Phospho-CASR (T888) Antibody

What is the calcium-sensing receptor (CASR) and why is threonine 888 (T888) significant?

The calcium-sensing receptor (CASR) is a G-protein-coupled receptor that plays a critical role in calcium homeostasis by sensing fluctuations in extracellular calcium concentration and modulating parathyroid hormone (PTH) production. CASR activates a phosphatidylinositol-calcium second messenger system through G-protein coupling . The receptor exists in both immature (140 kDa) core-glycosylated and mature (160 kDa) forms, with only the 160 kDa variant typically expressed on the cell membrane .

Threonine 888 (T888) is a key phosphorylation site in the intracellular domain of CASR that undergoes protein kinase C (PKC)-mediated phosphorylation. This site significantly influences agonist sensitivity of the receptor and contributes to CASR regulation . Phosphorylation at T888 creates a negative feedback loop that modulates receptor activity, making it a crucial target for studying CASR function in various physiological and pathological contexts.

How does Phospho-CASR (T888) Antibody specifically detect the phosphorylated form of the receptor?

Phospho-CASR (T888) antibodies are developed using synthesized peptides derived from human CASR around the phosphorylation site of T888. These antibodies are designed to specifically recognize the receptor only when phosphorylated at the T888 residue . The specificity is achieved through:

• Generation using immunogens containing the phosphorylated T888 residue (typically within amino acids 854-903 of human CASR)

• Affinity purification from rabbit antiserum using epitope-specific immunogen chromatography

• Validation through comparative analysis with non-phosphorylated controls or mutant CASR(T888A) that cannot be phosphorylated at this site

This high specificity enables researchers to distinguish between phosphorylated and non-phosphorylated forms of CASR, making these antibodies invaluable for studying the dynamics of receptor phosphorylation in response to various stimuli.

What are the optimal protocols for Western blotting using Phospho-CASR (T888) Antibody?

For effective Western blotting with Phospho-CASR (T888) Antibody, the following optimized protocol is recommended based on published methodologies:

Sample Preparation:

• Grow cells to 80-90% confluence in appropriate culture vessels

• Treat cells with experimental conditions in buffer containing 20 mM HEPES (pH 7.4), 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl₂, 0.5 mM MgCl₂, and 5.5 mM glucose

• Lyse cells on ice using RIPA buffer supplemented with 1 mM N-ethylmaleimide and phosphatase inhibitors

Immunoblotting Procedure:

• Mix protein extracts 3:1 with 4× Laemmli buffer containing β-mercaptoethanol and heat at 95°C for 10 min

• Separate proteins by SDS-PAGE on 4-15% gradient gels

• Transfer to PVDF membranes

• Block membranes in 5% blocking reagent in TBS (without detergent)

• Dilute primary antibody 1:500-1:2000 in blocking buffer containing 0.1% Tween-20

• Incubate with primary antibody for 2 hours at room temperature or overnight at 4°C

• Wash membranes thoroughly

• Incubate with appropriate secondary antibody diluted 1:10,000 in blocking buffer with 0.1% Tween-20 and 0.01% SDS

• Detect using an appropriate imaging system

Expected Results:
When blotting for Phospho-CASR (T888), expect to visualize two distinct bands:

• 160 kDa band representing mature glycosylated receptor (predominant at cell membrane)

• 140 kDa band representing core-glycosylated immature receptor

How should immunofluorescence experiments with Phospho-CASR (T888) Antibody be designed and optimized?

For immunofluorescence applications, the following methodological considerations are important:

Recommended Protocol:

• Culture cells on appropriate coverslips

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 for 10 minutes

• Block with 5% normal serum (matched to secondary antibody species) for 1 hour

• Dilute Phospho-CASR (T888) Antibody 1:200-1:1000 in blocking buffer

• Incubate overnight at 4°C in a humidified chamber

• Wash thoroughly with PBS

• Apply fluorescently-conjugated secondary antibody

• Counterstain nuclei if desired

• Mount slides with anti-fade mounting medium

Critical Controls:

• Negative control: CaR(T888A) mutant-expressing cells that cannot be phosphorylated at this site

• Phosphatase treatment: Pre-treatment of some samples with phosphatases to demonstrate phospho-specificity

• PKC activation: Positive control using PMA treatment (1 μM) which increases CaRT888 phosphorylation

What considerations are important when designing ELISA experiments with Phospho-CASR (T888) Antibody?

When using Phospho-CASR (T888) Antibody in ELISA applications, researchers should consider:

Recommended Dilution Range:

• ELISA applications typically require higher dilutions compared to other applications, with recommended dilutions around 1:40,000

Assay Design Considerations:

• Capture vs. Detection Format:

  • For sandwich ELISA, consider using a non-phospho-specific CASR antibody as capture antibody and Phospho-CASR (T888) as detection antibody

• Signal Amplification:

  • When detecting low levels of phosphorylated receptor, employ biotin-streptavidin amplification systems

• Sample Preparation:

  • Cellular lysates should be prepared with phosphatase inhibitors (calyculin, which blocks protein phosphatase 1/2A, is particularly important as it prevents dephosphorylation of CaRT888)

  • Prepare standards using lysates from cells treated with PMA to induce maximal phosphorylation

How can researchers study the dynamics of PKC-mediated CASR phosphorylation at T888?

The dynamics of PKC-mediated phosphorylation of CASR at T888 can be studied using the following approaches:

Time-Course Experiments:
Research has demonstrated that PMA treatment (1 μM) leads to time-dependent changes in CASR T888 phosphorylation. Significant increases can be detected within 2 minutes, reaching maximum levels by 10 minutes, followed by a decline from 15-20 minutes despite continued PMA presence . This indicates a dynamic regulatory mechanism controlling receptor phosphorylation.

Experimental Design:

• Treat cells with PMA (1 μM) for varying time points (0, 2, 5, 10, 15, and 20 minutes)

• Quantify phosphorylation levels by immunoblotting with Phospho-CASR (T888) Antibody

• Compare to total CASR levels using a non-phospho-specific antibody (e.g., ADD monoclonal antibody)

PKC Inhibition Studies:
To confirm the role of PKC in CASR phosphorylation:

• Pre-treat cells with PKC inhibitors prior to stimulation

• Compare acute vs. chronic PMA treatment (the latter causes PKC downregulation)

• Analyze both phospho-CASR and downstream signaling events

How does extracellular calcium concentration affect CASR T888 phosphorylation?

Research findings demonstrate a complex relationship between extracellular calcium levels and CASR T888 phosphorylation:

Key Experimental Findings:

• Raising extracellular Ca²⁺ concentration from 0.5 to 2.5 mM increases CaRT888 phosphorylation

• This effect is further potentiated by calcimimetics (such as NPS R-467)

• Higher extracellular Ca²⁺ concentrations (5 mM) can mimic these effects

• Calcilytic agents (NPS-89636) can abolish calcium-induced phosphorylation

Suggested Experimental Approach:

• Expose cells to varying extracellular calcium concentrations (0.5, 1.0, 2.5, 5.0 mM)

• Quantify phosphorylation using Phospho-CASR (T888) Antibody

• Monitor simultaneous changes in intracellular calcium using Fura-2/AM loading

• Correlate phosphorylation status with functional readouts such as intracellular calcium oscillations

What is the relationship between CASR T888 phosphorylation and intracellular calcium oscillations?

The phosphorylation state of CASR at T888 significantly influences intracellular calcium (Ca²⁺ᵢ) signaling patterns:

Mechanistic Relationships:

Experimental Design to Study This Relationship:

• Express wild-type CASR and CASR(T888A) mutant in appropriate cell models

• Monitor phosphorylation status using Phospho-CASR (T888) Antibody

• Simultaneously record Ca²⁺ᵢ oscillations using Fura-2/AM or other calcium indicators

• Manipulate phosphorylation/dephosphorylation dynamics using:

  • PKC activators (PMA)

  • PKC inhibitors

  • Phosphatase inhibitors (calyculin)

What are common issues when detecting phosphorylated CASR and how can they be resolved?

Researchers commonly encounter several technical challenges when working with Phospho-CASR (T888) Antibody:

How can researchers validate the specificity of phosphorylation detection?

Validating antibody specificity is crucial for reliable phosphorylation studies. Several approaches are recommended:

Essential Validation Experiments:

• Genetic Validation:

  • Compare signals between wild-type CASR and CASR(T888A) mutant-expressing cells

  • The T888A mutant should show no reactivity with the phospho-specific antibody

• Pharmacological Validation:

  • Treat cells with PKC activators (PMA) to increase phosphorylation

  • Use PKC inhibitors to block phosphorylation

  • Compare signal intensities under these conditions

• Dephosphorylation Controls:

  • Treat lysates with lambda phosphatase prior to immunoblotting

  • This should eliminate signal from a truly phospho-specific antibody

• Competing Peptide Controls:

  • Pre-incubate antibody with the phosphorylated peptide used as immunogen

  • This should block specific antibody binding

What experimental conditions affect the temporal dynamics of CASR T888 phosphorylation?

Understanding the temporal dynamics of CASR phosphorylation is essential for experimental design. Research has revealed several factors that influence these dynamics:

Time-Dependent Changes:
Studies have shown that PMA-induced phosphorylation of CASR at T888 follows a specific time course, with phosphorylation increasing significantly by 2 minutes, peaking at 10 minutes, and declining from 15-20 minutes despite continued PMA presence .

Factors Affecting Phosphorylation Dynamics:

• PKC Activation Method:

  • Phorbol esters (PMA) cause rapid but transient phosphorylation

  • Receptor-mediated PKC activation may follow different kinetics

• Calcium Concentration:

  • Moderate CaR stimulation (2.5 mM Ca²⁺) increases CaRT888 phosphorylation approximately 5-fold in CaR-transfected HEK-293 cells

  • Higher concentrations (5 mM Ca²⁺) produce more pronounced effects

• Phosphatase Activity:

  • Calyculin (protein phosphatase 1/2A inhibitor) blocks dephosphorylation of CaRT888

  • The balance between kinase and phosphatase activities determines the steady-state level of phosphorylation

Recommended Experimental Approach:

• Include multiple time points in phosphorylation studies (0, 2, 5, 10, 15, 20, 30 minutes)

• Consider the effects of both phosphorylation and dephosphorylation processes

• Include appropriate controls for each time point

• Normalize phospho-CASR signals to total CASR expression

How should researchers quantify and normalize Phospho-CASR (T888) signals in immunoblotting experiments?

Proper quantification and normalization are essential for reliable interpretation of phosphorylation data:

Recommended Quantification Procedure:

• Use semi-quantitative immunoblotting with the phospho-specific anti-CaR pT888 antibody

• Image blots using systems that provide linear detection range (e.g., infrared imaging systems)

• Analyze both the 160 kDa (mature) and 140 kDa (core-glycosylated) CASR bands separately

• Normalize phospho-specific signals to total CASR expression using a non-phospho-specific antibody

Normalization Approaches:

• Ratio Method: Calculate phospho-CASR/total CASR ratio for each sample

• Internal Control: Include a standard sample (e.g., PMA-treated cells) on each blot

• Fold Change: Express results as fold change relative to baseline conditions

• Total Protein Normalization: Use stain-free gel technology or total protein stains as alternative loading controls

How can contradictory results in phosphorylation studies be reconciled?

Researchers sometimes encounter contradictory results when studying CASR phosphorylation. Several factors may contribute to these discrepancies:

Common Sources of Contradictions:

• Cell Type Differences:

  • Different cell types may express distinct PKC isoforms or phosphatases

  • The ratio of 140 kDa to 160 kDa CASR may vary between cell types

  • Example: Results from HEK-293 cells may differ from those in parathyroid cells

• Methodology Variations:

  • Sample preparation techniques (particularly phosphatase inhibitor usage)

  • Antibody dilutions and incubation conditions

  • Detection methods (chemiluminescence vs. fluorescence)

• Temporal Dynamics:

  • The transient nature of phosphorylation means timing is critical

  • PMA-induced phosphorylation peaks at ~10 minutes and then declines

Reconciliation Strategies:

• Standardize experimental protocols across laboratories

• Include appropriate positive and negative controls

• Verify results using multiple techniques (e.g., immunoblotting, immunofluorescence)

• Consider the dynamics of phosphorylation/dephosphorylation cycles

What advanced research questions can be addressed using Phospho-CASR (T888) Antibody in disease models?

The Phospho-CASR (T888) Antibody can be utilized to investigate several important research questions related to disease states:

Potential Research Applications:

• Disorders of Calcium Homeostasis:

  • Investigate how CASR phosphorylation state changes in hypercalcemic or hypocalcemic conditions

  • Study the impact of disease-associated CASR mutations on T888 phosphorylation

  • Research implications for conditions like familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT)

• Cancer Research:

  • Examine how malignant transformation affects calcium-induced CASR phosphorylation

  • Normal primary keratinocytes and breast epithelial cells show inhibited proliferation at elevated extracellular calcium levels, but malignant transformations lose this responsiveness

  • Investigate if altered CASR phosphorylation contributes to this phenotype

• Neurodegenerative Diseases:

  • CASR is expressed in the nervous system and may be relevant to neurodegenerative conditions

  • Investigate if neuronal calcium dysregulation affects CASR phosphorylation status

  • Explore potential roles in excitotoxicity mechanisms

Methodological Approaches:

• Compare phosphorylation patterns between normal and diseased tissues

• Correlate phosphorylation status with disease progression markers

• Test effects of therapeutic compounds (calcimimetics, calcilytics) on CASR phosphorylation in disease models

• Combine phosphorylation analysis with functional assays to establish causality

By addressing these advanced research questions, scientists can gain deeper insights into the role of CASR phosphorylation in physiological and pathological processes, potentially leading to new therapeutic approaches for calcium-related disorders.