PTH1R Antibody

What is PTH1R and why is it important in research?

PTH1R (parathyroid hormone 1 receptor) is a glycoprotein with seven transmembrane domains featuring extracellular N-terminus and intracellular C-terminus regions. It functions as a receptor for both parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) . PTH1R signaling is primarily mediated by G proteins that activate adenylyl cyclase, triggering downstream signaling cascades essential for calcium homeostasis and bone metabolism . Research on PTH1R is particularly significant because it plays critical roles in bone turnover and calcium homeostasis, making it relevant to studies of osteoporosis, hypercalcemia, and related disorders . Additionally, PTH1R and PTHrP are associated with the differentiation of bone and cartilage during developmental stages, highlighting its importance in developmental biology research .

What criteria should guide PTH1R antibody selection for specific applications?

When selecting a PTH1R antibody, researchers should consider several factors based on their specific experimental needs. For Western blot applications, antibodies like 29115-1-AP have been validated at dilutions of 1:500-1:1000 with reactivity confirmed in human, mouse, and rat samples . For immunohistochemistry (IHC), the same antibody has been validated at dilutions of 1:50-1:500 . The observed molecular weight of PTH1R (70-80 kDa) compared to its calculated molecular weight (66 kDa) suggests post-translational modifications that may affect antibody recognition . Researchers should select antibodies that have been validated in their specific sample type and adjust dilutions according to their experimental system. For studying PTH1R in different species, confirm cross-reactivity with the target species before proceeding with experiments, as PTH1R antibodies can show varying degrees of cross-reactivity among human, mouse, and rat samples .

How do PTH1R antibodies differ from PTH1R autoantibodies found in clinical samples?

PTH1R antibodies used in research are deliberately generated immunoreagents designed to bind specific epitopes on the receptor for detection purposes. In contrast, PTH1R autoantibodies are pathological immunoglobulins that develop spontaneously in certain patients and can block the normal function of the receptor . Research-grade antibodies typically recognize specific sequences without interfering with function (unless designed as blocking antibodies), while autoantibodies identified in patients with PTH resistance primarily target the extracellular domain (ECD) of PTH1R and prevent normal hormone binding . Using Luciferase immunoprecipitation systems (LIPS) technology and cell-based assays measuring cAMP responses, researchers have demonstrated that these autoantibodies block PTH binding and signaling by more than 90% compared to control samples . Understanding these distinctions is crucial when designing experiments involving clinical samples that may contain interfering autoantibodies, which could confound research results if not properly accounted for in experimental design .

What are the optimal conditions for using PTH1R antibodies in Western Blot applications?

For Western Blot applications, the recommended dilution range for PTH1R antibodies like 29115-1-AP is 1:500-1:1000 . The expected molecular weight band should appear between 70-80 kDa, which is slightly higher than the calculated 66 kDa, likely due to post-translational modifications . When preparing samples, researchers should validate their antibody against positive controls known to express PTH1R, such as HeLa cells, HepG2 cells, mouse/rat kidney tissue, or mouse/rat liver tissue, which have been confirmed to express detectable levels of PTH1R . For optimal results, store the antibody according to manufacturer specifications (-20°C with 0.02% sodium azide and 50% glycerol at pH 7.3) and aliquot larger volumes to avoid repeated freeze-thaw cycles . It is advisable to titrate the antibody concentration in each specific testing system to determine optimal signal-to-noise ratio, as background levels can vary between different experimental setups and tissue types .

What specific protocols are recommended for PTH1R detection in immunohistochemistry?

For immunohistochemistry (IHC) applications with PTH1R antibodies, researchers should use dilutions ranging from 1:50-1:500, with specific optimization recommended for each tissue type . Antigen retrieval is a critical step that significantly impacts staining quality - for PTH1R detection, suggested protocols include using TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 . PTH1R antibodies have been successfully validated in human kidney tissue and human pancreatic cancer tissue, making these suitable positive controls . When optimizing IHC protocols, consider that PTH1R is a seven-transmembrane domain protein with complex structural elements that may require careful fixation and membrane permeabilization to preserve epitope accessibility . To ensure specificity, include appropriate negative controls such as isotype-matched irrelevant antibodies and, when possible, tissues known to lack PTH1R expression . For quantitative analysis, standardize image acquisition settings and develop consistent scoring criteria specific to subcellular PTH1R localization, which may vary depending on receptor internalization states .

How can researchers design experiments to study PTH1R signaling pathways?

To effectively study PTH1R signaling pathways, researchers should employ multiple complementary approaches. Cell-based assays measuring cAMP responses to PTH 1-34 (Forteo, Eli Lilly) in PTH1R-expressing cells provide valuable insights into receptor activation and downstream signaling . When designing these experiments, include dose-response curves to characterize the potency and efficacy of PTH binding to the receptor. For studying the receptor's structural dynamics during signaling, consider using Flag-tagged PTH1R constructs, which can be engineered by PCR overlap extension as described in published protocols . This approach involves generating three fragments using specific primers and PCR, followed by restriction enzyme digestion and subcloning into an appropriate vector like pcDNA3.1(+) . Comparing the signaling behavior of wild-type PTH1R to Flag-tagged versions will ensure that the epitope does not interfere with normal signaling processes . For more advanced studies, cryo-electron microscopy has revealed distinct conformational differences between PTH1R bound to different ligands (Teriparatide vs. Abaloparatide), which correlate with differences in signaling duration . Researchers can explore these conformational states through 3D variability analysis and site-directed mutagenesis to identify key residues involved in differential signaling patterns .

How can researchers investigate PTH1R autoimmunity mechanisms?

To investigate PTH1R autoimmunity mechanisms, researchers should implement a multi-faceted approach combining immunological, functional, and structural analyses. The Luciferase immunoprecipitation systems (LIPS) technology has proven effective for detecting autoantibodies against PTH1R in patient samples . This method involves constructing plasmids encoding light-emitting Gaussia luciferase fusion proteins for both PTH and PTH1R, transfecting them into Cos1 cells, and using the crude cell extracts for immunoprecipitation assays . To determine which domains of the receptor are targeted by autoantibodies, perform mapping experiments with PTH1R truncation mutants encompassing either the extracellular domain (PTH1R-ECD) or the transmembrane domain (PTH1R-TMD) . Functional analysis of the autoantibodies' blocking capacity can be assessed using cell-based assays measuring cAMP responses to PTH 1-34 after exposing PTH1R-expressing cells to purified IgG from patient samples . Additionally, researchers studying autoimmune mechanisms should consider analyzing B-cell subsets, particularly the CD21-low–CD38-negative B-cell subset, which has been reported to be enriched in patients with autoimmunity associated with autoantibody production .

What methods are optimal for studying the role of PTH1R in complex tissues?

Studying PTH1R in complex tissues requires specialized approaches that preserve spatial context while enabling functional analysis. For tissue-specific expression patterns, immunohistochemistry with optimized antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) provides reliable visualization of PTH1R distribution . When examining tissues with potential autoimmune involvement, consider combining IHC with analysis of CD21-low–CD38-negative B-cell subsets in nearby lymphoid structures, as these have been associated with autoantibody production targeting PTH1R . For functional studies in tissue contexts, ex vivo tissue slice cultures treated with PTH or other receptor ligands can provide insights into tissue-specific responses while maintaining the native cellular architecture. Advanced microscopy techniques such as multiphoton intravital imaging can be employed to study PTH1R dynamics in bone tissue in real-time. For molecular characterization in specific tissue regions, laser capture microdissection followed by RNA-sequencing or proteomics can reveal tissue-specific PTH1R signaling networks. When investigating PTH1R signaling in kidney tissues, researchers should be aware of potential confounding factors such as membranous glomerulonephritis, which has been associated with PTH1R autoantibodies in some cases , and consider appropriate controls to account for pathological conditions.

How do recently identified molecular mechanisms affect experimental design for PTH1R studies?

Recent structural and functional insights into PTH1R signaling necessitate several considerations for experimental design. The cryo-EM structures of PTH1R-Gs complexes bound to different peptide agonists have revealed that specific amino acid discrepancies between Teriparatide (PTH) and Abaloparatide (ABL) lead to distinct conformational states and signaling durations . Researchers designing mutation studies should focus on the six amino acid discrepancies within the N-terminal portions of these peptides (positions 1, 5, 8, 10, 11, and 14), with particular attention to positions 1, 10, and 11, which showed no obvious alteration to the detailed interactions despite sequence differences . For studying receptor activation mechanisms, it's important to note that substitution mutations of most N-terminal peptide residues, particularly residues 1-9, substantially reduce downstream cAMP signaling . When investigating PTH1R autoimmunity, experiments should account for the finding that autoantibodies primarily target the extracellular domain involved in PTH binding rather than the transmembrane domain . This domain-specific targeting suggests that blocking assays focusing on the extracellular domain will be most relevant for autoimmunity studies. Additionally, the association between PTH1R autoantibodies and other autoimmune manifestations indicates that experimental designs should consider the broader immunological context and potentially screen for other autoantibodies .

What are the latest techniques for engineering PTH1R constructs for research applications?

Engineering modified PTH1R constructs provides powerful tools for investigating receptor dynamics and signaling. Recent advances include the development of Flag-tagged PTH1R constructs with precise epitope placement for optimal functionality . This approach uses PCR overlap extension to generate three fragments: the first fragment (F1) of 0.3 kb size from the beginning of PTH1R to amino acid position 106 containing the Flag epitope and KpnI restriction site; the second fragment (F2) of 1.5 kb from amino acid position 100 to the end of PTH1R containing the last two amino acids of the Flag epitope; and the third fragment (F3) representing the complete sequence . After PCR amplification, these fragments are joined through overlap extension PCR and subcloned into expression vectors like pcDNA3.1(+) . When designing similar constructs, researchers should carefully consider epitope placement to avoid interfering with ligand binding or receptor function, particularly given the complex interaction between the N-terminal portions of PTH/ABL peptides and the transmembrane core of PTH1R . For advanced studies of receptor dynamics, FRET-based biosensors incorporating PTH1R can be designed to monitor conformational changes or protein-protein interactions in real-time. Utilizing the detailed structural information now available from cryo-EM studies, researchers can strategically introduce mutations or fluorescent tags at positions unlikely to disrupt the critical interactions between receptor domains and peptide ligands .

How can the study of PTH1R autoantibodies inform therapeutic development?

The identification of PTH1R-blocking autoantibodies in patients with acquired PTH resistance provides valuable insights for therapeutic development. These autoantibodies primarily target the extracellular domain (ECD) of PTH1R, the region involved in PTH binding, and can block PTH signaling by more than 90% . Researchers interested in developing treatments for autoimmune PTH resistance should consider strategies to either remove these autoantibodies or bypass their blocking effects. Potential approaches include designing modified PTH analogs that can bind to PTH1R even in the presence of autoantibodies, perhaps by inducing conformational changes in the receptor that reduce autoantibody binding affinity. Another strategy could involve targeting downstream components of the PTH signaling pathway to restore normal calcium homeostasis despite receptor blockade. The association between PTH1R autoantibodies and other autoimmune manifestations, including atypical membranous glomerulonephritis, suggests that broader immunomodulatory approaches might be beneficial . Researchers should also investigate the finding that patients with PTH1R autoantibodies had increased frequencies of the CD21-low–CD38-negative B-cell subset in peripheral blood, which might represent a specific therapeutic target . For developing screening tools, the LIPS technology used to detect PTH1R autoantibodies could be adapted for clinical diagnostic applications to identify patients who might benefit from specific immunotherapies .