CDCP1 Human

What is CDCP1 and what are its key structural features?

CDCP1, also known as Trask or CD318, is a type I transmembrane receptor with a large extracellular domain containing three CUB domains, a transmembrane domain, and a cytoplasmic domain with 5 tyrosine phosphorylation sites . Human CDCP1 exists in multiple forms: a full-length ~135 kDa form that can be cleaved into a ~70 kDa membrane-bound portion and a ~65 kDa circulating form . Additionally, a secreted-only form (Isoform 2) consisting of amino acids 30-343 of the extracellular domain can be expressed . Mature human Isoform 2 shares 86% and 85% amino acid sequence identity with mouse and rat CDCP1 Isoform 2, respectively .

Where is CDCP1 expressed in normal human tissues?

While CDCP1 is widely studied in cancer contexts, its distribution in normal tissues is less characterized. Research shows that CDCP1 is expressed on retinal pigment epithelial (RPE) cells in both humans and mice . This has been confirmed through multiple detection methods including Western blotting, immunofluorescent staining, and flow cytometry analysis . CDCP1 has also been found on stem cells, keratinocytes, and colonic epithelial cells . Understanding the normal tissue distribution is critical for interpreting experimental results and potential off-target effects in therapeutic development.

How can I detect CDCP1 expression in tissue samples?

Multiple complementary techniques can be employed to detect CDCP1:

• Western blotting: Using commercial antibodies like polyclonal anti-human CDCP1 antibodies can detect CDCP1 in tissue lysates . This approach allows differentiation between full-length and cleaved forms.

• Immunofluorescent staining: Monoclonal antibodies such as 9A2 have been used successfully for immunofluorescent staining of retinal sections . This approach allows visualization of CDCP1's spatial distribution within tissues.

• Flow cytometry: Multiple antibodies including clone CUB1 and 9A2 can detect cell surface CDCP1 . Flow cytometry is particularly useful for quantifying expression levels across different cell populations.

Each method provides different information, and combining approaches yields more comprehensive characterization.

What role does CDCP1 play in cancer progression and metastasis?

CDCP1 was originally identified from proteins involved in metastasis and has been associated with poor prognosis in multiple epithelial tumors, including lung, pancreatic, colorectal, renal, and ovarian carcinomas . It serves as a novel marker of aggressive human triple-negative breast cancers . Mechanistically, tyrosine phosphorylation of CDCP1's intracellular domain activates downstream signaling through Src-family kinases (SFKs), Akt, and PKCδ . Several extracellular forms of CDCP1 exhibit disease-specific expression in prostate cancer .

For researchers investigating CDCP1's role in cancer, it's important to consider both the membrane-bound and secreted forms, as they may have distinct functions in tumor progression and potential therapeutic implications.

How does CDCP1 interact with other cellular signaling pathways?

CDCP1 amplifies pro-tumorigenic signaling by other receptors including EGFR and HER2 . The phosphorylation state of CDCP1 regulates its effects on cell-cell and cell-substratum adhesion . Recent research has identified CDCP1 as a novel ligand of CD6, a surface marker and critical regulator of T cells . This interaction has implications for autoimmune conditions like multiple sclerosis and autoimmune uveitis .

Researchers should consider these interactions when designing experiments, particularly when studying the effects of CDCP1 inhibition, as there may be broader implications across multiple signaling networks.

What is the role of CDCP1 in retinal pigment epithelial (RPE) barrier integrity?

Recent studies have revealed that CDCP1 expressed on RPE cells interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption . This interaction leads to the opening of tight junctions, facilitating T cell infiltration and contributing to the development of uveitis . CDCP1-knockout (CDCP1-KO) mice developed attenuated retinal inflammation in a passive model of autoimmune uveitis compared to wild-type mice .

The experimental evidence for this includes:

• Disrupted tight junctions and infiltrating T cells were detected in RPE flat mounts from wild-type but not CDCP1-KO mice during experimental autoimmune uveitis (EAU) development

• CDCP1 on RPE cells was upregulated by IFN-γ both in vitro and after EAU induction in vivo

• CD6 stimulation induced increased RPE barrier permeability in wild-type but not CDCP1-knockdown (CDCP1-KD) RPE cells

• Activated T cells migrated through wild-type RPE monolayers more efficiently than through CDCP1-KD RPE monolayers

What are effective strategies for developing anti-CDCP1 antibodies for research?

When developing anti-CDCP1 antibodies for research purposes, several factors should be considered:

• Epitope selection: Target regions with high conservation between species if cross-reactivity is desired. The extracellular domain contains three CUB domains that could serve as targets .

• Antibody characterization: Thoroughly characterize antibodies using multiple methods:

  • Western blotting to confirm specificity and identify recognized isoforms

  • Flow cytometry to verify binding to native cell surface CDCP1

  • Competition assays to determine epitope relationships between antibodies

• Functional assessment: Anti-CDCP1 antibodies 10D7 and 41-2 have been characterized for their ability to induce loss of cell surface CDCP1 expression . When developing new antibodies, assess their functional properties including:

  • Internalization kinetics (can be tracked using pH-sensitive fluorescent dyes)

  • Binding competition with existing antibodies

  • Effects on CDCP1 signaling and cellular functions

How can I effectively generate and validate CDCP1 knockout or knockdown models?

Based on successful approaches in the literature:

• Knockout mouse models: CDCP1-KO mice have been generated and used to study CDCP1's role in retinal inflammation . When developing such models:

  • Confirm knockout by Western blotting of tissue lysates from wild-type and knockout animals

  • Verify absence of CDCP1 through immunofluorescent staining of relevant tissues

  • Use multiple antibodies targeting different epitopes to ensure complete knockout

• Cell line knockdown models: CDCP1-knockdown (CDCP1-KD) RPE cells have been developed to study barrier permeability and T cell migration . For similar approaches:

  • Use RNA interference (siRNA or shRNA) or CRISPR-Cas9 systems targeting CDCP1

  • Validate knockdown efficiency by both protein (Western blot, flow cytometry) and mRNA (qPCR) analyses

  • Include proper controls (scrambled sequences, wild-type cells) in all experiments

• Functional validation: Verify altered phenotypes in knockout/knockdown models:

  • For RPE cells, assess barrier integrity through permeability assays and tight junction protein staining

  • Evaluate cytoskeletal organization and focal adhesion formation

  • Measure T cell migration through cell monolayers

What imaging techniques are most informative for studying CDCP1 internalization and trafficking?

Live-cell imaging approaches provide valuable insights into CDCP1 dynamics:

• Spinning-disk confocal microscopy: This technique has been used successfully to track internalization and intracellular trafficking of GFP-tagged CDCP1 and antibody complexes . It offers advantages including:

  • Reduced phototoxicity compared to conventional confocal microscopy

  • Faster acquisition speeds suitable for dynamic processes

  • Good optical sectioning for 3D localization

• pH-sensitive fluorescent probes: Antibodies conjugated to pH-sensitive dyes (like pHrodo) allow selective visualization of internalized CDCP1 in acidic endosomal/lysosomal compartments . This approach has successfully demonstrated that anti-CDCP1 antibodies 10D7 and 41-2 become internalized into low pH vesicles .

• Subcellular colocalization: Co-staining with markers of different intracellular compartments (early endosomes, late endosomes, lysosomes) helps determine the trafficking pathway of internalized CDCP1.

How should I interpret conflicting data regarding CDCP1 expression in different cell types?

Researchers often encounter conflicting reports about CDCP1 expression, which may result from:

• Antibody specificity: Different antibodies may recognize different epitopes or isoforms. For example, antibody 4115 recognizes the intracellular carboxyl terminal of CDCP1, while others target extracellular domains . Always document which antibody was used and its target epitope.

• Detection method sensitivity: Flow cytometry may detect low levels of cell surface expression not visible by immunohistochemistry. Western blotting can reveal multiple isoforms that may be missed by other methods.

• Cell culture conditions: CDCP1 expression can be regulated by cytokines like IFN-γ . Document culture conditions, passage number, and confluence level.

• Post-translational modifications: The ~135 kDa full-length form can be cleaved into ~70 kDa membrane-bound and ~65 kDa circulating forms . Use antibodies that can distinguish these forms.

To address conflicting data, employ multiple detection methods and antibodies targeting different epitopes, and carefully document all experimental conditions.

What experimental controls are crucial when assessing CDCP1 function in barrier integrity studies?

When studying CDCP1's role in barrier integrity, include these essential controls:

Careful implementation of these controls helps distinguish direct CDCP1-mediated effects from secondary consequences and ensures reproducibility across different experimental systems.

What are the therapeutic implications of targeting CDCP1 in cancer and inflammatory diseases?

Anti-CDCP1 antibodies have shown promise in inhibiting cell migration and survival in vitro, and tumor growth and metastasis in vivo . Recent evidence suggests potential applications beyond cancer:

• Cancer immunotherapy: The development of anti-CDCP1 immuno-conjugates shows promise for both detection and inhibition of ovarian cancer . Zirconium 89-labelled 10D7 antibody has been detected by positron-emission tomography imaging of ovarian cancer patient-derived xenografts . Cytotoxin-conjugated 10D7 has demonstrated efficacy against ovarian cancer cells both in vitro and in vivo .

• Autoimmune disease modulation: Given CDCP1's role in RPE barrier integrity and T cell infiltration, targeting the CDCP1-CD6 interaction might represent a novel approach for treating autoimmune uveitis and potentially other T cell-mediated autoimmune conditions . CDCP1-KO mice showed protection against experimental autoimmune uveitis development .

• Biomarker development: CDCP1 is associated with poor prognosis in epithelial tumors and is a marker of aggressive triple-negative breast cancers . Several extracellular forms show disease-specific expression in prostate cancer . This suggests potential for development as a prognostic or diagnostic biomarker.

Researchers should consider how CDCP1's dual roles in cancer and inflammation might affect therapeutic strategies and potential off-target effects.