GPR150 Antibody

What are GPR15 and GPR155, and what cellular functions do they serve?

GPR15, also known as BOB, is a seven-transmembrane G protein-coupled receptor expressed in CD4+ T cells and alveolar macrophages. It functions as a cellular co-receptor for some isolates of HIV-1, HIV-2, and SIV through interactions with several viral envelope proteins . GPR15 has also been shown to play a protective role in antibody-mediated skin inflammation, potentially by limiting the recruitment of γδ T cells into the dermis .

GPR155, also known as Lysosomal cholesterol signaling protein (LYCHOS), is a cholesterol receptor on the lysosome that plays a role in the cholesterol-sensing lysosomal pathway and couples cholesterol concentration to MTORC1-dependent anabolic signaling .

What species reactivity can be expected when working with commercial GPR antibodies?

Most commercially available GPR15 antibodies show high sequence identity across species. Human GPR15 shares 96%-100% amino acid sequence identity with chimpanzee, macaque, and rhesus GPR15, and 76% with mouse GPR15 . The GPR155 antibody (12659-1-AP) from Proteintech has been tested for reactivity with human and mouse samples . When selecting an antibody for your research, confirm the species reactivity in the product documentation.

What applications are GPR antibodies typically used for in research settings?

GPR antibodies are commonly used in several research applications including:

Note: Optimal dilutions should be determined by each laboratory for each application .

How should researchers design flow cytometry experiments to detect GPR15 expression in transfected cell lines?

When designing flow cytometry experiments to detect GPR15 expression:

• Include appropriate controls, such as cells transfected with empty vectors or irrelevant proteins (e.g., eGFP alone)

• Use isotype control antibodies (e.g., Mouse IgG2B Flow Cytometry Isotype Control) to determine non-specific binding

• Select fluorophore-conjugated secondary antibodies compatible with your flow cytometer configuration

• Consider using HEK293 transfection systems as positive controls, as they have been validated for GPR15 detection

• For visualization of results, compare staining of GPR15-transfected versus non-transfected cells in histogram format

The scientific literature shows successful detection of GPR15 in HEK293 human cell lines transfected with human GPR15 and eGFP using Mouse Anti-Human GPR15 Monoclonal Antibody (MAB3654) followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody .

What considerations are important when investigating GPR15 expression changes in inflammatory conditions?

When studying GPR15 expression in inflammatory conditions, researchers should consider:

• Regulatory dynamics: GPR15 and its ligand GPR15L show inverse regulation during inflammation. GPR15 mRNA is expressed at relatively high levels in naïve wild-type skin but significantly decreases in inflamed skin, while GPR15L is barely detectable in naïve skin but markedly upregulated in inflamed skin .

• Cell population changes: In murine models of autoimmune skin inflammation, GPR15+ cells in lymph nodes increase slightly during disease progression. While GPR15+ cells are mainly CD3-CD19- under naïve conditions, a significant proportion expresses CD3 during inflammation, with GPR15 mostly expressed on CD8+ cells .

• Tissue-specific effects: GPR15 can play different roles depending on the tissue and inflammatory context. In antibody-mediated skin inflammation, GPR15 appears to be protective, as GPR15-deficient mice show exacerbated disease .

• Experimental controls: Include time-course analyses to capture dynamic expression changes, and compare multiple tissue sites to understand tissue-specific regulation.

How can researchers accurately quantify subepidermal cleft formation in skin inflammation models when studying GPR15 function?

To accurately quantify subepidermal cleft formation in skin inflammation models:

• Collect tissue samples at standardized time points (e.g., day 14 in BP-like EBA models)

• Use consistent histological processing and staining protocols

• Measure the extent of subepidermal clefts using digital morphometry

• Normalize measurements to total epidermal length

• Perform blinded analysis to prevent observer bias

• Include appropriate controls (wild-type versus knockout animals)

• Correlate cleft formation with clinical disease scores and immune cell infiltration

Research has shown that GPR15-deficient mice exhibit significantly more pronounced subepidermal cleft formation compared to wild-type mice in the BP-like EBA model, suggesting a higher activity of neutrophils, the major drivers of cleft formation, in the dermis of GPR15-deficient mice .

What is the optimal protocol for immunohistochemical detection of GPR155 in brain tissue samples?

For optimal immunohistochemical detection of GPR155 in brain tissue:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

• Immunostaining procedure:

  • Block endogenous peroxidase and non-specific binding

  • Dilute GPR155 antibody (e.g., 12659-1-AP) at 1:50-1:500

  • Incubate at 4°C overnight

  • Apply appropriate detection system

  • Counterstain with hematoxylin

• Validation and controls:

  • Include positive control tissues (mouse or human brain)

  • Include negative controls (omitting primary antibody)

  • Consider dual staining with neuronal markers for colocalization studies

This protocol has been verified for both human and mouse brain tissue samples .

What techniques should be employed to distinguish GPR15-expressing cell populations in lymphoid tissues?

To distinguish GPR15-expressing cell populations in lymphoid tissues:

• Multi-parameter flow cytometry panel design:

  • Include GPR15 antibody

  • Add lineage markers: CD3 (T cells), CD19 (B cells)

  • Add subset markers: CD4, CD8 (T cell subsets)

  • Consider additional markers for innate lymphoid cells and dendritic cells

• Gating strategy:

  • First gate on live cells (using viability dye)

  • Identify GPR15+ population

  • Determine co-expression with lineage markers

  • Further characterize positive populations

• Analysis considerations:

  • Approximately 2-3% of living cells express GPR15 in lymphoid tissues

  • GPR15+ cells are mainly CD3-CD19- under naïve conditions

  • During inflammation, a significant proportion of GPR15+ cells express CD3

  • GPR15 is predominantly expressed on CD8+ T cells during inflammation

  • There is typically no co-expression with CD19 (B cell marker)

• Validation approaches:

  • Compare expression patterns in inflamed versus non-inflamed tissues

  • Use GPR15-deficient samples as negative controls

This approach has been validated in studies of inguinal lymph nodes and spleen tissues in mouse models of skin inflammation .

How does GPR15 mechanistically counteract antibody-mediated skin inflammation?

The protective mechanism of GPR15 in antibody-mediated skin inflammation appears to involve:

• Regulation of γδ T cell recruitment: GPR15-deficient (Gpr15−/−) mice show significantly increased accumulation of γδ T cells in the dermis during experimental epidermolysis bullosa acquisita (BP-like EBA), compared to wild-type mice .

• Inverse regulation with GPR15L: During skin inflammation, GPR15 expression decreases while its ligand GPR15L is markedly upregulated, suggesting a feedback regulatory mechanism .

• Effects on subepidermal cleft formation: GPR15-deficient mice exhibit more pronounced subepidermal cleft formation, the histopathological correlate of blisters and erosions in BP-like EBA .

• Specific T cell populations: GPR15 expression during inflammation is predominantly on CD8+ T cells, though the exact mechanism by which these cells might mediate protection remains under investigation .

The research suggests that enhancing GPR15 activity could represent a novel therapeutic approach for pemphigoid diseases, including bullous pemphigoid-like epidermolysis bullosa acquisita .

What are the technical challenges in studying the interaction between GPR15 and viral envelope proteins?

Studying GPR15 interactions with viral envelope proteins presents several technical challenges:

• Protein expression and purification:

  • GPR15 is a seven-transmembrane G protein-coupled receptor, making it difficult to express and purify in its native conformation

  • Viral envelope proteins often require proper glycosylation for functional interactions

• Interaction assay design:

  • Direct binding assays may not reflect the complex membrane environment required for physiological interactions

  • Cell-based assays need to control for co-receptor expression patterns

• Specificity determination:

  • GPR15 functions as a co-receptor for some but not all isolates of HIV-1, HIV-2, and SIV

  • Determining the structural basis for this selectivity requires extensive mutational analysis

• Physiological relevance:

  • In vitro binding studies need to be validated in relevant primary cell types

  • The high sequence conservation of GPR15 across primates (96-100% identity) suggests important functional constraints that should be considered in experimental design

Researchers should consider using systems biology approaches combining structural biology, cell-based assays, and in vivo models to comprehensively characterize these interactions .

What strategies can resolve discrepancies in observed molecular weights when detecting GPR155 by Western blot?

When addressing molecular weight discrepancies in GPR155 Western blots:

These strategies will help ensure accurate interpretation of Western blot results when studying GPR155 .

How can researchers optimize storage and handling of anti-GPR antibodies to maintain reactivity?

To maintain optimal reactivity of anti-GPR antibodies:

• Storage conditions:

  • Store at -20°C for long-term stability

  • Avoid repeated freeze-thaw cycles by aliquoting upon receipt

  • For short-term storage (up to 1 month), 2-8°C under sterile conditions is acceptable after reconstitution

  • For medium-term storage (up to 6 months), -20 to -70°C under sterile conditions after reconstitution is recommended

• Reconstitution practices:

  • Use sterile techniques when handling antibodies

  • Follow manufacturer's specific reconstitution instructions

  • For antibodies supplied with 50% glycerol, aliquoting is unnecessary for -20°C storage

• Working solution preparation:

  • Dilute in appropriate buffer immediately before use

  • Discard unused working solutions

  • Maintain cold chain during experimental procedures

• Quality control measures:

  • Test reactivity periodically with positive controls

  • Record lot numbers and expiration dates

  • Monitor performance across experiments for consistency

Proper storage and handling will maximize antibody shelf life and ensure reproducible experimental results across studies .