EC50 and Emax values for signaling responses (cAMP accumulation, calcium signaling, β-arrestin recruitment) between mutant and wild-type receptors. For instance, research has shown that the PTH1R-R186H mutant had comparable potency but PTH1R-V204E showed reduced response-maxima compared to PTH1R-WT .

• Surface expression analysis: Quantify cell surface expression using methods like antibody binding to extracellular tags (e.g., HA tag). Previous studies demonstrated that while PTH1R-R186H showed comparable antibody binding to PTH1R-WT, binding was significantly reduced for PTH1R-V204E (44% of wild-type levels) .

• Ligand binding affinity: Assess binding affinities with various ligands, as mutations can affect binding of different PTH analogs differently. For example, the potency of cAMP signaling induced by PTH(1-11) was reduced by ninefold and threefold for PTH1R-R186H and PTH1R-V204E, respectively, compared to PTH1R-WT .

• Statistical analysis: Apply appropriate statistical methods to determine significance of observed differences. Include sufficient replicates (typically n≥3) and report p-values.

• Structure-function correlations: Map mutations to known structural domains of PTH1R and correlate with observed functional effects. Both R186H and V204E mutations map to the first transmembrane helix (TM1) of PTH1R, which contains important residues for ligand binding and receptor activation .

• Pathophysiological relevance: Correlate in vitro findings with clinical phenotypes associated with the mutations, such as primary failure of tooth eruption (PFE) or pseudohypoparathyroidism .

How can I differentiate between technical artifacts and true biological effects when using HRP-conjugated PTH1R antibodies?

To differentiate between technical artifacts and true biological effects when using HRP-conjugated PTH1R antibodies, implement these methodological approaches:

• Multiple detection systems: Validate findings using different detection systems. Research has shown that PTH-HRP constructions support multiple peroxidase co-substrates (TrueBlue™, TMB, luminol, biotin-phenol with streptavidin-Qdots), which can help distinguish true signals from artifacts .

• Competition controls: Include competition controls with excess unconjugated antibody or PTH 1-34 to demonstrate specificity of binding. Specific binding should be competitively displaced .

• Dose-response relationships: Establish dose-response curves for both antibody concentration and substrate exposure time. True biological effects typically show predictable dose-dependent relationships.

• Comparison across cell types: Test the antibody in multiple cell lines with known differential expression of PTH1R. The signal should correlate with expected expression patterns.

• Knockout/knockdown validation: Use genetic approaches to modulate PTH1R expression and confirm corresponding changes in antibody binding.

• Substrate controls: Include controls for potential direct interaction between your experimental compounds and the HRP substrate system, as some compounds can directly affect peroxidase activity.

• Alternative detection methods: Validate key findings using non-HRP methods such as fluorescence-based detection or non-conjugated primary antibodies with separate secondary detection.

How are PTH1R antibodies being used to study biased signaling and functional selectivity?

PTH1R antibodies are increasingly being utilized to investigate biased signaling and functional selectivity through several advanced approaches:

• Antibody-mediated receptor modulation: Single chain Fv antibodies (scFvhFc) targeting the extracellular domain (ECD) of PTH1R have been identified that can act as β-arrestin 2 antagonists while allowing canonical G protein signaling, thereby serving as valuable tools to characterize PTH1R signaling bias .

• Pathway-specific readouts: Researchers use PTH1R antibodies in combination with assays that separately measure G protein activation (cAMP accumulation), β-arrestin recruitment, and calcium signaling to determine how different ligands or receptor modifications affect signaling bias .

• RAMP interaction studies: PTH1R antibodies help investigate how Receptor Activity-Modifying Proteins (RAMPs) modulate receptor signaling. FRET imaging with labeled antibodies has revealed that PTH1R preferentially interacts with RAMP2 and, to a lesser extent, RAMP3, with differential effects on various signaling pathways .

• Ligand-specific effects: Different PTH and PTHrP-derived ligands (PTH(1-34), PTHrP(1-34), PTH(1-84), ZP2307) can induce distinct patterns of signaling bias, which can be detected using antibody-based assays for different pathways .

• G protein subtype activation analysis: Antibody-capture scintillation proximity assays have demonstrated that modulators like RAMP2 can differentially affect G protein activation patterns in a ligand-dependent manner .

What novel detection systems can be coupled with HRP-conjugated antibodies for PTH1R research?

Several innovative detection systems can be coupled with HRP-conjugated antibodies for enhanced PTH1R research:

• Proximity labeling applications: HRP-conjugated antibodies can be used with biotin-phenol substrates for proximity labeling, allowing identification of proteins in close proximity to PTH1R through subsequent streptavidin-based pulldown and mass spectrometry analysis. This approach has been demonstrated with PTH-HRP fusion proteins and biotin-phenol with streptavidin-Qdots .

• Multiplexed detection systems: Combining HRP-conjugated antibodies with spectrally distinct fluorophores or quantum dots allows simultaneous detection of multiple targets, enabling co-localization studies of PTH1R with interacting proteins or trafficking markers.

• Super-resolution microscopy compatibility: Newer HRP substrates can generate products compatible with super-resolution microscopy techniques, enabling nanoscale visualization of PTH1R distribution and clustering.

• Microfluidic platforms: Integration of HRP-conjugated antibodies with microfluidic systems allows real-time monitoring of PTH1R activity in controlled microenvironments with minimal sample consumption.

• Bioluminescence resonance energy transfer (BRET): HRP-mediated chemiluminescence can serve as a donor in BRET systems when coupled with appropriate acceptor fluorophores, enabling sensitive detection of protein-protein interactions involving PTH1R.

• Digital pathology applications: Machine learning algorithms can be applied to analyze HRP-based immunohistochemistry images of PTH1R, enabling quantitative assessment of receptor expression patterns across different tissues and disease states.

How can PTH1R antibodies contribute to understanding rare bone disorders and calcium metabolism diseases?

PTH1R antibodies provide valuable tools for investigating rare bone disorders and calcium metabolism diseases through several research approaches:

• Mutation-specific antibodies: Developing antibodies that specifically recognize common PTH1R mutations, such as R186H and V204E associated with primary failure of tooth eruption (PFE), enables direct detection of mutant receptors in patient samples .

• Autoantibody detection: LIPS technology using PTH1R-luciferase fusion proteins has successfully identified PTH1R-blocking IgG autoantibodies in patients with PTH resistance, providing diagnostic tools for autoimmune forms of hypoparathyroidism .

• Tissue expression mapping: Immunohistochemistry with PTH1R antibodies can map receptor expression in various tissues in both normal and pathological states, helping to understand the tissue-specific manifestations of PTH1R-related disorders .

• Therapeutic monitoring: In patients receiving PTH analog therapies for osteoporosis or hypoparathyroidism, monitoring PTH1R expression and localization can help understand treatment responses or resistance.

• Developmental studies: PTH1R antibodies enable visualization of receptor expression during skeletal development, providing insights into disorders like Blomstrand osteochondrodysplasia and other developmental bone diseases .

• Signaling pathway analysis: In disorders associated with PTH resistance, antibody-based assays can help distinguish between receptor-level defects and downstream signaling abnormalities, guiding more targeted therapeutic approaches.