PTH1R Antibody, Biotin conjugated

What is PTH1R and what is its biological significance in research contexts?

PTH1R (parathyroid hormone 1 receptor) is a G-protein coupled receptor that serves as the primary receptor for both parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). It plays critical roles in bone metabolism, calcium homeostasis, and cellular differentiation processes .

From a research perspective, PTH1R is significant because it mediates multiple signaling pathways, primarily through G protein activation of adenylyl cyclase and the phosphatidylinositol-calcium second messenger system . PTH1R is expressed on multiple cell types including hepatocytes, renal epithelium, smooth muscle cells, osteoblasts, and chondrocytes, making it relevant to studies in bone biology, endocrinology, and developmental biology .

Structurally, mature human PTH1R is a 7-transmembrane glycoprotein 567 amino acids in length with a calculated molecular weight of 66 kDa, though it is typically observed at 70-80 kDa due to post-translational modifications . The receptor contains a ligand-binding N-terminal extracellular region (amino acids 27-188) and a 130 amino acid cytoplasmic C-terminal domain responsible for intracellular signaling .

What are the key characteristics of biotin-conjugated PTH1R antibodies?

Biotin-conjugated PTH1R antibodies are immunological tools designed with the following key specifications:

• Conjugation chemistry: These antibodies have biotin molecules covalently attached, enabling strong interaction with streptavidin/avidin systems while maintaining antibody specificity .

• Reactivity profile: Available antibodies show reactivity with different species, with some specific to human PTH1R (such as bsm-61833r-biotin) and others demonstrating cross-reactivity with mouse and rat specimens (such as 29115-1-AP) .

• Molecular recognition: These antibodies typically target specific epitopes within the PTH1R structure, often focusing on the extracellular N-terminal domain where ligand binding occurs (e.g., Tyr23-Met189 region) .

• Storage requirements: Most biotin-conjugated PTH1R antibodies require storage at -20°C and can maintain stability for approximately 12 months under proper conditions .

• Application versatility: These antibodies are validated for various experimental applications including Western blotting (WB) and immunoprecipitation (IP), with appropriate dilution ranges specific to each application .

How does PTH1R signaling affect bone and adipose tissue differentiation?

PTH1R signaling plays a central role in the regulation of mesenchymal progenitor cell differentiation, with significant impacts on both osteogenesis and adipogenesis:

• Osteogenic regulation: Intermittent PTH treatment via PTH1R activation increases bone formation by enhancing both osteoblast numbers and function, resulting in higher bone mass. This mechanism has been demonstrated to promote lineage allocation of skeletal stromal cells toward osteoblasts at the expense of adipogenesis .

• Adipogenic suppression: Research confirms that PTH treatment reduces bone marrow adiposity primarily through shifting lineage allocation of progenitor cells. Conditional deletion of PTH1R in mesenchymal progenitors produces the opposite effect - reduced osteoblast differentiation and enhanced marrow adipogenesis .

• Molecular signaling pathways: PTH1R primarily signals through:

  • PKA pathway - crucial for osteogenic effects and suppression of adipogenic factors

  • PKC pathway - with lesser involvement in adipogenic regulation

  This has been confirmed through inhibitor studies showing that PKA inhibitors (e.g., H89) block PTH-mediated suppression of adipogenic factors like Zfp467, while PKC inhibitors (e.g., Go6983) have minimal effect .

• Transcriptional regulation: PTH1R activation downregulates expression of Zfp467 (a pro-adipogenic factor) via the PKA pathway. Conversely, genetic deletion of Zfp467 increases PTH1R expression by approximately 40%, establishing a positive feedback mechanism that enhances cellular responsiveness to PTH .

What are the optimal storage and handling protocols for biotin-conjugated PTH1R antibodies?

For maximum stability and experimental reproducibility, biotin-conjugated PTH1R antibodies require specific storage and handling procedures:

Handling precautions:

• Always centrifuge vials briefly before opening to collect all material at the bottom.

• When preparing working dilutions, use aseptic technique in a clean environment.

• Allow antibody to equilibrate to room temperature before opening to prevent condensation that could introduce contaminants.

• Document all usage including freeze-thaw cycles to track potential degradation .

What are the validated application protocols and optimal dilutions for biotin-conjugated PTH1R antibodies?

The biotin-conjugated PTH1R antibodies have been validated for specific applications with empirically determined optimal dilution ranges:

Western Blotting (WB) Protocol:

• Sample preparation: Lyse cells in RIPA buffer with protease inhibitors

• Protein separation: Load 20-40 μg protein per lane on 8-10% SDS-PAGE gel

• Expected band size: 70-80 kDa (despite calculated MW of 66 kDa)

• Recommended dilution: 1:300-1:5000 (specific to antibody catalog number)

• Detection system: Streptavidin-HRP system preferred for optimal signal

Immunoprecipitation (IP) Protocol:

• Lysate preparation: 200-500 μg total protein in 200-500 μl lysis buffer

• Antibody amount: 1-2 μg per reaction

• Pre-clearing: Incubate lysate with protein A/G beads before adding antibody

• Capture method: Use streptavidin-conjugated beads for efficient capture

Sample types validated:

• Cell lines: HeLa, HepG2

• Tissues: Mouse kidney, mouse liver, rat kidney, rat liver

• Primary cells: Bone marrow stromal cells (BMSCs), calvarial osteoblasts (COBs)

Optimization recommendations:
Each laboratory should perform titration experiments to determine optimal concentration for their specific sample type and application. Begin with the manufacturer's recommended dilution range and adjust based on signal-to-noise ratio .

How can researchers effectively validate the specificity of biotin-conjugated PTH1R antibodies?

Validating antibody specificity is crucial for experimental reproducibility and accurate data interpretation. For biotin-conjugated PTH1R antibodies, implement these validation strategies:

• Positive and negative control samples:

  • Positive controls: Tissues/cells known to express PTH1R (kidney, liver, osteoblasts)

  • Negative controls: Use PTH1R knockout samples or siRNA-treated cells to confirm signal reduction

  • Compare observed molecular weight (70-80 kDa) with expected size (calculated 66 kDa)

• Cross-validation with multiple detection methods:

  • Compare results from different antibody clones targeting distinct PTH1R epitopes

  • Use orthogonal methods such as mRNA expression analysis to confirm protein expression patterns

  • Validate with both N-terminal and C-terminal targeting antibodies when possible

• Signal specificity controls:

  • Pre-adsorption test: Pre-incubate antibody with immunizing peptide to block specific binding

  • Secondary-only controls: Verify absence of non-specific binding from detection system

  • Isotype controls: Use non-specific IgG of same isotype to confirm specificity

• Functional validation:

  • Confirm antibody detects changes in PTH1R expression following experimental manipulation

  • Use siRNA to knockdown PTH1R expression and verify corresponding reduction in antibody signal

  • Validate that forskolin treatment (which affects PTH1R signaling) produces expected changes in antibody-detected signals

• Biotin-specific considerations:

  • Test for endogenous biotin: Include streptavidin-only controls to detect endogenous biotinylated proteins

  • Validate that biotin conjugation hasn't compromised epitope recognition using parallel experiments with unconjugated antibody

How do receptor activity-modifying proteins (RAMPs) influence PTH1R signaling and antibody detection?

Recent research reveals complex interactions between PTH1R and receptor activity-modifying proteins (RAMPs) that significantly impact receptor function and potentially antibody detection:

RAMP interactions with PTH1R:

• PTH1R preferentially interacts with RAMP2 and, to a lesser extent, RAMP3, but shows minimal interaction with RAMP1

• These interactions create receptor complexes with altered properties, including:

  • RAMP2-PTH1R complexes show enhanced signaling responses

  • RAMP3-PTH1R complexes demonstrate attenuated signaling

Impact on receptor expression and localization:

• RAMP3 co-expression significantly reduces cell surface expression of PTH1R

• This reduction may affect antibody accessibility to extracellular epitopes

• Researchers should consider RAMP expression profiles in their experimental systems when interpreting antibody staining patterns

Differential effects on ligand responses:

• RAMP2 significantly enhances PTH1R-mediated signaling responses to multiple ligands:

  • PTH (1-34)

  • PTHrP (1-34)

  • PTH (1-84)

  • PTH (1-17) analogue ZP2307

• RAMP3 co-expression attenuates or abolishes these responses

• These findings suggest that RAMP expression could create microheterogeneity in PTH1R conformation that might affect antibody binding efficiency

Methodological implications:

• When using PTH1R antibodies, researchers should consider documenting RAMP expression in their experimental system

• Validation experiments should include assessment of how RAMP co-expression might affect antibody binding characteristics

• Differential binding of antibodies to RAMP-associated versus free PTH1R could potentially be exploited to distinguish receptor populations

What approaches can researchers use to investigate PTH1R-mediated signaling pathways?

Investigating PTH1R signaling requires sophisticated methodological approaches to capture the complexity of downstream pathway activation:

cAMP signaling assessment:

• Real-time cAMP monitoring:

  • Utilize FRET-based sensors (e.g., EPAC-based sensors) for live-cell imaging

  • Measure PKA activity using fluorescent reporters such as AKAR (A-kinase activity reporter)

  • These approaches revealed enhanced cAMP production in Zfp467-/- cells in response to exogenous PTH

• Endpoint cAMP measurements:

  • ELISA-based detection of total cAMP accumulation following ligand stimulation

  • Competitive immunoassays using labeled cAMP tracers

  • Include phosphodiesterase inhibitors (e.g., IBMX) to prevent cAMP degradation during assays

G-protein subtype activation analysis:

• Bioluminescence resonance energy transfer (BRET):

  • Allows real-time monitoring of G-protein activation in live cells

  • Research demonstrates RAMP2 differentially modulates G-protein activation by PTH1R in a ligand-dependent manner

  • PTH (1-34) and PTHrP (1-34) induce distinct patterns of G-protein subtype activation

• G-protein selective inhibitors:

  • Use pertussis toxin to block Gαi-mediated signaling

  • Apply YM-254890 to inhibit Gαq/11 pathways

  • Employ PKA inhibitors (e.g., H89) or PKC inhibitors (e.g., Go6983) to dissect downstream effectors

Transcriptional regulation assessment:

• Promoter analysis techniques:

  • Dual fluorescence reporter assays to study promoter activity

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding

  • These approaches have revealed NFκB1 binding to the P2 promoter of PTH1R following Zfp467 deletion

• Gene expression analysis:

  • qRT-PCR for targeted gene expression analysis

  • RNA-seq for global transcriptional profiling

  • Western blotting using phospho-specific antibodies to detect activated signaling proteins

Metabolic analysis:

• Glycolytic activity measurement:

  • Seahorse XF analyzers to measure extracellular acidification rate

  • Lactate production assays to quantify glycolytic output

  • PTH1R activation has been shown to increase glycolysis in Zfp467-/- cells

How can researchers investigate the relationship between PTH1R signaling and bone/adipose tissue differentiation?

Investigating the complex relationship between PTH1R signaling and differentiation of mesenchymal progenitors requires multifaceted experimental approaches:

In vitro differentiation models:

• Osteoblast differentiation assays:

  • Calvarial osteoblast (COB) culture with osteogenic media (β-glycerophosphate, ascorbic acid, dexamethasone)

  • Assess mineralization (Alizarin Red staining)

  • Measure osteoblastic markers (RUNX2, OSX, OCN) by qRT-PCR and Western blot

  • Compare wild-type cells with PTH1R knockdown or overexpression models

• Adipogenic differentiation models:

  • Bone marrow stromal cell (BMSC) culture with adipogenic media (insulin, dexamethasone, IBMX)

  • Assess lipid accumulation (Oil Red O staining)

  • Measure adipogenic markers (PPARγ, C/EBPα, adiponectin) by qRT-PCR and Western blot

  • Compare differentiation efficiency with and without PTH treatment

Molecular mechanism investigation:

• Gene silencing approaches:

  • siRNA knockdown of PTH1R (demonstrated to increase Zfp467 expression)

  • shRNA for stable knockdown in long-term differentiation studies

  • CRISPR-Cas9 for complete knockout models

• Pathway dissection:

  • Pharmacological activation with forskolin (which suppresses Zfp467 expression)

  • Pathway inhibitors: H89 for PKA, Go6983 for PKC

  • Time-course experiments to elucidate signaling kinetics

Lineage allocation analysis:

• Progenitor cell fate tracking:

  • Pulse-chase experiments with BrdU or EdU labeling

  • Lineage tracing using Cre-lox systems (Prrx1-Cre for mesenchymal progenitors)

  • Single-cell RNA sequencing to identify differentiation trajectories

• Transcription factor dynamics:

  • ChIP-seq to identify genome-wide binding patterns of key transcription factors

  • Confocal immunofluorescence to track nuclear translocation of transcription factors like NFκB1

  • Protein-protein interaction studies (co-immunoprecipitation, proximity ligation assay)

Translational relevance:

• Analyze bone microarchitecture in animal models (microCT)

• Assess marrow adiposity (osmium tetroxide staining, histology)

• Correlate findings with human studies showing PTH treatment reduces bone marrow adiposity

What are common technical challenges with biotin-conjugated antibodies and how can they be resolved?

Researchers working with biotin-conjugated PTH1R antibodies may encounter several technical challenges that can affect experimental results:

High background signal in Western blots and immunostaining:

Weak or absent signal:

Inconsistent molecular weight:

Signal detection issues:

How do model systems and species differences affect PTH1R antibody performance?

Understanding species-specific and model-dependent factors is crucial for selecting appropriate PTH1R antibodies and interpreting results accurately:

Species cross-reactivity considerations:

• Human vs. rodent PTH1R:

  • Human PTH1R shares 88% amino acid identity with mouse PTH1R over amino acids 1-189

  • Some antibodies show broad cross-reactivity (human, mouse, rat) while others are species-specific

  • When using antibodies across species, validation is essential as epitope conservation may vary

• Species-specific isoforms:

  • PTH1R has several isoforms that may be differentially expressed across species and tissues

  • Alternative splicing patterns can affect epitope availability

  • Researchers should verify which isoforms their antibodies detect in their specific model system

Cell/tissue-specific expression patterns:

Model system-specific factors:

• Cell culture models:

  • Expression levels may change with passage number and culture conditions

  • PTH1R expression increases during osteoblastic differentiation

  • Consider time-course analysis when working with differentiation models

• Animal models:

  • Genetic models with PTH1R modifications (e.g., Prrx1Cre;Pth1r fl/fl mice) may need special consideration for antibody selection

  • Different mouse strains may have minor variations in PTH1R sequence

  • Age-dependent expression patterns should be considered when selecting samples

• Primary vs. immortalized cells:

  • Primary cells (BMSCs, COBs) often show more physiologically relevant PTH1R expression patterns

  • Cell lines may have altered post-translational modifications

  • Experimental validation in multiple systems strengthens findings

How can researchers interpret complex PTH1R signaling data in the context of bone and adipose tissue regulation?

Interpreting complex PTH1R signaling data requires consideration of multiple factors that influence receptor function and downstream effects:

Temporal dynamics of PTH1R signaling:

• Acute vs. chronic PTH exposure:

  • Intermittent PTH treatment increases bone formation

  • Continuous PTH exposure can stimulate bone resorption

  • Experiments should carefully control timing and duration of PTH treatments

• Signaling kinetics:

  • PKA pathway activation occurs rapidly (within minutes)

  • Transcriptional changes in factors like Zfp467 can be detected within 10 minutes of PTH treatment

  • Later transcriptional programs affecting differentiation require hours to days to manifest

Integration with other signaling pathways:

• PTH1R-RAMP interactions:

  • RAMP2 enhances while RAMP3 attenuates PTH1R signaling responses

  • These modulatory proteins create receptor heterogeneity that affects interpretation of results

  • Researchers should consider documenting RAMP expression in their experimental systems

• Feedback mechanisms:

  • PTH1R activation via PKA downregulates Zfp467

  • Zfp467 deletion increases PTH1R expression by ~40%

  • This establishes a positive feedback loop that enhances cellular responsiveness to PTH

  • Such feedback loops can complicate interpretation of cause-effect relationships

• Transcriptional network analysis:

  • NFκB1 binds to the P2 promoter of PTH1R and increases its transcription in Zfp467-deficient cells

  • Multiple transcription factors may compete or cooperate to regulate PTH1R expression

  • Systems biology approaches may be needed to fully interpret complex regulatory networks

Cell-type specific responses:

Translational relevance:

• Correlation with in vivo phenotypes:

  • Zfp467 deletion results in increased trabecular bone volume and reduced marrow adipose tissue

  • These phenotypes should be considered when interpreting in vitro signaling data

  • Disconnect between signaling outcomes and tissue-level effects may indicate involvement of additional mechanisms

• Human relevance:

  • PTH treatment in humans reduces bone marrow adiposity through lineage allocation shifts

  • Species differences in signaling amplitude or kinetics may exist

  • Validation in human-derived cells strengthens translational potential of findings

How are biotinylated PTH1R antibodies being used to advance understanding of bone-fat communication?

Biotinylated PTH1R antibodies are enabling innovative research approaches to elucidate the complex communication between bone and adipose tissues:

Spatial proteomic applications:

• Multiplexed imaging approaches combining biotinylated PTH1R antibodies with other tissue markers

• Co-localization studies between PTH1R and factors like Zfp467 to understand spatial regulation within the bone marrow niche

• High-resolution mapping of receptor distribution within heterogeneous tissues

Single-cell analysis:

• Flow cytometry-based approaches using biotinylated antibodies to isolate PTH1R-expressing cell populations

• Integration with single-cell RNA-sequencing to characterize transcriptional profiles of specific PTH1R-expressing subpopulations

• These approaches are revealing previously unrecognized heterogeneity in mesenchymal lineage cells

Mechanistic investigations:

• Detailed studies showing how PTH1R activation via PKA pathway suppresses adipogenic factors like Zfp467

• Identification of NFκB1 as a key mediator binding to the P2 promoter of PTH1R, increasing its transcription

• Characterization of a positive feedback loop where suppression of Zfp467 leads to increased PTH1R expression

Therapeutic implications:

• Understanding PTH-mediated suppression of bone marrow adiposity offers potential therapeutic strategies for osteoporosis

• PTH treatment reduces bone marrow adiposity in humans through lineage allocation shifts

• Identifying specific downstream mediators may provide novel therapeutic targets with fewer side effects

What novel methodologies are being developed for studying PTH1R-RAMP complex formation?

Recent research has revealed important interactions between PTH1R and receptor activity-modifying proteins (RAMPs), driving development of specialized methodologies:

Protein-protein interaction detection:

• Resonance energy transfer techniques (FRET/BRET) to detect and quantify PTH1R-RAMP interactions in live cells

• Proximity ligation assays to visualize receptor complexes in fixed tissues

• These approaches have demonstrated preferential interaction of PTH1R with RAMP2 and to a lesser extent RAMP3

Functional characterization methods:

• Sophisticated signaling assays showing that RAMP2 enhances while RAMP3 attenuates PTH1R-mediated responses

• G-protein subtype-specific biosensors revealing that RAMP2 differentially modulates G-protein activation in a ligand-dependent manner

• PTH (1-34) and PTHrP (1-34) induce distinct patterns of G-protein activation when complexed with RAMPs

Complex-specific antibody development:

• Biotinylated antibodies detecting specific conformations of PTH1R-RAMP complexes

• Epitope mapping to identify complex-specific regions

• These tools aim to distinguish between "free" PTH1R and RAMP-associated receptor populations

Structural biology approaches:

• Cryo-electron microscopy of PTH1R-RAMP complexes

• Computational modeling of interaction interfaces

• These structural insights guide development of complex-specific pharmacological agents

How might biotinylated PTH1R antibodies contribute to developing targeted bone therapeutics?

Biotinylated PTH1R antibodies are advancing development of bone-targeted therapeutics through several innovative approaches:

Diagnostic applications:

• Development of imaging protocols using biotinylated antibodies to assess PTH1R expression in patient samples

• Correlation of receptor expression patterns with disease progression and treatment response

• These diagnostics could help identify patients most likely to respond to PTH-based therapies

Drug delivery strategies:

• Creation of antibody-drug conjugates targeting PTH1R-expressing cells

• Biotinylated antibodies coupled with streptavidin-linked therapeutic payloads

• These approaches allow targeted delivery of osteoanabolic agents to specific cell populations

Therapeutic monitoring:

• Using biotinylated antibodies to track changes in PTH1R expression during treatment

• Assessment of receptor internalization and recycling in response to therapeutic agents

• These methods provide mechanistic insights into drug efficacy and resistance

Novel therapeutic design:

• Development of antibody-based PTH1R modulators with distinct signaling properties

• Design of agents that disrupt or enhance specific PTH1R-RAMP interactions

• This precision approach aims to activate beneficial signaling pathways while avoiding unwanted effects

Precision medicine approaches:

• Understanding how genetic variations like Gly121Glu, Ala122Thr, and Arg255His impair PTH1R signaling

• Development of mutation-specific therapeutic strategies

• These personalized approaches could improve outcomes in patients with receptor variants