{"gene":"CEACAM3","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1996,"finding":"CGM1a (CEACAM3) expressed on neutrophils acts as a direct receptor for gonococcal opacity (Opa) proteins; HeLa cells expressing CGM1a bind and internalize OpaI+ E. coli, and monoclonal antibody COL-1 against CGM1 blocks this interaction.","method":"Ligand binding assay with PMN lysates, HeLa cell transfection with CGM1a, bacterial internalization assay, monoclonal antibody blocking","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding demonstrated with transfected cells, antibody blockade, and confirmed by multiple orthogonal methods; independently replicated by subsequent studies","pmids":["8962144"],"is_preprint":false},{"year":1993,"finding":"CGM1a (CEACAM3) contains an ITAM-like consensus motif in its cytoplasmic domain (encoded by the Cyt1 exon) and a shorter splice variant CGM1c lacking this motif; the ITAM-like sequence is implicated in signal transduction.","method":"Gene cloning, exon mapping, sequence analysis of splice variants","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — sequence-based identification of ITAM-like motif confirmed functionally in subsequent studies; original evidence is sequence analysis only","pmids":["8508798"],"is_preprint":false},{"year":2001,"finding":"The ITAM-like sequence in the CEACAM3 cytoplasmic domain is required for phagocytosis of Opa+ gonococci; mutation of the ITAM tyrosines abolishes signal transduction and bacterial uptake. CEACAM3-ITAM-mediated phagocytosis depends on Syk kinase and phospholipase C activity and triggers calcium flux and cell death.","method":"ITAM tyrosine mutagenesis in CGM1a, DT40 cell reconstitution, kinase inhibitors, pharmacological inhibition of PLC, calcium flux measurement, bacterial uptake assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis plus genetic reconstitution (DT40 cells), pharmacological dissection, replicated in multiple subsequent studies","pmids":["11278708"],"is_preprint":false},{"year":2001,"finding":"CEACAM3 cytoplasmic domain physically associates with calprotectin from granulocyte extracts; this interaction is calcium-modulated but phosphorylation-independent. CEACAM3 cytoplasmic domain is phosphorylated in vitro by PKC, casein kinase I, and Src kinase.","method":"Recombinant CEACAM3 cytoplasmic domain pulldown from granulocyte lysates, in vitro kinase assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single pulldown experiment, no reciprocal validation, single lab","pmids":["11708798"],"is_preprint":false},{"year":2003,"finding":"CEACAM3 is tyrosine-phosphorylated by a Src family kinase-dependent process upon gonococcal Opa protein engagement. This phosphorylation is necessary for efficient bacterial uptake; ITAM mutagenesis or kinase inhibitors reduce but do not abolish uptake. Ligated CEACAM3 recruits to a cytoskeleton-containing fraction, and actin polymerization foci form at bacterial attachment sites; cytochalasin D blocks all uptake.","method":"CEACAM3 ITAM mutagenesis, Src kinase inhibitors, HeLa-CEACAM3 transfection, subcellular fractionation, actin staining, cytochalasin D treatment, bacterial uptake assay","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis plus pharmacological inhibition plus subcellular fractionation plus cytoskeletal disruption; multiple orthogonal methods, replicated concept","pmids":["12864848"],"is_preprint":false},{"year":2004,"finding":"CEACAM3-mediated phagocytosis of Neisseria, Moraxella, and Haemophilus species is opsonin-independent and triggers membrane recruitment and GTP-loading of Rac. This requires the ITAM-like cytoplasmic sequence and Src family kinase phosphorylation. Dominant-negative Rac blocks CEACAM3- but not CEACAM6-mediated uptake. Blockage of CEACAM3-mediated events reduces primary granulocyte bactericidal capacity.","method":"Dominant-negative Rac expression, Src kinase inhibitors, ITAM mutagenesis, Rac GTP-loading assay, primary granulocyte bacterial killing assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic tools (dominant-negative, mutagenesis), biochemical GTP-loading assay, and primary cell functional assay; multiple orthogonal methods","pmids":["14707113"],"is_preprint":false},{"year":1996,"finding":"Antibody crosslinking of CD66d (CEACAM3) on neutrophils stimulates increased adhesion to endothelial cells via CD11/CD18 upregulation, requiring extracellular calcium; CD66d independently transmits signals from CD66a, b, and c.","method":"Neutrophil adhesion assay to HUVEC monolayers, CD66 monoclonal antibodies, CD18 blocking antibody, calcium chelation, sequential desensitization experiments","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional neutrophil assays with blocking antibodies; single lab but multiple CD66 members tested in parallel","pmids":["8699114"],"is_preprint":false},{"year":2007,"finding":"The guanine nucleotide exchange factor Vav directly associates with phosphorylated Tyr230 of CEACAM3 via its SH2 domain, colocalizes with CEACAM3 at bacterial contact sites, and is required for Rac GTP-loading and bacterial internalization. This represents a direct ITAM-to-GEF coupling that bypasses canonical ITAM signaling.","method":"Dominant-negative Vav overexpression, siRNA knockdown, Vav1/Vav2-deficient cell reconstitution, co-immunoprecipitation, SH2 domain direct binding assay with phosphopeptides, colocalization microscopy, TAT-mediated transduction in primary granulocytes","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct SH2-phosphopeptide binding, reciprocal co-IP, genetic knockout rescue, primary cell functional assay; multiple orthogonal methods","pmids":["17339478"],"is_preprint":false},{"year":2007,"finding":"Syk kinase is recruited to the CEACAM3 cytoplasmic ITAM upon bacterial binding, resulting in mutual phosphorylation of CEACAM3 and Syk. Syk is required for bacterial killing responses including the oxidative burst and degranulation, and for internalization when cargo size is large or ligand density is below a threshold.","method":"Co-immunoprecipitation, kinase inhibition, Syk-deficient cell studies, oxidative burst assay, degranulation assay, bacterial killing assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP plus genetic deficiency plus multiple functional assays (ROS, degranulation, killing); replicated concept from DT40 study","pmids":["17506820"],"is_preprint":false},{"year":2011,"finding":"The SH2 domains of the PI3K regulatory subunit bind directly to phospho-Tyr230 of CEACAM3. PI3K is rapidly recruited to CEACAM3 upon bacterial binding (confirmed by FRET). PI3K activity is dispensable for bacterial uptake but is critical for the oxidative burst and intracellular bacterial killing.","method":"SH2 domain binding assay, FRET analysis in intact cells, PI3K inhibitors, reactive oxygen species measurement, intracellular bacterial degradation assay, primary granulocyte experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct SH2-phosphosite binding plus FRET in intact cells plus pharmacological dissection of functional consequences; multiple orthogonal methods","pmids":["21216968"],"is_preprint":false},{"year":2012,"finding":"Adaptor proteins Nck1 and Nck2 bind to tyrosine-phosphorylated CEACAM3 via their SH2 domains in a phosphorylation-dependent manner. Nck constitutively associates with WAVE2 and recruits the WAVE complex to phosphorylated CEACAM3. Nck knockdown or genetic deletion, dominant-negative WAVE2, and WAVE/Nap1 knockdown all impair lamellipodia formation and bacterial internalization.","method":"Biochemical pulldown and co-IP, RNAi knockdown, Nck1/2 genetic deletion, dominant-negative WAVE2, shRNA knockdown, microscopy of lamellipodia, bacterial uptake assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphorylation-dependent SH2 binding, genetic deletion with rescue, multiple knockdown approaches, functional lamellipodia and phagocytosis readouts","pmids":["22448228"],"is_preprint":false},{"year":2012,"finding":"Grb14 SH2 domain binds to the CEACAM3 ITAM-like sequence and is recruited to bacteria-host cell contact sites (confirmed by FRET-FLIM). Grb14 acts as a negative regulator: its knockdown enhances and its overexpression reduces CEACAM3-mediated bacterial phagocytosis.","method":"SH2 domain microarray screen, biochemical co-IP, FRET-FLIM in intact cells, RNAi knockdown, overexpression, bacterial uptake assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — SH2 microarray plus FRET-FLIM direct association plus bidirectional genetic perturbation (KD and OE); multiple orthogonal methods single lab","pmids":["22948154"],"is_preprint":false},{"year":2016,"finding":"Moraxella catarrhalis UspA1 engages CEACAM3, triggering NF-κB activation via Syk and CARD9 pathway in a manner dependent on CEACAM3 ITAM phosphorylation. This leads to neutrophil degranulation, ROS production, and chemokine secretion.","method":"siRNA knockdown of CEACAM3/Syk/CARD9, kinase inhibitors, blocking antibodies, NF-κB luciferase reporter assay, chromatin immunoprecipitation, ELISA for chemokines, ROS assay, degranulation assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA plus pharmacological inhibition plus reporter assay plus ChIP; multiple orthogonal methods in single lab","pmids":["27038042"],"is_preprint":false},{"year":2017,"finding":"CEACAM3 cross-linking alone is insufficient to induce cytokine production; the inflammatory response requires integration with TLR signaling. Using CEABAC mouse neutrophils, genetic knockout shows Bcl10 (but not Malt1 uniquely) enables synergy between TLR4 and CEACAM3 for cytokine production, while Rac2, Bcl10, and Malt1 are dispensable for gonococcal engulfment but required for CEACAM3-mediated cytokine production.","method":"CEABAC transgenic mouse model, genetic knockout of Rac2/Bcl10/Malt1, ex vivo neutrophil infection, cytokine measurements, bacterial engulfment assay","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple knockouts in defined in vivo model; separates phagocytic and inflammatory arms mechanistically","pmids":["28886618"],"is_preprint":false},{"year":2022,"finding":"The receptor-type protein tyrosine phosphatase PTPRJ directly dephosphorylates the cytoplasmic tyrosine residues of CEACAM3 (demonstrated with purified recombinant PTPRJ on full-length CEACAM3 and phosphopeptide substrates). PTPRJ depletion enhances CEACAM3-mediated phagocytosis and lamellipodia formation, while constitutively active PTPRJ reduces bacterial uptake. PTPRJ-deficient human phagocytes exhibit exaggerated lamellipodia and enhanced opsonin-independent phagocytosis.","method":"In vitro phosphatase assay with recombinant PTPRJ and full-length CEACAM3, synthetic phosphopeptide substrate assay, PTPRJ depletion/overexpression in cells, primary human phagocyte PTPRJ knockdown, lamellipodia quantification, bacterial uptake assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstituted phosphatase assay on full-length CEACAM3 plus gain/loss-of-function in intact cells plus primary human phagocytes; multiple orthogonal methods","pmids":["35850306"],"is_preprint":false},{"year":2023,"finding":"A genome-wide CRISPR/Cas9 screen identified CYRI-B (a Rac-GTP-sequestering protein) as a negative regulator of CEACAM3-mediated phagocytosis. CYRI-B knockout in HL-60 cells enhances CEACAM3-downstream Rac GTP-loading and PAK phosphorylation, leading to increased bacterial phagocytosis; complementation reverts the phenotype.","method":"Genome-wide CRISPR/Cas9 screen, next-generation sequencing of sgRNA enrichment, clonal CYRI-B knockout HL-60 cells, genetic complementation, Rac GTP-loading assay, PAK phosphorylation assay, bacterial uptake assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genome-wide genetic screen confirmed by clonal knockout plus biochemical validation plus genetic rescue; multiple orthogonal approaches in single lab","pmids":["37264948"],"is_preprint":false},{"year":2016,"finding":"LOS lgtF mutant gonococci expressing OpaI cannot interact functionally with CEACAM3 to promote phagocytosis in HeLa-CEACAM3 cells or trigger chemiluminescence in neutrophils, indicating that the alpha-oligosaccharide moiety of LOS is required for Opa protein to adopt the conformation needed for CEACAM3 engagement.","method":"HeLa-CEACAM3 bacterial uptake assay, neutrophil chemiluminescence assay, LOS lgtF mutant bacteria","journal":"Journal of Huazhong University of Science and Technology","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, two complementary functional assays but no direct binding measurement; establishes LOS structural requirement for Opa-CEACAM3 interaction","pmids":["27376801"],"is_preprint":false}],"current_model":"CEACAM3 is a granulocyte-restricted, immunoglobulin-superfamily transmembrane receptor that mediates opsonin-independent phagocytosis of human-specific Gram-negative pathogens (Neisseria, Moraxella, Haemophilus) by binding their Opa/UspA1 adhesins via its extracellular IgV-like domain; upon engagement, Src family kinases phosphorylate a cytoplasmic ITAM-like motif (principally Tyr230), creating docking sites for Syk, Vav (GEF for Rac), PI3K, Nck (which recruits the WAVE complex), and the inhibitory adaptors Grb14 and CYRI-B, collectively orchestrating Rac-GTP-driven actin polymerization and lamellipodia-based bacterial engulfment; bacterial killing is further promoted by Syk-dependent oxidative burst and degranulation, while PI3K activity is specifically required for intracellular pathogen destruction; termination of signaling is achieved by the phosphatase PTPRJ, which directly dephosphorylates CEACAM3's cytoplasmic tyrosines, and by CYRI-B, which sequesters Rac-GTP; inflammatory cytokine production additionally requires co-engagement of TLRs and the Bcl10 scaffold."},"narrative":{"mechanistic_narrative":"CEACAM3 (CGM1a/CD66d) is a granulocyte-expressed immunoglobulin-superfamily receptor that drives opsonin-independent phagocytosis of human-restricted Gram-negative pathogens by directly binding bacterial adhesins, serving as a single-receptor phagocytic and pro-inflammatory module in neutrophils [PMID:8962144, PMID:14707113]. Its extracellular domain functions as a direct receptor for gonococcal Opa proteins, mediating bacterial binding and internalization, and engages Neisseria, Moraxella, and Haemophilus species [PMID:8962144, PMID:14707113]; functional Opa engagement additionally depends on the alpha-oligosaccharide of bacterial LOS to present the adhesin in a competent conformation [PMID:27376801]. Signaling is routed through a cytoplasmic ITAM-like motif (Cyt1 exon) that, upon bacterial engagement, is tyrosine-phosphorylated by Src family kinases—principally at Tyr230—an event required for efficient uptake [PMID:8508798, PMID:11278708, PMID:12864848]. The phosphorylated ITAM serves as a docking platform: Vav binds via its SH2 domain to couple the receptor directly to Rac GTP-loading and actin-driven internalization [PMID:14707113, PMID:17339478]; Nck binds phospho-CEACAM3 and recruits the WAVE complex to build lamellipodia for engulfment [PMID:22448228]; Syk is recruited and is required for the oxidative burst, degranulation, and uptake of large cargo [PMID:17506820]; and the PI3K regulatory subunit binds phospho-Tyr230 to drive the oxidative burst and intracellular bacterial killing without being required for uptake itself [PMID:21216968]. Signaling is restrained by negative regulators that dock on or downstream of the receptor: the SH2 adaptor Grb14 binds the ITAM [PMID:22948154], the phosphatase PTPRJ directly dephosphorylates CEACAM3's cytoplasmic tyrosines [PMID:35850306], and the Rac-GTP-sequestering protein CYRI-B limits Rac/PAK signaling [PMID:37264948], each constraining phagocytosis and lamellipodia formation. The inflammatory output is mechanistically separable from engulfment: cytokine production requires integration with TLR signaling through a Syk–CARD9–NF-κB axis and the Bcl10 scaffold, whereas engulfment proceeds independently of these factors [PMID:27038042, PMID:28886618].","teleology":[{"year":1993,"claim":"Defined the molecular feature that would make CEACAM3 a signaling receptor by identifying an ITAM-like motif in its cytoplasmic tail, distinguishing it from a non-signaling splice variant.","evidence":"Gene cloning, exon mapping, and splice-variant sequence analysis","pmids":["8508798"],"confidence":"Medium","gaps":["Motif identified by sequence only, with no functional demonstration at this stage","Did not establish which tyrosines are functionally phosphorylated"]},{"year":1996,"claim":"Established CEACAM3 as a direct cell-surface receptor for bacterial Opa proteins, answering whether neutrophils possess a dedicated adhesin receptor.","evidence":"PMN lysate ligand binding, HeLa transfection with internalization assay, and monoclonal antibody blockade","pmids":["8962144"],"confidence":"High","gaps":["Did not map the binding site within the extracellular domain","Downstream signaling not yet defined"]},{"year":1996,"claim":"Showed CEACAM3 (CD66d) crosslinking transmits independent activating signals in neutrophils, raising integrin-mediated adhesion via CD11/CD18.","evidence":"Neutrophil adhesion to HUVEC with CD66 antibodies, CD18 blockade, and calcium chelation","pmids":["8699114"],"confidence":"Medium","gaps":["Antibody crosslinking does not establish the physiological bacterial trigger","Molecular link to integrin upregulation not defined"]},{"year":2001,"claim":"Demonstrated that the ITAM-like motif is functionally required for phagocytosis and that uptake depends on Syk and PLC, converting a sequence motif into a defined signaling requirement.","evidence":"ITAM tyrosine mutagenesis, DT40 reconstitution, kinase/PLC inhibitors, calcium flux and uptake assays","pmids":["11278708"],"confidence":"High","gaps":["Direct SH2 partners docking on the ITAM not yet identified","Connection to actin machinery unresolved"]},{"year":2001,"claim":"Reported a calcium-modulated, phosphorylation-independent association of the cytoplasmic domain with calprotectin and in vitro phosphorylation by multiple kinases.","evidence":"Recombinant cytoplasmic-domain pulldown from granulocyte lysate and in vitro kinase assays","pmids":["11708798"],"confidence":"Medium","gaps":["Single pulldown without reciprocal validation","Functional role of calprotectin binding in phagocytosis not established","In vitro kinases may not reflect the in vivo Src-dependent event"]},{"year":2003,"claim":"Linked Src-family-kinase-dependent tyrosine phosphorylation of CEACAM3 to actin-based uptake, connecting the receptor to the cytoskeleton.","evidence":"ITAM mutagenesis, Src inhibitors, subcellular fractionation, actin staining, and cytochalasin D treatment","pmids":["12864848"],"confidence":"High","gaps":["The actin-nucleating machinery downstream was not identified","ITAM-independent residual uptake component left unexplained"]},{"year":2004,"claim":"Identified Rac GTP-loading as the core actin-regulatory output of CEACAM3 and showed the receptor confers genuine bactericidal capacity across multiple human pathogens.","evidence":"Dominant-negative Rac, Src inhibitors, ITAM mutagenesis, Rac GTP-loading and primary granulocyte killing assays","pmids":["14707113"],"confidence":"High","gaps":["The GEF coupling the ITAM to Rac was not yet identified","Distinct from CEACAM6, mechanism of selectivity unresolved"]},{"year":2007,"claim":"Resolved how CEACAM3 activates Rac by showing Vav binds phospho-Tyr230 directly and is required for Rac loading, establishing a direct ITAM-to-GEF coupling.","evidence":"SH2-phosphopeptide binding, co-IP, Vav1/Vav2-deficient cell rescue, colocalization, and primary granulocyte transduction","pmids":["17339478"],"confidence":"High","gaps":["Other ITAM-docking effectors not yet mapped","Quantitative contribution of Vav versus other GEFs unclear"]},{"year":2007,"claim":"Defined Syk's role at the ITAM as required for microbicidal effector functions and for uptake under demanding cargo/ligand conditions.","evidence":"Co-IP, kinase inhibition, Syk-deficient cells, oxidative burst, degranulation, and killing assays","pmids":["17506820"],"confidence":"High","gaps":["Whether Syk and Vav act in series or parallel not resolved","Substrates of Syk in this pathway not enumerated"]},{"year":2011,"claim":"Separated uptake from killing by showing PI3K binds phospho-Tyr230 and is dispensable for engulfment but required for the oxidative burst and intracellular killing.","evidence":"SH2 binding assay, FRET in intact cells, PI3K inhibitors, ROS and intracellular degradation assays in primary granulocytes","pmids":["21216968"],"confidence":"High","gaps":["Downstream PI3K effectors not identified","Mechanism of intracellular killing beyond ROS not detailed"]},{"year":2012,"claim":"Connected phospho-CEACAM3 to the actin-nucleation machinery via Nck-mediated recruitment of the WAVE complex, explaining lamellipodial engulfment.","evidence":"Co-IP/pulldown, Nck1/2 deletion with rescue, dominant-negative WAVE2, WAVE/Nap1 knockdown, lamellipodia microscopy and uptake assays","pmids":["22448228"],"confidence":"High","gaps":["Coordination between Nck-WAVE and Vav-Rac branches not resolved","Spatial hierarchy of effector recruitment unknown"]},{"year":2012,"claim":"Identified Grb14 as an ITAM-docking negative regulator, establishing that the same phosphomotif recruits both activating and inhibitory adaptors.","evidence":"SH2 microarray screen, co-IP, FRET-FLIM, knockdown and overexpression with uptake assays","pmids":["22948154"],"confidence":"High","gaps":["Mechanism by which Grb14 dampens signaling not defined","Competition with activating SH2 effectors not directly measured"]},{"year":2016,"claim":"Extended CEACAM3 signaling to transcriptional inflammatory output by linking UspA1 engagement to NF-κB via a Syk–CARD9 axis.","evidence":"siRNA of CEACAM3/Syk/CARD9, inhibitors, NF-κB reporter, ChIP, chemokine ELISA, ROS and degranulation assays","pmids":["27038042"],"confidence":"High","gaps":["Did not establish requirement for co-receptor input","Intermediate steps between Syk and CARD9 unmapped"]},{"year":2016,"claim":"Showed the bacterial LOS alpha-oligosaccharide is required for Opa to functionally engage CEACAM3, defining a structural prerequisite on the ligand side.","evidence":"HeLa-CEACAM3 uptake and neutrophil chemiluminescence assays with LOS lgtF mutant bacteria","pmids":["27376801"],"confidence":"Medium","gaps":["No direct binding measurement to confirm conformational model","Single lab, functional readouts only"]},{"year":2017,"claim":"Mechanistically separated the phagocytic and inflammatory arms by showing cytokine production requires TLR co-engagement and Bcl10, while engulfment does not.","evidence":"CEABAC transgenic mice, Rac2/Bcl10/Malt1 knockouts, ex vivo neutrophil infection, cytokine and engulfment assays","pmids":["28886618"],"confidence":"High","gaps":["Molecular nature of TLR-CEACAM3 integration not defined","Role of Malt1 left ambiguous"]},{"year":2022,"claim":"Identified the direct off-switch by demonstrating PTPRJ dephosphorylates CEACAM3 cytoplasmic tyrosines to restrain phagocytosis and lamellipodia.","evidence":"In vitro phosphatase assay on full-length CEACAM3 and phosphopeptides, PTPRJ depletion/overexpression, primary human phagocyte knockdown with lamellipodia and uptake quantification","pmids":["35850306"],"confidence":"High","gaps":["Site preference of PTPRJ on the ITAM not detailed","Regulation of PTPRJ recruitment to CEACAM3 unknown"]},{"year":2023,"claim":"Added a downstream brake by identifying CYRI-B, via a genome-wide screen, as a Rac-GTP sequester that limits CEACAM3 phagocytosis.","evidence":"Genome-wide CRISPR screen, clonal CYRI-B knockout in HL-60, complementation, Rac GTP-loading and PAK phosphorylation assays, uptake assay","pmids":["37264948"],"confidence":"High","gaps":["Whether CYRI-B acts on a CEACAM3-specific Rac pool not resolved","Interplay with PTPRJ and Grb14 in signal termination not defined"]},{"year":null,"claim":"How the activating (Vav, Nck-WAVE, Syk, PI3K) and inhibitory (Grb14, PTPRJ, CYRI-B) modules are coordinated in space and time on a single ITAM to set phagocytic threshold versus inflammatory output remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No integrated structural or kinetic model of competing SH2 effector occupancy","Stoichiometry and ordering of activating versus inhibitory adaptor recruitment unknown","Physiological setting that tips the balance between engulfment and cytokine production undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[0,5,16]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[2,4,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7,9,10,11]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,5,6]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[4,10]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,8,12,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,7,9]}],"complexes":[],"partners":["VAV1","SYK","NCK1","PIK3R1","GRB14","PTPRJ","CYRI-B"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P40198","full_name":"Cell adhesion molecule CEACAM3","aliases":["Carcinoembryonic antigen CGM1","Carcinoembryonic antigen-related cell adhesion molecule 3","CEA cell adhesion molecule 3"],"length_aa":252,"mass_kda":27.1,"function":"Major granulocyte receptor mediating recognition and efficient opsonin-independent phagocytosis of CEACAM-binding microorganisms, including Neissiria, Moxarella and Haemophilus species, thus playing an important role in the clearance of pathogens by the innate immune system. Responsible for RAC1 stimulation in the course of pathogen phagocytosis","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/P40198/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CEACAM3","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CEACAM3","total_profiled":1310},"omim":[{"mim_id":"609142","title":"CEA CELL ADHESION MOLECULE 3; CEACAM3","url":"https://www.omim.org/entry/609142"},{"mim_id":"603855","title":"CYSTIC FIBROSIS, MODIFIER OF, 1; CFM1","url":"https://www.omim.org/entry/603855"},{"mim_id":"163980","title":"CEA CELL ADHESION MOLECULE 6; CEACAM6","url":"https://www.omim.org/entry/163980"},{"mim_id":"114890","title":"CEA CELL ADHESION MOLECULE 5; CEACAM5","url":"https://www.omim.org/entry/114890"},{"mim_id":"112205","title":"CD79A ANTIGEN; CD79A","url":"https://www.omim.org/entry/112205"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"bone marrow","ntpm":41.8}],"url":"https://www.proteinatlas.org/search/CEACAM3"},"hgnc":{"alias_symbol":["CD66d","CGM1a"],"prev_symbol":["CGM1"]},"alphafold":{"accession":"P40198","domains":[{"cath_id":"2.60.40.10","chopping":"38-142","consensus_level":"high","plddt":96.4681,"start":38,"end":142},{"cath_id":"1.20.5","chopping":"148-182","consensus_level":"medium","plddt":89.1737,"start":148,"end":182}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P40198","model_url":"https://alphafold.ebi.ac.uk/files/AF-P40198-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P40198-F1-predicted_aligned_error_v6.png","plddt_mean":78.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CEACAM3","jax_strain_url":"https://www.jax.org/strain/search?query=CEACAM3"},"sequence":{"accession":"P40198","fasta_url":"https://rest.uniprot.org/uniprotkb/P40198.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P40198/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P40198"}},"corpus_meta":[{"pmid":"8962144","id":"PMC_8962144","title":"CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8962144","citation_count":148,"is_preprint":false},{"pmid":"14707113","id":"PMC_14707113","title":"Granulocyte CEACAM3 is a phagocytic receptor of the innate immune system that mediates recognition and elimination of human-specific pathogens.","date":"2004","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/14707113","citation_count":131,"is_preprint":false},{"pmid":"8699114","id":"PMC_8699114","title":"CD66a, CD66b, CD66c, and CD66d each independently stimulate neutrophils.","date":"1996","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/8699114","citation_count":120,"is_preprint":false},{"pmid":"12864848","id":"PMC_12864848","title":"Immunoreceptor tyrosine-based activation motif phosphorylation during engulfment of Neisseria gonorrhoeae by the neutrophil-restricted CEACAM3 (CD66d) receptor.","date":"2003","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/12864848","citation_count":71,"is_preprint":false},{"pmid":"8508798","id":"PMC_8508798","title":"Genomic organization, splice variants and expression of CGM1, a CD66-related member of the carcinoembryonic antigen gene family.","date":"1993","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8508798","citation_count":67,"is_preprint":false},{"pmid":"17506820","id":"PMC_17506820","title":"The specific innate immune receptor CEACAM3 triggers neutrophil bactericidal activities via a Syk kinase-dependent pathway.","date":"2007","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/17506820","citation_count":56,"is_preprint":false},{"pmid":"11278708","id":"PMC_11278708","title":"The CGM1a (CEACAM3/CD66d)-mediated phagocytic pathway of Neisseria gonorrhoeae expressing opacity proteins is also the pathway to cell death.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11278708","citation_count":51,"is_preprint":false},{"pmid":"17339478","id":"PMC_17339478","title":"The granulocyte receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) directly associates with Vav to promote phagocytosis of human pathogens.","date":"2007","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/17339478","citation_count":44,"is_preprint":false},{"pmid":"18606569","id":"PMC_18606569","title":"CEACAM3: an innate immune receptor directed against human-restricted bacterial pathogens.","date":"2008","source":"International journal of medical microbiology : IJMM","url":"https://pubmed.ncbi.nlm.nih.gov/18606569","citation_count":38,"is_preprint":false},{"pmid":"22448228","id":"PMC_22448228","title":"The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22448228","citation_count":36,"is_preprint":false},{"pmid":"27038042","id":"PMC_27038042","title":"Moraxella catarrhalis induces CEACAM3-Syk-CARD9-dependent activation of human granulocytes.","date":"2016","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/27038042","citation_count":29,"is_preprint":false},{"pmid":"21216968","id":"PMC_21216968","title":"Phosphatidylinositol 3'-kinase activity is critical for initiating the oxidative burst and bacterial destruction during CEACAM3-mediated phagocytosis.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21216968","citation_count":28,"is_preprint":false},{"pmid":"22469950","id":"PMC_22469950","title":"HemITAM signaling by CEACAM3, a human granulocyte receptor recognizing bacterial pathogens.","date":"2012","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/22469950","citation_count":25,"is_preprint":false},{"pmid":"32117212","id":"PMC_32117212","title":"CEACAM3-A Prim(at)e Invention for Opsonin-Independent Phagocytosis of Bacteria.","date":"2020","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/32117212","citation_count":19,"is_preprint":false},{"pmid":"11708798","id":"PMC_11708798","title":"The microbial receptor CEACAM3 is linked to the calprotectin complex in granulocytes.","date":"2001","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11708798","citation_count":12,"is_preprint":false},{"pmid":"28886618","id":"PMC_28886618","title":"Bcl10 synergistically links CEACAM3 and TLR-dependent inflammatory signalling.","date":"2017","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/28886618","citation_count":9,"is_preprint":false},{"pmid":"35850306","id":"PMC_35850306","title":"Phagocytosis mediated by the human granulocyte receptor CEACAM3 is limited by the receptor-type protein tyrosine phosphatase PTPRJ.","date":"2022","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35850306","citation_count":7,"is_preprint":false},{"pmid":"22948154","id":"PMC_22948154","title":"Grb14 is a negative regulator of CEACAM3-mediated phagocytosis of pathogenic bacteria.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22948154","citation_count":7,"is_preprint":false},{"pmid":"32606071","id":"PMC_32606071","title":"CEACAM3 decreases asthma exacerbations and modulates respiratory syncytial virus latent infection in children.","date":"2020","source":"Thorax","url":"https://pubmed.ncbi.nlm.nih.gov/32606071","citation_count":6,"is_preprint":false},{"pmid":"34847408","id":"PMC_34847408","title":"Antibody ligation of CEACAM1, CEACAM3, and CEACAM6, differentially enhance the cytokine release of human neutrophils in responses to Candida albicans.","date":"2021","source":"Cellular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34847408","citation_count":6,"is_preprint":false},{"pmid":"38215579","id":"PMC_38215579","title":"Controling the cytoskeleton during CEACAM3-mediated phagocytosis.","date":"2024","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38215579","citation_count":4,"is_preprint":false},{"pmid":"26556959","id":"PMC_26556959","title":"The Trend of CEACAM3 Blood Expression as Number Index of the CTCs in the Colorectal Cancer Perioperative Course.","date":"2015","source":"Mediators of 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Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban","url":"https://pubmed.ncbi.nlm.nih.gov/27376801","citation_count":3,"is_preprint":false},{"pmid":"37264948","id":"PMC_37264948","title":"A genome-wide genetic screen identifies CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis.","date":"2023","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/37264948","citation_count":2,"is_preprint":false},{"pmid":"40876590","id":"PMC_40876590","title":"Cgm1 is a β-galactoside α-(1 → 4)-mannosyltransferase involved in the biosynthesis of capsular glucuronoxylomannogalactan in Cryptococcus neoformans.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40876590","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.29.646107","title":"Expression of human CEACAMs promotes inflammation and organ damage during systemic  <i>Candida albicans</i>  infection in mice","date":"2025-04-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.29.646107","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.10.27.684817","title":"Replication-competent SIVcpz CRISPR screen identifies barriers to successful cross-species transmission","date":"2025-10-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.27.684817","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":30766,"output_tokens":4821,"usd":0.141434,"retried_sync":true,"stage1_stop_reason":"refusal"},"stage2":{"model":"claude-opus-4-8","input_tokens":13005,"output_tokens":5202,"usd":0.097537,"stage2_stop_reason":"end_turn"},"total_usd":0.238971,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"CGM1a (CEACAM3) expressed on neutrophils acts as a direct receptor for gonococcal opacity (Opa) proteins; HeLa cells expressing CGM1a bind and internalize OpaI+ E. coli, and monoclonal antibody COL-1 against CGM1 blocks this interaction.\",\n      \"method\": \"Ligand binding assay with PMN lysates, HeLa cell transfection with CGM1a, bacterial internalization assay, monoclonal antibody blocking\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding demonstrated with transfected cells, antibody blockade, and confirmed by multiple orthogonal methods; independently replicated by subsequent studies\",\n      \"pmids\": [\"8962144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"CGM1a (CEACAM3) contains an ITAM-like consensus motif in its cytoplasmic domain (encoded by the Cyt1 exon) and a shorter splice variant CGM1c lacking this motif; the ITAM-like sequence is implicated in signal transduction.\",\n      \"method\": \"Gene cloning, exon mapping, sequence analysis of splice variants\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — sequence-based identification of ITAM-like motif confirmed functionally in subsequent studies; original evidence is sequence analysis only\",\n      \"pmids\": [\"8508798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The ITAM-like sequence in the CEACAM3 cytoplasmic domain is required for phagocytosis of Opa+ gonococci; mutation of the ITAM tyrosines abolishes signal transduction and bacterial uptake. CEACAM3-ITAM-mediated phagocytosis depends on Syk kinase and phospholipase C activity and triggers calcium flux and cell death.\",\n      \"method\": \"ITAM tyrosine mutagenesis in CGM1a, DT40 cell reconstitution, kinase inhibitors, pharmacological inhibition of PLC, calcium flux measurement, bacterial uptake assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis plus genetic reconstitution (DT40 cells), pharmacological dissection, replicated in multiple subsequent studies\",\n      \"pmids\": [\"11278708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CEACAM3 cytoplasmic domain physically associates with calprotectin from granulocyte extracts; this interaction is calcium-modulated but phosphorylation-independent. CEACAM3 cytoplasmic domain is phosphorylated in vitro by PKC, casein kinase I, and Src kinase.\",\n      \"method\": \"Recombinant CEACAM3 cytoplasmic domain pulldown from granulocyte lysates, in vitro kinase assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown experiment, no reciprocal validation, single lab\",\n      \"pmids\": [\"11708798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CEACAM3 is tyrosine-phosphorylated by a Src family kinase-dependent process upon gonococcal Opa protein engagement. This phosphorylation is necessary for efficient bacterial uptake; ITAM mutagenesis or kinase inhibitors reduce but do not abolish uptake. Ligated CEACAM3 recruits to a cytoskeleton-containing fraction, and actin polymerization foci form at bacterial attachment sites; cytochalasin D blocks all uptake.\",\n      \"method\": \"CEACAM3 ITAM mutagenesis, Src kinase inhibitors, HeLa-CEACAM3 transfection, subcellular fractionation, actin staining, cytochalasin D treatment, bacterial uptake assay\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis plus pharmacological inhibition plus subcellular fractionation plus cytoskeletal disruption; multiple orthogonal methods, replicated concept\",\n      \"pmids\": [\"12864848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CEACAM3-mediated phagocytosis of Neisseria, Moraxella, and Haemophilus species is opsonin-independent and triggers membrane recruitment and GTP-loading of Rac. This requires the ITAM-like cytoplasmic sequence and Src family kinase phosphorylation. Dominant-negative Rac blocks CEACAM3- but not CEACAM6-mediated uptake. Blockage of CEACAM3-mediated events reduces primary granulocyte bactericidal capacity.\",\n      \"method\": \"Dominant-negative Rac expression, Src kinase inhibitors, ITAM mutagenesis, Rac GTP-loading assay, primary granulocyte bacterial killing assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic tools (dominant-negative, mutagenesis), biochemical GTP-loading assay, and primary cell functional assay; multiple orthogonal methods\",\n      \"pmids\": [\"14707113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Antibody crosslinking of CD66d (CEACAM3) on neutrophils stimulates increased adhesion to endothelial cells via CD11/CD18 upregulation, requiring extracellular calcium; CD66d independently transmits signals from CD66a, b, and c.\",\n      \"method\": \"Neutrophil adhesion assay to HUVEC monolayers, CD66 monoclonal antibodies, CD18 blocking antibody, calcium chelation, sequential desensitization experiments\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional neutrophil assays with blocking antibodies; single lab but multiple CD66 members tested in parallel\",\n      \"pmids\": [\"8699114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The guanine nucleotide exchange factor Vav directly associates with phosphorylated Tyr230 of CEACAM3 via its SH2 domain, colocalizes with CEACAM3 at bacterial contact sites, and is required for Rac GTP-loading and bacterial internalization. This represents a direct ITAM-to-GEF coupling that bypasses canonical ITAM signaling.\",\n      \"method\": \"Dominant-negative Vav overexpression, siRNA knockdown, Vav1/Vav2-deficient cell reconstitution, co-immunoprecipitation, SH2 domain direct binding assay with phosphopeptides, colocalization microscopy, TAT-mediated transduction in primary granulocytes\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct SH2-phosphopeptide binding, reciprocal co-IP, genetic knockout rescue, primary cell functional assay; multiple orthogonal methods\",\n      \"pmids\": [\"17339478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Syk kinase is recruited to the CEACAM3 cytoplasmic ITAM upon bacterial binding, resulting in mutual phosphorylation of CEACAM3 and Syk. Syk is required for bacterial killing responses including the oxidative burst and degranulation, and for internalization when cargo size is large or ligand density is below a threshold.\",\n      \"method\": \"Co-immunoprecipitation, kinase inhibition, Syk-deficient cell studies, oxidative burst assay, degranulation assay, bacterial killing assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP plus genetic deficiency plus multiple functional assays (ROS, degranulation, killing); replicated concept from DT40 study\",\n      \"pmids\": [\"17506820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The SH2 domains of the PI3K regulatory subunit bind directly to phospho-Tyr230 of CEACAM3. PI3K is rapidly recruited to CEACAM3 upon bacterial binding (confirmed by FRET). PI3K activity is dispensable for bacterial uptake but is critical for the oxidative burst and intracellular bacterial killing.\",\n      \"method\": \"SH2 domain binding assay, FRET analysis in intact cells, PI3K inhibitors, reactive oxygen species measurement, intracellular bacterial degradation assay, primary granulocyte experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct SH2-phosphosite binding plus FRET in intact cells plus pharmacological dissection of functional consequences; multiple orthogonal methods\",\n      \"pmids\": [\"21216968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Adaptor proteins Nck1 and Nck2 bind to tyrosine-phosphorylated CEACAM3 via their SH2 domains in a phosphorylation-dependent manner. Nck constitutively associates with WAVE2 and recruits the WAVE complex to phosphorylated CEACAM3. Nck knockdown or genetic deletion, dominant-negative WAVE2, and WAVE/Nap1 knockdown all impair lamellipodia formation and bacterial internalization.\",\n      \"method\": \"Biochemical pulldown and co-IP, RNAi knockdown, Nck1/2 genetic deletion, dominant-negative WAVE2, shRNA knockdown, microscopy of lamellipodia, bacterial uptake assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphorylation-dependent SH2 binding, genetic deletion with rescue, multiple knockdown approaches, functional lamellipodia and phagocytosis readouts\",\n      \"pmids\": [\"22448228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Grb14 SH2 domain binds to the CEACAM3 ITAM-like sequence and is recruited to bacteria-host cell contact sites (confirmed by FRET-FLIM). Grb14 acts as a negative regulator: its knockdown enhances and its overexpression reduces CEACAM3-mediated bacterial phagocytosis.\",\n      \"method\": \"SH2 domain microarray screen, biochemical co-IP, FRET-FLIM in intact cells, RNAi knockdown, overexpression, bacterial uptake assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SH2 microarray plus FRET-FLIM direct association plus bidirectional genetic perturbation (KD and OE); multiple orthogonal methods single lab\",\n      \"pmids\": [\"22948154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Moraxella catarrhalis UspA1 engages CEACAM3, triggering NF-κB activation via Syk and CARD9 pathway in a manner dependent on CEACAM3 ITAM phosphorylation. This leads to neutrophil degranulation, ROS production, and chemokine secretion.\",\n      \"method\": \"siRNA knockdown of CEACAM3/Syk/CARD9, kinase inhibitors, blocking antibodies, NF-κB luciferase reporter assay, chromatin immunoprecipitation, ELISA for chemokines, ROS assay, degranulation assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus pharmacological inhibition plus reporter assay plus ChIP; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"27038042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CEACAM3 cross-linking alone is insufficient to induce cytokine production; the inflammatory response requires integration with TLR signaling. Using CEABAC mouse neutrophils, genetic knockout shows Bcl10 (but not Malt1 uniquely) enables synergy between TLR4 and CEACAM3 for cytokine production, while Rac2, Bcl10, and Malt1 are dispensable for gonococcal engulfment but required for CEACAM3-mediated cytokine production.\",\n      \"method\": \"CEABAC transgenic mouse model, genetic knockout of Rac2/Bcl10/Malt1, ex vivo neutrophil infection, cytokine measurements, bacterial engulfment assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple knockouts in defined in vivo model; separates phagocytic and inflammatory arms mechanistically\",\n      \"pmids\": [\"28886618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The receptor-type protein tyrosine phosphatase PTPRJ directly dephosphorylates the cytoplasmic tyrosine residues of CEACAM3 (demonstrated with purified recombinant PTPRJ on full-length CEACAM3 and phosphopeptide substrates). PTPRJ depletion enhances CEACAM3-mediated phagocytosis and lamellipodia formation, while constitutively active PTPRJ reduces bacterial uptake. PTPRJ-deficient human phagocytes exhibit exaggerated lamellipodia and enhanced opsonin-independent phagocytosis.\",\n      \"method\": \"In vitro phosphatase assay with recombinant PTPRJ and full-length CEACAM3, synthetic phosphopeptide substrate assay, PTPRJ depletion/overexpression in cells, primary human phagocyte PTPRJ knockdown, lamellipodia quantification, bacterial uptake assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstituted phosphatase assay on full-length CEACAM3 plus gain/loss-of-function in intact cells plus primary human phagocytes; multiple orthogonal methods\",\n      \"pmids\": [\"35850306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A genome-wide CRISPR/Cas9 screen identified CYRI-B (a Rac-GTP-sequestering protein) as a negative regulator of CEACAM3-mediated phagocytosis. CYRI-B knockout in HL-60 cells enhances CEACAM3-downstream Rac GTP-loading and PAK phosphorylation, leading to increased bacterial phagocytosis; complementation reverts the phenotype.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen, next-generation sequencing of sgRNA enrichment, clonal CYRI-B knockout HL-60 cells, genetic complementation, Rac GTP-loading assay, PAK phosphorylation assay, bacterial uptake assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide genetic screen confirmed by clonal knockout plus biochemical validation plus genetic rescue; multiple orthogonal approaches in single lab\",\n      \"pmids\": [\"37264948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LOS lgtF mutant gonococci expressing OpaI cannot interact functionally with CEACAM3 to promote phagocytosis in HeLa-CEACAM3 cells or trigger chemiluminescence in neutrophils, indicating that the alpha-oligosaccharide moiety of LOS is required for Opa protein to adopt the conformation needed for CEACAM3 engagement.\",\n      \"method\": \"HeLa-CEACAM3 bacterial uptake assay, neutrophil chemiluminescence assay, LOS lgtF mutant bacteria\",\n      \"journal\": \"Journal of Huazhong University of Science and Technology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, two complementary functional assays but no direct binding measurement; establishes LOS structural requirement for Opa-CEACAM3 interaction\",\n      \"pmids\": [\"27376801\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CEACAM3 is a granulocyte-restricted, immunoglobulin-superfamily transmembrane receptor that mediates opsonin-independent phagocytosis of human-specific Gram-negative pathogens (Neisseria, Moraxella, Haemophilus) by binding their Opa/UspA1 adhesins via its extracellular IgV-like domain; upon engagement, Src family kinases phosphorylate a cytoplasmic ITAM-like motif (principally Tyr230), creating docking sites for Syk, Vav (GEF for Rac), PI3K, Nck (which recruits the WAVE complex), and the inhibitory adaptors Grb14 and CYRI-B, collectively orchestrating Rac-GTP-driven actin polymerization and lamellipodia-based bacterial engulfment; bacterial killing is further promoted by Syk-dependent oxidative burst and degranulation, while PI3K activity is specifically required for intracellular pathogen destruction; termination of signaling is achieved by the phosphatase PTPRJ, which directly dephosphorylates CEACAM3's cytoplasmic tyrosines, and by CYRI-B, which sequesters Rac-GTP; inflammatory cytokine production additionally requires co-engagement of TLRs and the Bcl10 scaffold.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CEACAM3 (CGM1a/CD66d) is a granulocyte-expressed immunoglobulin-superfamily receptor that drives opsonin-independent phagocytosis of human-restricted Gram-negative pathogens by directly binding bacterial adhesins, serving as a single-receptor phagocytic and pro-inflammatory module in neutrophils [#0, #5]. Its extracellular domain functions as a direct receptor for gonococcal Opa proteins, mediating bacterial binding and internalization, and engages Neisseria, Moraxella, and Haemophilus species [#0, #5]; functional Opa engagement additionally depends on the alpha-oligosaccharide of bacterial LOS to present the adhesin in a competent conformation [#16]. Signaling is routed through a cytoplasmic ITAM-like motif (Cyt1 exon) that, upon bacterial engagement, is tyrosine-phosphorylated by Src family kinases—principally at Tyr230—an event required for efficient uptake [#1, #2, #4]. The phosphorylated ITAM serves as a docking platform: Vav binds via its SH2 domain to couple the receptor directly to Rac GTP-loading and actin-driven internalization [#5, #7]; Nck binds phospho-CEACAM3 and recruits the WAVE complex to build lamellipodia for engulfment [#10]; Syk is recruited and is required for the oxidative burst, degranulation, and uptake of large cargo [#8]; and the PI3K regulatory subunit binds phospho-Tyr230 to drive the oxidative burst and intracellular bacterial killing without being required for uptake itself [#9]. Signaling is restrained by negative regulators that dock on or downstream of the receptor: the SH2 adaptor Grb14 binds the ITAM [#11], the phosphatase PTPRJ directly dephosphorylates CEACAM3's cytoplasmic tyrosines [#14], and the Rac-GTP-sequestering protein CYRI-B limits Rac/PAK signaling [#15], each constraining phagocytosis and lamellipodia formation. The inflammatory output is mechanistically separable from engulfment: cytokine production requires integration with TLR signaling through a Syk–CARD9–NF-κB axis and the Bcl10 scaffold, whereas engulfment proceeds independently of these factors [#12, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Defined the molecular feature that would make CEACAM3 a signaling receptor by identifying an ITAM-like motif in its cytoplasmic tail, distinguishing it from a non-signaling splice variant.\",\n      \"evidence\": \"Gene cloning, exon mapping, and splice-variant sequence analysis\",\n      \"pmids\": [\"8508798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Motif identified by sequence only, with no functional demonstration at this stage\", \"Did not establish which tyrosines are functionally phosphorylated\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Established CEACAM3 as a direct cell-surface receptor for bacterial Opa proteins, answering whether neutrophils possess a dedicated adhesin receptor.\",\n      \"evidence\": \"PMN lysate ligand binding, HeLa transfection with internalization assay, and monoclonal antibody blockade\",\n      \"pmids\": [\"8962144\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the binding site within the extracellular domain\", \"Downstream signaling not yet defined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showed CEACAM3 (CD66d) crosslinking transmits independent activating signals in neutrophils, raising integrin-mediated adhesion via CD11/CD18.\",\n      \"evidence\": \"Neutrophil adhesion to HUVEC with CD66 antibodies, CD18 blockade, and calcium chelation\",\n      \"pmids\": [\"8699114\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Antibody crosslinking does not establish the physiological bacterial trigger\", \"Molecular link to integrin upregulation not defined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated that the ITAM-like motif is functionally required for phagocytosis and that uptake depends on Syk and PLC, converting a sequence motif into a defined signaling requirement.\",\n      \"evidence\": \"ITAM tyrosine mutagenesis, DT40 reconstitution, kinase/PLC inhibitors, calcium flux and uptake assays\",\n      \"pmids\": [\"11278708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct SH2 partners docking on the ITAM not yet identified\", \"Connection to actin machinery unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Reported a calcium-modulated, phosphorylation-independent association of the cytoplasmic domain with calprotectin and in vitro phosphorylation by multiple kinases.\",\n      \"evidence\": \"Recombinant cytoplasmic-domain pulldown from granulocyte lysate and in vitro kinase assays\",\n      \"pmids\": [\"11708798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single pulldown without reciprocal validation\", \"Functional role of calprotectin binding in phagocytosis not established\", \"In vitro kinases may not reflect the in vivo Src-dependent event\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Linked Src-family-kinase-dependent tyrosine phosphorylation of CEACAM3 to actin-based uptake, connecting the receptor to the cytoskeleton.\",\n      \"evidence\": \"ITAM mutagenesis, Src inhibitors, subcellular fractionation, actin staining, and cytochalasin D treatment\",\n      \"pmids\": [\"12864848\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The actin-nucleating machinery downstream was not identified\", \"ITAM-independent residual uptake component left unexplained\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified Rac GTP-loading as the core actin-regulatory output of CEACAM3 and showed the receptor confers genuine bactericidal capacity across multiple human pathogens.\",\n      \"evidence\": \"Dominant-negative Rac, Src inhibitors, ITAM mutagenesis, Rac GTP-loading and primary granulocyte killing assays\",\n      \"pmids\": [\"14707113\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The GEF coupling the ITAM to Rac was not yet identified\", \"Distinct from CEACAM6, mechanism of selectivity unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved how CEACAM3 activates Rac by showing Vav binds phospho-Tyr230 directly and is required for Rac loading, establishing a direct ITAM-to-GEF coupling.\",\n      \"evidence\": \"SH2-phosphopeptide binding, co-IP, Vav1/Vav2-deficient cell rescue, colocalization, and primary granulocyte transduction\",\n      \"pmids\": [\"17339478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other ITAM-docking effectors not yet mapped\", \"Quantitative contribution of Vav versus other GEFs unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined Syk's role at the ITAM as required for microbicidal effector functions and for uptake under demanding cargo/ligand conditions.\",\n      \"evidence\": \"Co-IP, kinase inhibition, Syk-deficient cells, oxidative burst, degranulation, and killing assays\",\n      \"pmids\": [\"17506820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Syk and Vav act in series or parallel not resolved\", \"Substrates of Syk in this pathway not enumerated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Separated uptake from killing by showing PI3K binds phospho-Tyr230 and is dispensable for engulfment but required for the oxidative burst and intracellular killing.\",\n      \"evidence\": \"SH2 binding assay, FRET in intact cells, PI3K inhibitors, ROS and intracellular degradation assays in primary granulocytes\",\n      \"pmids\": [\"21216968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream PI3K effectors not identified\", \"Mechanism of intracellular killing beyond ROS not detailed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected phospho-CEACAM3 to the actin-nucleation machinery via Nck-mediated recruitment of the WAVE complex, explaining lamellipodial engulfment.\",\n      \"evidence\": \"Co-IP/pulldown, Nck1/2 deletion with rescue, dominant-negative WAVE2, WAVE/Nap1 knockdown, lamellipodia microscopy and uptake assays\",\n      \"pmids\": [\"22448228\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between Nck-WAVE and Vav-Rac branches not resolved\", \"Spatial hierarchy of effector recruitment unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified Grb14 as an ITAM-docking negative regulator, establishing that the same phosphomotif recruits both activating and inhibitory adaptors.\",\n      \"evidence\": \"SH2 microarray screen, co-IP, FRET-FLIM, knockdown and overexpression with uptake assays\",\n      \"pmids\": [\"22948154\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Grb14 dampens signaling not defined\", \"Competition with activating SH2 effectors not directly measured\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended CEACAM3 signaling to transcriptional inflammatory output by linking UspA1 engagement to NF-κB via a Syk–CARD9 axis.\",\n      \"evidence\": \"siRNA of CEACAM3/Syk/CARD9, inhibitors, NF-κB reporter, ChIP, chemokine ELISA, ROS and degranulation assays\",\n      \"pmids\": [\"27038042\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish requirement for co-receptor input\", \"Intermediate steps between Syk and CARD9 unmapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed the bacterial LOS alpha-oligosaccharide is required for Opa to functionally engage CEACAM3, defining a structural prerequisite on the ligand side.\",\n      \"evidence\": \"HeLa-CEACAM3 uptake and neutrophil chemiluminescence assays with LOS lgtF mutant bacteria\",\n      \"pmids\": [\"27376801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct binding measurement to confirm conformational model\", \"Single lab, functional readouts only\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mechanistically separated the phagocytic and inflammatory arms by showing cytokine production requires TLR co-engagement and Bcl10, while engulfment does not.\",\n      \"evidence\": \"CEABAC transgenic mice, Rac2/Bcl10/Malt1 knockouts, ex vivo neutrophil infection, cytokine and engulfment assays\",\n      \"pmids\": [\"28886618\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of TLR-CEACAM3 integration not defined\", \"Role of Malt1 left ambiguous\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified the direct off-switch by demonstrating PTPRJ dephosphorylates CEACAM3 cytoplasmic tyrosines to restrain phagocytosis and lamellipodia.\",\n      \"evidence\": \"In vitro phosphatase assay on full-length CEACAM3 and phosphopeptides, PTPRJ depletion/overexpression, primary human phagocyte knockdown with lamellipodia and uptake quantification\",\n      \"pmids\": [\"35850306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Site preference of PTPRJ on the ITAM not detailed\", \"Regulation of PTPRJ recruitment to CEACAM3 unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Added a downstream brake by identifying CYRI-B, via a genome-wide screen, as a Rac-GTP sequester that limits CEACAM3 phagocytosis.\",\n      \"evidence\": \"Genome-wide CRISPR screen, clonal CYRI-B knockout in HL-60, complementation, Rac GTP-loading and PAK phosphorylation assays, uptake assay\",\n      \"pmids\": [\"37264948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CYRI-B acts on a CEACAM3-specific Rac pool not resolved\", \"Interplay with PTPRJ and Grb14 in signal termination not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the activating (Vav, Nck-WAVE, Syk, PI3K) and inhibitory (Grb14, PTPRJ, CYRI-B) modules are coordinated in space and time on a single ITAM to set phagocytic threshold versus inflammatory output remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No integrated structural or kinetic model of competing SH2 effector occupancy\", \"Stoichiometry and ordering of activating versus inhibitory adaptor recruitment unknown\", \"Physiological setting that tips the balance between engulfment and cytokine production undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [0, 5, 16]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [2, 4, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7, 9, 10, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 8, 12, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 7, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"VAV1\", \"SYK\", \"NCK1\", \"PIK3R1\", \"GRB14\", \"PTPRJ\", \"CYRI-B\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}