VIPR2 Antibody

What is VIPR2 and what cellular functions does it regulate?

VIPR2, also known as Vasoactive Intestinal Peptide Receptor 2, is a member of the Class II family of G-protein coupled receptors (GPCRs). It exhibits high affinity for both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP), two structurally related neuropeptides with significant biological functions . As a class II GPCR, VIPR2 possesses distinctive structural features including large N-terminal extracellular domains containing 10 highly conserved amino acids (including six cysteines), putative N-terminal leader sequences, and several potential N-glycosylation sites . VIPR2 demonstrates wide tissue distribution, with expression documented in the uterus, prostate, gastrointestinal tract smooth muscle, seminal vesicles, skin, blood vessels, thymus, and various brain regions including the thalamus . Recent research has revealed VIPR2's significant role in regulating tumor cell migration through PI3K/PI(3,4,5)P3 pathway activation . This signaling pathway influences critical cellular processes including lamellipodium formation, actin filament remodeling, and cancer cell motility, suggesting VIPR2 as a potential therapeutic target for inhibiting metastatic progression .

What epitope characteristics define commonly available VIPR2 antibodies?

The epitope specifications of VIPR2 antibodies are crucial for experimental planning and interpretation. One extensively characterized VIPR2 antibody targets the peptide sequence CRFHLEIQEEETK, corresponding to amino acid residues 25-37 of human VIPR2 (Accession P41587) . This epitope is located in the extracellular, N-terminal region of the receptor, making it particularly valuable for detecting cell surface expression without permeabilization requirements . The antibody's extracellular targeting capability enables detection of VIPR2 in its native conformation on live cells, facilitating studies of receptor distribution, trafficking, and ligand interactions under physiological conditions. When selecting a VIPR2 antibody, researchers should carefully evaluate whether the epitope recognition aligns with experimental objectives, particularly considering whether extracellular versus intracellular domains need to be targeted, and whether the epitope sequence is conserved across species if cross-reactivity is desired . Some experimental approaches may benefit from antibodies recognizing post-translationally modified forms of VIPR2, necessitating specialized antibody selection based on specific research questions.

What is the expression pattern of VIPR2 across different tissues and cell lines?

VIPR2 exhibits a specific but widespread expression pattern across multiple tissue types. In peripheral tissues, VIPR2 is prominently expressed in the uterus, prostate, gastrointestinal tract smooth muscle, seminal vesicles, skin, blood vessels, and thymus . Within the central nervous system, VIPR2 expression has been documented in various brain regions, with notable presence in the thalamus and suprachiasmatic nucleus . At the cellular level, research has demonstrated VIPR2 expression in multiple cell types including human melanoma (Malme-3M) cells, human T-cell leukemia (Jurkat) cells, and rat pheochromocytoma (PC12) cells . VIPR2 has also been detected in breast cancer cell lines including MDA-MB-231 and MCF-7, where it plays important roles in cell migration . The expression pattern analysis is supported by multiple detection methodologies including Western blot analysis of rat and mouse brain lysates and human melanoma cell lysates, flow cytometry detection in Jurkat cells, and immunocytochemistry in PC12 cells . Understanding this expression pattern is essential for selecting appropriate experimental models when studying VIPR2 function and for interpreting findings within relevant physiological contexts.

What protocols are optimal for detecting VIPR2 using Western blot analysis?

For optimal Western blot detection of VIPR2, researchers should implement a carefully validated protocol that accounts for the protein's specific characteristics. Based on published methodologies, the following procedure is recommended: prepare lysates from relevant tissues (such as rat or mouse brain) or cell lines (such as human Malme-3M melanoma cells) using standard protein extraction buffers containing protease inhibitors . For protein separation, use standard SDS-PAGE with an appropriate percentage acrylamide gel (typically 8-10%) to effectively resolve VIPR2's molecular weight. Following electrophoresis, transfer proteins to a PVDF or nitrocellulose membrane using standard transfer conditions. For immunoblotting, block membranes with 5% non-fat dry milk or BSA in TBST, and then incubate with anti-VIPR2 antibody at an optimized dilution ratio (approximately 1:600 for Anti-VPAC2 (VIPR2) extracellular Antibody has been validated) . Following primary antibody incubation, wash membranes thoroughly with TBST and incubate with an appropriate HRP-conjugated secondary antibody. For development, use enhanced chemiluminescence detection systems. Critical controls should include a blocking peptide competition assay, where the antibody is pre-incubated with the immunizing peptide prior to membrane probing, which should abolish specific bands if the antibody is truly specific . This systematic approach ensures reliable VIPR2 detection while minimizing non-specific signals.

How can flow cytometry be effectively used to detect cell surface VIPR2?

Flow cytometry offers a powerful approach for quantifying and characterizing cell surface VIPR2 expression across different cell populations. For effective detection, implement the following optimized protocol: begin with viable, healthy cells (such as human Jurkat T-cell leukemia cells) in single-cell suspension at approximately 1×10^6 cells/mL . Wash cells in flow cytometry buffer (PBS containing 2% FBS and 0.1% sodium azide) to remove serum proteins. For cell surface VIPR2 detection, it is crucial that cells remain non-permeabilized to preserve membrane integrity. Incubate cells with anti-VIPR2 extracellular antibody at an optimized concentration (approximately 2.5 μg has been validated for some applications) . Following primary antibody incubation, wash cells thoroughly and incubate with a fluorophore-conjugated secondary antibody, such as goat-anti-rabbit-APC . After washing to remove unbound secondary antibody, analyze samples using appropriate cytometer settings with properly compensated fluorescence channels. Essential controls must include: unstained cells to establish autofluorescence baseline; secondary antibody-only samples to assess non-specific binding; and when possible, a known VIPR2-negative cell line as a biological negative control . For comprehensive analysis, researchers may combine VIPR2 detection with markers for specific cell subpopulations to characterize differential expression patterns across heterogeneous samples. This methodical approach enables reliable quantification of VIPR2 surface expression levels.

What are the critical parameters for VIPR2 immunocytochemistry experiments?

Successful immunocytochemistry for VIPR2 detection requires attention to several critical parameters. First, cell preparation is crucial: for cell surface detection in intact cells (which is optimal for VIPR2's extracellular domain), cells should be gently fixed with 4% paraformaldehyde to preserve membrane protein conformation while maintaining cell morphology . When working with adherent cell lines like rat pheochromocytoma (PC12) cells, culture cells on appropriate substrates such as fibronectin-coated coverslips or chamber slides to promote proper attachment and morphology . For primary antibody incubation, optimize dilution ratios through preliminary experiments (a 1:50 dilution has been validated for certain applications) . Select appropriate fluorophore-conjugated secondary antibodies (such as goat anti-rabbit-AlexaFluor-594) that provide sufficient signal strength while minimizing background . Include proper controls including secondary-antibody-only samples and, when available, VIPR2-knockdown cells as negative controls. For colocalization studies, carefully select compatible fluorophores with minimal spectral overlap and implement appropriate imaging parameters. When examining VIPR2 distribution in migrating cells or at leading edges, consider live-cell imaging approaches to capture dynamic receptor distribution . For fixed samples, confocal microscopy is recommended for precise subcellular localization analysis, particularly when examining VIPR2 colocalization with proteins like WAVE2 at lamellipodia . These optimized parameters ensure reliable visualization of VIPR2 distribution and interactions in cellular contexts.

How does VIPR2 signaling regulate tumor cell migration mechanisms?

VIPR2 signaling exerts significant regulatory control over tumor cell migration through multiple interconnected molecular mechanisms. Upon binding its ligand vasoactive intestinal peptide (VIP), VIPR2 activates downstream signaling pathways that culminate in enhanced cell motility . A primary mechanism involves VIPR2-mediated activation of PI3-kinase (PI3K), particularly the PI3Kγ isoform, which catalyzes the conversion of PI(4,5)P2 to PI(3,4,5)P3 at the plasma membrane . This increased PI(3,4,5)P3 generation is critical for migration, as evidenced by the significant attenuation of VIP-induced cell migration following treatment with either ZSTK474 (a pan-PI3K inhibitor) or AS605240 (a PI3Kγ-selective inhibitor) . Mechanistically, PI(3,4,5)P3 accumulation at the plasma membrane promotes the translocation of WAVE2 (WASP family verprolin homologous protein 2) to the cell periphery, particularly at lamellipodia . At these sites, WAVE2 facilitates interaction with the ARP2/3 complex, driving actin nucleation and polymerization essential for lamellipodium extension . Experimental evidence from both silencing and overexpression studies demonstrates VIPR2's critical role in this process—VIPR2-silenced cells show significantly reduced lamellipodia formation, decreased WAVE2 membrane localization, and impaired interaction between WAVE2, ARP3, and actin . Conversely, VIPR2 overexpression enhances these processes, promoting robust lamellipodium extension and increased migration speed . This comprehensive mechanistic pathway positions VIPR2 as a potential therapeutic target for inhibiting cancer metastasis.

What methods effectively quantify VIPR2-mediated effects on cellular migration?

Rigorous quantification of VIPR2-mediated effects on cellular migration requires implementing multiple complementary methodologies. The scratch assay (wound healing assay) provides a straightforward approach for assessing collective cell migration in VIPR2-modulated cells . For this method, cells (such as MDA-MB-231 transfected with VIPR2 siRNA or expressing VIPR2-EGFP) are grown to confluence on fibronectin-coated (50 μg/mL) surfaces, wounded with a sterile pipette tip, and then monitored for 24 hours in serum-free medium containing 100 nM VIP and 50 mM HEPES (pH 7.4) . Migration distance is measured at multiple randomly selected points along the wound edge, and migration speed is calculated by dividing distance by time . For more detailed analysis of leading edge dynamics, time-lapse microscopy with kymography analysis provides insights into lamellipodium extension rates . In this approach, cells at wound edges are recorded every 2 minutes for 60 minutes following VIP stimulation, and kymographs are generated using ImageJ's MultipleKymograph plugin . Transwell migration assays offer quantitative assessment of chemotactic migration, where cells are placed in upper chambers and allowed to migrate toward VIP (200 nM) in lower chambers for 48 hours . For precise quantification, migrated cells are counted in multiple randomly selected fields . Finally, random migration assays track individual cell movements over time, enabling calculation of parameters like migration distance and speed . These complementary approaches provide comprehensive characterization of how VIPR2 modulates different aspects of cell migration, from collective movement to individual cell dynamics.

What is the relationship between VIPR2 and WAVE2 in lamellipodium formation?

The functional relationship between VIPR2 and WAVE2 represents a critical mechanism in lamellipodium formation during cell migration. Immunocytochemistry and live-cell imaging studies reveal that upon VIP stimulation, VIPR2 undergoes redistribution to concentrate at the plasma membrane of extending lamellipodia . Concurrently, WAVE2—a key regulator of actin nucleation—shows significant accumulation at these same membrane regions . Quantitative analysis demonstrates substantially stronger WAVE2 signals at the plasma membrane in VIPR2-overexpressing cells compared to control cells following VIP stimulation, with clear co-localization between VIPR2-EGFP and WAVE2 signals specifically at lamellipodia . This spatial association is functionally significant, as VIPR2 silencing markedly reduces WAVE2 recruitment to the plasma membrane despite VIP stimulation . Mechanistically, this relationship depends on PI(3,4,5)P3 production—VIPR2 activation stimulates PI3Kγ activity, generating PI(3,4,5)P3 at the plasma membrane, which serves as a docking site for WAVE2 . Once recruited to the membrane, WAVE2 interacts with the ARP2/3 complex to promote actin nucleation and polymerization essential for lamellipodium extension . Pull-down assays further demonstrate that VIPR2 silencing substantially reduces the interaction between WAVE2, ARP3, and actin, confirming VIPR2's critical role in assembling this molecular machinery . Together, these findings establish that VIPR2 functions as an upstream regulator of WAVE2 localization and activity during lamellipodium formation, coordinating the spatial organization of actin polymerization machinery required for directional cell migration.

What controls are essential for validating VIPR2 antibody specificity?

Rigorous validation of VIPR2 antibody specificity requires implementing multiple complementary controls. The peptide competition assay represents a fundamental control where the antibody is pre-incubated with the immunizing peptide (such as VPAC2/VIPR2 extracellular Blocking Peptide) prior to application in the experimental system . In Western blot applications, this control should eliminate or significantly reduce specific bands corresponding to VIPR2, as demonstrated in analyses of rat brain, mouse brain, and human Malme-3M melanoma cell lysates . For immunocytochemistry and flow cytometry applications, parallel samples treated with peptide-blocked antibody should show minimal staining compared to unblocked antibody samples . Genetic controls provide another powerful validation approach—cells transfected with VIPR2-specific siRNA should exhibit reduced antibody signal in all detection platforms, while cells overexpressing VIPR2 should demonstrate enhanced signal intensity . When available, multiple antibodies targeting different VIPR2 epitopes should yield consistent staining patterns and protein detection. Species cross-reactivity testing is also valuable, as demonstrated by comparative analysis of VIPR2 detection in rat, mouse, and human samples . Additionally, correlation between protein detection methods (Western blot, ICC, flow cytometry) and mRNA expression data provides further validation of specificity. Implementation of these comprehensive controls ensures confident interpretation of VIPR2 detection results and minimizes the risk of experimental artifacts from non-specific antibody interactions.

How can researchers resolve inconsistent results when using VIPR2 antibody across different cell lines?

Resolving inconsistent VIPR2 antibody results across different cell lines requires systematic analysis of several variables. First, evaluate baseline VIPR2 expression levels in each cell line through independent methods such as RT-qPCR, as expression variability may explain detection inconsistencies. Consider species-specific differences in the VIPR2 epitope sequence, particularly when working with cell lines from different organisms, as this may affect antibody binding efficiency . Optimize sample preparation protocols for each cell line—different lysis buffers, fixation methods, or permeabilization protocols may be required to effectively expose VIPR2 epitopes while maintaining protein integrity. For Western blot applications, adjust protein loading amounts based on expected VIPR2 expression levels, and optimize primary antibody concentration and incubation time for each cell type . When performing flow cytometry or immunocytochemistry, cell-specific autofluorescence or high background may necessitate adjustments to blocking conditions and antibody dilutions . Additionally, consider the activation state of VIPR2 in different cell contexts, as receptor internalization or conformational changes following ligand binding may affect antibody accessibility. For cells with low VIPR2 expression, signal amplification strategies such as tyramide signal amplification or more sensitive detection systems may be required. Finally, verify antibody performance using positive controls (e.g., VIPR2-overexpressing cells) alongside experimental samples to confirm detection capability under identical conditions . This methodical troubleshooting approach should identify the specific variables causing inconsistencies and enable optimization of detection protocols for each cell line.

What technical challenges exist when studying VIPR2-protein interactions through co-immunoprecipitation?

Investigating VIPR2-protein interactions through co-immunoprecipitation presents several technical challenges requiring careful methodological consideration. As a membrane-bound G-protein coupled receptor, VIPR2 contains hydrophobic transmembrane domains that complicate extraction while maintaining native protein-protein interactions . Researchers should optimize lysis conditions using buffers containing appropriate detergents (such as CHAPS, digitonin, or NP-40) at concentrations that solubilize VIPR2 without disrupting protein-protein interactions. Cross-linking approaches may help preserve transient or weak interactions before cell lysis. The choice of antibody is critical—antibodies targeting VIPR2's extracellular domain may be less effective for co-IP if the epitope is involved in protein interactions . Conversely, antibodies against intracellular domains must be accessible following membrane solubilization. When studying specific interactions like those between VIPR2 and WAVE2, consider the subcellular localization of the interaction, which may require isolation of specific membrane fractions or implementation of proximity ligation assays as complementary approaches . Non-specific binding presents another challenge; implement stringent controls including IgG control immunoprecipitations and, when available, VIPR2-knockout or knockdown samples . For interactions that depend on receptor activation state, careful timing of VIP stimulation before lysis is essential, as demonstrated in studies of VIPR2's influence on WAVE2-ARP3-actin interactions . Finally, verification of identified interactions through reciprocal co-IP (pulling down the interacting partner and blotting for VIPR2) provides stronger evidence of specific association. These methodological considerations help overcome the inherent challenges of studying VIPR2's protein interaction network.

How can VIPR2 antibodies be used to evaluate potential therapeutic interventions in cancer?

VIPR2 antibodies offer valuable tools for evaluating cancer therapeutic interventions through multiple experimental approaches. Immunohistochemical analysis using VIPR2 antibodies enables assessment of receptor expression across patient tumor samples, facilitating correlation between expression levels and clinical outcomes or response to therapy . In xenograft tumor models, VIPR2 antibody staining of tumor sections before and after treatment with candidate therapeutic agents can reveal changes in receptor expression, localization, or downstream signaling pathway activation . For evaluating VIPR2-targeted therapies such as antagonists (e.g., KS-133), antibodies provide essential tools for confirming target engagement—flow cytometry or immunocytochemistry can detect whether the antagonist alters receptor internalization or surface expression patterns . In mechanistic studies, VIPR2 antibodies enable monitoring of therapy-induced changes in receptor-protein interactions or downstream effectors like WAVE2 localization and PI3K pathway activation . Importantly, VIPR2's established role in promoting cancer cell migration suggests its potential utility as a biomarker for metastatic potential . Through transwell migration assays combined with VIPR2 immunodetection, researchers can quantitatively assess how candidate therapeutics modulate VIPR2-dependent cell migration . The dose-dependent inhibition of VIP-induced migration by the VIPR2 antagonist KS-133 demonstrates the feasibility of this approach . These diverse applications of VIPR2 antibodies in therapeutic evaluation provide both mechanistic insights and translational opportunities for developing cancer interventions targeting VIPR2 signaling pathways.

What technical approaches best elucidate the spatiotemporal dynamics of VIPR2 during cell migration?

Elucidating VIPR2's spatiotemporal dynamics during cell migration requires sophisticated imaging techniques and molecular tools. Live-cell imaging with fluorescently tagged VIPR2 (such as VIPR2-EGFP) provides direct visualization of receptor trafficking during migration . For optimal results, researchers should use stable expression systems with expression levels close to endogenous, imaged using spinning disk confocal microscopy with environmental control to maintain physiological conditions . Time-lapse imaging combined with kymography analysis effectively quantifies VIPR2 redistribution at leading edges following VIP stimulation . To specifically visualize active receptors, researchers can implement FRET-based biosensors that report conformational changes upon ligand binding or bimolecular fluorescence complementation (BiFC) to detect VIPR2 interactions with downstream effectors like G-proteins. Super-resolution microscopy techniques including STORM or PALM offer nanoscale resolution of VIPR2 organization within specialized membrane structures like lamellipodia . For correlating VIPR2 dynamics with functional outcomes, simultaneous imaging of PI(3,4,5)P3 (using PH domain biosensors) and actin dynamics (using LifeAct) alongside VIPR2 reveals mechanistic relationships between receptor activation, signaling intermediates, and cytoskeletal rearrangements . Optogenetic approaches enable precise spatiotemporal control of VIPR2 activation to determine how localized receptor signaling influences directional migration. Finally, correlative light and electron microscopy (CLEM) provides ultrastructural context for VIPR2 localization within membrane microdomains during migration. These advanced technical approaches collectively provide comprehensive characterization of how VIPR2 dynamics orchestrate the spatiotemporal coordination of migratory machinery.

What are the emerging research directions for VIPR2 beyond cancer cell migration?

Emerging research directions for VIPR2 extend beyond its established role in cancer cell migration, spanning multiple biological systems and disease contexts. In neuroscience, VIPR2's expression in brain regions such as the thalamus and suprachiasmatic nucleus suggests important functions in neuronal circuits, potentially involving neuroprotection, circadian rhythm regulation, and neurodevelopmental processes . The receptor's widespread expression in immune tissues including the thymus points toward immunomodulatory functions that merit investigation, particularly regarding VIP's known anti-inflammatory effects . VIPR2's presence in vascular tissues indicates potential roles in regulating vascular tone, permeability, and angiogenesis—processes relevant to both physiological and pathological conditions . Within endocrine systems, VIPR2 likely participates in hormone secretion regulation, metabolic control, and tissue homeostasis . Given VIPR2's established involvement in PI3K signaling, investigating its contribution to other PI3K-dependent cellular processes beyond migration, such as survival, proliferation, and metabolic regulation, represents another promising research direction . From a therapeutic perspective, VIPR2-selective antagonists like KS-133 demonstrate the feasibility of targeting this receptor, warranting exploration in disease models beyond cancer, including inflammatory, neurodegenerative, and metabolic disorders . Additionally, the overlapping but distinct functions of VIPR1 and VIPR2 suggest important receptor subtype-specific biological roles that require further elucidation through selective genetic and pharmacological tools . These diverse research directions highlight VIPR2's broad biological significance and therapeutic potential across multiple physiological systems and disease contexts.