kdelr3 Antibody

What is KDELR3 and what is its primary function in cells?

KDELR3 (KDEL Endoplasmic Reticulum Protein Retention Receptor 3) is part of the KDEL receptor family responsible for retrieving soluble proteins containing the C-terminal KDEL tetrapeptide signal from the cis-Golgi back to the endoplasmic reticulum (ER). Unlike the traditional view of these receptors functioning exclusively between ER and Golgi, recent evidence suggests KDELR3 may also be expressed at the plasma membrane . The protein consists of 214 amino acids with a calculated molecular weight of 25 kDa, though it typically appears as a 28 kDa band in Western blots . KDELR3 plays a role in maintaining ER homeostasis by preventing the secretion of ER-resident chaperones and enzymes, though it appears to have more specialized functions compared to other KDEL receptors, particularly in signaling pathways rather than primarily in protein retrieval .

How does KDELR3 differ functionally from other KDEL receptors?

In HeLa cells, KDELR3 plays a minor role in client retrieval compared to KDELR2, which is the primary receptor responsible for preventing secretion of ER-resident proteins . Instead, KDELR3 serves a distinct signaling role that opposes that of KDELR1. Specifically, KDELR3 inhibits the accumulation of AGR2 transcripts, while KDELR1 stimulates AGR2 production . This functional specialization suggests that the three KDEL receptors have evolved distinct roles beyond simple protein retrieval. Experiments involving single and combined knockdowns of KDEL receptors demonstrate that KDELR3 silencing leads to increased AGR2 levels without causing substantial secretion of ER-resident proteins, highlighting its primary role in signaling rather than retention .

What applications are KDELR3 antibodies most commonly used for?

KDELR3 antibodies are primarily utilized for:

• Western Blotting (WB): For detection of endogenous KDELR3 protein, typically observed at 28 kDa

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of KDELR3

• Immunocytochemistry (ICC) and Immunofluorescence (IF): For cellular localization studies

• Immunohistochemistry (IHC): For tissue expression analysis

Most commercially available KDELR3 antibodies are rabbit polyclonal antibodies that have been validated for human and mouse samples, with predicted reactivity across multiple species including rat, cow, dog, horse, pig, rabbit, and guinea pig .

How can researchers distinguish between potential artifacts from KDELR3 overexpression and physiological functions?

The study of KDELR3 has been complicated by potential artifacts arising from overexpression and the lack of antibodies efficiently discriminating between the three KDEL receptors . To address this challenge, researchers should:

• Use CRISPR-Cas9 genome editing techniques to introduce tags at the C-terminal end of endogenous KDELR3, as demonstrated for KDELR1 . This approach maintains endogenous expression levels while enabling detection.

• Implement surface biotinylation protocols to specifically detect cell surface expression of KDELR3, distinguishing between internal and plasma membrane pools .

• Validate KDELR3 knockdown efficiency at both mRNA (RT-qPCR) and protein levels (Western blot), while simultaneously monitoring expression of other KDEL receptors to account for potential compensatory mechanisms .

• Use multiple cell lines to verify findings, as KDEL receptor stoichiometry varies across tissues, potentially reflecting cell-type specific functions .

• Employ epistasis analysis through combined knockdowns of KDELR3 with potential interacting partners to establish functional pathways .

What is the significance of KDELR3 overexpression in glioma and its potential as a prognostic biomarker?

KDELR3 has been identified as markedly overexpressed in glioma, with expression levels positively correlating with clinical progression . A comprehensive study integrating data from 1127 glioma samples from multiple sources (including The Cancer Genome Atlas and Chinese Glioma Genome Atlas) demonstrated that KDELR3 serves as an independent predictor of adverse outcomes in glioma patients .

Gene set enrichment analysis revealed that KDELR3 expression in glioma is associated with several key signaling pathways:

• Cytokine-cytokine receptor interaction

• Extracellular matrix (ECM)-receptor interaction

• Complement and coagulation cascades

These findings suggest that KDELR3 may play roles beyond ER protein retention in cancer progression, potentially influencing tumor microenvironment interactions and inflammatory responses. Researchers investigating KDELR3 as a prognostic marker should consider these associated pathways and validate findings using both Kaplan-Meier survival analysis and Cox proportional hazards regression models to establish independent prognostic value .

How does KDELR3 regulate AGR2 production and what are the implications for cellular homeostasis?

KDELR3 plays a critical inhibitory role in regulating AGR2 production at the pre-translational level. Research has demonstrated that:

• KDELR3 knockdown leads to significant increases in AGR2 mRNA and protein levels, without causing AGR2 secretion into the medium .

• The regulatory mechanism appears independent of ER stress, as levels of BiP and PDI mRNAs do not change significantly upon KDELR3 knockdown .

• ERp44, a folding assistant that cycles between ER and cis-Golgi, appears to be epistatic with KDELR3 in the same pathway regulating AGR2, as simultaneous knockdown of both does not yield additive effects .

• KDELR1 and KDELR3 have opposing effects on AGR2 regulation, with KDELR1 stimulating and KDELR3 inhibiting AGR2 transcript accumulation .

This regulatory circuit has significant implications for cellular homeostasis, as AGR2 is known to play roles in protein folding, cell migration, and cancer progression. The balance between KDELR1 and KDELR3 signaling may therefore represent an important control mechanism for cellular adaptation to changing protein synthesis demands.

What are the optimal conditions for using KDELR3 antibodies in Western blotting applications?

For optimal Western blotting results with KDELR3 antibodies, researchers should consider the following protocol specifications:

Sample preparation:

• Use whole cell lysates from relevant cell lines (NIH/3T3 cells have shown consistent positive results)

• Include appropriate controls (KDELR3 knockdown samples as negative controls)

Antibody dilution:

• Recommended dilution range: 1:500-1:2000 for most commercial KDELR3 antibodies

• Optimal dilution should be determined empirically for each experimental system

Detection:

• Expected molecular weight: 28 kDa (observed) vs. 25 kDa (calculated)

• For tagged KDELR3 constructs, adjust expected size accordingly (e.g., 3xFlag-mCherry tagged KDELR3 appears at ~52 kDa)

Storage conditions:

• Store antibodies at -20°C (standard antibody preparations) or -80°C (PBS-only formulations)

• Avoid repeated freeze-thaw cycles by preparing small aliquots

Commercial antibodies have been validated using cell lysates as positive controls, and their specificity should be verified in each experimental system through appropriate controls .

How can researchers effectively study the differential roles of KDELR3 versus other KDEL receptors?

To effectively differentiate the functions of KDELR3 from other KDEL receptors, researchers should implement the following methodological approaches:

• Single and combined knockdown experiments:

  • Use specific siRNAs targeting individual KDEL receptors

  • Create double knockdowns (e.g., KDELR1+2, KDELR1+3, KDELR2+3) and triple knockdowns (KDELR1+2+3)

  • Validate knockdown efficiency using RT-qPCR for each receptor

• Secretion assays:

  • Collect cell culture medium and cell lysates

  • Analyze presence of ER-resident proteins (BiP, ERp44, PDI, AGR2) by Western blotting

  • Compare secretion patterns across different knockdown combinations

• Transcript analysis:

  • Perform RT-qPCR to quantify mRNA levels of potential target genes (e.g., AGR2)

  • Use this approach to distinguish between translational and transcriptional effects

• Epistasis analysis:

  • Knockdown potential interactors (e.g., ERp44) alone and in combination with KDELR3

  • Analyze whether effects are additive (independent pathways) or non-additive (same pathway)

• Localization studies:

  • Use CRISPR-Cas9 to tag endogenous KDELR3 with fluorescent proteins

  • Employ surface biotinylation to distinguish cell surface from internal pools

These approaches have successfully revealed that KDELR3 plays a minor role in client retrieval compared to KDELR2, but has a significant role in regulating AGR2 transcription that opposes the effect of KDELR1 .

What controls should be included when validating KDELR3 antibody specificity?

When validating KDELR3 antibody specificity, the following controls are essential:

• Genetic knockdown/knockout controls:

  • siRNA or shRNA-mediated KDELR3 knockdown samples

  • CRISPR-Cas9-generated KDELR3 knockout cells

  • These provide negative controls to confirm antibody specificity

• Overexpression controls:

  • Cells transfected with tagged KDELR3 constructs (e.g., HA-Halo-3xFlag-KDELR3)

  • These provide positive controls with known molecular weights

• Cross-reactivity controls:

  • Samples with knockdown of other KDEL receptors (KDELR1, KDELR2)

  • These help assess potential cross-reactivity with homologous proteins

• Endogenous tagged reference:

  • If available, cell lines with endogenously tagged KDELR3 (using CRISPR-Cas9)

  • These provide a standard for comparing expression levels

• Multiple detection methods:

  • Validate findings using at least two independent methods (e.g., Western blot and immunofluorescence)

  • This confirms antibody specificity across different applications

The validation process should include careful quantification and statistical analysis to ensure reliable results.

How should researchers interpret discrepancies between KDELR3's calculated and observed molecular weights?

The calculated molecular weight of KDELR3 is 25 kDa based on its 214 amino acid sequence, but it is consistently observed at approximately 28 kDa in Western blot analyses . This discrepancy may be attributed to several factors that researchers should consider when interpreting their results:

• Post-translational modifications: As a membrane protein involved in protein-protein interactions, KDELR3 may undergo glycosylation or other modifications that increase its apparent molecular weight.

• Protein structure contributions: The seven-transmembrane domain structure of KDEL receptors can affect migration patterns in SDS-PAGE.

• Technical variations: Different gel systems, running buffers, and protein standards can influence the apparent molecular weight.

To address these discrepancies, researchers should:

• Include both molecular weight markers and positive controls on each blot

• Consider using gradient gels for better resolution around the size range of interest

• Perform validation with tagged versions of KDELR3 where the exact size increment is known

• Treat samples with glycosidases or phosphatases to assess contribution of these modifications to mobility shifts

How can researchers reconcile contradictory findings about KDELR3's subcellular localization?

The traditional view held that KDEL receptors function exclusively between the ER and Golgi, but recent evidence suggests KDELR3 may also be expressed at the plasma membrane . When confronted with contradictory findings regarding KDELR3 localization, researchers should consider:

• Cell type-specific differences: The relative abundance and localization of KDEL receptors vary across different tissues and cell lines . Findings in one cell type may not generalize to others.

• Technical approaches: Different localization techniques (immunofluorescence vs. surface biotinylation vs. subcellular fractionation) may have varying sensitivities for detecting low-abundance surface pools.

• Physiological state: KDELR3 localization may be dynamic and responsive to cellular conditions such as ER stress or secretory load.

• Antibody specificity: The lack of antibodies efficiently discriminating between the three KDEL receptors has impeded precise clarification of their localization .

• Overexpression artifacts: Studies using overexpressed KDELR3 may not reflect physiological localization patterns .

To reconcile contradictory findings, researchers should:

• Use complementary approaches to validate localization (e.g., combining biochemical fractionation with imaging)

• Tag endogenous KDELR3 using CRISPR-Cas9 to avoid overexpression artifacts

• Perform careful kinetic studies to capture dynamic localization changes

• Consider cell type-specific differences when comparing across studies

What methodological challenges might affect data reproducibility when studying KDELR3 in different cell types?

Several methodological challenges can impact data reproducibility when studying KDELR3 across different cell types:

• Differential expression levels: The abundance and relative stoichiometry of the three KDELRs vary in different tissues , which may affect:

  • Antibody detection sensitivity

  • Functional compensation following knockdown

  • Phenotypic outcomes of perturbations

• Interaction partner variations: The availability of KDELR3 interaction partners (like ERp44) may differ between cell types, affecting observed functions and localization .

• Transfection/transduction efficiency: Different cell types have varying susceptibilities to transfection/transduction, impacting knockdown or overexpression studies.

• Cell-specific signaling contexts: The signaling pathways interacting with KDELR3 may be differentially active across cell types.

• Technical considerations:

  • Antibody accessibility may vary with different fixation/permeabilization protocols

  • Protein extraction efficiency may differ based on cell architecture and membrane composition

  • Background signal levels can vary across cell types

To address these challenges, researchers should:

• Include appropriate cellular controls specific to each cell type

• Validate knockdown/overexpression efficiency in each cell system

• Adjust antibody concentrations and detection methods for each cell type

• Consider using multiple cell lines to establish generalizability of findings

• Standardize protocols across cell types where possible, documenting necessary modifications