NT5E (CD73) Mouse

What is the physiological role of NT5E (CD73) in mice?

NT5E (CD73) functions as a key enzyme in the purinergic signaling pathway, catalyzing the hydrolysis of extracellular AMP to adenosine and inorganic phosphate. This reaction is largely irreversible, unlike the upstream enzymatic reactions involving CD39/ENTPD1 . In mice, CD73 plays crucial homeostatic roles in multiple tissue types. It is abundantly expressed on epithelial and endothelial cells, neurons, glia, myocytes, and fibroblasts . NT5E activity is essential for maintaining tissue integrity, particularly endothelial and epithelial barrier functions, and facilitating recovery following hypoxia, ischemia/reperfusion, and inflammatory injury in tissues including the brain, heart, lung, kidney, liver, and digestive system . Importantly, adenosine generated through NT5E activity serves as a negative feedback regulator, restricting inflammatory immune responses and protecting against excessive tissue damage .

How do NT5E (CD73) knockout mouse models differ from wild-type mice phenotypically?

NT5E knockout (Nt5e^-/-) mice exhibit several distinct phenotypic differences compared to wild-type counterparts. Most notably, these mice display increased vascular leakage in response to normobaric hypoxia across multiple tissues including lung, liver, gut, muscle, heart, kidney, and brain . They show heightened susceptibility to cardiovascular, respiratory, gastrointestinal and liver injury, primarily because they lack the adaptive mechanisms provided by extracellular adenosine during hypoxic stress .

Interestingly, despite the severe vascular phenotype observed in humans with NT5E mutations (CALJA/ACDC disease), mouse models of CD73 deficiency do not fully recapitulate the human disease phenotype, particularly regarding vascular calcification . Sex differences are also apparent in Nt5e^-/- mice, with females showing dramatically lower frequencies of spontaneous transient adenosine events in the hippocampus compared to wild-type, while males show compensatory upregulation of tissue-nonspecific alkaline phosphatase (TNAP) . This suggests female mice are more reliant on CD73 for adenosine production, likely due to estrogen-mediated regulation of CD73 expression and activity .

What tissue distribution pattern does NT5E (CD73) exhibit in wild-type mice?

NT5E (CD73) demonstrates a broad but variable tissue distribution pattern in wild-type mice. High expression levels are observed in epithelial cells of the respiratory tract, smooth muscle cells, cardiac myocytes, neurons, glia, and fibroblasts . Particularly notable NT5E expression occurs in tissues including the smooth muscle, female reproductive system, liver, and gastrointestinal tract .

Within the immune system, NT5E is expressed at significant levels on regulatory T cells (T~reg~) and at even higher levels on anergic CD4+ T cells, playing important roles in immunosuppression and self-tolerance . The expression pattern aligns with CD73's diverse physiological functions in maintaining tissue barrier integrity, regulating epithelial ion transport for mucosal hydration, controlling inflammatory responses, and adapting to hypoxic conditions . Notably, there are qualitative differences in NT5E function depending on the expressing cell type, with lymphocyte-expressed NT5E showing greater susceptibility to phosphatidylinositol phospholipase and displaying signal transduction capabilities not observed in epithelial cell-expressed NT5E .

How should researchers design experiments to investigate sex-specific differences in NT5E (CD73) function?

When investigating sex-specific differences in NT5E (CD73) function, researchers should implement the following methodological approaches:

• Balanced cohort design: Include equal numbers of age-matched male and female mice in both experimental (Nt5e^-/-) and control (wild-type) groups. Historical data suggests most in vivo studies on CD73 have been conducted only in male mice or without explicitly considering biological sex as a variable .

• Hormonal status assessment: Measure estrogen levels and consider estrous cycle staging in female mice, as estrogen receptors (ERα and ERβ) positively regulate CD73 expression and activity in tissues such as the hippocampus . Consider ovariectomy with estrogen replacement as an experimental approach to directly assess estrogen effects.

• Tissue-specific analyses: Compare CD73 expression and activity across multiple tissues in both sexes. Focus on tissues known to have sex-specific differences, such as:

  • Hippocampus: Examine spontaneous transient adenosine events, as female Nt5e^-/- mice show dramatically lower frequencies compared to wild-type, while male mice show compensatory TNAP upregulation .

  • Cardiovascular tissues: Investigate differences in vascular responses to hypoxia and inflammatory stressors.

• Compensatory mechanism investigation: Assess expression and activity of alternative enzymes that may compensate for CD73 deficiency in a sex-specific manner, particularly TNAP, which shows upregulation in male but not female Nt5e^-/- mice .

• Functional readouts: Include both molecular and physiological endpoints such as adenosine measurements, tissue integrity assessments, and responses to hypoxic or inflammatory challenges to comprehensively capture sex differences in CD73 function.

What are the most appropriate control groups for experiments using NT5E (CD73) knockout mice?

When designing experiments with NT5E (CD73) knockout mice, researchers should include the following control groups to ensure robust and interpretable results:

• Wild-type littermate controls: These should be age-matched and sex-matched with the experimental knockout mice and derived from the same breeding colony to minimize genetic background effects. This is especially important given the complex phenotypic effects of CD73 deficiency that can be influenced by genetic background .

• Heterozygous (Nt5e^+/-) controls: Including heterozygous mice allows assessment of gene dosage effects, which is particularly relevant for enzymes like CD73 where partial activity might be sufficient for certain functions.

• Tissue-specific knockout controls: When using conditional knockout models, include mice with the Cre recombinase but without floxed CD73 alleles to control for any Cre-mediated effects independent of CD73 deletion.

• Pharmacological controls: In experiments using CD73 inhibitors, include vehicle-treated wild-type mice and inhibitor-treated knockout mice to distinguish between on-target and off-target effects of the compounds.

• Sham-operated or unstressed controls: For experiments involving hypoxia, ischemia/reperfusion, or inflammatory challenges, include both genotypes (WT and KO) without stress induction to establish proper baselines, as CD73-deficient mice show increased susceptibility to these stressors .

• Cross-genotype bone marrow chimeras: To distinguish between hematopoietic and non-hematopoietic CD73 functions, consider using bone marrow transplantation between wild-type and knockout mice to create chimeric animals with selective CD73 deficiency.

What methods are recommended for measuring adenosine production in NT5E (CD73) mouse models?

To accurately measure adenosine production in NT5E (CD73) mouse models, researchers should consider the following methodological approaches:

• High-performance liquid chromatography (HPLC): This technique provides quantitative measurement of adenosine in tissue extracts or body fluids with high sensitivity. For NT5E mouse models, samples should be collected with adenosine deaminase inhibitors to prevent rapid adenosine degradation.

• Enzyme-based colorimetric/fluorometric assays: These assays measure CD73 enzyme activity by quantifying the conversion of AMP to adenosine. The protocol should include:

  • Tissue homogenate or cell preparation in the presence of enzyme inhibitors

  • Incubation with AMP substrate

  • Measurement of inorganic phosphate release (colorimetric) or adenosine formation (HPLC)

  • Parallel samples with CD73 inhibitors to determine specificity

• In situ enzyme histochemistry: This technique visualizes CD73 enzymatic activity in tissue sections through lead phosphate precipitation. The protocol involves:

  • Fresh-frozen tissue sections

  • Incubation with AMP substrate and lead nitrate

  • Conversion of lead phosphate to lead sulfide for visualization

  • Counterstaining for tissue architecture

• Microelectrode biosensors: For real-time measurement of adenosine in living tissue, especially in brain regions where CD73 contributes to spontaneous transient adenosine events that show sex-specific differences in Nt5e^-/- mice .

• Mass spectrometry: Provides highly sensitive and specific measurement of adenosine and related metabolites, allowing for comprehensive purinergic metabolite profiling.

For all methods, researchers must consider the extremely short half-life of extracellular adenosine (seconds) and implement appropriate sample handling procedures, including the use of adenosine transport inhibitors (e.g., dipyridamole) and adenosine deaminase inhibitors (e.g., EHNA) to preserve adenosine for measurement.

How should researchers interpret conflicting phenotypes between human NT5E mutations and mouse models?

The discrepancy between human NT5E mutation phenotypes (CALJA/ACDC disease) and mouse NT5E knockout models presents a significant challenge for researchers. To properly interpret these conflicting phenotypes, consider the following analytical approaches:

How can researchers distinguish between enzymatic and non-enzymatic functions of NT5E (CD73) in experimental data?

Distinguishing between enzymatic and non-enzymatic functions of NT5E (CD73) requires careful experimental design and interpretation. Researchers should implement the following strategies:

• Comparison of enzyme-dead mutants vs. complete knockouts: Engineer mouse models expressing catalytically inactive CD73 (point mutations in the active site) and compare their phenotype to complete CD73 knockout mice. Differences would indicate non-enzymatic functions.

• Selective pharmacological approaches: Use:

  • Specific CD73 enzyme inhibitors (e.g., APCP) to block catalytic activity without affecting protein expression

  • Adenosine receptor antagonists to block downstream adenosine signaling

  • Exogenous adenosine or adenosine analogs for rescue experiments

• Functional domain analysis: CD73 can act as a receptor molecule mediating cell-cell adhesion between lymphocytes and endothelial cells, and can interact with extracellular matrix components independent of its enzymatic activity . These interactions occur even when enzymatic activity is blocked by concanavalin A . Experiments should:

  • Test CD73-ECM interactions (fibronectin, tenascin C, collagen 1) with and without enzymatic inhibitors

  • Assess cell adhesion and migration phenotypes that may be independent of adenosine production

• Signal transduction analysis: CD73 can facilitate cellular signal transduction despite lacking intracellular signaling domains, possibly by associating with src protein kinases . Researchers should:

  • Examine phosphorylation cascades following CD73 crosslinking

  • Compare signaling events with enzymatically active vs. inactive CD73

  • Investigate cell type-specific differences (lymphocyte CD73 shows signal transduction activity while epithelial cell CD73 does not)

• Domain-specific antibodies or blocking peptides: Use reagents that target specific regions of CD73 to selectively inhibit particular functions while leaving others intact.

What factors should be considered when analyzing tissue-specific differences in NT5E (CD73) function?

When analyzing tissue-specific differences in NT5E (CD73) function, researchers should consider the following factors for comprehensive data interpretation:

• Baseline expression patterns: NT5E expression varies significantly across tissues, with highest human expression in arteries and notable expression in smooth muscle, female reproductive system, liver, and gastrointestinal tract . Analysis should include:

  • Quantitative assessment of baseline CD73 expression at mRNA and protein levels across tissues

  • Evaluation of enzymatic activity that may not directly correlate with expression levels

  • Cell type-specific expression within heterogeneous tissues

• Microenvironmental regulation: CD73 expression and function are regulated by microenvironmental factors including:

  • Oxygen tension: CD73 is hypoxia-inducible through HIF-1α binding to the NT5E promoter

  • Hormonal influences: Estrogen receptors (ERα and ERβ) positively regulate CD73 expression in tissues like the hippocampus

  • Inflammatory mediators: These affect CD73 expression in a tissue-specific manner

• Compensatory mechanisms: Alternative pathways for adenosine production may exist in different tissues, including:

  • Tissue-nonspecific alkaline phosphatase (TNAP), which shows compensatory upregulation in male but not female Nt5e^-/- mice hippocampus

  • Other ectonucleotidases with tissue-specific expression patterns

• Qualitative functional differences: The same protein may have distinct functions depending on cellular context:

  • Lymphocyte-expressed CD73 shows greater susceptibility to phosphatidylinositol phospholipase and can transduce signals, unlike epithelial cell-expressed CD73

  • CD73 shedding from cell surfaces occurs in lymphocytes but not epithelial cells

• Downstream signaling variations: Different tissues express distinct adenosine receptor subtypes (A1, A2A, A2B, A3) that mediate diverse and sometimes opposing effects of adenosine generated by CD73.

• Specialized tissue functions: Consider how CD73 contributes to tissue-specific roles, such as:

  • Epithelial ion transport and mucosal hydration in respiratory tract

  • Endothelial barrier "resealing" in vascular tissues

  • Neuronal synaptic transmission in the brain

How can NT5E (CD73) mouse models be utilized to study cancer immunotherapy approaches?

NT5E (CD73) mouse models offer valuable platforms for investigating cancer immunotherapy approaches targeting the adenosinergic pathway. Researchers should consider the following methodological strategies:

• Tumor microenvironment (TME) evaluation: NT5E knockout mice can be used to study how host CD73 deficiency affects:

  • Tumor-infiltrating lymphocyte populations and their functional status

  • Myeloid-derived suppressor cell (MDSC) recruitment and activity

  • Regulatory T cell (T~reg~) function within the TME

  • Adenosine levels in the TME measured using microdialysis or metabolomic approaches

• Combination therapy models: Evaluate CD73-targeting approaches in combination with:

  • Immune checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Adenosine receptor antagonists (A2A, A2B receptor blockade)

  • Conventional therapies (radiation, chemotherapy)

  These experiments should include detailed immune phenotyping and assessment of long-term survival outcomes.

• Cell-specific knockout approaches: Generate conditional knockout mice with CD73 deletion in specific cell populations:

  • T~reg~-specific deletion using Foxp3-Cre

  • Tumor cell-specific deletion using tumor-specific promoters

  • Endothelial cell-specific deletion using Tie2-Cre

  This allows dissection of the relative contribution of CD73 from different cellular sources to tumor immune evasion.

• Therapeutic antibody evaluation: Test anti-CD73 blocking antibodies in wild-type tumor-bearing mice. Previous studies have shown that injection of blocking NT5E-specific antibodies into tumor-bearing mice resulted in reduced outgrowth of NT5E-expressing tumors across various tumor entities . Compare with:

  • Small molecule CD73 inhibitors

  • CD73 knockout mice (as positive controls)

  • Combination approaches targeting both CD73 and CD39

• Metastasis models: Utilize CD73 mouse models to investigate both immune and non-immune mechanisms of CD73 in metastasis:

  • Experimental metastasis assays (intravenous injection)

  • Spontaneous metastasis from orthotopic tumors

  • Cell adhesion and migration assays to assess CD73-extracellular matrix interactions that facilitate tumor cell dissemination independent of enzymatic activity

These approaches can inform the ongoing clinical development of multiple CD73-targeting antibodies and small-molecule inhibitors currently being tested in human clinical trials .

What approaches should be used to investigate the role of NT5E (CD73) in hypoxia and ischemia/reperfusion injury models?

To investigate NT5E (CD73) in hypoxia and ischemia/reperfusion injury models, researchers should implement these methodological approaches:

• Tissue-specific hypoxia models: Compare wild-type and Nt5e^-/- mice in various hypoxia models:

  • Global normobaric hypoxia chamber exposure (10-12% O₂)

  • Tissue-specific ischemia models (myocardial, cerebral, renal, hepatic)

  • Varying durations of hypoxia to assess acute vs. chronic responses

  Multiple endpoints should be measured, including tissue adenosine levels, vascular permeability, inflammatory markers, and functional recovery metrics.

• Molecular regulation analysis: Investigate the hypoxia-CD73 regulatory axis:

  • Chromatin immunoprecipitation to confirm HIF-1α binding to the NT5E promoter under hypoxic conditions

  • Analysis of NT5E mRNA and protein expression kinetics during hypoxia

  • Assessment of post-translational modifications of CD73 under hypoxic conditions

  • Evaluation of microRNA-mediated regulation in hypoxic environments

• Conditional and inducible models: Use tissue-specific, inducible CD73 knockout or overexpression models to determine:

  • Critical windows for CD73-mediated protection

  • Tissue-specific contributions to systemic responses

  • Therapeutic potential of timed CD73 modulation

• Pharmacological interventions: Test CD73-targeted therapies in hypoxia and ischemia/reperfusion models:

  • CD73 activators or recombinant CD73 administration prior to reperfusion

  • Adenosine receptor agonists as potential bypass therapies

  • CD73 inhibitors to determine potential detrimental effects in these models

• Vascular permeability assessment: Given that Nt5e^-/- mice display vascular leakage in response to normobaric hypoxia in multiple tissues , comprehensive vascular permeability should be evaluated using:

  • Evans blue dye extravasation assays

  • Intravital microscopy

  • Transendothelial electrical resistance measurements

• Multi-omics profiling: Compare wild-type and CD73-deficient tissues following hypoxia using:

  • Transcriptomics to identify differentially expressed genes

  • Proteomics to assess hypoxia-responsive protein networks

  • Metabolomics focusing on purinergic pathway metabolites

This comprehensive approach will help elucidate the mechanisms by which CD73 contributes to adaptive responses to hypoxia and potential therapeutic applications for ischemia/reperfusion injuries.

How can researchers study the interplay between NT5E (CD73) and regulatory T cell function in mouse models?

To effectively study the interplay between NT5E (CD73) and regulatory T cell (T~reg~) function in mouse models, researchers should employ the following methodological approaches:

• Cell-specific genetic models: Generate and characterize:

  • T~reg~-specific CD73 knockout mice (Foxp3-Cre × NT5E^fl/fl^)

  • CD73-reporter mice crossed to Foxp3-reporter strains to simultaneously track CD73 expression and T~reg~ identity

  • Inducible systems to temporally control CD73 deletion in T~regs~

• In vivo suppression assays: Compare the suppressive capacity of wild-type versus CD73-deficient T~regs~ in:

  • Adoptive transfer models of colitis

  • Graft-versus-host disease models

  • Tumor immune suppression models

  • Autoimmune disease models (EAE, diabetes)

• Metabolic profiling: Analyze the adenosinergic pathway in T~regs~ and their target cells:

  • Quantify adenosine production by T~regs~ in different activation states

  • Measure expression of adenosine receptors on target effector cells

  • Assess cAMP levels in effector cells following co-culture with wild-type vs. CD73-deficient T~regs~

  • Investigate metabolic adaptations in T~regs~ lacking CD73

• T~reg~ apoptosis and adenosine release: Study the recently described phenomenon where T~regs~ undergoing apoptosis within the metabolically abnormal tumor microenvironment release substantial amounts of ATP that is degraded by nucleotidases of the faded T~reg~, resulting in accumulating adenosine levels . Experimental approaches should include:

  • Tracking T~reg~ survival and turnover in tissues

  • Measuring extracellular ATP and adenosine in areas of T~reg~ apoptosis

  • Comparing wild-type and CD73-deficient T~regs~ in their capacity to generate suppressive adenosine during apoptosis

• Integration with other suppressive mechanisms: Determine how CD73-adenosine axis interacts with other T~reg~ suppressive mechanisms:

  • CTLA-4-mediated suppression

  • IL-10 and TGF-β production

  • Metabolic competition

• T~reg~ development and stability: Assess how CD73 deficiency affects:

  • Thymic versus peripheral T~reg~ development

  • T~reg~ stability in inflammatory environments

  • Lineage plasticity and potential conversion to effector phenotypes

These approaches will provide mechanistic insights into how CD73 contributes to T~reg~-mediated immunosuppression, which has implications for understanding self-tolerance in healthy individuals, protection of the fetus from maternal immune attack during pregnancy , and developing cancer immunotherapies targeting the CD73-adenosine axis.

What are the key technical considerations for accurately measuring NT5E (CD73) expression in mouse tissues?

Accurate measurement of NT5E (CD73) expression in mouse tissues requires attention to several technical considerations:

• Antibody selection and validation:

  • Use antibodies validated for specific applications (IHC, flow cytometry, Western blot)

  • Validate antibody specificity using Nt5e^-/- tissues as negative controls

  • Be aware that some commercial antibodies may cross-react with other ectonucleotidases

  • Consider the conformational state of CD73 when selecting antibodies, as CD73 can exhibit open or closed conformations during substrate cleavage

• Potential shedding artifacts: Account for CD73 shedding from cell surfaces, which can create detection challenges:

  • Reports indicate antibody binding to CD73 can trigger shedding from lymphocytes but not epithelial cells

  • Similar observations were reported with murine B16F10 melanoma cells showing intracellular but not surface CD73 expression due to shedding

  • Consider measuring soluble CD73 in culture supernatants or body fluids

• mRNA versus protein analysis:

  • Compare mRNA expression (qPCR, RNA-seq) with protein levels (Western blot, flow cytometry)

  • Be aware of post-transcriptional regulation by microRNAs that may cause discrepancies between mRNA and protein levels

  • Consider chromatin immunoprecipitation to examine transcription factor binding to the NT5E promoter

• Tissue processing considerations:

  • Fresh tissues are preferable for enzymatic activity assays

  • Fixation can affect epitope accessibility for immunohistochemistry

  • Cryopreservation is generally superior to formalin fixation for CD73 detection

  • Single-cell preparations for flow cytometry may alter surface expression through enzymatic digestion

• Cell type-specific analysis:

  • Use cell sorting or single-cell approaches to account for heterogeneous expression within tissues

  • Consider co-staining with cell type-specific markers

  • Use tissue-specific reporter mice or conditional knockout models to track expression in specific cell populations

• Activity-based detection: Consider enzyme activity assays in addition to expression analysis:

  • Malachite green assay for phosphate release

  • Lead precipitation methods for tissue sections

  • HPLC-based activity measurements

How should researchers address potential compensatory mechanisms in NT5E (CD73) deficient mice?

Addressing compensatory mechanisms in NT5E (CD73) deficient mice requires systematic investigation using these approaches:

• Comprehensive ectonucleotidase profiling:

  • Measure expression and activity of alternative adenosine-generating enzymes, particularly:

    • Tissue-nonspecific alkaline phosphatase (TNAP), which shows compensatory upregulation in male but not female Nt5e^-/- mice hippocampus

    • CD39/ENTPD1 and other members of the ectonucleoside triphosphate diphosphohydrolase family

    • Prostatic acid phosphatase (PAP)

    • Ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs)

  • Compare enzyme activities across multiple tissues, as compensatory mechanisms may be tissue-specific

• Temporal analysis:

  • Examine compensatory changes across different developmental stages

  • Consider both acute and chronic compensation following CD73 deletion

  • Use inducible knockout models to distinguish between developmental compensation and acute responses

• Genetic approaches:

  • Generate double knockout mice lacking both CD73 and potential compensatory enzymes (e.g., Nt5e^-/- × TNAP^+/-^)

  • Use shRNA or CRISPR approaches to knockdown compensatory enzymes in CD73-deficient backgrounds

  • Consider triple adenosine receptor knockout mice to assess collective roles in adenosine signaling

• Functional adenosine signaling assessment:

  • Measure tissue adenosine levels to determine if compensation maintains normal adenosine concentrations

  • Assess adenosine receptor activation and downstream signaling pathways

  • Compare physiological responses to adenosine receptor agonists in wild-type and CD73-deficient tissues

• Sex-specific analysis:

  • Explicitly examine compensatory mechanisms in both male and female mice

  • Consider hormonal influences, as female mice appear more reliant on CD73 for adenosine production, while males show greater compensation

  • Perform gonadectomy studies to determine the role of sex hormones in compensation

• Comparative multi-omics:

  • RNA-seq to identify upregulated transcripts potentially involved in compensation

  • Proteomics to detect changes in protein expression and post-translational modifications

  • Metabolomics to assess changes in adenosine and related metabolites

What methods are recommended for studying the dual enzymatic and non-enzymatic functions of NT5E (CD73)?

To effectively study the dual enzymatic and non-enzymatic functions of NT5E (CD73), researchers should employ the following methodological approaches:

• Structure-function analysis using mutant constructs:

  • Generate point mutations that specifically abolish enzymatic activity without affecting protein folding or expression

  • Create domain deletion mutants to identify regions responsible for non-enzymatic functions

  • Express these constructs in CD73-deficient cells or mice for functional rescue experiments

• Enzymatic activity assays:

  • Malachite green phosphate detection assay to quantify released inorganic phosphate

  • HPLC analysis to measure AMP depletion and adenosine formation

  • Enzyme kinetic studies with varying substrate concentrations

  • Comparison of activity in native versus denatured or fixed samples to distinguish enzyme-dependent effects

• Non-enzymatic function assessment:

  • Cell adhesion assays to measure CD73-mediated cell-cell and cell-matrix interactions

  • Binding assays with extracellular matrix components (fibronectin, tenascin C, collagen 1)

  • Signal transduction studies examining src kinase activation and downstream pathways

  • Immunoprecipitation to identify protein binding partners

• Pharmacological dissection:

  • Use concanavalin A to block enzymatic activity while preserving non-enzymatic functions

  • Apply specific CD73 inhibitors (APCP) and compare effects to knockout models

  • Use adenosine receptor antagonists to block downstream effects of enzymatic activity

  • Apply recombinant soluble CD73 with and without enzymatic activity

• Compartment-specific analysis:

  • Compare membrane-bound versus soluble CD73 functions

  • Investigate intracellular versus extracellular roles

  • Examine CD73 in different membrane microdomains (lipid rafts, caveolae)

• Biophysical approaches:

  • Surface plasmon resonance to quantify CD73 interactions with other proteins

  • Förster resonance energy transfer (FRET) to study CD73 conformational changes and protein-protein interactions

  • Atomic force microscopy to examine cell surface distribution and clustering

• Cell type-specific functional assessment:

  • Compare lymphocyte CD73 (which can transduce signals) with epithelial cell CD73 (which lacks this capability)

  • Investigate mechanisms of CD73 shedding from lymphocytes but not epithelial cells

  • Examine cell migration, which is facilitated by CD73-tenascin C interactions independent of enzymatic activity

These complementary approaches will allow researchers to comprehensively characterize both the adenosine-generating enzymatic activity of CD73 and its non-enzymatic roles in cell adhesion, migration, and signal transduction.