Recombinant Mouse Vasoactive intestinal polypeptide receptor 1 (Vipr1)

What is the biological function of Vipr1 in mouse immune system?

Vipr1 (also known as VPAC-1) is a G-protein-coupled receptor highly expressed in the immune system that modulates diverse T cell functions. It binds vasoactive intestinal peptide (VIP) and activates primarily cAMP-dependent signaling pathways. In lymphocytes, Vipr1 signaling regulates chemotaxis, proliferation, apoptosis, and differentiation processes . The receptor plays an immunomodulatory role, often mediating anti-inflammatory effects. The human VPAC-1 5'-flanking region contains four high-affinity Ikaros consensus sequences, and studies have shown that Ikaros native protein from T cell nuclear extracts recognizes binding motifs in the VPAC-1 promoter in a sequence-specific manner . This regulatory system may represent a dominant determinant of VPAC-1 expression in immune responses.

How does Vipr1 expression vary across different mouse tissues?

Vipr1 demonstrates tissue-specific expression patterns that correlate with its diverse physiological roles. The receptor is abundantly expressed in:

• Immune tissues: Thymus, spleen, and lymphoid organs

• Central nervous system: Cerebral cortex and hypothalamus

• Digestive system: Intestinal tissues

• Respiratory system: Lung epithelial cells

• Liver: Hepatocytes (with reduced expression in hepatocellular carcinoma)

Expression levels can be significantly altered in pathological conditions. For instance, in hepatocellular carcinoma (HCC), Vipr1 expression is downregulated compared to normal liver tissues . This downregulation correlates with advanced clinical stages, tumor growth, recurrence, and poor outcomes of HCC clinically . The differential expression across tissues reflects the diverse physiological roles of VIP signaling in neuronal function, immune regulation, and metabolic processes.

What signaling pathways are activated by Vipr1 in mouse cells?

Vipr1 activation triggers multiple signaling cascades:

Research in HCC cells has revealed that VIP treatment upregulates argininosuccinate synthase (ASS1) and subsequently suppresses CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) phosphorylation in an mTOR/p70S6K signaling-dependent manner . This mechanism helps explain how Vipr1 activation inhibits cancer progression by modulating arginine metabolism and pyrimidine synthesis.

How does Vipr1 activation impact cancer progression in mouse models?

Vipr1 activation demonstrates significant anti-tumor effects in mouse cancer models, particularly in hepatocellular carcinoma (HCC):

In xenograft mouse models using HCCLM3 cells (human HCC cell line), tumors with VIPR1 overexpression grew significantly slower than control tumors . Treatment with VIP further reduced tumor growth, with more pronounced inhibition in VIPR1-overexpressing tumors. Ki-67 immunohistochemistry staining confirmed that VIP treatment markedly reduced HCC proliferation in these models .

Regarding metastasis, bioluminescence imaging studies showed that VIPR1-overexpressing groups had significantly fewer metastatic foci in liver and lung tissues compared to control groups . VIP treatment further restricted metastasis formation, suggesting that exogenous VIP administration suppresses HCC metastasis through VIPR1 activation.

Mechanistically, transcriptome sequencing analyses revealed that Vipr1 activation by VIP regulates arginine biosynthesis. VIP treatment partially restores the expression of argininosuccinate synthase (ASS1), a key enzyme in arginine anabolism, and inhibits de novo pyrimidine synthesis by downregulating CAD activation . This modulation of metabolic pathways represents a novel mechanism through which Vipr1 exerts its anti-tumor effects.

What experimental approaches are most effective for studying Vipr1 antagonism?

Studying Vipr1 antagonism requires specialized techniques spanning from peptide design to functional assays:

Antagonist Design and Characterization:

• Computational modeling approaches have been used to predict binding affinities of novel peptide sequences to VIP receptors

• Structural analysis reveals that amino acid substitutions in VIP antagonists can create new interactions with receptor residues not seen with natural ligands

• For example, the N9D substitution in antagonist peptides creates novel interactions with Y98 in VPAC1

Receptor Binding Assays:

• Competitive binding assays using radiolabeled VIP (typically [125I]-VIP) can quantify antagonist binding affinity

• Surface plasmon resonance provides real-time binding kinetics

• Structural studies suggest that antagonist binding may produce subtle conformational changes that inhibit downstream signaling

Functional Characterization:

• cAMP accumulation assays measure inhibition of VIP-induced signaling

• Calcium flux assays detect antagonist effects on VIP-stimulated calcium mobilization

• Phosphorylation status of downstream targets (CREB, ERK1/2) can be monitored via Western blotting

In Vivo Applications:

• VIP receptor antagonists have demonstrated the ability to confer protective immunity to tumor re-challenge in mouse models

• Antagonist stability and pharmacokinetics must be optimized for in vivo studies

• Routes of administration affect biodistribution and targeting of specific immune compartments

How is Vipr1 gene expression regulated in mouse tissues?

Vipr1 expression is regulated through complex transcriptional mechanisms:

Transcriptional Control:
The human VPAC-1 5'-flanking region (1.4 kb) contains four high-affinity Ikaros (IK) consensus sequences . Ikaros proteins from T cell nuclear extracts and recombinant IK-1 and IK-2 proteins recognize these binding motifs in electrophoretic mobility shift assays through sequence-specific mechanisms . Studies with stable NIH-3T3 clones overexpressing IK-1 or IK-2 isoforms demonstrated that both isoforms suppress endogenous VPAC-1 expression by 50-93% .

Luciferase reporter assays with nested deletions of the VPAC-1 promoter identified two major IK-2 binding domains at -1076 to -623 bp and at -222 to -35 bp . Since both Ikaros and VPAC-1 are highly expressed in T cells, this regulatory system likely plays a dominant role in controlling VPAC-1 expression during immune responses.

Pathological Regulation:
In hepatocellular carcinoma, Vipr1 expression is significantly downregulated compared to normal liver tissue . This downregulation correlates with advanced clinical stages, tumor growth, recurrence, and poor outcomes . The mechanisms driving this downregulation in cancer may involve epigenetic modifications, although specific details remain to be fully elucidated.

What methodologies are most reliable for detecting recombinant mouse Vipr1 in experimental systems?

Multiple complementary approaches provide robust detection of recombinant mouse Vipr1:

Antibody-Based Detection:

• Western Blotting: Rabbit-anti-mouse VPAC1 antibodies have been characterized for detecting Vipr1, with expected molecular weight of 52-58 kDa

• Immunohistochemistry: Fixed tissue sections can be probed with validated anti-Vipr1 antibodies with appropriate antigen retrieval techniques

• Flow Cytometry: Surface staining protocols using fluorescently-labeled antibodies can quantify receptor expression in cell populations

Functional Detection:

• Radioligand Binding: [125I]-VIP binding to membrane preparations can quantify receptor density

• cAMP Accumulation: VIP-stimulated cAMP production serves as a functional readout of receptor activity

• Calcium Mobilization: Fura-2 or Fluo-4 loaded cells can detect VIP-induced calcium responses

Genetic Detection:

• RT-PCR/qPCR: Primers spanning exon junctions prevent genomic DNA amplification

• RNA-Seq: Provides comprehensive transcriptomic context and identifies splice variants

• In Situ Hybridization: Preserves spatial information about Vipr1 expression in tissues

When validating recombinant mouse Vipr1 detection, it is essential to use multiple orthogonal methods to confirm both expression and functional activity of the receptor.

How does Vipr1 contribute to arginine metabolism in hepatocellular carcinoma?

Vipr1 plays a critical role in regulating arginine metabolism in hepatocellular carcinoma (HCC):

Metabolic Pathway Regulation:
Activation of Vipr1 by VIP markedly impacts arginine biosynthesis and related metabolic pathways. Mechanistic studies in cultured HCC cells have demonstrated that VIP treatment partially restores the expression of argininosuccinate synthase (ASS1), a key enzyme in arginine anabolism . ASS1 catalyzes the conversion of citrulline and aspartate to argininosuccinate in the urea cycle.

Pyrimidine Synthesis Inhibition:
Vipr1 activation inhibits de novo pyrimidine synthesis by downregulating the activation of CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) . This effect is mediated through the suppression of CAD phosphorylation in an mTOR/p70S6K signaling-dependent manner. By reducing pyrimidine synthesis, Vipr1 activation limits the availability of nucleotides required for cancer cell proliferation.

Clinical Correlations:
Human HCC samples exhibit downregulation of ASS1 but upregulation of CAD phosphorylation . Importantly, Vipr1 levels positively correlate with ASS1 levels and serum levels of urea (the end product of the urea cycle and arginine metabolism) in HCC patients . This suggests that the loss of Vipr1 expression in HCC facilitates CAD phosphorylation and tumor progression by altering arginine metabolism.

These findings identify a novel metabolic mechanism through which Vipr1 exerts its tumor-suppressive effects and suggest that restoration of Vipr1 and treatment with Vipr1 agonists may represent a promising approach for HCC treatment.

What expression systems yield functional recombinant mouse Vipr1 for in vitro studies?

Several expression systems can be employed to produce functional recombinant mouse Vipr1, each with distinct advantages:

For most functional studies, mammalian expression systems (particularly HEK293 or CHO cells) are preferred as they provide the most physiologically relevant post-translational modifications and membrane environment for proper receptor folding and function. When using these systems, expression can be optimized by:

• Incorporating a cleavable signal sequence at the N-terminus

• Adding affinity tags (His, FLAG) for purification

• Including a fluorescent protein fusion for localization studies

• Optimizing codon usage for the expression host

• Using inducible promoters to control expression levels

The choice of expression system should be guided by the specific experimental requirements, balancing yield, functionality, and resource constraints.

What are the critical parameters for designing Vipr1 knockout or knockdown models?

Creating effective Vipr1 knockout or knockdown models requires careful design considerations:

CRISPR/Cas9 Gene Editing Approach:

• Target the Vipr1 gene with guide RNAs directed at critical exons (preferably early exons)

• Design multiple guide RNAs to increase efficiency and verify specificity using alignment tools

• Consider potential off-target effects by performing whole-genome sequencing

• For conditional knockouts, design loxP sites flanking critical exons to enable tissue-specific deletion

Target Selection:
When designing Vipr1 knockout strategies, consider targeting:

• Exons encoding the ligand-binding domain

• Regions encoding transmembrane domains critical for receptor structure

• Sequences encoding G-protein coupling domains

Validation Strategy:
Comprehensive validation of Vipr1 knockout models should include:

• Genomic DNA sequencing to confirm mutations

• RT-PCR and Western blotting to verify absence of Vipr1 mRNA and protein

• Functional assays measuring cAMP responses to VIP stimulation

• Phenotypic characterization in immune, metabolic, and cancer contexts

Knockdown Alternatives:
For situations where complete knockout is not desirable:

• shRNA-mediated knockdown using lentiviral vectors

• Antisense oligonucleotides for temporary knockdown

• Dominant-negative constructs that interfere with receptor function

The validation of knockout or knockdown efficiency is critical before proceeding with phenotypic analyses. The choice between these approaches depends on the specific research question, required level of gene suppression, and whether temporal or spatial control is necessary.

How should recombinant Vipr1 be stabilized for structural and functional studies?

Maintaining the stability of recombinant mouse Vipr1 requires optimized conditions:

Buffer Composition:
For membrane preparations containing Vipr1:

• 50 mM HEPES or Tris-HCl, pH 7.4

• 150 mM NaCl (physiological ionic strength)

• 5 mM MgCl2 (stabilizes G protein coupling)

• 10% glycerol as cryoprotectant

• Complete protease inhibitor cocktail

For solubilized receptors:

• Mild detergents: DDM (0.1%), CHAPS (0.5%), or digitonin (0.5-1%)

• Addition of cholesterol hemisuccinate (CHS, 0.01%) to mimic membrane environment

• Lower pH (7.0-7.2) to reduce deamidation

Ligand Stabilization:
Adding ligands can significantly enhance Vipr1 stability:

• VIP or antagonist peptides (100 μM) occupy the binding site and stabilize conformation

• Antagonists like those described in the research often provide greater stabilization than agonists

Storage Conditions:

• Aliquot in single-use volumes to avoid freeze-thaw cycles

• Flash freeze in liquid nitrogen

• Store at -80°C (stable for 6-12 months)

• For short-term storage (1-2 weeks), 4°C is preferable to freeze-thaw

Alternative Stabilization Approaches:
For challenging applications like crystallography:

• Thermostabilizing mutations based on computational prediction

• Fusion partners (T4 lysozyme, BRIL) to stabilize flexible regions

• Nanobodies or antibody fragments that lock preferred conformations

When preparing Vipr1 for structural studies, it's important to note that AlphaFold modeling has shown limitations in predicting the conformation of intracellular regions of human VPAC1, particularly when comparing binding of VIP versus antagonists like ANT308 . These limitations underscore the need for experimental approaches to complement computational predictions.

What binding assays best characterize Vipr1 interactions with agonists and antagonists?

Multiple complementary binding assays provide comprehensive characterization of Vipr1 ligand interactions:

Radioligand Binding Assays:

• Saturation Binding: Using [125I]-VIP to determine Bmax (receptor density) and Kd (affinity)

• Competition Binding: Displacement of radiolabeled VIP by unlabeled ligands to determine Ki values

• Association/Dissociation Kinetics: Measure kon and koff rates

• Advantages: Gold standard for affinity determination, established methodology

• Limitations: Requires radioactive materials, labor-intensive

Non-Radioactive Alternatives:

• Fluorescence-based binding: Using fluorescently-labeled VIP analogs

• Surface Plasmon Resonance: Real-time binding kinetics without labeling

• Time-Resolved FRET: Using lanthanide-labeled ligands for higher sensitivity

• Microscale Thermophoresis: Detects binding-induced changes in thermophoretic mobility

Computational Approaches:
Research indicates that in silico screening has been used to identify peptides with high predicted binding affinities to VPAC1 . These computational approaches can complement experimental methods, especially for initial screening of potential ligands.

Structural Considerations:
The amino acid side chains of VIP and its antagonists form extensive networks of engagement with residues in the receptors . Substitutions in antagonist peptides can create new interactions with receptor residues not seen with natural ligands. For example, the N9D substitution in the ANT300 and ANT308 antagonists interacts with Y98 in VPAC1, creating a novel binding interaction .

When characterizing new agonists or antagonists, it's important to use multiple orthogonal binding assays to confirm interaction with Vipr1 and determine structure-activity relationships.

What cell-based assays best measure functional responses of recombinant Vipr1?

Several complementary functional assays provide comprehensive assessment of Vipr1 signaling:

cAMP Pathway Assays:

• ELISA-based cAMP detection: Quantifies total cAMP accumulation after VIP stimulation

• Real-time cAMP sensors: FRET-based reporters (Epac-camps) for temporal resolution

• Protocol optimization: Include phosphodiesterase inhibitor (IBMX, 100 μM)

• Controls: Forskolin (direct adenylyl cyclase activator) as positive control

Downstream Signaling Assays:

• CREB Phosphorylation: Western blot using phospho-CREB (Ser133) antibodies

• PKA substrate phosphorylation: Using anti-phospho-(Ser/Thr) PKA substrate antibodies

• Reporter Gene Assays: CRE-luciferase constructs for transcriptional readout

• ERK1/2 Activation: Detects MAPK pathway engagement via phospho-specific antibodies

Receptor Regulatory Mechanisms:

• Internalization Assays: Flow cytometry or ELISA to measure surface receptor loss

• β-Arrestin Recruitment: BRET-based assays between receptor and β-arrestin

• Receptor Phosphorylation: Phospho-specific antibodies against receptor residues

Metabolic Function Assays:
Based on the role of Vipr1 in regulating arginine metabolism in HCC, specific functional assays include:

• ASS1 Expression Analysis: qPCR and Western blot to measure upregulation

• CAD Phosphorylation: Western blot with phospho-specific antibodies

• Urea Production: Colorimetric assays to measure the end product of arginine metabolism

• Pyrimidine Synthesis: Metabolic labeling to measure de novo nucleotide production

When validating recombinant Vipr1 function, it is essential to perform multiple orthogonal assays that assess both immediate receptor coupling (cAMP, calcium) and downstream responses (gene expression, metabolic changes). Research has shown that VIP treatment upregulates ASS1 and suppresses CAD phosphorylation in an mTOR/p70S6K signaling-dependent manner in HCC cells , demonstrating the complexity of Vipr1 signaling networks.