KISS1R Antibody

What is KISS1R and why is it important in research?

KISS1R (also known as GPR54, AXOR12, or HOT7T175) is a G-protein coupled receptor that binds to kisspeptins, most notably kisspeptin-54 (KP-54), which are peptides derived from the KISS1 metastasis suppressor protein. KISS1R is critically important in research for several reasons:

The receptor plays a fundamental role in reproductive physiology, being essential for normal gonadotropin-releasing hormone (GnRH) function and puberty onset. In the hypothalamus, the KiSS1/KISS1R system serves as a pivotal regulator of the gonadotropic axis both during development and adulthood . Beyond reproduction, KISS1R has significant implications in cancer research, as it mediates many of the metastasis suppressor functions of KISS1 in various cancer types. The KISS1/KISS1R signaling pathway has demonstrated both tumor-suppressive and potentially tumor-promoting effects depending on the cancer context, making it a complex but promising target for cancer therapeutics and prognostic assessments .

What types of KISS1R antibodies are available for research applications?

Researchers have access to several types of KISS1R antibodies, with polyclonal antibodies being commonly used in multiple applications. Based on available information:

Polyclonal antibodies targeting KISS1R are available from commercial sources, including those derived from immunization of rabbits with KLH-conjugated synthetic peptides from human KISS1R. These antibodies typically target specific regions of the receptor, such as amino acid ranges 121-200 of the 398-amino acid protein . Commercial KISS1R antibodies are validated for multiple applications including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunohistochemistry on frozen sections (IHC-F) . Many commercially available antibodies demonstrate cross-reactivity with KISS1R from multiple species including human, mouse, and rat, with predicted reactivity to dog, sheep, pig, and guinea pig KISS1R .

How can I validate the specificity of a KISS1R antibody for my research?

Validating antibody specificity is critical for obtaining reliable results, especially given reported concerns about the specificity of commercially available KISS1R antibodies. A comprehensive validation approach includes:

Positive and negative controls: Use cell lines or tissues with known high expression (e.g., certain breast cancer cell lines, hypothalamic tissue) and those lacking KISS1R expression. Multiple studies indicate significant discrepancies in results may be attributed to inadequate antibody validation .

Knockdown/knockout validation: As emphasized in the literature, proper validation should involve knock-out and/or knock-down approaches to verify antibody specificity. This is particularly important as the review by Prentice et al. noted that "many of the commercially-available KISS1R antibodies in use are not highly specific" .

Peptide competition: Pre-incubating the antibody with the immunizing peptide should abolish specific staining in Western blots or immunohistochemistry.

Multiple detection methods: Cross-validate your findings using different techniques (e.g., if using IHC, confirm with Western blotting) and, ideally, different antibodies targeting distinct epitopes of KISS1R.

Correlation with mRNA expression: Compare protein detection with mRNA levels using RT-qPCR or in situ hybridization, as "positive correlation between mRNA and protein data strengthens a given study" .

What are the optimal conditions for using KISS1R antibodies in immunohistochemistry?

For optimal immunohistochemical detection of KISS1R in tissues, researchers should consider the following methodological recommendations:

Tissue preparation: Both paraffin-embedded (IHC-P) and frozen sections (IHC-F) can be used for KISS1R detection, with specific commercial antibodies validated for both applications . When examining breast cancer tissues, studies have successfully detected KISS1R in ductal carcinoma in situ and invasive ductal carcinoma, noting higher cytoplasmic staining in tumors and surrounding cells compared to normal tissue .

Antigen retrieval: Heat-induced epitope retrieval is typically required for formalin-fixed tissues, though specific buffer conditions should be optimized.

Blocking and antibody dilution: Use appropriate blocking with bovine serum albumin (BSA) or serum matching the secondary antibody host. Optimal dilutions vary by antibody preparation, with some commercial antibodies supplied at 1μg/μl concentration .

Controls and validation: Include positive controls such as human breast cancer tissue, where KISS1R has been well-documented, and negative controls omitting primary antibody. For accurate interpretation, it's critical to verify antibody specificity, as emphasized in multiple studies highlighting discrepancies potentially resulting from non-specific antibody binding .

How should I optimize Western blot protocols for KISS1R detection?

Western blotting for KISS1R requires careful optimization due to the membrane-associated nature of this G-protein coupled receptor:

Sample preparation: Cell membrane fractionation can improve detection of this membrane-localized receptor . Use appropriate lysis buffers containing protease inhibitors, and consider specialized membrane protein extraction methods.

Protein denaturation: Due to KISS1R's multiple transmembrane domains, avoid excessive heating that might cause protein aggregation.

Blocking and antibody incubation: Use 1-5% BSA in TBS-T for blocking and antibody dilutions rather than milk, which can interfere with detection of some membrane proteins. Commercial KISS1R antibodies may be supplied in specialized storage buffers (e.g., "0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol") .

Detection method: Enhanced chemiluminescence (ECL) with appropriate exposure times is generally suitable for KISS1R detection.

Molecular weight verification: KISS1R is expected to appear at approximately 43-45 kDa, though post-translational modifications may affect migration.

What factors should be considered when analyzing KISS1R expression in cancer tissues?

When analyzing KISS1R expression in cancer tissues, researchers should consider several critical factors that may influence interpretation:

Cancer type-specific expression patterns: KISS1R expression varies significantly between cancer types and even within the same cancer type at different stages. For example, in pancreatic cancer, KISS1R expression is higher in cancer tissue compared to adjacent normal tissue . In ovarian cancer, expression patterns correlate with prognosis .

Correlation with clinicopathological parameters: Studies have shown varying correlations between KISS1R expression and tumor grade, size, lymph node status, and histological type. For example, in breast cancer, some studies found no significant correlation with tumor grade or size, possibly due to small sample sizes (n = 43) .

Patient treatment history: Consider whether patients received neo-adjuvant chemotherapy or surgical intervention, as these factors can complicate data interpretation and contribute to discrepant findings .

Co-expression with KISS1: Analyze both KISS1 and KISS1R expression, as their relative levels and co-expression patterns provide more comprehensive prognostic information. In breast cancer, patients with high expression of both genes had the shortest relapse-free survival .

Subcellular localization: Document whether KISS1R staining is predominantly cytoplasmic, membranous, or nuclear, as localization may have functional implications.

How can I investigate potential non-canonical signaling of KISS1R in my experimental system?

Investigating non-canonical KISS1R signaling requires sophisticated approaches, as evidence suggests KISS1 may function through KISS1R-independent mechanisms in some contexts:

Cell model selection: Carefully select cell models that either express or lack KISS1R expression. Several studies demonstrate that "re-expression or over-expression of KISS1 results in phenotypic changes" even in cells that do not express detectable KISS1R .

Pathway analysis: Investigate multiple downstream signaling molecules beyond the canonical Gq/11-PLC-Ca²⁺ pathway. Evidence suggests KISS1 may affect alternative pathways, including "stabilizing the master of mitochondrial biogenesis PGC1α, inhibition of AMPK, and downregulation of PPARα" .

Knockdown/knockout experiments: Employ KISS1R knockdown or knockout approaches to determine whether observed effects of kisspeptin treatment persist in the absence of the receptor, which would suggest non-canonical mechanisms.

Alternative mediators: Explore potential alternative mediators such as PKCα, which has been implicated in KISS1R-independent metastasis suppression in ovarian and prostate cancer cells .

Dose and time-dependency: Test various concentrations of kisspeptins and exposure times, as "drug (i.e., KP or KP mimic) concentration and exposure time vary widely" across studies and may activate different signaling pathways .

How do I reconcile conflicting data on KISS1R's role as a tumor suppressor versus promoter?

The literature contains seemingly contradictory findings regarding KISS1R's role in cancer, requiring careful analysis to reconcile these differences:

Cancer-type specificity: KISS1/KISS1R functions appear to be "cancer context-specific and may be further modulated by other factors such as the micro-environment" . For example, KISS1R signaling appears tumor-suppressive in pancreatic, ovarian, and colorectal cancers but potentially tumor-promoting in certain breast cancers .

Stage-dependent effects: Evidence suggests a biphasic pattern in some cancers: "an initial upregulation of KISS1/KISS1R expression to suppress tumor progression" in early stages, followed by decreased expression in advanced disease . This is particularly evident in pancreatic, ovarian, and colorectal cancers.

Hormone receptor status: In breast cancer, KISS1/KISS1R expression and function correlate with estrogen receptor status. Studies have shown that "ERα signaling downregulates KISS/KISS1R levels," and in triple-negative breast cancer where ERα is absent, elevated KISS1/KISS1R expression associates with poor outcomes .

Methodological differences: Discrepancies may stem from "different experimental procedures and use of different reagents," particularly antibodies with varying specificity . When comparing studies, carefully evaluate whether they measured full-length KISS1 or smaller peptides, as these may have opposite relationships with cancer progression .

Sample size and clinical data completeness: Consider the statistical power of each study, as "the size of clinical cohorts remains a great challenge and care must be placed on the over-interpretation of data based on small clinical populations" .

What are the most reliable biomarkers to use alongside KISS1R for cancer prognosis assessment?

For comprehensive cancer prognosis assessment, KISS1R should be evaluated alongside complementary biomarkers:

Circulating kisspeptin levels: Plasma kisspeptin concentrations may serve as non-invasive biomarkers. In ovarian cancer, stage I patients showed significantly higher plasma kisspeptin levels (25.1 ±15.2 pmol/L) compared to advanced stages (11.8 ±10.3 pmol/L) and healthy controls (13.1 ±6.92 pmol/L) .

Estrogen receptor status: In breast cancer, the relationship between KISS1R and prognosis is significantly influenced by ER status. Studies have shown that "treatment of ERα-positive MCF7 and T47D breast cancer cells with tamoxifen... stimulated KISS1/KISS1R expression," indicating regulatory interactions .

Metastasis-associated markers: Combine KISS1R assessment with markers of invasion and metastasis, such as matrix metalloproteinases (MMPs). Research has shown that KISS1 expression can repress NF-κB translocation, thereby reducing MMP9 expression in certain cell lines .

Epigenetic modifications: In colorectal, bladder, and renal cell carcinomas, decreased KISS1 expression due to hypermethylation correlates with lower survival rates and increased metastasis . Therefore, methylation status of the KISS1 promoter may provide additional prognostic information.

What are the key methodological considerations for developing KISS1R-targeted therapeutics?

Developing KISS1R-targeted therapeutics requires addressing several methodological challenges:

Peptide stability and delivery: Native kisspeptins have limited half-lives in circulation. Researchers should focus on developing modified peptides or mimetics with improved stability and tissue penetration while maintaining receptor specificity.

Receptor specificity: Ensure that therapeutic candidates selectively activate KISS1R without affecting other GPCRs. This requires comprehensive screening against related receptors and off-target binding assessment.

Cancer context-specific effects: Given that KISS1R signaling can be either tumor-suppressive or tumor-promoting depending on the cancer type, therapeutics must be carefully tailored to specific cancer contexts. As noted in the literature, "the roles (that is, suppressor or promoter) these molecules play are cancer context-specific" .

Combination approaches: Consider combining KISS1R-targeted therapeutics with other treatments based on cancer type and molecular profile. For instance, in breast cancer where tamoxifen treatment affects KISS1/KISS1R expression , sequential or combination therapies might be explored.

Biomarker development: Develop companion diagnostics to identify patients most likely to benefit from KISS1R-targeted therapies, based on expression patterns of KISS1, KISS1R, and related pathway components.

How can I design experiments to investigate the relationship between circulating kisspeptins and local tissue KISS1R signaling?

Designing experiments to elucidate the relationship between circulating kisspeptins and local KISS1R signaling requires multi-faceted approaches:

Patient cohort studies: Collect matched plasma and tissue samples from cancer patients to correlate circulating kisspeptin levels with tissue KISS1R expression and activation. Previous studies have measured plasma kisspeptin in ovarian cancer patients, finding stage-dependent differences .

Ex vivo tissue exposure: Expose fresh patient-derived tissue slices to various concentrations of kisspeptins and assess receptor activation and downstream signaling events.

In vivo models with controlled kisspeptin administration: Develop animal models where circulating kisspeptin levels can be experimentally manipulated, followed by assessment of tissue-specific KISS1R signaling in multiple organs.

Cell-specific reporters: Engineer cell lines with KISS1R signaling reporters to monitor activation in response to exogenous kisspeptins versus locally produced peptides.

Microdialysis approaches: In appropriate model systems, use microdialysis to measure local tissue concentrations of kisspeptins in relation to circulating levels.

These experiments would address fundamental questions raised in the literature, such as: "Do circulating plasma kisspeptin modify cancerous tissue? Do tumors produce kisspeptin in situ? Do normal cells produce kisspeptin to minimize metastasis?"

What are the most promising approaches for improving the specificity of KISS1R antibodies for research applications?

Improving KISS1R antibody specificity is crucial for advancing research in this field, particularly given documented concerns about current antibodies:

Multi-epitope validation strategy: Generate antibodies against multiple discrete epitopes of KISS1R and validate concordance between detection methods. This addresses concerns that "many of the commercially-available KISS1R antibodies in use are not highly specific" .

Knockout validation systems: Develop comprehensive validation systems using CRISPR/Cas9-mediated KISS1R knockout cell lines and tissues as negative controls. This approach is specifically recommended in the literature, which notes that "antibody specificity should be verified using knock-out and/or knock-down approaches" .

Cross-species epitope selection: Target highly conserved regions of KISS1R across species to develop antibodies with broader research applications. Current commercial antibodies show reactivity across human, mouse, and rat, with predicted reactivity to additional species .

Recombinant antibody technology: Utilize phage display or similar technologies to develop highly specific recombinant antibodies or antibody fragments with enhanced specificity.

Application-specific optimization: Recognize that different applications (WB, IHC, ELISA) may require differently optimized antibodies, and validate each antibody specifically for its intended application rather than assuming cross-application reliability.