HCAR3/HCAR2 Antibody

What are HCAR2 and HCAR3 receptors and their role in human physiology?

HCAR2 (Hydroxycarboxylic acid receptor 2, also known as GPR109A) belongs to the class A G protein-coupled receptor family and plays key roles in regulating lipolysis and free fatty acid formation in humans . HCAR2 is deeply involved in multiple pathophysiological processes and serves as an attractive target for treating cardiovascular, neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases . HCAR3, a closely related receptor, shares structural similarities with HCAR2 but exhibits distinct ligand selectivity profiles.

Both receptors are activated by specific hydroxycarboxylic acids, with HCAR2 being notably activated by niacin (vitamin B3) and its derivatives. Upon activation, HCAR2 primarily couples to Gi/o proteins to inhibit adenylate cyclase and cyclic AMP signaling . This signaling cascade mediates various physiological responses including anti-lipolytic effects in adipocytes and anti-inflammatory effects in immune cells.

How do HCAR2 and HCAR3 differ in their expression patterns?

HCAR2 shows a tissue-specific expression pattern that is particularly important for its physiological functions. In the brain, HCAR2 is expressed selectively by microglia and is robustly induced by amyloid pathology in Alzheimer's disease (AD) . This neurological expression pattern suggests a specialized role in neuroimmune regulation.

Experimental evidence from 5xFAD amyloidogenic mouse models shows that HCAR2 expression increases in the hippocampus and cortex during periods of active plaque deposition (between 4-6 months of age) in both female and male mice . Analysis involving microglia depletion using CSF1R antagonist PLX5622 demonstrated that approximately 70% reduction in cortical microglia correspondingly reduced HCAR2 mRNA expression, confirming the microglia-specific expression of this receptor .

HCAR3 expression patterns differ somewhat from HCAR2, which partially explains their different roles in physiological processes. These expression differences are important considerations when designing experiments using antibodies against these receptors.

What are the general applications of HCAR2/HCAR3 antibodies in research?

HCAR2/HCAR3 antibodies are essential tools in multiple research applications:

• Immunohistochemistry (IHC): Used to visualize receptor expression in tissue sections, particularly for studying microglial HCAR2 expression in relation to amyloid plaques in AD models

• Western Blotting: For quantitative assessment of receptor protein levels in tissue or cell lysates

• Flow Cytometry: To analyze receptor expression in specific cell populations (e.g., microglia CD11b+ cells)

• Immunoprecipitation: For studying protein-protein interactions involving HCAR2/HCAR3

• Validation of Genetic Manipulations: To confirm successful knockout or knockdown of receptor expression

When selecting antibodies for these applications, researchers should consider specificity between HCAR2 and HCAR3, which share structural similarities. Validation of antibody specificity is critical, especially given that many GPCR antibodies demonstrate nonspecific immunoreactivity .

How can I validate the specificity of HCAR2/HCAR3 antibodies?

Given the structural similarities between HCAR2 and HCAR3, antibody specificity validation is crucial. The following approaches are recommended:

• Genetic Knockdown Controls: Perform siRNA-mediated knockdown of HCAR2/HCAR3 to confirm antibody specificity, as demonstrated with the human microglial cell line HMC3 . The literature shows that siRNA-mediated knockdown of HCAR2 resulted in a corresponding reduction of HCAR2 immunoreactivity, confirming antibody specificity .

• Knockout Tissue Controls: Use tissue from HCAR2-/- or HCAR3-/- animals as negative controls for antibody validation.

• Peptide Competition Assays: Pre-incubate the antibody with the immunizing peptide to block specific binding.

• Cross-Reactivity Testing: Test the antibody against cells/tissues expressing only HCAR2 or HCAR3 to evaluate cross-reactivity.

• Multiple Antibody Validation: Use multiple antibodies targeting different epitopes of the same receptor to confirm consistent staining patterns.

It's worth noting that antibody performance can vary significantly between applications (e.g., an antibody that works well for Western blotting may not be suitable for IHC). Therefore, validation should be performed for each specific application.

What methodological considerations are important when using HCAR2/HCAR3 antibodies for immunohistochemistry?

Successful immunohistochemical detection of HCAR2/HCAR3 requires careful consideration of the following factors:

• Fixation Protocol: For brain tissue, 4% paraformaldehyde fixation is commonly used, but optimization may be required for specific antibodies.

• Antigen Retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) may be necessary to unmask antigens.

• Blocking Strategy: Thorough blocking with appropriate serum (5-10%) plus BSA (1-3%) is crucial to reduce nonspecific binding.

• Antibody Dilution: Titration experiments should be performed to determine optimal antibody concentration.

• Incubation Conditions: Overnight incubation at 4°C often yields better results than shorter incubations at room temperature.

• Detection System: Consider fluorescent secondary antibodies for co-localization studies or HRP-based systems for chromogenic detection.

• Proper Controls: Include positive controls (tissues known to express the target), negative controls (tissues not expressing the target), and secondary-only controls.

When studying microglial HCAR2 expression in AD models, co-staining with microglial markers (Iba1) and amyloid markers is recommended to demonstrate the relationship between HCAR2 expression and pathology .

How does HCAR2 contribute to microglial function in Alzheimer's disease?

HCAR2 appears to play a critical role in the microglial response to amyloid pathology in Alzheimer's disease. Research using the 5xFAD mouse model has provided several key insights:

• Microglial Activation: Genetic inactivation of HCAR2 in 5xFAD mice results in impairment of the microglial response to amyloid deposition .

• Gene Expression Changes: HCAR2 knockout leads to deficits in the expression of genes involved in microglial activation and phagocytosis, including SPP1, CST7, CD68, TREM2, TYROBP, and AXL .

• Phagocytic Function: HCAR2-deficient microglia show reduced uptake of amyloid-β (Aβ), contributing to exacerbated amyloid burden .

• Plaque Envelopment: Loss of HCAR2 impairs the ability of microglia to surround and contain amyloid plaques .

• Neuroprotection: HCAR2 activation with niacin (Niaspan) reduces plaque burden, attenuates neuronal loss, and rescues working memory deficits in 5xFAD mice .

These findings suggest that HCAR2 is required for an efficient and neuroprotective response of microglia to amyloid pathology, making it a promising therapeutic target for AD .

What signaling mechanisms differentiate HCAR2 from HCAR3 activation?

Despite structural similarities, HCAR2 and HCAR3 exhibit distinct signaling properties that can be studied using antibodies specific to each receptor or their downstream signaling components:

• Ligand Selectivity: While niacin and acipimox can activate both HCAR2 and HCAR3 receptors, they show much higher affinity for HCAR2 . In contrast, MK-6892 is a highly subtype-specific agonist of HCAR2 that barely activates HCAR3 .

• Key Residues: Structural studies have identified three key residues (R111^3.36, S179^45.52, and Y284^7.43) that form the basis of a general pharmacophore for HCAR2 agonists . The mutation of R111^3.36 to alanine significantly reduces the agonistic activity of niacin and acipimox .

• G Protein Coupling: Both receptors primarily couple to Gi proteins, but may differ in their coupling efficiency or ability to recruit other signaling partners like β-arrestins .

• Downstream Effects: HCAR2 activation in microglia promotes a protective phenotype that enhances phagocytosis and amyloid clearance , while the functional consequences of HCAR3 activation in these cells are less well-characterized.

Understanding these differences is crucial for designing selective therapeutic approaches targeting either receptor.

What experimental approaches can be used to study HCAR2/HCAR3 activation in cellular models?

Several experimental approaches can be employed to study HCAR2/HCAR3 activation:

• cAMP Accumulation Assays: Since both receptors couple to Gi proteins to inhibit adenylyl cyclase, measuring decreases in cAMP production following receptor activation is a standard approach .

• GTP Turnover Assays: These assays directly measure G protein activation and can be used to compare the efficacy of different agonists .

• β-Arrestin Recruitment Assays: NanoBiT assays can be used to measure ligand-induced recruitment of β-arrestin1 to HCAR2/HCAR3 .

• Receptor Internalization: Antibody-based approaches can be used to track receptor internalization following activation.

• Calcium Mobilization: Although not directly coupled to calcium signaling, secondary calcium responses can sometimes be measured.

• Phosphorylation of Downstream Targets: Western blotting with phospho-specific antibodies can be used to assess activation of downstream signaling pathways.

• Gene Expression Analysis: qPCR or RNA-seq can be used to identify genes regulated by HCAR2/HCAR3 signaling .

When designing these experiments, appropriate positive controls (known receptor agonists) and negative controls (receptor antagonists or cells not expressing the receptors) should be included.

How do structural studies of HCAR2/HCAR3 inform antibody development and therapeutic targeting?

Recent structural studies of HCAR2 have provided valuable insights that can inform both antibody development and therapeutic strategies:

• Cryo-EM Structures: Four cryo-EM structures of human HCAR2–Gi1 complexes have been determined: HCAR2 bound to niacin (2.69 Å), acipimox (3.23 Å), MK-6892 (3.25 Å), and in apo form (3.28 Å) . These structures reveal the detailed architecture of the receptor's binding pocket and its interactions with different ligands.

• Binding Pocket Characteristics: The ligand binding pocket of HCAR2 involves key residues like R111^3.36, S179^45.52, and Y284^7.43 that interact with various agonists . Notably, MK-6892 binding reveals an extended binding pocket compared to other agonists .

• Ligand Selectivity Determinants: Structural studies have identified residues that determine ligand selectivity between HCAR2 and HCAR3 . For example, N86^2.63 in HCAR2 corresponds to tyrosine in HCAR3, and mutation of this residue affects receptor activation .

• Antibody Epitope Selection: These structural insights can guide the development of antibodies targeting specific epitopes that may differentiate between HCAR2 and HCAR3, or between different conformational states of these receptors.

• Therapeutic Development: Understanding the structural basis of ligand recognition can accelerate the development of HCAR2-targeting drugs with greater efficacy, higher selectivity, and fewer side effects .

What are the key considerations for developing conformation-specific antibodies for HCAR2/HCAR3?

Developing conformation-specific antibodies that selectively recognize active or inactive states of HCAR2/HCAR3 requires specialized approaches:

• Epitope Selection: Target regions that undergo significant conformational changes upon receptor activation, such as the intracellular loops or the DRY motif region.

• Immunization Strategy: Use purified receptors locked in specific conformational states through the use of nanobodies, G proteins, or conformation-stabilizing mutations.

• Screening Methods: Implement differential screening approaches to identify antibodies that bind preferentially to one conformational state over another.

• Validation in Cellular Systems: Confirm that antibodies recognize the appropriate receptor state in cellular contexts where receptor activation can be manipulated.

• Functional Testing: Assess whether the antibodies themselves affect receptor function, as some conformation-specific antibodies can act as allosteric modulators.

These conformation-specific antibodies can be valuable tools for studying receptor activation dynamics and for developing novel therapeutic approaches that selectively modulate specific signaling pathways.

How is HCAR2 expression altered in neurodegenerative disease models?

HCAR2 expression undergoes significant changes in neurodegenerative disease models, particularly in Alzheimer's disease:

• Induction in AD Mouse Models: HCAR2 expression is increased in the hippocampus and cortex of 5xFAD animals during active plaque deposition (between 4-6 months of age) in both females and males . Similar induction is observed in the APPPS1 amyloidogenic mouse model .

• Cell Type Specificity: The increased expression is specific to microglia, as demonstrated by microglia depletion experiments using CSF1R antagonists .

• Plaque Association: Transcriptomic analysis of sorted microglia from the APPPS1 model revealed that HCAR2 mRNA was selectively increased in microglia associated with neuritic Aβ plaques (Clec7a+) compared with microglia not associated with Aβ plaques (Clec7a-) .

• Tau Pathology: HCAR2 expression also increases in microglia of PS19 tauopathy mice, suggesting that tau pathology can also induce HCAR2 in AD .

• Human AD Brain: Analysis of human transcriptomic datasets revealed increased HCAR2 expression in the dorsolateral prefrontal cortex of patients with AD compared to non-demented controls . This finding was validated by real-time qPCR and immunohistochemistry, which confirmed that HCAR2 protein expression is selective for Iba1-positive cells (microglia) and significantly increases in AD .

These findings suggest that HCAR2 upregulation is part of the microglial response to pathology in neurodegenerative diseases.

What therapeutic potential does targeting HCAR2 hold for neurodegenerative diseases?

The therapeutic potential of targeting HCAR2 in neurodegenerative diseases, particularly Alzheimer's disease, is supported by several lines of evidence:

• Niacin as a Therapeutic Agent: Increased dietary intake of niacin has been correlated with reduced risk of AD . Administration of Niaspan (an FDA-approved formulation of niacin) to 5xFAD mice leads to reduced plaque burden, attenuated neuronal loss, and rescue of working memory deficits .

• Microglial Response Modulation: Activation of HCAR2 stimulates a broad and complex protective response mediated by microglia, leading to decreased plaque burden and reduced neuronal pathology .

• HCAR2 Requirement for Microglial Function: Genetic inactivation of HCAR2 in 5xFAD mice impairs the microglial response to amyloid deposition, including deficits in gene expression, proliferation, and phagocytosis, ultimately leading to exacerbation of amyloid burden and cognitive deficits .

• Translational Potential: Given that Niaspan is already FDA-approved for treating dyslipidemia, its repurposing for AD represents a potentially expedited path to clinical application .

• Targeting Specific Pathways: The identification of specific signaling pathways downstream of HCAR2 may enable the development of more selective therapeutic agents with fewer side effects .

These findings support HCAR2 activation as a promising therapeutic strategy for AD that specifically targets the neuroimmune response .

How can I troubleshoot inconsistent results with HCAR2/HCAR3 antibodies?

Inconsistent results with HCAR2/HCAR3 antibodies can arise from various sources. Here are approaches to identify and address common issues:

• Antibody Validation Issues:

  • Verify antibody specificity through knockout/knockdown controls

  • Use positive controls (tissues/cells known to express the target)

  • Test multiple antibodies targeting different epitopes

  • Sequence verification of your experimental model (check for mutations in the epitope region)

• Technical Factors:

  • Optimize fixation conditions (overfixation can mask epitopes)

  • Try different antigen retrieval methods

  • Adjust blocking conditions to reduce background

  • Titrate antibody concentration to find optimal working dilution

  • Extend incubation times or adjust temperature

• Sample Preparation:

  • Ensure consistent sample handling and processing

  • Control for post-mortem interval in human samples

  • Consider the region-specific expression of receptors

  • Account for disease state or treatment effects on receptor expression

• Detection Systems:

  • Try alternative detection methods (fluorescent vs. chromogenic)

  • Use amplification systems for low-abundance targets

  • Ensure secondary antibodies are appropriate for your primary antibody

• Data Analysis:

  • Use consistent quantification methods

  • Control for background signal

  • Blind analysis to avoid bias

  • Include appropriate statistical tests

Remember that many GPCR antibodies show nonspecific immunoreactivity , so rigorous validation is essential.

What are the latest developments in HCAR2/HCAR3 research methodologies?

Recent advances in research methodologies are expanding our understanding of HCAR2/HCAR3 biology:

• Cryo-EM Structural Studies: High-resolution cryo-EM structures of HCAR2-Gi complexes with various ligands have provided unprecedented insights into receptor activation mechanisms and ligand recognition .

• Single-Cell Transcriptomics: These approaches allow for detailed analysis of HCAR2/HCAR3 expression in heterogeneous cell populations and identification of cell-specific responses to receptor activation.

• CRISPR/Cas9 Gene Editing: Precise genetic manipulation enables the study of receptor function in various cellular contexts and the creation of refined animal models.

• Designer Receptors (DREADDs): Modified versions of HCAR2/HCAR3 that respond to synthetic ligands can be used to selectively activate specific signaling pathways.

• Proximity Labeling Techniques: Methods like BioID or APEX2 can identify novel protein interaction partners of HCAR2/HCAR3 in different activation states.

• Nanobody Technology: The development of nanobodies against specific conformational states of HCAR2/HCAR3 provides new tools for studying receptor dynamics.

• In vivo Imaging: New techniques allow visualization of receptor activation in living organisms, providing insights into the temporal and spatial dynamics of signaling.

• Computational Approaches: Molecular dynamics simulations combined with structural data provide insights into receptor dynamics and ligand interactions that are difficult to study experimentally .

These methodological advances promise to accelerate our understanding of HCAR2/HCAR3 biology and the development of therapeutic strategies targeting these receptors.