RXFP1 Antibody

What is RXFP1 and why is it an important target for antibody-based research?

RXFP1 is a G protein-coupled receptor also known as Relaxin Receptor 1, LGR7, or relaxin/insulin-like family peptide receptor 1. This receptor is structurally complex with an N-terminal extracellular domain containing an LDLa module and 10 leucine-rich repeats (LRRs), followed by seven transmembrane domains. RXFP1 has a molecular weight of approximately 87 kilodaltons .

The receptor is primarily activated by relaxin-2, an important regulator of reproductive and cardiovascular physiology. Research interest in RXFP1 has grown substantially due to its emerging roles in fibrotic diseases, making antibodies against this receptor valuable tools for:

• Studying receptor expression and localization in normal and diseased tissues

• Investigating changes in expression levels during disease progression

• Elucidating signaling mechanisms unique to this receptor

• Validating potential therapeutic approaches targeting the relaxin/RXFP1 axis

What applications are RXFP1 antibodies validated for in research settings?

Based on available research data, RXFP1 antibodies have been validated for multiple experimental applications:

When designing experiments, researchers should note that optimal dilutions are application-dependent and should be determined empirically for each specific experimental system . It's particularly important to validate antibodies when studying tissues with low RXFP1 expression levels to ensure sufficient sensitivity and specificity.

How is RXFP1 expression distributed across tissue types?

RXFP1 expression varies significantly across tissue types, with important implications for experimental design:

• Normal tissues: Expression has been detected in reproductive tissues (uterus, testes), brain, heart, and vascular tissues

• Disease tissues: Upregulated expression has been observed in fibrotic scarring in liver tissues from patients with non-alcoholic steatohepatitis (NASH) and autoimmune hepatitis (AIH)

• Cell types: Expression is particularly notable in activated hepatic stellate cells (HSCs), with much lower expression in quiescent HSCs

When designing antibody-based experiments, consider that RXFP1 expression levels may be 400-fold lower in primary human hepatic stellate cells and LX-2 cell lines compared to overexpressing recombinant systems like HEK293-RXFP1 cells . This significant difference necessitates careful optimization of detection methods and potentially signal amplification approaches.

What methodological approaches can overcome the challenges of detecting low RXFP1 expression?

Detecting endogenous RXFP1 presents significant challenges due to its relatively low expression in many tissue types. Research has demonstrated several methodological approaches to address this issue:

• Signal amplification techniques: When using immunohistochemistry, consider biotin-streptavidin amplification systems or tyramide signal amplification to enhance detection sensitivity .

• Recombinant expression systems: For mechanistic studies, the BacMam expression system has been successfully used to adjust RXFP1 expression levels in HEK293 cells, increasing signal windows from 1.4 (in endogenous systems) to greater than 3 in the HEK293-RXFP1 cells .

• Complementary methods: Combine antibody-based protein detection with mRNA analysis techniques such as in situ hybridization to confirm expression patterns. This approach was successfully employed to detect RXFP1 mRNA in fibrotic liver tissues .

• Antigen retrieval optimization: For FFPE tissues, studies have shown that different antigen retrieval methods significantly affect RXFP1 detection. TE buffer at pH 9.0 may yield better results than citrate buffer at pH 6.0 for certain antibodies .

These techniques must be carefully validated for each experimental system, as the specific microenvironment of different tissues can affect epitope accessibility and antibody binding.

How do alternative splicing variants of RXFP1 impact antibody selection and experimental interpretation?

Alternative splicing of RXFP1 has emerged as an important consideration in experimental design. Research has identified multiple splice variants with potential functional consequences, particularly in fibrotic diseases .

When selecting antibodies and designing experiments:

• Consider epitope location: Choose antibodies targeting conserved regions if detecting all variants is desired, or select epitopes specific to particular splice variants for differential analysis.

• Validate with molecular weight analysis: Western blots should be carefully analyzed for bands of unexpected molecular weights, which may represent splice variants rather than non-specific binding.

• Complementary transcript analysis: Combine protein detection with RT-PCR using primers spanning potential splice junctions to correlate protein expression with specific transcript variants.

• Functional correlations: When studying receptor signaling, be aware that splice variants may exhibit altered signaling properties. In fibrotic tissues, reduced RXFP1 expression and alternative splicing have been associated with diminished responsiveness to relaxin treatment .

Recent studies suggest that splice variants may act as important regulators of canonical RXFP1 expression in different fibrotic diseases, potentially explaining the limited efficacy of relaxin-based therapies in clinical trials for systemic sclerosis despite promising preclinical data .

What are the key considerations for validating RXFP1 antibody specificity?

Validating antibody specificity is particularly critical for RXFP1 research due to its structural complexity and relatively low expression levels. Comprehensive validation should include:

• Positive and negative control tissues: Use tissues with known RXFP1 expression profiles. Research has validated human brain tissue, human testis tissue, mouse testis tissue, and mouse brain tissue as positive controls for certain antibodies .

• Recombinant expression systems: Comparing wild-type cells to those overexpressing RXFP1 can validate specificity, as demonstrated in studies using HEK293 cells with BacMam-induced RXFP1 expression .

• Knockout/knockdown controls: Where possible, use genetic approaches to reduce or eliminate RXFP1 expression as a negative control.

• Peptide competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining.

• Cross-validation with multiple antibodies: Use antibodies targeting different epitopes of RXFP1 to confirm results.

• Correlation with functional assays: Confirm that antibody-detected RXFP1 correlates with functional responses such as cAMP generation in response to relaxin or ML290 stimulation .

How can RXFP1 antibodies be used to investigate the unique activation mechanisms of this receptor?

RXFP1 has a unique activation mechanism among GPCRs, involving its LDLa module and an autoinhibition mechanism. Antibodies are valuable tools for investigating these mechanisms:

• Domain-specific antibodies: Antibodies targeting the LDLa module, extracellular loop 2 (ECL2), or the leucine-rich repeat domain can help elucidate domain-specific functions in receptor activation.

• Conformation-specific antibodies: Recent research has revealed that RXFP1 signals through a mechanism of autoinhibition, wherein the receptor's ECL2 occupies the orthosteric site in the active state but is inhibited by the ectodomain in the absence of relaxin-2 . Conformation-specific antibodies could help detect these different states.

• Co-immunoprecipitation studies: Antibodies can be used to investigate interactions between RXFP1 and other proteins, including G proteins, revealing how specific residues like Leu-7, Tyr-9, and Lys-17 in the LDLa module contribute to receptor activation through hydrophobic interactions .

• Mutation analysis support: Antibodies can validate the expression and localization of RXFP1 mutants in structure-function studies. For example, studies have shown that mutations of Phe564 and Leu566 in ECL2 almost completely abolished RXFP1 signaling in response to relaxin-2 .

How do experimental findings using RXFP1 antibodies inform therapeutic approaches for fibrotic diseases?

Research using RXFP1 antibodies has revealed important insights for therapeutic development:

• Expression correlation with disease stage: Immunohistochemical studies have demonstrated a positive correlation between RXFP1 transcript levels and the stage of fibrosis in liver disease, suggesting potential utility as a biomarker .

• Cellular targets for therapy: Antibody-based studies have identified activated hepatic stellate cells as the primary targets of relaxin in liver fibrosis, with upregulated RXFP1 expression compared to quiescent HSCs .

• Mechanism of action studies: Investigations of small molecule RXFP1 agonists such as ML290 have used antibodies to validate RXFP1 expression in target cells. These studies reveal potential disconnects between cAMP activation and changes in expression of fibrotic markers, highlighting the need to understand cell- and tissue-specific signaling mechanisms .

• Therapeutic resistance mechanisms: Immunohistochemical analyses have revealed that reduced RXFP1 expression in fibrotic lung and skin tissues may explain the lack of response to exogenous relaxin treatments in clinical trials, despite promising preclinical data .

These findings suggest that future therapeutic approaches may need to include strategies to upregulate RXFP1 expression or develop biased agonists that can activate signaling pathways even in tissues with low receptor expression or altered splice variant profiles.

What are the recommended fixation and antigen retrieval protocols for RXFP1 immunohistochemistry?

Optimal detection of RXFP1 in tissue sections requires careful consideration of fixation and antigen retrieval methods:

• Fixation: Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used for RXFP1 immunohistochemistry in multiple studies .

• Antigen retrieval methods:

  • TE buffer at pH 9.0 is recommended for some antibodies

  • Alternative antigen retrieval with citrate buffer at pH 6.0 may be performed, but comparative studies suggest potentially lower sensitivity

  • Heat-induced epitope retrieval is generally more effective than enzymatic methods

• Section thickness: 4-5 μm sections are typically used for optimal antibody penetration and signal-to-noise ratio.

• Controls: Include appropriate positive control tissues (such as human uterus endometrium, which has been validated for some RXFP1 antibodies) .

An example protocol validated for RXFP1 detection in human tissue:

• Deparaffinize and rehydrate tissue sections

• Perform heat-induced antigen retrieval using TE buffer (pH 9.0) for 20 minutes

• Block endogenous peroxidase with 3% H₂O₂

• Apply primary RXFP1 antibody at 10 μg/ml and incubate overnight at 4°C

• Apply appropriate secondary antibody and detection system

• Counterstain, dehydrate, and mount

How can researchers effectively use RXFP1 antibodies to study receptor dimerization and signaling complexes?

RXFP1 dimerization and complex formation with other signaling molecules are emerging as important regulatory mechanisms. Effective study of these phenomena requires specialized approaches:

• Co-immunoprecipitation: Using RXFP1 antibodies for pull-down experiments can identify interaction partners. This approach requires antibodies with high specificity and affinity for native conformations of the receptor.

• Proximity ligation assays (PLA): This technique can detect protein-protein interactions within 40 nm distance in situ, making it valuable for studying RXFP1 homo- and heterodimerization in native tissue contexts.

• Bioluminescence/fluorescence resonance energy transfer (BRET/FRET): These techniques can be used in conjunction with antibody validation to study receptor dimerization dynamics in live cells.

• Cross-linking studies: Chemical cross-linking followed by immunoprecipitation with RXFP1 antibodies can stabilize transient interactions for subsequent analysis.

Research has shown that receptor dimerization plays important roles in relaxin/RXFP1 signaling, and these molecular interactions may have significant implications for developing therapeutics to restore relaxin/RXFP1 signaling in diseases . Careful selection of antibodies that do not interfere with dimerization interfaces is essential for these studies.

What considerations should guide the selection of RXFP1 antibodies for studying both human and animal models?

When designing studies that span human and animal models, antibody selection requires careful consideration of species cross-reactivity and epitope conservation:

When conducting comparative studies, it is advisable to validate antibody performance in each species and application independently, even when suppliers claim cross-reactivity.

How can researchers correlate RXFP1 antibody-based protein detection with functional receptor activity?

Correlating protein expression with functional activity is crucial for understanding RXFP1 biology. Several methodological approaches facilitate this correlation:

• Parallel cAMP assays: RXFP1 activation increases intracellular cAMP levels. Research has demonstrated that endogenous RXFP1 levels in primary cells correlate with cAMP responses to relaxin stimulation, with pEC₅₀ values of approximately 9.0 in LX-2 and primary HSCs compared to 11.0 in overexpressing HEK293-RXFP1 cells .

• Phosphorylation-specific antibodies: RXFP1 activation leads to phosphorylation of downstream signaling molecules. Antibodies detecting these phosphorylated proteins can be used alongside total RXFP1 detection.

• Gene expression analysis: RXFP1 activation affects expression of target genes. Correlating receptor detection with changes in these genes provides functional validation.

• Mutational analysis: Studies have shown that mutations in key residues (e.g., Phe564 and Leu566 in ECL2) abolish signaling despite normal receptor expression . Comparing antibody detection of wild-type and mutant receptors with their signaling capabilities can reveal structure-function relationships.

• Small molecule agonist response: ML290 and its derivatives activate RXFP1 through a mechanism distinct from relaxin. Comparing antibody detection with responses to both relaxin and ML290 can provide insights into receptor conformation and signaling bias .