PTH2R Antibody

What is PTH2R and why is it important in neuroscience research?

PTH2R (parathyroid hormone 2 receptor) is a G-protein-coupled receptor belonging to the class B subfamily that includes receptors for glucagon-GHRH-VIP family peptides, calcitonin, and CRF. In neuroscience, PTH2R has significant research value because:

• It is abundantly expressed in specific brain regions, particularly in the central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular and periventricular nuclei, medial geniculate, and pontine tegmentum .

• PTH2R and its ligand, tuberoinfundibular peptide of 39 residues (TIP39), constitute a neuromodulator system implicated in endocrine and nociceptive regulations .

• Evidence suggests PTH2R plays roles in fear, anxiety, reproductive behaviors, pituitary hormone release, and nociception .

The receptor's structure includes a long extracellular N-terminal domain (approximately 150 amino acids) important for ligand binding, seven transmembrane domains arranged in a circular configuration, three extracellular and three intracellular loops, and a C-terminal tail of about 130 amino acids extending intracellularly .

What are the primary experimental applications for PTH2R antibodies?

Based on current research practices, PTH2R antibodies have several key applications:

Researchers should note that PTH2R antibodies have been validated for detecting the receptor in human, rat, and mouse samples, with observed molecular weights of approximately 62-66 kDa .

What is the anatomical distribution of PTH2R in the brain?

PTH2R shows a distinctive distribution pattern in the brain that is highly conserved between rodents and primates:

In primates, PTH2R-immunoreactive fibers are abundant in:

• Medial preoptic area

• Hypothalamic paraventricular, periventricular and infundibular (arcuate) nuclei

• Lateral hypothalamic area

• Median eminence

• Thalamic paraventricular nucleus

• Periaqueductal gray

• Lateral parabrachial nucleus

• Nucleus of the solitary tract

• Sensory trigeminal nuclei

• Medullary dorsal reticular nucleus

• Dorsal horn of the spinal cord

In situ hybridization studies have shown high levels of PTH2R expression in the central amygdaloid nucleus, medial preoptic area, hypothalamic paraventricular and periventricular nuclei, medial geniculate, and pontine tegmentum . This distribution pattern supports its proposed roles in endocrine regulation and nociception.

What technical considerations are important when performing double immunolabeling with PTH2R antibodies?

Double immunolabeling with PTH2R antibodies requires careful optimization:

Protocol overview for PTH2R and neurochemical marker co-localization:

• Sequential immunolabeling approach:

  • First, perform PTH2R immunolabeling using dilute anti-PTH2R primary antiserum (1:40,000 is recommended to avoid antibody cross-reactivity)

  • Visualize using fluorescent secondary antibodies (e.g., FITC-tyramide)

  • For second marker detection, perform antigen retrieval if needed (e.g., citric acid buffer, pH 6.0 at 90°C for 15 min)

  • Apply second primary antibody (e.g., anti-VGLUT2 at 1:10,000)

  • Visualize using a different fluorophore (e.g., Alexa Fluor 594)

• Critical considerations:

  • Antibody specificity must be validated through appropriate controls

  • Cross-reactivity between primary and secondary antibodies must be eliminated

  • For PTH2R co-localization with somatostatin or CRH, concentrate on hypothalamic regions, particularly the periventricular and paraventricular nuclei

  • When examining co-localization with glutamatergic markers, focus on regions with high VGLUT2 expression

In primate studies, this approach has successfully demonstrated that PTH2R fibers are glutamatergic and that TIP39 may directly influence hypophysiotropic somatostatin-containing neurons and indirectly influence corticotropin-releasing hormone-containing neurons .

How should researchers compare the specificity and sensitivity of different PTH2R antibodies for primate versus rodent studies?

When comparing PTH2R antibodies for cross-species studies, researchers should consider:

Critical evaluation parameters:

• Epitope conservation:

  • Analyze sequence homology between target species

  • Note that human and rat PTH2R have approximately 84% amino acid sequence identity

  • Examine immunogen sequences: antibodies targeting the extracellular domain (e.g., amino acids 125-137 in rat) show good cross-reactivity

• Validation evidence:

  • Western blot validation showing appropriate molecular weight (62-66 kDa)

  • Immunohistochemical labeling matching known distribution patterns

  • Blocking peptide controls demonstrating specificity

• Cross-species reactivity data:

  Antibody Type	Human	Rat	Mouse	Monkey	Reference
  Extracellular domain	Yes	Yes	Yes	Yes
  C-Terminal	Yes	Variable	Variable	Yes
  N-Terminal	Yes	Yes	Yes	Yes

• Application-specific performance:

  • Some antibodies perform better in WB than IHC across species

  • Fixation sensitivity may differ between species tissues

  • Antibodies targeting phosphorylated epitopes (e.g., pThr2) may have higher species specificity

Researchers should ideally validate each antibody for their specific application and species, rather than relying solely on manufacturer claims.

What are the most effective methods for detecting PTH2R expression at the mRNA level in brain tissue?

For mRNA detection of PTH2R in brain tissue, researchers should consider these methodological approaches:

• RT-PCR analysis:

  • Tissue preservation is critical: RNA degradation should be assessed by examining 28S to 18S rRNA ratio on denaturing gels

  • DNase treatment is essential to eliminate genomic DNA contamination

  • Recommended primer design:

    • Primers spanning exon-exon junctions to avoid genomic amplification

    • For human PTH2R: 5′-CAATTGCTTGGCTGTAGCTTT-3′ and 5′-ACAAAATCAATTTGCAGACACAA-3′ (440 bp product)

    • Include housekeeping genes (e.g., GAPDH) as internal controls

  • PCR conditions: 95°C for 3 min, followed by cycles of 95°C for 0.5 min, 60°C for 0.5 min and 72°C for 1 min

  • Cycle numbers: 38 cycles for PTH2R, 33 cycles for GAPDH

• In situ hybridization histochemistry:

  • For sensitive detection of PTH2R mRNA in specific cell populations

  • Riboprobe generation using (35S)UTP labeling

  • Exposure time optimization: approximately 3 weeks at 4°C for autoradiographic detection

  • Section thickness considerations: 12 μm for frozen sections is optimal

  • Post-hybridization processing: counterstaining with Giemsa improves cellular resolution

• Key considerations:

  • PTH2R mRNA detection efficiency varies by brain region

  • Age-dependent expression has been observed: TIP39 mRNA may decline beyond late postnatal ages in some species

  • Regional expression patterns should be compared with protein distribution to confirm translation

In primates, these approaches have successfully identified PTH2R expression in multiple brain regions, with high expression in the septum, caudate nucleus, medial geniculate body, hypothalamus, pretectal area, pontine tegmentum, and cerebellar cortex .

How should researchers interpret discrepancies between PTH2R immunohistochemistry and in situ hybridization results?

Discrepancies between protein and mRNA detection methods are common and require careful interpretation:

Common discrepancy patterns and their interpretations:

• High mRNA but low protein detection:

  • Possible explanations: post-transcriptional regulation, rapid protein turnover, or transport to distal regions

  • For PTH2R, this pattern is observed in some thalamic and hypothalamic neurons

  • Methodological consideration: antibody sensitivity may be insufficient for low abundance proteins

• Low mRNA but high protein detection:

  • Possible explanations: protein accumulation in terminals, low mRNA turnover, or detection of transported protein

  • For PTH2R, dense fiber networks are observed in regions with few PTH2R-expressing cell bodies

  • Analysis approach: examine projection patterns from known PTH2R-expressing neuronal populations

• Completely mismatched patterns:

  • Technical considerations:

    • Antibody specificity should be re-validated with appropriate controls

    • Probe specificity for in situ hybridization should be confirmed with sense probes

    • Different sensitivity thresholds between methods

Recommended validation approach:

• Perform multiple antibody labeling with antibodies targeting different epitopes

• Use transgenic reporter systems where available (e.g., β-galactosidase expression driven by PTH2R promoter)

• Correlate findings with functional studies or receptor binding assays

• Use additional approaches like RNAscope for more sensitive mRNA detection

• Consider species differences when comparing literature data

The literature indicates that in primates, PTH2R protein distribution (detected by immunocytochemistry) generally corresponds well with mRNA distribution patterns, though protein is more widely distributed due to axonal transport .

What controls are essential for validating PTH2R antibody specificity in neuroanatomical studies?

Rigorous controls are necessary to ensure reliable PTH2R antibody labeling:

Essential control experiments:

• Peptide absorption controls:

  • Pre-incubate antibody with immunizing peptide (blocking peptide)

  • This should abolish specific labeling in all applications

  • Example: Anti-PTH2R antibodies pre-incubated with the immunizing peptide should show no labeling in Western blot of brain membranes

• Tissue-specific controls:

  • Positive control tissues with known PTH2R expression:

    • Hypothalamic paraventricular and periventricular nuclei

    • Central amygdaloid nucleus

    • Medial preoptic area

    • Pontine tegmentum

  • Negative control regions with minimal PTH2R expression:

    • Ventral thalamus

    • Mediodorsal thalamic nucleus

    • Pulvinar

    • Substantia nigra

    • Pontine nuclei

• Methodological controls:

  • Omission of primary antibody

  • Use of non-immune serum from same species as primary antibody

  • Use of secondary antibody alone

• Genetic validation:

  • Where available, tissues from PTH2R knockout animals

  • Comparison with reporter systems (e.g., β-galactosidase in knock-in mice with β-galactosidase driven by the PTH2R promoter)

• Cross-validation:

  • Compare labeling patterns using multiple antibodies targeting different epitopes

  • Correlation with in situ hybridization patterns

  • Functional validation where possible (e.g., receptor binding studies)

When reporting results, researchers should explicitly state which controls were performed and include control images in publications to allow readers to assess specificity claims.

How can researchers resolve inconsistent Western blot results when detecting PTH2R in different tissue preparations?

Inconsistencies in Western blot detection of PTH2R can be addressed through systematic troubleshooting:

Common issues and solutions:

• Variable molecular weight detection (62-66 kDa range):

  • Cause: Post-translational modifications, particularly glycosylation

  • Solution: Enzymatic deglycosylation treatments prior to electrophoresis

  • Interpretation: Compare with theoretical weight of 62 kDa

• Tissue preparation considerations:

  • For brain tissue: Membrane fractionation significantly improves detection

  • Protocol recommendation:

    • Homogenize fresh tissue in ice-cold buffer containing protease inhibitors

    • Centrifuge at low speed to remove debris

    • Ultracentrifuge supernatant to isolate membrane fraction

    • Solubilize in appropriate detergent buffer

• Extraction and loading optimization:

  Tissue Type	Recommended Extraction	Loading Amount	Reference
  Brain	RIPA buffer with protease inhibitors	50-75 μg
  Testis	Stronger detergents (e.g., SDS)	30-50 μg
  Cell lines	Gentle NP-40 based buffers	20-40 μg

• Antibody selection based on application:

  • For brain samples: Antibodies targeting extracellular domain show better performance

  • For peripheral tissues: C-terminal antibodies may provide cleaner results

  • Dilution optimization: Starting at 1:500 and titrating as needed

• Detection system optimization:

  • Enhanced chemiluminescence with longer exposure times

  • Consider using signal enhancers for low abundance tissues

  • Fluorescent secondary antibodies for quantitative analysis

By systematically addressing these variables, researchers can achieve consistent Western blot detection of PTH2R across various tissue types and experimental conditions.

What methodological approaches are most effective for studying PTH2R-TIP39 interactions in specific brain circuits?

To study PTH2R-TIP39 interactions in neural circuits, researchers should employ complementary approaches:

Circuit-specific analytical methods:

• Anatomical co-localization studies:

  • Double immunofluorescence labeling for PTH2R and TIP39

  • Confocal microscopy to resolve synaptic relationships

  • Triple labeling with neuronal subtype markers

  • Electron microscopy for ultrastructural localization

  • Key finding: Subregional distribution of TIP39- and PTH2R-immunoreactive fibers shows remarkable similarities in rats and mice

• Functional pathway mapping:

  • Circuit-specific optogenetic activation of TIP39-expressing neurons

  • Calcium imaging in PTH2R-expressing neurons following pathway stimulation

  • Ex vivo electrophysiology in brain slices with application of TIP39

  • Focus regions:

    • Thalamic subparafascicular area (source of TIP39)

    • Pontine medial paralemniscal nucleus (source of TIP39)

    • Hypothalamic paraventricular and periventricular nuclei (rich in PTH2R)

• Molecular interaction studies:

  • Receptor binding assays with labeled TIP39

  • Co-immunoprecipitation of receptor complexes

  • FRET-based approaches for monitoring ligand-receptor interactions

  • Receptor signaling analysis (cAMP measurement, as PTH2R activates adenylate cyclase)

• Circuit manipulation approaches:

  • Local infusion of PTH2R antagonists in specific brain regions

  • Viral-mediated knockdown of PTH2R in targeted brain areas

  • Designer receptors exclusively activated by designer drugs (DREADDs) in TIP39 neurons

Research has demonstrated that PTH2R fibers are glutamatergic, suggesting that TIP39 may directly influence hypophysiotropic somatostatin-containing neurons and indirectly influence corticotropin-releasing hormone-containing neurons .

How do PTH2R expression and function in primate brains compare to those in rodent models?

Comparative analysis reveals both similarities and differences in PTH2R systems between primates and rodents:

Cross-species comparison:

These comparative insights are crucial for translating findings from rodent models to primate and human applications, particularly for potential therapeutic targeting of the PTH2R-TIP39 system.

What are the most promising approaches for studying the role of PTH2R in neuroendocrine regulation and pain processing?

Research into PTH2R's role in neuroendocrine and nociceptive functions benefits from specialized methodological approaches:

Neuroendocrine regulation studies:

• Hypothalamic-pituitary axis investigation:

  • Double-labeling studies targeting:

    • PTH2R and somatostatin in periventricular nucleus

    • PTH2R and CRH in paraventricular nucleus

  • Functional approaches:

    • Measurement of hormone release following TIP39 administration

    • In vivo microdialysis in median eminence during PTH2R manipulation

    • Transgenic models with conditional PTH2R deletion in specific hypothalamic nuclei

• Key anatomical relationships identified:

  • PTH2R-immunoreactive fibers in the median eminence largely contain somatostatin

  • PTH2R-ir fiber terminals closely appose CRH-ir perikarya but do not co-localize

  • These findings suggest TIP39 may directly influence hypophysiotropic somatostatin-containing neurons and indirectly influence CRH-containing neurons

Pain processing approaches:

• Nociceptive circuit analysis:

  • Targeted investigation of PTH2R in pain-related regions:

    • Periaqueductal gray

    • Lateral parabrachial nucleus

    • Sensory trigeminal nuclei

    • Spinal cord dorsal horn (lamina II)

  • Methodologies:

    • Electrophysiological recording in these regions during nociceptive stimulation

    • Behavioral assessment following localized PTH2R manipulation

    • Calcium imaging in PTH2R-expressing neurons during noxious stimulation

• Translational pain studies:

  • Comparative analysis in rodent and primate models

  • Pharmacological manipulation with PTH2R antagonists in pain models

  • Assessment of PTH2R expression changes in chronic pain conditions

• Mechanism elucidation:

  • Intracellular signaling pathway analysis downstream of PTH2R activation

  • Co-localization with other nociception-related receptors and transmitters

  • Cross-talk with established pain modulatory systems (opioid, cannabinoid)

The PTH2R system's involvement in both neuroendocrine and nociceptive functions suggests potential for developing targeted therapeutics for endocrine disorders and pain conditions, with the advantage of a more restricted distribution compared to many other receptor systems.

What emerging technologies might enhance PTH2R antibody applications in neuroscience research?

Several cutting-edge technologies show promise for advancing PTH2R research:

• Advanced microscopy approaches:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization of PTH2R

  • Expansion microscopy for enhanced visualization of PTH2R in dense fiber networks

  • Lightsheet microscopy for whole-brain mapping of PTH2R distribution

  • These methods could resolve subcellular localization questions that remain challenging with conventional microscopy

• Single-cell technologies:

  • Single-cell RNA sequencing to identify all cell types expressing PTH2R

  • Spatial transcriptomics to map PTH2R expression while preserving anatomical context

  • Patch-seq combining electrophysiology, morphology, and transcriptomics of PTH2R neurons

  • These approaches could reveal previously unidentified PTH2R-expressing cell populations

• Antibody engineering enhancements:

  • Development of recombinant nanobodies against PTH2R for improved tissue penetration

  • Site-specific conjugation strategies for more consistent labeling

  • Photoactivatable antibodies for spatiotemporal control of PTH2R detection

  • These tools could overcome current limitations in antibody specificity and sensitivity

• In vivo applications:

  • Development of PET tracers based on PTH2R antibody fragments

  • Genetically encoded sensors for monitoring PTH2R activation dynamics

  • CRISPR-based approaches for endogenous tagging of PTH2R

  • These methods could enable non-invasive monitoring of PTH2R function

By integrating these emerging technologies with established approaches, researchers can develop a more comprehensive understanding of PTH2R distribution, dynamics, and function in complex neural circuits.

How should researchers design studies to investigate potential species differences in PTH2R antibody specificity and sensitivity?

A systematic approach to evaluating species differences should include:

• Comprehensive epitope analysis:

  • Sequence alignment of PTH2R across target species (human, non-human primates, rodents)

  • Epitope mapping to identify conserved versus variable regions

  • Custom antibody development targeting highly conserved epitopes

  • Experimental design should include at least 3-4 species for meaningful comparison

• Multi-platform validation strategy:

  Validation Approach	Methodology	Comparative Measures
  Genetic validation	Transfection of species-specific PTH2R constructs	Signal intensity, background
  Protein detection	Western blot of tissues from multiple species	Band pattern, intensity, MW
  Tissue distribution	Side-by-side IHC of comparable brain regions	Signal:noise ratio, pattern
  Functional validation	Receptor internalization assays	Antibody-induced signaling

• Quantitative sensitivity assessment:

  • Titration experiments across species samples

  • Calculation of detection limits for each species

  • Signal:noise ratio comparison at standardized antibody concentrations

  • These measures provide objective comparison of antibody performance

• Cross-reactivity profiling:

  • Testing against closely related receptors (e.g., PTH1R)

  • Mass spectrometry identification of all proteins immunoprecipitated by the antibody

  • Comparing non-specific binding profiles across species

What are the key considerations for designing studies examining the relationship between PTH2R dysfunction and neurological or psychiatric disorders?

Investigating PTH2R in disease contexts requires thoughtful experimental design:

• Candidate disorder selection based on PTH2R distribution:

  • Anxiety disorders (amygdala, bed nucleus of stria terminalis expression)

  • Pain disorders (expression in nociceptive pathways)

  • Neuroendocrine disorders (hypothalamic expression)

  • Selection should be guided by known PTH2R functions in fear, anxiety, reproductive behaviors, pituitary hormone release, and nociception

• Human tissue analysis approaches:

  • Post-mortem brain tissue examination:

    • Compare PTH2R expression in control vs. disorder cases

    • Consider comorbidities and medication effects

    • Age-matched controls are essential (given developmental regulation)

  • Genetic association studies:

    • PTH2R polymorphism analysis in disorder cohorts

    • Functional characterization of identified variants

• Animal model development:

  • Conditional knockout strategies targeting PTH2R in specific circuits

  • Circuit-specific expression manipulation using viral vectors

  • Behavioral assessment focusing on:

    • Anxiety-like behaviors

    • Pain sensitivity

    • Neuroendocrine function

    • Social and reproductive behaviors

• Translational considerations:

  • Comparative studies in rodents and non-human primates

  • Pharmacological tools:

    • Development of brain-penetrant PTH2R modulators

    • PET ligands for non-invasive receptor quantification

  • Biomarker exploration:

    • Cerebrospinal fluid TIP39 levels in patient populations

    • Correlation with symptom severity

• Methodological challenges:

  • PTH2R antibody specificity must be extensively validated in disease tissues

  • Post-translational modifications may alter antibody recognition

  • TIP39 expression changes with age and potentially disease state

  • Control for effects of common medications on PTH2R expression