Phospho-OPRM1 (Ser375) Antibody

Basic Research Questions

• What is the Phospho-OPRM1 (Ser375) Antibody and what epitope does it recognize?

  Phospho-OPRM1 (Ser375) antibodies specifically detect the μ-opioid receptor (OPRM1) only when phosphorylated at serine residue 375. This phosphorylation site represents a crucial regulatory element in receptor function. Most commercially available antibodies are generated using synthetic phosphopeptides containing the sequence around Ser375 (typically amino acids 341-390) . Importantly, some antibodies detect what has been historically referenced as Ser375 but is annotated as Ser377 in certain species or databases . The antibody is highly specific for the phosphorylated form and does not recognize the non-phosphorylated receptor, allowing researchers to monitor dynamic phosphorylation events in response to various agonists.

• What are the validated applications for Phospho-OPRM1 (Ser375) Antibody?

  Phospho-OPRM1 (Ser375) antibodies have been validated for multiple research applications:

  Application	Typical Dilution Range	Notes
  Western Blotting (WB)	1:500-1:2000	Most common application
  Immunoprecipitation (IP)	1:100	Useful for enriching phosphorylated receptor
  Immunohistochemistry (IHC)	1:100-1:300	For tissue localization studies
  Immunofluorescence (IF)	1:50-1:200	Useful for subcellular localization
  ELISA	1:20000	For quantitative analysis

  When using these antibodies, appropriate controls should be included to verify specificity, particularly when applying them to new experimental systems.

• What species reactivity has been verified for commercial Phospho-OPRM1 (Ser375) antibodies?

  Commercial Phospho-OPRM1 (Ser375) antibodies show varied species reactivity profiles:

  Antibody Source	Verified Species	Predicted Cross-Reactivity
  Cell Signaling (#3451)	Mouse	Limited to mouse
  AFG Scientific (A46530)	Human, Mouse, Rat	Not specified
  St. John's Labs (STJ91113)	Human, Mouse, Rat	Pig, Bovine, Sheep, Rabbit, Dog, Chicken, Xenopus
  Affinity Biosciences (AF4420)	Human, Mouse	Pig, Bovine, Sheep, Rabbit, Dog, Chicken, Xenopus

  Cross-reactivity predictions are typically based on 100% sequence homology in the antigenic region but require experimental validation . When working with species not explicitly verified, researchers should conduct preliminary validation studies.

• What is the expected molecular weight pattern for phosphorylated OPRM1 in Western blotting?

  The molecular weight of phosphorylated OPRM1 observed in Western blotting varies depending on experimental conditions:

  • Typically appears as bands between 70-90 kDa in most experimental systems

  • Some suppliers list the calculated molecular weight at approximately 45-49 kDa

  • The apparent molecular weight differences are attributed to post-translational modifications, particularly glycosylation

  Researchers should be aware that the migration pattern may vary between tissue types and cell lines. When performing Western blots, including positive controls (cells expressing known OPRM1 levels) is recommended for accurate band identification.

• What are the proper storage and handling conditions for Phospho-OPRM1 (Ser375) antibodies?

  Proper storage and handling are critical for maintaining antibody activity:

  • Store at -20°C or -80°C as recommended by the manufacturer

  • Most commercial formulations contain 50% glycerol to prevent freezing solid at -20°C

  • Avoid repeated freeze-thaw cycles to maintain antibody integrity

  • Some manufacturers do not recommend aliquoting the antibody

  • Most formulations contain preservatives (e.g., 0.02% sodium azide) and stabilizers (e.g., 0.5% BSA)

  Following these storage recommendations will help maintain antibody performance over time and ensure experimental reproducibility.

Advanced Research Questions

• What is the functional significance of OPRM1 phosphorylation at Ser375 in opioid receptor regulation?

  Ser375 phosphorylation represents a critical molecular switch in μ-opioid receptor signaling:

  • Serves as a primary phosphorylation site mediating agonist-induced receptor desensitization

  • Acts as a prerequisite for β-arrestin recruitment and subsequent receptor internalization

  • Different opioid agonists induce varying degrees of Ser375 phosphorylation, with morphine notably producing minimal phosphorylation compared to other agonists like DAMGO, etorphine, and fentanyl

  • Is primarily mediated by G protein-coupled receptor kinase 2 (GRK2)

  • Determines which downstream signaling pathways are activated following receptor stimulation

  • Creates a biased signaling mechanism where phosphorylation status dictates G-protein versus β-arrestin pathway engagement

  Understanding this phosphorylation event is crucial for elucidating mechanisms of opioid tolerance, dependence, and side effect profiles of different opioid drugs.

• How does agonist-specific OPRM1 phosphorylation at Ser375 influence downstream signaling pathways?

  Different opioid agonists produce distinct phosphorylation patterns that direct signaling:

  • Strong agonists (DAMGO, etorphine) induce robust Ser375 phosphorylation, leading to efficient β-arrestin recruitment and receptor internalization

  • Fentanyl induces intermediate levels of Ser375 phosphorylation, resulting in moderate β-arrestin recruitment

  • Morphine produces minimal Ser375 phosphorylation, leading to preferential activation of PKCε pathway rather than β-arrestin pathway

  • The phosphorylation status determines whether ERK activation occurs via G-protein-dependent or β-arrestin-dependent mechanisms

  • Phosphorylation at Ser375 specifically influences G-protein-dependent but not β-arrestin-dependent activation of the ERK pathway

  These findings demonstrate that receptor phosphorylation acts as a molecular switch determining which signaling cascades are activated following receptor stimulation, contributing to the diverse pharmacological profiles of different opioids.

• What experimental approaches can be used to modulate OPRM1 phosphorylation for mechanistic studies?

  Several strategies can be employed to experimentally manipulate OPRM1 phosphorylation:

  • Genetic approaches:

    • Expression of phosphorylation-deficient mutants (S375A) using adenoviral vectors (AdS375A)

    • Expression of "3A" triple mutant (mutation of all three C-terminal phosphorylation sites)

    • Overexpression of wild-type GRK2 (increases phosphorylation) or dominant-negative GRK2-K220R (decreases phosphorylation)

  • Pharmacological approaches:

    • Use of PKC subtype-specific inhibitors to dissect phosphorylation pathways

    • Treatment with opioid receptor antagonists (naloxone, Cys2-Tyr3-Orn5-Pen7-amide) to block agonist-induced phosphorylation

    • Manipulation of membrane cholesterol content to modulate receptor phosphorylation efficiency

  These approaches provide powerful tools for investigating the causal relationship between receptor phosphorylation and downstream signaling events in both cell lines and primary neuronal cultures.

• What methodological considerations are critical when studying OPRM1 phosphorylation in heterologous expression systems versus native tissues?

  Different experimental systems require specific methodological considerations:

  Heterologous Expression Systems:

  • Adenoviral expression systems allow controlled receptor expression levels (typical target: ~2.5×10^9 infectious units/ml)

  • Multiplicity of infection (MOI) should be optimized to achieve physiologically relevant expression levels

  • HEK293 cells are commonly used but may have different signaling characteristics than neuronal cells

  • Receptor expression should be verified using [³H]diprenorphine binding assays

  Primary Neuronal Cultures:

  • Hippocampal neurons provide a more physiologically relevant system but have lower endogenous receptor levels (~0.048 pmol/mg protein compared to ~0.45 pmol/mg protein after viral expression)

  • Adenoviral infection efficiency should be confirmed by immunofluorescence (HA-tag staining relative to DAPI-positive cells)

  • Primary cultures from both wild-type and β-arrestin2^(-/-) mice can help dissect phosphorylation-dependent signaling mechanisms

  Tissue Samples:

  • Rapid post-mortem processing is essential to prevent dephosphorylation

  • Comprehensive phosphatase inhibitor cocktails must be included in all extraction buffers

  • Regional heterogeneity of receptor expression must be considered when analyzing brain tissue

  These considerations help ensure that experimental findings accurately reflect physiological phosphorylation mechanisms.

• How can researchers ensure specificity and sensitivity when detecting phosphorylated OPRM1 in complex samples?

  Several strategies enhance specificity and sensitivity of phosphorylated OPRM1 detection:

  • Sample Preparation:

    • Include comprehensive phosphatase inhibitor cocktails in all buffers (sodium fluoride, sodium pyrophosphate, sodium vanadate)

    • Use appropriate detergents for membrane protein extraction (1% Brij-98, 0.25% sodium deoxycholate)

    • Consider immunoprecipitation before Western blotting for enrichment of phosphorylated receptor

  • Antibody Validation:

    • Use phosphorylation-deficient mutants (S375A) as negative controls

    • Compare phospho-specific antibody (P375OPRM1) with total phospho-Ser/Thr antibodies (Pan)

    • Perform antibody validation using phosphopeptide competition assays

    • Use both phospho-specific and total receptor antibodies for normalization

  • Signal Detection:

    • Normalize phospho-specific signal to total receptor levels

    • Compare results from multiple antibodies when possible

    • Include positive controls (cells/tissues treated with strong OPRM1 agonists like DAMGO)

  These approaches maximize both specificity and sensitivity when detecting phosphorylated OPRM1 in experimental systems.

• What is the relationship between OPRM1 phosphorylation and membrane microdomains in signaling efficiency?

  Membrane microenvironment significantly influences OPRM1 phosphorylation and signaling:

  • Cholesterol content in membrane microdomains affects both receptor localization and signaling efficacy

  • Cholesterol not only influences OPRM1 localization in lipid rafts but also enhances the recruitment of β-arrestins following phosphorylation

  • Receptor compartmentalization in lipid rafts modulates accessibility to kinases (like GRK2) and phosphatases

  • The efficiency of phosphorylation-dependent signaling pathways varies between cell types with different membrane compositions

  • Manipulating membrane cholesterol content experimentally can alter the balance between G-protein and β-arrestin signaling pathways

  These findings highlight the importance of considering membrane microenvironment when studying receptor phosphorylation and its functional consequences.

• How does OPRM1 Ser375 phosphorylation contribute to the antidepressant mechanisms of ketamine?

  Recent studies have revealed a connection between OPRM1 phosphorylation and ketamine's antidepressant effects:

  • The endogenous opioid system in the medial prefrontal cortex (mPFC) is implicated in ketamine's rapid antidepressant actions

  • Ketamine treatment increases β-endorphin levels and OPRM1 gene expression in the mPFC

  • Blocking opioid receptors with naltrexone (systemically or directly in the mPFC) prevents both behavioral and molecular effects of ketamine

  • Neutralization of β-endorphin in the mPFC using specific antibodies abolishes ketamine's antidepressant-like effects

  • Phosphorylation of OPRM1 at Ser375 likely serves as a molecular readout of receptor activation in this pathway

  These findings suggest that monitoring OPRM1 phosphorylation status could provide mechanistic insights into novel rapid-acting antidepressant mechanisms and potentially identify new therapeutic targets.

• What are the technical challenges in quantifying OPRM1 phosphorylation kinetics and how can they be addressed?

  Accurately measuring phosphorylation kinetics presents several technical challenges:

  • Challenge: Rapid phosphorylation/dephosphorylation dynamics

    • Solution: Use time-course experiments with precise timing of agonist application and cell lysis

    • Solution: Include phosphatase inhibitors immediately upon lysis to prevent post-lysis dephosphorylation

  • Challenge: Variable receptor expression levels between samples

    • Solution: Normalize phospho-specific signal to total receptor levels

    • Solution: Use adenoviral expression systems with controlled MOI to standardize receptor expression

  • Challenge: Antibody specificity across phosphorylation sites

    • Solution: Compare results using site-specific antibodies (Ser375) versus pan-phosphorylation antibodies

    • Solution: Include phosphorylation-deficient mutants as controls

  • Challenge: Correlation of phosphorylation with functional outcomes

    • Solution: Couple phosphorylation measurements with functional assays (calcium signaling, ERK activation)

    • Solution: Use both wild-type receptors and phosphorylation site mutants to establish causality

  Addressing these challenges enables more accurate quantification of phosphorylation dynamics and their functional consequences.

• How do different patterns of OPRM1 phosphorylation contribute to opioid tolerance and dependence?

  OPRM1 phosphorylation patterns have significant implications for opioid tolerance development:

  • Agonists that strongly induce Ser375 phosphorylation (like DAMGO and etorphine) typically produce rapid receptor desensitization and internalization

  • Morphine's limited ability to induce Ser375 phosphorylation correlates with its distinct tolerance profile

  • Phosphorylation-deficient mutants (S375A) show altered desensitization properties and direct different opioid agonists to the PKCε pathway

  • The balance between receptor phosphorylation, internalization, and recycling influences the development of tolerance

  • Chronic agonist exposure leads to sustained phosphorylation patterns that differ from acute administration

  Understanding these phosphorylation-dependent mechanisms may contribute to the development of novel opioid drugs with improved tolerance profiles or strategies to mitigate tolerance to existing opioids.

• What emerging techniques are advancing our understanding of OPRM1 phosphorylation dynamics in vivo?

  Several cutting-edge approaches are enhancing in vivo phosphorylation studies:

  • Phospho-specific antibodies with improved sensitivity:
    Newer generation antibodies can detect endogenous phosphorylated receptors in brain tissue without requiring overexpression systems

  • Genetic approaches:
    Generation of knock-in mice expressing phosphorylation-deficient OPRM1 mutants (S375A) allows for in vivo assessment of phosphorylation's role in behavior

  • Phosphoproteomics:
    Mass spectrometry-based approaches can identify multiple phosphorylation sites simultaneously and provide quantitative measurements across experimental conditions

  • Real-time imaging:
    Phosphorylation biosensors based on FRET technology allow visualization of phosphorylation events in living cells and potentially in vivo

  • Single-cell analysis:
    Techniques to measure phosphorylation in individual neurons within neural circuits provide spatial resolution of signaling events

  These emerging approaches promise to reveal new insights into how dynamic phosphorylation regulates OPRM1 function in physiologically relevant contexts.