GPR107 Antibody

What is GPR107 and why is it important in biological research?

GPR107 (G protein-coupled receptor 107), also known as LUSTR1 or KIAA1624, is a seven-transmembrane protein belonging to the LU7TM family. It functions as a multi-pass membrane protein involved in critical cellular processes including signal transduction, cell growth regulation, and intracellular trafficking . GPR107 has gained significant research interest because:

• It is essential for embryonic development (GPR107 knockout in mice is embryonically lethal)

• It plays a role in receptor-mediated endocytosis and recycling

• It has been implicated in several pathological conditions including prostate cancer and diabetic nephropathy

• It serves as a host factor for bacterial toxin intoxication (e.g., Pseudomonas aeruginosa exotoxin A)

GPR107 represents a promising research target for understanding fundamental cellular mechanisms and developing potential therapeutic strategies for diseases involving membrane trafficking and signal transduction.

To maintain optimal antibody performance and extend shelf life:

• Store concentrated antibody stocks at -20°C for long-term storage (up to one year)

• For frequent use and short-term storage, keep at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce binding efficiency

• Most GPR107 antibodies are supplied in liquid format containing PBS with 50% glycerol, 0.5% BSA, and 0.02-0.1% sodium azide as preservative

• When using for experimental procedures, dilute antibodies in fresh buffer according to the specific application requirements

• Follow manufacturer validation data for optimal performance in your specific application

What are the common immunogen strategies used for GPR107 antibody production?

GPR107 antibodies are typically generated using the following immunogen strategies:

• Synthetic peptides derived from the internal region of human GPR107

• Fusion proteins with specific GPR107 regions (e.g., GPR107 Fusion Protein Ag18649)

• Immunogens targeting specific epitopes within amino acid ranges 141-190 of the human GPR107 sequence

These strategies help ensure antibody specificity while optimizing recognition of the target protein in its native conformation or under denatured conditions, depending on the intended application.

What role does GPR107 play in prostate cancer progression and how can antibodies help investigate this mechanism?

GPR107 has been identified as a potential diagnostic, prognostic, and therapeutic target in prostate cancer (PCa), particularly in aggressive castration-resistant prostate cancer (CRPC). Research indicates that:

• GPR107 is overexpressed in PCa and associated with advanced disease stage, vascular invasion, and metastasis

• GPR107 silencing inhibits proliferation and migration rates in androgen-independent PCa cells

• GPR107 knockdown alters key oncogenic signaling pathways including KI67/CDKN2D/MMP9/PRPF40A and AKT signaling

• Neuronostatin (NST), which may interact with GPR107, inhibits proliferation/migration only in androgen-independent PCa cells

Methodological approach for GPR107 investigation in cancer:

• Use GPR107 antibodies for expression analysis in tissue microarrays comparing normal prostate, PCa, and CRPC samples

• Employ siRNA/shRNA techniques for GPR107 silencing followed by functional assays (proliferation, migration, invasion)

• Analyze downstream signaling pathway alterations through phospho-specific antibodies and Western blot

• Investigate potential GPR107-NST interaction through co-immunoprecipitation studies

• Validate findings in patient-derived xenograft models

This research direction holds promise for developing novel therapeutic strategies targeting GPR107 in aggressive prostate cancer.

How does GPR107 contribute to diabetic nephropathy, and what experimental approaches can elucidate its mechanism?

Recent research has uncovered a critical role for GPR107 in diabetic nephropathy (DN), particularly in regulating collagen type IV (COL4) balance in podocytes:

• GPR107 expression is significantly decreased in renal tissues from DN patients and streptozocin (STZ)-induced DN mice

• GPR107-deficient mice with STZ-induced DN exhibit more severe kidney damage, marked by increased glomerular basement membrane (GBM) thickening and COL4 accumulation

• GPR107 deficiency under high-glucose conditions promotes COL4 accumulation in the extracellular matrix (ECM) of podocytes through increased COL4 production and decreased degradation

• Mechanistically, GPR107 facilitates angiotensin II receptor type 1 (AT1R) internalization through clathrin-mediated endocytosis in podocytes

Experimental approaches to investigate GPR107 in DN:

• Generate GPR107-deficient cell lines using CRISPR-Cas9 technology with appropriate primers (forward: 5'-CAGAGGAGACCACGTTAGAAGT-3', reverse: 5'-CTGGGCTCAGGTGAAGAGATG-3')

• Perform co-immunoprecipitation to detect protein interactions between GPR107, AT1R, and clathrin

• Employ transmission electron microscopy to measure GBM thickness

• Conduct rescue experiments by transfecting GPR107-deficient cells with plasmids containing GPR107 cDNA

• Use inhibitors like Losartan (10 μM) or SKF-96365 (40 μM) to block AT1R or calcium channels in podocytes

These findings suggest GPR107 as a potential therapeutic target for diabetic nephropathy.

What is the significance of GPR107 in bacterial toxin intoxication and host-pathogen interactions?

GPR107 plays a critical role in bacterial toxin intoxication, particularly with Pseudomonas aeruginosa exotoxin A (PE):

• Genome-wide genetic screens identified GPR107 as an essential host factor for PE intoxication

• GPR107 functions in intracellular trafficking steps necessary for toxin action

• GPR107 may act as a receptor that associates with G-proteins at the Golgi to regulate membrane transport

• GPR107 knockout cells show resistance to multiple bacterial toxins including PE, Campylobacter jejuni cytolethal distending toxin (CDT), and ricin

Experimental approaches for investigating GPR107 in toxin biology:

• Generate GPR107-depleted cell lines using gene-trap (GT) mutagenesis or CRISPR-Cas9 technology

• Design targeted disruption with sequences such as GGTGCCATCCTCTTCCCAG (GPR107 CRISPR target)

• Verify knockout through RT-PCR and genomic DNA analysis

• Perform toxin challenge assays comparing wildtype and GPR107-deficient cells

• Conduct rescue experiments by reintroducing GPR107 cDNA variants to determine functional domains

Understanding GPR107's role in toxin trafficking provides insights into both pathogen mechanisms and fundamental cellular trafficking pathways.

How does GPR107 interact with neuronostatin signaling and what experimental approaches can validate this relationship?

GPR107 has been identified as a promising candidate receptor for neuronostatin (NST), a peptide derived from the somatostatin gene:

• NST treatment inhibits proliferation and migration specifically in androgen-independent prostate cancer cells through potential GPR107 interaction

• NST decreases GPR107 expression exclusively in androgen-independent PCa cells, suggesting negative feedback regulation

• The NST/GPR107 interaction impacts cAMP-independent protein kinase A (PKA) phosphorylation

Experimental validation approaches:

• Perform co-immunoprecipitation studies using GPR107 antibodies to detect NST-GPR107 binding

• Conduct competitive binding assays with labeled NST in presence/absence of GPR107 antibodies

• Employ FRET or BRET techniques to visualize direct interactions in live cells

• Use phospho-specific antibodies to monitor downstream signaling cascades (particularly PKA phosphorylation at T197)

• Validate findings through GPR107 silencing/overexpression approaches followed by NST treatment

This relationship between NST and GPR107 presents opportunities for targeted interventions in various pathophysiological contexts, including prostate cancer and cardiovascular regulation.

What are the key considerations for Western blot optimization when using GPR107 antibodies?

For optimal Western blot results when detecting GPR107:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent protein degradation

• Protein loading: Load approximately 100 picograms total protein per sample

• Blocking: Perform overnight blocking with 5% BSA to minimize background

• Primary antibody incubation: Optimal dilution ranges from 1:500-1:2000; incubate overnight at 4°C

• Secondary antibody: Use HRP-linked antibodies at 1:1000-1:5000 dilution for 1-2 hours at room temperature

• Detection system: Choose enhanced chemiluminescence (ECL) or fluorescence-based detection depending on required sensitivity

• Expected molecular weight: Approximately 67 kDa (calculated molecular weight: 66990 Da)

• Controls: Include both positive controls (tissues/cells known to express GPR107) and negative controls (GPR107 knockout cells if available)

For challenging applications, consider using the SNAP ID Western blot analysis system with GPR107 antibody diluted at 1:500 .

How can researchers effectively validate GPR107 antibody specificity?

Thorough validation of GPR107 antibody specificity is critical for reliable experimental results:

• Genetic validation approaches:

  • Compare antibody reactivity between wildtype and GPR107 knockout/knockdown cells

  • Perform antibody testing on cells overexpressing tagged GPR107 constructs

  • Use siRNA/shRNA-mediated knockdown to confirm signal reduction correlates with GPR107 depletion

• Biochemical validation approaches:

  • Conduct peptide competition assays using the immunogen peptide

  • Perform Western blot analysis to confirm detection of a single band at the expected molecular weight

  • Test multiple antibodies targeting different GPR107 epitopes to confirm consistent results

• Imaging validation approaches:

  • Compare immunofluorescence patterns with subcellular markers appropriate for GPR107 localization

  • Co-localize GPR107 with known interaction partners such as clathrin or AT1R

  • Verify specificity through signal loss in GPR107-deficient cells

Proper validation ensures experimental rigor and reproducibility in GPR107 research.

What are the key technical considerations for studying GPR107 in endocytosis and trafficking pathways?

To effectively study GPR107's role in endocytosis and trafficking:

• Cell model selection:

  • Choose appropriate cell lines with endogenous GPR107 expression or establish stable GPR107-expressing cell lines

  • Consider cell-specific contexts where GPR107 function has been implicated (e.g., podocytes for AT1R trafficking, KBM7 cells for toxin studies)

• Visualization strategies:

  • Generate fluorescently-tagged GPR107 constructs for live-cell imaging

  • Perform co-localization studies with endocytic markers (e.g., clathrin, transferrin, EEA1)

  • Employ super-resolution microscopy for detailed trafficking analysis

• Functional assays:

  • Conduct transferrin uptake and recycling assays in GPR107-depleted cells

  • Perform cargo-specific endocytosis assays (e.g., AT1R internalization in podocytes)

  • Use toxin challenge assays (e.g., P. aeruginosa exotoxin A) as functional readouts

• Molecular interaction analysis:

  • Implement co-immunoprecipitation to confirm interactions with trafficking machinery

  • Verify protein-protein interactions using proximity ligation assays

  • Conduct membrane fractionation to analyze GPR107 distribution

By combining these approaches, researchers can comprehensively characterize GPR107's role in membrane trafficking pathways.

What genetic tools are available for GPR107 functional studies?

Researchers can utilize several genetic approaches to study GPR107 function:

• CRISPR-Cas9 genome editing:

  • Target sequence for GPR107 knockout: GGTGCCATCCTCTTCCCAG

  • Verify knockout through genomic PCR using specific primers:
    Forward: 5'-CAGAGGAGACCACGTTAGAAGT-3'
    Reverse: 5'-CTGGGCTCAGGTGAAGAGATG-3'

• RNA interference approaches:

  • siRNA for transient knockdown

  • shRNA for stable knockdown through lentiviral transduction

• Overexpression systems:

  • Generate stable cell lines expressing wildtype GPR107

  • Create tagged versions (HA, Flag, GFP) for detection and interaction studies

  • Develop rescue cell lines by reintroducing GPR107 into knockout backgrounds

• Animal models:

  • Note that complete GPR107 knockout in mice is embryonically lethal

  • Consider conditional knockout approaches for tissue-specific studies

  • STZ-induced diabetic nephropathy model in GPR107-deficient mice has been established

These genetic tools provide versatile approaches for investigating GPR107 function across different experimental contexts.

What precautions should be taken when interpreting GPR107 antibody results across different species?

When working with GPR107 antibodies across species boundaries:

• Species reactivity verification:

  • Commercially available GPR107 antibodies typically target human and/or mouse GPR107

  • Sequence conservation analysis is essential when extending to other species

  • Validate antibody cross-reactivity through Western blot of multiple species samples

• Epitope conservation considerations:

  • Examine sequence homology in the target epitope region across species

  • For antibodies targeting synthetic peptides from internal regions, verify peptide conservation

  • Consider using antibodies raised against highly conserved domains for cross-species studies

• Control implementation:

  • Include positive controls from validated species

  • Whenever possible, include GPR107-deficient samples as negative controls

  • Consider using recombinant GPR107 from the species of interest as additional controls

• Assay-specific optimization:

  • Adjust antibody concentrations and incubation conditions for cross-species applications

  • Optimize blocking and washing conditions to minimize non-specific binding

  • Validate antibody performance in each application context for the target species

Careful attention to these considerations helps ensure reliable cross-species interpretations in GPR107 research.

Understanding these methodological nuances ensures optimal experimental design and interpretation when studying GPR107 across different research contexts.