Phospho-CCR5 (Ser349) Antibody

What is Phospho-CCR5 (Ser349) Antibody and what does it specifically detect?

Phospho-CCR5 (Ser349) antibody specifically detects endogenous levels of CCR5 only when phosphorylated at serine 349. This antibody binds to the phosphorylated form of the receptor but not to unphosphorylated CCR5 . CCR5 is a G protein-coupled receptor that functions as a chemokine receptor and HIV-1 coreceptor. The phosphorylation at Ser349 is a critical regulatory mechanism following ligand binding, particularly after stimulation with chemokines like RANTES (CCL5) .

Methodologically, these antibodies are typically produced by immunizing rabbits with synthetic phosphopeptides corresponding to the region around Ser349 (peptide sequence E-I-S(p)-V-G derived from human CCR5), conjugated to carrier proteins like KLH . The antibodies are then purified using affinity chromatography with phospho-specific peptides, and importantly, non-phospho-specific antibodies are removed through additional chromatography steps using non-phosphopeptides .

What role does phosphorylation at Ser349 play in CCR5 function?

Phosphorylation of CCR5 at Ser349 serves as a key regulatory mechanism in receptor function through multiple pathways:

The significance of this phosphorylation is demonstrated through experiments with kinase inhibitors like staurosporine, which reduces CCR5 internalization and signalosome formation by preventing phosphorylation events .

What are the primary applications for Phospho-CCR5 (Ser349) Antibody in research?

Phospho-CCR5 (Ser349) antibodies are versatile tools with several important research applications:

• Western Blotting: The most common application, typically using dilutions of 1:500-1:1000 . These antibodies can detect phosphorylated CCR5 in cell lysates, enabling quantitative assessment of receptor phosphorylation status under different experimental conditions .

• Immunocytochemistry/Immunofluorescence: Used to visualize the cellular distribution and trafficking of phosphorylated CCR5 in fixed cells, providing spatial information about receptor localization after stimulation with ligands like RANTES or antibodies .

• Flow Cytometry/Phospho-Flow: Particularly useful for quantifying phosphorylation levels in heterogeneous cell populations. This technique allows kinetic analysis of CCR5 phosphorylation after stimulation, as demonstrated in studies with CHO-CCR5 cells treated with chemokines .

• Immunoprecipitation: Used in conjunction with other techniques to isolate CCR5 signalosomes and identify interacting proteins. This approach has revealed critical interactions between phosphorylated CCR5, β-arrestin, ERK1, and trafficking molecules like Rab5 .

• ELISA: Some antibodies are compatible with ELISA-based detection systems for quantitative analysis of phosphorylated CCR5 levels .

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods for optimal results.

How can Phospho-CCR5 (Ser349) Antibody be used to investigate HIV-1 infection mechanisms?

Phospho-CCR5 (Ser349) antibodies provide valuable tools for investigating the molecular mechanisms of HIV-1 entry and infection through several experimental approaches:

• Receptor conformation studies: CCR5 conformation affects its function as an HIV-1 coreceptor. Monitoring phosphorylation status can provide insights into how different receptor conformations influence HIV-1 binding and entry. This is particularly important given that CCR5 exhibits "conformational masking" where certain epitopes are only accessible in specific states .

• Analysis of CCR5 antagonist effects: CCR5 antagonists used in HIV treatment (like maraviroc) work by inducing conformational changes in CCR5. Researchers can use phospho-specific antibodies to determine whether these drugs alter phosphorylation patterns at Ser349, potentially contributing to their antiviral mechanisms .

• Signalosome dynamics: Following the formation of CCR5 signalosomes using phospho-specific antibodies can reveal how HIV-1 infection affects normal CCR5 signaling. Studies have shown that receptor phosphorylation status is crucial for the formation of stable β-arrestin2/ERK1 complexes with internalized CCR5 .

• Natural resistance mechanisms: Some long-term non-progressors (LTNPs) produce natural antibodies against CCR5 that induce prolonged internalization of the receptor. Phospho-CCR5 (Ser349) antibodies can be used to investigate whether these natural antibodies alter the phosphorylation state of CCR5, contributing to HIV resistance .

A particularly informative experimental design involves comparing CCR5 phosphorylation patterns in cells from HIV-resistant individuals (such as those with the CCR5Δ32 mutation) versus normal controls during HIV exposure.

What is the relationship between CCR5 phosphorylation at Ser349 and β-arrestin recruitment?

The relationship between CCR5 phosphorylation at Ser349 and β-arrestin recruitment represents a critical regulatory mechanism with several important features:

• Sequential signaling process: Following ligand binding (e.g., RANTES/CCL5), GRK-mediated phosphorylation of CCR5 at Ser349 creates binding sites for β-arrestin proteins. This phosphorylation-dependent recruitment is part of the desensitization process that regulates receptor signaling .

• Phosphorylation-dependent signalosome formation: Experimental evidence shows that staurosporine (a broad-spectrum kinase inhibitor) treatment significantly reduces the association between CCR5, β-arrestin1/2, and ERK1. This indicates that phosphorylation is required for stable signalosome assembly .

• Two distinct mechanisms: Research has revealed two mechanisms for CCR5 internalization:

  • A phosphorylation-dependent mechanism involving β-arrestin2 and ERK1

  • A phosphorylation-independent mechanism that is less efficient but still operational

This dual regulation is demonstrated in studies using CCR5-S4A mutants, where four serine residues in the C-terminal domain were mutated to alanine. These mutants still underwent internalization after treatment with RANTES derivatives, although less efficiently than wild-type CCR5 .

A comparative table summarizing the effects of phosphorylation on CCR5 internalization and β-arrestin recruitment:

These findings highlight the nuanced role of phosphorylation in regulating CCR5 trafficking and signal transduction.

How do different post-translational modifications of CCR5 interact with phosphorylation at Ser349?

CCR5 undergoes multiple post-translational modifications (PTMs) that can interact with or influence phosphorylation at Ser349, creating a complex regulatory network:

These interacting PTMs create distinct subpopulations of CCR5 on the cell surface with varying functional properties. Methodologically, researchers can investigate these interactions using combinations of inhibitors (phosphorylation inhibitors, palmitoylation inhibitors) along with mutant receptors that lack specific modification sites to delineate their interdependence.

What methodological considerations are important for phospho-flow cytometry using Phospho-CCR5 (Ser349) Antibody?

Phospho-flow cytometry using Phospho-CCR5 (Ser349) antibodies requires careful consideration of several methodological parameters:

• Cell fixation and permeabilization: Phosphorylation detection requires access to intracellular epitopes. Standard protocols involve:

  • Fixation with formaldehyde (typically 2-4%)

  • Permeabilization with methanol or detergent-based reagents

  • The specific fixation/permeabilization protocol significantly impacts antibody performance and must be optimized

• Kinetic considerations: Phosphorylation is a dynamic process with rapid onset and variable duration. The E11/19 monoclonal antibody has been used successfully for kinetic analysis of Ser349 phosphorylation, revealing distinct temporal patterns following different stimuli .

• Signal amplification: Phosphorylation signals can be relatively weak. Consider:

  • Secondary antibody selection for optimal signal-to-noise ratio

  • Use of biotin-streptavidin amplification systems where necessary

  • Ensure minimal compensation spillover by careful fluorophore selection

• Controls and validation:

  • Negative controls: Unstimulated cells and isotype controls are essential

  • Positive controls: RANTES/CCL5 stimulation (10 nM) reliably induces Ser349 phosphorylation

  • Specificity controls: Pretreatment with phosphatase or blocking with specific phosphopeptides

  • Staurosporine (50 nM) can serve as an additional control by inhibiting phosphorylation

• Cell type considerations: Different cell lines exhibit varying baseline and stimulated phosphorylation levels. CHO-CCR5 cells and R5-SupT1 clones (both L23 and M10) have been validated for phospho-flow assays targeting CCR5 Ser349 .

A typical workflow involves:

• Pre-treatment with inhibitors (if applicable)

• Stimulation with agonist (e.g., RANTES/CCL5, typically 30 min)

• Rapid fixation to preserve phosphorylation state

• Permeabilization

• Staining with phospho-specific antibody

• Analysis focusing on shifts in median fluorescence intensity

How can researchers distinguish between different phosphorylated forms of CCR5 in experimental systems?

Distinguishing between different phosphorylated forms of CCR5 requires sophisticated experimental approaches:

• Phospho-specific antibodies: Beyond Ser349, CCR5 can be phosphorylated at multiple serine and threonine residues. Researchers can use:

  • Site-specific phospho-antibodies (where available)

  • Custom antibody development against specific phosphorylation sites

  • E11/19 monoclonal antibody specifically recognizes phosphorylated Ser349

• Mutational analysis: Systematic mutation of potential phosphorylation sites:

  • Serine-to-alanine mutations (S→A) prevent phosphorylation

  • Serine-to-aspartate mutations (S→D) can mimic constitutive phosphorylation

  • The CCR5-S4A mutant with four C-terminal serines mutated has been used to study phosphorylation-independent mechanisms

• Mass spectrometry approaches:

  • Phospho-proteomics can identify and quantify multiple phosphorylation sites simultaneously

  • Requires careful sample preparation, including enrichment of phosphopeptides

  • Can reveal temporal patterns of multi-site phosphorylation

• Differential inhibitor sensitivity:

  • GRK inhibitors specifically reduce Ser349 phosphorylation

  • PKC inhibitors affect other phosphorylation sites

  • Staurosporine (pan-kinase inhibitor) affects all phosphorylation sites

• Two-dimensional phosphopeptide mapping:

  • Can resolve complex patterns of phosphorylation

  • Particularly useful for comparing wild-type and mutant receptors

  • Helps identify kinase-specific phosphorylation signatures

An example experimental approach could involve:

• Expressing wild-type CCR5 and specific serine-to-alanine mutants

• Stimulating with different ligands (RANTES/CCL5, MIP-1α, MIP-1β)

• Analyzing phosphorylation patterns using phospho-specific antibodies

• Confirming with mass spectrometry to identify all phosphorylation sites

• Correlating phosphorylation patterns with functional outcomes (internalization, signaling)

What controls should be included when using Phospho-CCR5 (Ser349) Antibody?

Proper experimental controls are essential for reliable results with Phospho-CCR5 (Ser349) antibodies:

• Positive controls:

  • RANTES/CCL5 stimulation (10 nM, 30 min) reliably induces Ser349 phosphorylation

  • A549 cells have been validated for detecting phospho-CCR5 by Western blot

  • CHO-CCR5 cells provide consistent phosphorylation responses

• Negative controls:

  • Antigen-specific peptide blocking: Pre-incubating the antibody with the immunizing phosphopeptide should eliminate specific staining

  • Non-phosphorylated peptide competition should not affect staining

  • CCR5-negative cells (e.g., parental MT-2 cells without CCR5 expression)

• Specificity controls:

  • Lambda phosphatase treatment: Samples treated with this enzyme should show reduced or eliminated signal

  • CCR5-S4A mutant cells, where Ser349 has been mutated to alanine

  • Staurosporine treatment (50 nM) inhibits phosphorylation and can serve as a kinase inhibition control

• Antibody controls:

  • Isotype control antibodies at matching concentrations

  • Secondary antibody-only controls

  • For monoclonal antibodies, alternative clones targeting the same epitope

For Western blot applications specifically, loading controls and molecular weight markers are essential to verify protein quantity and size. The anticipated molecular weight of CCR5 is approximately 40-41 kDa as indicated in product information sheets .

How can researchers optimize Western blotting protocols for Phospho-CCR5 (Ser349) detection?

Optimizing Western blotting for Phospho-CCR5 (Ser349) detection requires attention to several critical parameters:

• Sample preparation:

  • Rapid lysis with phosphatase inhibitors (Na₃VO₄, NaF) is crucial to preserve phosphorylation states

  • Maintain cold temperatures throughout processing

  • For membrane proteins like CCR5, use appropriate detergents (e.g., 1% Nonidet P-40)

  • Consider enriching membrane fractions to increase signal

• Antibody selection and dilution:

  • Polyclonal antibodies typically used at 1:500-1:1000 dilution

  • Monoclonal antibodies may require different dilutions

  • Both rabbit polyclonal and rabbit monoclonal formats are available

• Signal optimization:

  • Enhanced chemiluminescence (ECL) detection systems are commonly used

  • Consider using PVDF membranes which may retain phosphoproteins better than nitrocellulose

  • Longer primary antibody incubation (overnight at 4°C) can improve sensitivity

  • Milk-based blocking buffers may contain phosphatases; BSA-based blockers are preferred

• Stimulation conditions:

  • Optimal RANTES/CCL5 concentration: 10 nM

  • Stimulation time: 30 minutes for initial phosphorylation

  • For sustained phosphorylation studies, longer time courses (up to 150 min) have been used

• Common challenges and solutions:

  • High background: Increase washing steps, optimize blocking, reduce antibody concentration

  • Weak signal: Increase protein loading, extend exposure time, use signal amplification systems

  • Multiple bands: Verify specificity with peptide competition, consider receptor dimerization or glycosylation variants

A detailed troubleshooting table for Western blot detection:

What are the key differences between monoclonal and polyclonal Phospho-CCR5 (Ser349) antibodies?

Understanding the differences between monoclonal and polyclonal Phospho-CCR5 (Ser349) antibodies helps researchers select the appropriate tool for their specific application:

Monoclonal Antibodies (e.g., E11/19):

• Production method: Generated from single B-cell clones, typically using chimeric recombinant technology for rabbit monoclonals

• Specificity: Highly specific to a single epitope around phosphorylated Ser349

• Consistency: Minimal lot-to-lot variation

• Applications: Particularly valuable for:

  • Flow cytometry where high specificity is required

  • Comparative quantitative analysis across experiments

  • Detection of specific phosphorylated forms without cross-reactivity

• Example: The E11/19 monoclonal antibody specifically recognizes phosphoserine 349 and has been validated for flow cytometry applications

Polyclonal Antibodies:

• Production method: Generated by immunizing rabbits with synthetic phosphopeptides and KLH conjugates, followed by affinity purification

• Specificity: Recognize multiple epitopes around the phosphorylated Ser349 region

• Sensitivity: Often provide stronger signals by binding multiple epitopes per molecule

• Applications: Particularly valuable for:

  • Western blotting

  • Immunoprecipitation

  • Applications requiring signal amplification

• Examples: Multiple commercial polyclonal antibodies are available with validated performance in Western blotting applications

Comparative performance:

The choice between monoclonal and polyclonal antibodies should be guided by the specific experimental requirements, with monoclonals preferred for highly specific detection and quantification, while polyclonals may offer advantages in sensitivity for applications like Western blotting.

How has Phospho-CCR5 (Ser349) research contributed to understanding HIV persistence and latency?

Research on CCR5 phosphorylation at Ser349 has provided several insights into HIV persistence and latency mechanisms:

• Receptor desensitization and recycling: Phosphorylation at Ser349 regulates CCR5 internalization and recycling, which impacts HIV entry. Studies using phospho-specific antibodies have revealed that:

  • Different HIV-1 strains may differentially affect CCR5 phosphorylation patterns

  • Persistent phosphorylation leads to prolonged CCR5 internalization, potentially creating temporarily resistant states

• Natural resistance mechanisms: Natural antibodies against CCR5 found in Long-Term Non-Progressors (LTNPs) induce long-lasting CCR5 internalization (up to 48 hours), involving β-arrestin2 and ERK1 recruitment. The phosphorylation status of CCR5 is crucial for this protective mechanism .

• Signalosome dynamics and HIV latency: The formation of stable signalosomes containing phosphorylated CCR5, β-arrestin2, and ERK1 may influence cellular signaling pathways that affect HIV latency. Key findings include:

  • The phosphorylation status of both CCR5 and ERK1 is necessary for driving internalized CCR5 into early endosomes

  • Blocking phosphorylation with staurosporine disrupts this process

• Therapeutic implications: Understanding CCR5 phosphorylation mechanisms has informed development of:

  • CCR5 antagonists that may indirectly affect phosphorylation status

  • Gene editing approaches targeting CCR5

  • Novel approaches to induce CCR5 downregulation

Researchers are currently exploring how manipulating CCR5 phosphorylation could potentially be exploited to develop new HIV treatment strategies, particularly for purging latent viral reservoirs or enhancing natural resistance mechanisms.

What cutting-edge methods are being developed for studying CCR5 phosphorylation dynamics?

Researchers are employing increasingly sophisticated methodologies to study CCR5 phosphorylation dynamics:

• Live-cell imaging with phospho-specific biosensors:

  • FRET-based biosensors for real-time visualization of phosphorylation events

  • Genetically encoded indicators that report on kinase activity near CCR5

  • These approaches allow temporal and spatial resolution of phosphorylation events following receptor stimulation

• Single-molecule tracking:

  • Following individual CCR5 molecules and their phosphorylation state in living cells

  • Correlating molecular motion patterns with phosphorylation status

  • Revealing heterogeneity in receptor behavior based on modification state

• Advanced mass spectrometry methods:

  • Absolute quantification (AQUA) of phosphorylation stoichiometry

  • Targeted proteomics approaches for monitoring multiple phosphorylation sites

  • Combining phosphoproteomics with interactome analysis to identify phospho-dependent binding partners

  • Temporal analysis of phosphorylation/dephosphorylation cycles

• CRISPR-based screening approaches:

  • Systematic identification of kinases and phosphatases regulating CCR5 phosphorylation

  • Generation of cell lines with modified phosphorylation sites using precise genome editing

  • Creation of phosphorylation-specific reporter cell lines

• Structural biology methods:

  • Cryo-EM studies of CCR5 in different phosphorylation states

  • NMR analysis of phosphorylated receptor domains

  • Molecular dynamics simulations to predict effects of phosphorylation on receptor conformations

These emerging technologies promise to provide unprecedented insights into how CCR5 phosphorylation regulates receptor function in normal immune responses and during pathological conditions like HIV infection.

How might understanding CCR5 phosphorylation inform development of new therapeutic strategies?

Understanding CCR5 phosphorylation mechanisms opens several avenues for novel therapeutic development:

• Enhanced CCR5 antagonists:

  • Current CCR5 antagonists like maraviroc primarily block HIV binding

  • Next-generation antagonists could be designed to induce specific phosphorylation patterns that promote prolonged internalization

  • Compounds that stabilize CCR5 signalosomes might provide more durable receptor downregulation

• Kinase modulation strategies:

  • Targeted inhibition or activation of specific kinases (like GRKs) that phosphorylate CCR5

  • Manipulation of phosphatase activity to control receptor recycling

  • Studies with staurosporine demonstrate the principle that manipulating phosphorylation can significantly affect CCR5 trafficking and function

• Phosphorylation-mimetic peptides:

  • Synthetic peptides that mimic phosphorylated CCR5 C-terminal regions

  • Could potentially compete with natural targets of phospho-binding domains

  • Might disrupt specific signaling pathways downstream of CCR5

• Antibody-based therapeutics:

  • Antibodies that recognize and stabilize phosphorylated conformations of CCR5

  • Engineered antibodies that induce phosphorylation-dependent internalization

  • Lessons from natural anti-CCR5 antibodies found in LTNPs could inform design

• Therapeutic combinations:

  • Synergistic approaches combining CCR5 antagonists with kinase modulators

  • Targeting multiple post-translational modifications simultaneously (phosphorylation, sulfation, palmitoylation)

  • Personalized approaches based on patient-specific CCR5 expression and modification patterns

The phosphorylation status of CCR5 affects not only HIV entry but also inflammatory and immune functions, suggesting that phosphorylation-targeting therapeutics might have applications beyond HIV in conditions like inflammatory diseases, cancers, and autoimmune disorders where CCR5 plays significant roles .