{"gene":"ALCAM","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":1995,"finding":"ALCAM (CD166) was identified as a ligand for CD6: COS cells transfected with ALCAM cDNA bound a CD6 immunoglobulin fusion protein (CD6-Rg), and an ALCAM-Rg fusion protein bound COS cell transfectants expressing CD6, establishing a direct heterophilic ALCAM–CD6 receptor–ligand interaction.","method":"COS cell transfection, immunoglobulin fusion protein binding assay, antibody-blocking studies","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — reciprocal binding demonstrated with soluble fusion proteins and blocking antibodies; foundational paper independently replicated across multiple subsequent labs.","pmids":["7760007"],"is_preprint":false},{"year":1997,"finding":"The CD6-binding site of ALCAM maps to the N-terminal Ig-like domain, and mutagenesis of hALCAM identified residues critical for CD6 binding on the predicted A'GFCC'C\" β-sheet of this domain; all critical residues are conserved in mouse ALCAM, explaining cross-species CD6/ALCAM interaction.","method":"Truncation constructs of ALCAM extracellular region, binding assays with soluble CD6, site-directed mutagenesis, molecular modeling","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis plus truncation binding assays in a single study, findings replicated by subsequent structural work.","pmids":["9209500"],"is_preprint":false},{"year":1998,"finding":"ALCAM (MEMD) mediates homophilic (ALCAM–ALCAM) cell-cell clustering in CD6-negative melanoma cells; transfection of ALCAM into ALCAM-negative melanoma cells restored cell-cell interaction, demonstrating that ALCAM expression is sufficient for homophilic adhesion in this context.","method":"Transfection experiments, FACS-based aggregation assays","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function transfection with functional adhesion readout in a single lab; supported by correlation data.","pmids":["9502422"],"is_preprint":false},{"year":2000,"finding":"CD6–CD166 heterophilic interaction has a KD of ~0.4–1.0 µM with fast off-rate (Koff ≥0.4 s⁻¹); homophilic ALCAM–ALCAM interaction is ~100-fold weaker (KD ~29–48 µM, Koff ≥5.3 s⁻¹), demonstrating that heterophilic binding is substantially stronger than homophilic binding.","method":"In vitro binding assays with soluble recombinant proteins (surface plasmon resonance / biophysical characterization)","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — quantitative biophysical measurement with soluble recombinant proteins; replicated in the 2015 structural study.","pmids":["15048703"],"is_preprint":false},{"year":2000,"finding":"α-catenin is required to recruit ALCAM to cell–cell contacts; prostate cancer cell lines lacking α-catenin show cytoplasmic ALCAM staining, whereas transfection of α-N-catenin restores ALCAM localization to cell-cell junctions alongside E-cadherin.","method":"α-catenin transfection into α-catenin-null cell lines, immunofluorescence localization","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function rescue experiment with defined subcellular localization readout; single lab.","pmids":["10673383"],"is_preprint":false},{"year":2001,"finding":"Homophilic ALCAM–ALCAM cell adhesion requires two structurally and functionally distinct modules: (1) ligand binding mediated by the membrane-distal N-terminal Ig domain, and (2) avidity control through ALCAM clustering involving membrane-proximal Ig domains. A transmembrane deletion mutant lacking the ligand-binding domain inhibited cell-cell adhesion by interfering with ALCAM avidity without affecting soluble homophilic ligand binding.","method":"Amino-terminally deleted ALCAM constructs, monoclonal antibody blocking, co-expression of dominant-negative mutant, cell adhesion assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure-function analysis with multiple deletion constructs, dominant-negative interference, and blocking antibodies; mechanistically rigorous single-lab study.","pmids":["11306570"],"is_preprint":false},{"year":2001,"finding":"ALCAM expression on yolk sac endothelium supports hematopoietic progenitor cell development; ALCAM-transfected adult endothelial cells (EOMA) supported hematopoietic progenitor development compared to vector controls, and ALCAM was found to be involved in capillary tube formation and hemangioblast differentiation.","method":"ALCAM transfection into EOMA endothelial cells, hematopoietic progenitor co-culture assay, immunohistochemistry","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function transfection with functional hematopoietic readout; single lab.","pmids":["11568000"],"is_preprint":false},{"year":2004,"finding":"CD6–ALCAM interactions are required not only for establishing initial DC–T-cell contact but also for sustaining T-cell proliferation; ALCAM-blocking antibodies and recombinant ALCAM-Fc proteins strongly and sustainably inhibited DC-induced T-cell proliferation, and simultaneous crosslinking of CD6 and CD3 induced proliferation comparable to CD3+CD28 co-stimulation.","method":"Antibody-blocking assays, ALCAM-Fc recombinant protein inhibition, CD6/CD3 co-crosslinking, T-cell proliferation assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (blocking antibody, soluble Fc protein, crosslinking) with defined proliferative readout; replicated conceptually across multiple studies.","pmids":["16352806"],"is_preprint":false},{"year":2005,"finding":"ALCAM controls MMP-2 activation in melanoma: truncation of ALCAM (dominant-negative) severely impaired pro-MMP-2 activation by reducing MT1-MMP transcript levels and impairing MT1-MMP processing. ALCAM depletion by RNAi recapitulated this failure of the proteolytic cascade mainly through incomplete MT1-MMP processing. Extensive cell–cell contacts, wild-type ALCAM, and cell–matrix interactions were all required for efficient MMP-2 activation.","method":"Dominant-negative ALCAM truncation mutant, RNA interference (RNAi), nude mouse xenograft model, 2D and 3D collagen-gel cultures, zymography/MMP-2 activation assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Strong — two independent loss-of-function approaches (truncation mutant + RNAi) with in vitro and in vivo validation in a single rigorous study.","pmids":["16204050"],"is_preprint":false},{"year":2005,"finding":"ALCAM undergoes ligand engagement-induced internalization via a clathrin-mediated pathway (colocalizing with clathrin but not caveolin) and recycles back to the cell surface, as shown by surface biotinylation and recycling assays. This endocytic pathway enables intracellular delivery of ALCAM-targeted immunotoxins.","method":"Phage display antibody-induced internalization, immunofluorescence colocalization with clathrin/caveolin, surface biotinylation, recycling assay, immunotoxin kill assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (colocalization, biochemical recycling assay, functional immunotoxin delivery) in a single study.","pmids":["15769845"],"is_preprint":false},{"year":2006,"finding":"CD6 and CD166 are recruited together to the center of the immunological synapse between γδ T cells and antigen-loaded tumor cells, colocalizing with γδ TCR/CD3. CD166 transfection into a CD166-negative tumor line markedly enhanced γδ T cell activation, while shRNA-mediated CD166 knockdown reduced it, demonstrating that CD6–CD166 engagement at the synapse is required for γδ T cell activation by nonpeptide antigen-presenting tumor cells.","method":"CD166 cDNA transfection, shRNA knockdown, immunofluorescence of immunological synapse, T-cell activation assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain- and loss-of-function with defined synapse localization readout; multiple orthogonal approaches.","pmids":["16818742"],"is_preprint":false},{"year":2006,"finding":"ALCAM gene silencing in breast cancer cells (MCF-7) reduced BCL-2 protein levels and triggered apoptosis (caspase-7 activation, PARP cleavage) and autophagy (MAP1LC3, Beclin1 upregulation), indicating ALCAM supports cell survival downstream of BCL-2.","method":"ALCAM gene silencing (siRNA), laser scanning cytometry, Western blotting for apoptosis/autophagy markers","journal":"Medical science monitor","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, loss-of-function with defined molecular markers but no mechanistic pathway reconstitution.","pmids":["16865058"],"is_preprint":false},{"year":2007,"finding":"NDRG2 expressed in dendritic cells prevents down-regulation of ALCAM during monocyte-to-DC differentiation; NDRG2 siRNA knockdown specifically reduced ALCAM expression in differentiating DCs and diminished their ability to induce T cell proliferation, while NDRG2 overexpression in U937 cells conferred resistance to GM-CSF/IL-4-induced ALCAM reduction.","method":"RNA interference of NDRG2, NDRG2 overexpression in U937, flow cytometry for ALCAM, T-cell proliferation assay","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with defined ALCAM expression and T-cell functional readout; single lab.","pmids":["17911180"],"is_preprint":false},{"year":2008,"finding":"DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis in Xenopus laevis; loss-of-function reduced expression of first-heart-field markers (Tbx20, TnIc) but not second-heart-field markers (Isl-1, BMP-4), caused defective cell adhesion and cardiac morphogenesis, and DM-GRASP expression rescued the phenotype caused by loss of non-canonical Wnt11-R signaling, demonstrating functional coupling between ALCAM and Wnt11-R during cardiac development.","method":"Xenopus laevis loss-of-function (morpholino knockdown), rescue experiments with DM-GRASP expression, in situ hybridization for cardiac marker genes","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (rescue experiment) with orthogonal molecular readouts in a developmental model organism; single lab.","pmids":["18598690"],"is_preprint":false},{"year":2011,"finding":"NF-κB P50/P65 heterodimer activates both CD166/ALCAM and miR-9-1 transcription after serum deprivation. miR-9, induced with a delay, represses ALCAM protein translation via its 3'-UTR, creating a negative auto-regulatory loop. miR-9 also promotes cell migration partly via inhibition of CD166.","method":"Luciferase reporter assay, NF-κB inhibition/knockdown, miRNA overexpression and inhibitor experiments, Western blotting, qRT-PCR","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assays plus miRNA gain/loss-of-function with defined molecular endpoints; single lab.","pmids":["21572107"],"is_preprint":false},{"year":2012,"finding":"ALCAM directly associates with the tetraspanin CD9 and ADAM17/TACE on the leukocyte surface; CD9 upregulates both homophilic and heterophilic ALCAM-mediated adhesion by (1) promoting ALCAM clustering and (2) inhibiting ADAM17 sheddase activity to increase ALCAM surface expression.","method":"Confocal microscopy colocalization, co-immunoprecipitation, cell adhesion and migration/proliferation functional assays, ADAM17 activity assays","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, colocalization, and multiple functional assays demonstrating dual mechanism; single lab with multiple orthogonal methods.","pmids":["23052204"],"is_preprint":false},{"year":2012,"finding":"ALCAM regulates motility, invasiveness, and adherens junction formation in uveal melanoma; shRNA knockdown of ALCAM reduced cell motility and invasion and disrupted adherens junction formation, while ALCAM overexpression enhanced recruitment of β-catenin and N-cadherin to adherens junctions. ALCAM is necessary but not sufficient to promote metastasis-associated behaviors.","method":"shRNA stable knockdown, stable overexpression, gap-closure motility assay, transwell invasion assay, immunostaining for adherens junction components","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with multiple functional readouts; single lab.","pmids":["22745734"],"is_preprint":false},{"year":2012,"finding":"ALCAM mRNA is locally translated in retinal ganglion cell axonal growth cones, regulated by the 3'-UTR and dependent on ERK and TOR kinase activity. Local growth cone translation of ALCAM is required for enhanced axon elongation on ALCAM substrate, rapid compensation for experimentally induced ALCAM internalization, and axonal preference for ALCAM-containing lanes.","method":"Isolated growth cone translation assay, 3'-UTR reporter constructs, kinase inhibitors (ERK, TOR), ALCAM internalization assay, axon choice assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution of local translation in isolated growth cones plus multiple functional assays; mechanistically rigorous single-lab study.","pmids":["22421359"],"is_preprint":false},{"year":2013,"finding":"ALCAM regulates long-term hematopoietic stem cell (HSC) self-renewal and engraftment; Alcam-/- mice show reduced long-term repopulating capacity and engraftment efficiency, age-associated expansion of CD150hi LT-HSCs with myeloid-biased output, and premature elevation of age-associated genes (Selp, Clu, Cdc42, Foxo3).","method":"Alcam knockout mouse model, serial transplantation assays, in vitro replating, gene expression analysis","journal":"Stem cells","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with defined functional HSC phenotype across multiple in vivo readouts; single lab.","pmids":["23280653"],"is_preprint":false},{"year":2013,"finding":"ALCAM mediates adhesion, migration, and tube formation in lymphatic endothelial cells (LECs) and supports dendritic cell adhesion to lymphatic endothelium. ALCAM knockout mice have reduced LEC numbers, defects in organized lymphatic vessel network formation, and compromised DC migration from lung to draining lymph nodes.","method":"ALCAM knockout mouse, in vitro LEC adhesion/migration/tube formation assays, DC migration assay in vivo and in vitro","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with multiple in vitro and in vivo functional readouts; multiple orthogonal methods.","pmids":["23169771"],"is_preprint":false},{"year":2013,"finding":"S100B binds CD166/ALCAM and induces dose- and time-dependent NF-κB activation in endothelial cells. siRNA knockdown of CD166/ALCAM completely inhibited S100B-induced NF-κB activation in RAGE-/- cells. In vivo, ALCAM siRNA attenuated delayed-type hypersensitivity (DTH) by ~40–50%; ALCAM-/- mice showed compensatory RAGE upregulation.","method":"siRNA knockdown, NF-κB reporter/activation assay, ALCAM-/- and RAGE-/- mouse DTH model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function in vitro and in vivo with defined signaling readout; genetic knockout with compensatory cross-regulation revealed.","pmids":["23729438"],"is_preprint":false},{"year":2014,"finding":"ALCAM is shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β signaling, and this ectodomain shedding is required for effective bone metastasis; shRNA knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished skeletal dissemination and tumor growth in bone, associated with increased apoptosis and decreased proliferation.","method":"Biochemical shedding assays, ADAM17 identification by inhibitor and siRNA, ALCAM shRNA knockdown, in vivo bone metastasis model (intratibial engraftment), IHC for caspase-3 and Ki67","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical identification of ADAM17 as sheddase plus in vivo loss-of-function with multiple mechanistic readouts; single lab, multiple orthogonal methods.","pmids":["24385212"],"is_preprint":false},{"year":2014,"finding":"ALCAM intracellular domain coupling to the actin cortex does not affect the affinity of individual ALCAM–CD6 bonds, but does control ALCAM recruitment to adhesion sites and membrane tether formation. Linking ALCAM to the actin cortex stiffens the cortex and strengthens overall cell adhesion to CD6 at the immunological synapse.","method":"Single-cell force spectroscopy (SCFS), TIRF microscopy, ALCAM cytoplasmic tail deletion mutants, actin cytoskeleton perturbation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative biophysical measurement (SCFS) combined with defined ALCAM mutants and imaging; mechanistically rigorous.","pmids":["24496453"],"is_preprint":false},{"year":2014,"finding":"CD166 promotes anti-apoptotic signaling in liver cancer via PI3K/AKT: AKT upregulates CD166 expression post-transcriptionally, and CD166 in turn promotes AKT expression and activity (positive feedback). CD166 also activates YAP through transcriptional regulation via CREB and post-transcriptional stabilization via AMOT130 inhibition. CD9 enhances CD166-mediated YAP regulation by facilitating CD166–CD166 homophilic interaction.","method":"CD166 siRNA knockdown, AKT overexpression rescue, subcellular fractionation, co-immunoprecipitation, luciferase reporter, tissue microarray","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple loss/gain-of-function approaches with defined signaling readouts; single lab.","pmids":["24482231"],"is_preprint":false},{"year":2014,"finding":"CD166 regulates MCAM protein stability by suppressing the ubiquitin E3 ligases βTrCP and Smurf1 through PI3K/AKT and c-Raf/MEK/ERK signaling, thereby protecting MCAM from proteasomal degradation.","method":"CD166 knockdown/overexpression, ubiquitination assays, E3 ligase overexpression, pathway inhibitors (PI3K, MEK), co-immunoprecipitation, tissue microarray","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway dissection with multiple loss/gain-of-function and ubiquitination readouts; single lab.","pmids":["26004137"],"is_preprint":false},{"year":2014,"finding":"CD166 regulates FOXO protein stability and subcellular localization through AKT: CD166 overexpression accelerates FOXO ubiquitination and degradation and shifts FOXO from nucleus to cytoplasm, while CD166 knockdown reduces FOXO phosphorylation. AKT overexpression rescues CD166 knockdown-induced FOXO dephosphorylation and anti-carcinogenic effects, placing AKT between CD166 and FOXO.","method":"CD166 knockdown/overexpression, subcellular fractionation, ubiquitination assay, AKT overexpression rescue, in vitro and in vivo tumor assays","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis rescue (AKT overexpression) with biochemical and localization readouts; single lab.","pmids":["24891117"],"is_preprint":false},{"year":2015,"finding":"Crystal structures of the three SRCR domains of CD6 and the two N-terminal Ig domains of CD166 were solved by X-ray crystallography. Structural analysis revealed the CD6/CD166 binding interface, showed that a SNP in CD6 introduces glycosylation that sterically hinders the CD6/CD166 interaction, and native mass spectrometry demonstrated competition between heterophilic CD6-CD166 and homophilic CD166-CD166 interactions.","method":"X-ray crystallography, native mass spectrometry, SNP glycosylation analysis","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structures of both binding partners with functional validation by native MS; definitive structural evidence.","pmids":["26146185"],"is_preprint":false},{"year":2016,"finding":"ILT3 (LILRB4) binds CD166/ALCAM directly; CRISPR-Cas9 knockout of CD166 abolished ILT3.Fc binding and its tumor-inhibitory effect. ILT3.Fc binding to CD166 inhibits tumor cell growth through inactivation of the p70 S6 kinase (p70S6K) signaling pathway.","method":"Flow cytometry, mass spectrometry, Biacore (SPR), CD166 knockdown by nucleofection and CRISPR-Cas9 KO, p70S6K signaling assay, in vitro and in vivo tumor growth assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding confirmed by SPR, CRISPR KO abolishes effect, signaling mechanism identified; multiple orthogonal methods.","pmids":["29263213"],"is_preprint":false},{"year":2016,"finding":"CD166 in multiple myeloma cells inhibits osteoblastogenesis by suppressing Runx2 gene expression in osteoblast progenitors, and promotes osteoclastogenesis by activating TRAF6-dependent signaling in osteoclast progenitors; CD166 silencing reduced skeletal dissemination and osteolytic lesion formation in vivo.","method":"CD166 siRNA/shRNA knockdown in MM cell lines, intratibial engraftment model, ex vivo bone organ culture, osteoblast/osteoclast differentiation assays, Runx2 and TRAF6 signaling analysis","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with defined in vitro and in vivo mechanistic readouts across multiple cell types; single lab, multiple orthogonal methods.","pmids":["27634757"],"is_preprint":false},{"year":2017,"finding":"ALCAM knockout mice develop more severe EAE (experimental autoimmune encephalomyelitis) with increased CNS-infiltrating leukocytes; passive transfer experiments linked this to absence of ALCAM on blood-brain barrier endothelial cells. ALCAM KO mice also show reduced expression of BBB tight junction proteins and increased CNS blood vessel permeability, establishing ALCAM as a component required for tight junction assembly and BBB integrity.","method":"ALCAM knockout mouse, active and passive EAE model, phenotypic characterization, tight junction protein expression, in vitro BBB permeability assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with in vivo disease model, passive transfer epistasis, and molecular characterization of junction proteins; multiple orthogonal methods.","pmids":["28069965"],"is_preprint":false},{"year":2016,"finding":"ALCAM mediates preferential diapedesis of CD4+ Th1 cells (but not Th17 cells) across the human BBB in vitro; antibody-mediated ALCAM inhibition reduced Th1 but not Th17 diapedesis under static conditions. ALCAM also contributes to rolling, adhesion, and diapedesis of CD14+ monocytes across the human BBB under flow and static conditions.","method":"Anti-ALCAM antibody blocking, ALCAM-/- in vitro BBB model, leukocyte transmigration assays under static and flow conditions","journal":"Journal of cerebral blood flow and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — antibody blocking plus ALCAM-/- BBB model with defined cell-type-specific readouts; single lab.","pmids":["28273717"],"is_preprint":false},{"year":2017,"finding":"CD166 loss in intestinal crypts reduces active-cycling Lgr5+ ISC numbers; homeostasis is maintained by transit-amplifying compartment expansion (not slow-cycling Bmi1+ ISC stimulation). Loss of active-cycling ISCs is coupled to defective Paneth cell terminal differentiation linked to reduced Wnt3 ligand expression and depleted nuclear β-catenin in CD166-/- Paneth cells.","method":"CD166-/- mouse, immunohistochemistry, flow cytometry, gene expression analysis, enteroid culture","journal":"Cellular and molecular gastroenterology and hepatology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with multiple orthogonal methods revealing ISC niche signaling mechanism; Wnt pathway placement established.","pmids":["28462380"],"is_preprint":false},{"year":2017,"finding":"ALCAM mediates DC migration through afferent lymphatics and promotes allospecific immune reactions; anti-murine ALCAM blocking antibody reduced DC transmigration across lymphatic endothelial monolayers, DC emigration from human skin explants, lymphangiogenic processes in vitro, and prevented corneal allograft rejection by retaining DCs in the cornea.","method":"Blocking monoclonal antibody, in vitro DC transmigration assay, human skin explant emigration, in vivo corneal allograft model, developmental lymphangiogenesis assay","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple in vitro and in vivo functional assays with defined blocking reagent; multiple orthogonal methods in a single rigorous study.","pmids":["31031759"],"is_preprint":false},{"year":2017,"finding":"E3 ubiquitin ligase CHIP directly regulates ALCAM protein stability through the ubiquitin proteasome system; CHIP negatively correlates with CD166 in head and neck cancer samples, and CHIP expression represses cancer stem-like cell characteristics via targeting CD166 for degradation.","method":"Co-immunoprecipitation, ubiquitin proteasome inhibitor assay, CHIP overexpression/knockdown, Western blotting, tissue microarray","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional ubiquitination assay with defined CSC readout; single lab.","pmids":["28279658"],"is_preprint":false},{"year":2019,"finding":"PRMT1 interacts with ALCAM directly (confirmed by co-immunoprecipitation and LC-MS); PRMT1 silencing reduced ALCAM protein levels and suppressed melanoma tumor growth and metastasis, while re-expression of ALCAM in PRMT1-silenced cells restored colony formation and metastatic ability, placing ALCAM downstream of PRMT1.","method":"LC-MS/MS, co-immunoprecipitation, PRMT1 shRNA knockdown, ALCAM rescue overexpression, colony formation and migration assays","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical interaction confirmed by co-IP and LC-MS, epistasis rescue experiment; single lab.","pmids":["27175582"],"is_preprint":false},{"year":2020,"finding":"CD166/ALCAM is internalized via a clathrin-independent endocytic pathway driven by endophilin-A3 (not A1 or A2 isoforms) and extracellular galectin-8. Endophilin-A3 physically interacts with CD166-containing early endocytic carriers. This endocytic modality modulates CD166 surface abundance and regulates adhesive and migratory properties of cancer cells.","method":"Endophilin-A isoform-specific knockdown, galectin-8 perturbation, live-cell endocytic carrier imaging, co-immunoprecipitation, cell adhesion and migration assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — isoform-specific knockdown demonstrating selectivity, physical interaction data, and functional consequences on adhesion/migration; multiple orthogonal methods.","pmids":["32193381"],"is_preprint":false},{"year":2020,"finding":"SOSTDC1 interacts with ALCAM/CD166 (identified by immunoprecipitation and mass spectrometry, confirmed by confocal microscopy and competition ELISA); this interaction involves the N-terminal region of SOSTDC1, which contains a sequence similar to the CD6-binding motif for ALCAM. ALCAM also interacts with α2β1 and α1β1 integrins. Knockdown of either SOSTDC1 or ALCAM, or antibody blockade, reduces invasion by inhibiting Src and PI3K/AKT signaling.","method":"Co-immunoprecipitation, mass spectrometry, confocal microscopy, competition ELISA, SOSTDC1/ALCAM knockdown, invasion assay, Src/PI3K-AKT signaling readout, in vivo liver metastasis model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical interaction confirmed by multiple orthogonal methods (co-IP, MS, ELISA, imaging), pathway placement by loss-of-function, in vivo validation.","pmids":["32801337"],"is_preprint":false},{"year":2016,"finding":"Galectin-8 interacts with ALCAM/CD166 in a glycosylation-dependent manner (demonstrated by surface plasmon resonance with recombinant glycosylated ALCAM ectodomain and endogenous ALCAM from breast cancer cells); ALCAM-silenced cells showed reduced binding to Gal-8. Exogenous Gal-8 caused ALCAM surface segregation/trapping at the cell surface.","method":"Surface plasmon resonance (SPR) binding assay, ALCAM siRNA knockdown, SPR binding of ALCAM-silenced cells, internalization/surface localization assay","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct biophysical binding measurement (SPR) with glycosylation dependency demonstrated, plus functional silencing; rigorous single-lab study.","pmids":["27130882"],"is_preprint":false},{"year":2022,"finding":"ALCAM/CD166 is involved in binding and uptake of cancer-derived extracellular vesicles (EVs) by recipient cancer cells; ALCAM participates in EV docking and subsequent uptake, demonstrated in colorectal and ovarian cancer cell systems.","method":"ALCAM expression manipulation, EV binding and uptake assays, flow cytometry","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — functional assay with defined molecular readout; single lab, mechanistic detail limited to abstract description.","pmids":["35628559"],"is_preprint":false},{"year":2022,"finding":"The CD6/ALCAM pathway promotes lupus nephritis (LN) via T cell-mediated responses; ALCAM is expressed by renal structural cells while CD6 is exclusive to T cells in the LN kidney. Antibody blockade of CD6 in murine lupus and immune-complex glomerulonephritis models significantly decreased immune cell infiltration, inflammatory markers, and disease measures.","method":"Immunophenotyping of LN kidney cells, anti-CD6 antibody blockade in spontaneous lupus and immune-complex glomerulonephritis mouse models, uALCAM ELISA in patient cohorts","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo antibody blockade in two mouse models with defined immunological and disease readouts; replicated in two model systems and corroborated by large human cohort data.","pmids":["34981775"],"is_preprint":false},{"year":2024,"finding":"Hypoxia promotes ALCAM expression in macrophages via HIF-1α binding to the ALCAM promoter; ALCAMhigh macrophages co-localize with exhausted CD8+ T cells in the tumor spatial microenvironment and promote T cell exhaustion. HIF-1α inhibition reduces ALCAM expression in macrophages and potentiates T cell anti-tumor function.","method":"Bulk, single-cell, and spatial transcriptomics integration, HIF-1α ChIP on ALCAM promoter, HIF-1α inhibitor experiments, preclinical immunotherapy models","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — HIF-1α promoter binding demonstrated, functional consequence shown with HIF-1α inhibitor in preclinical model; single study.","pmids":["38956900"],"is_preprint":false}],"current_model":"ALCAM/CD166 is a transmembrane IgSF cell adhesion molecule that engages in strong heterophilic interactions with CD6 (KD ~0.4–1 µM, mediated by its membrane-distal N-terminal Ig domain) and weaker homophilic ALCAM–ALCAM interactions (KD ~30–48 µM, dependent on membrane-proximal domain-driven clustering); its crystal structure with CD6 is solved. At the immunological synapse, ALCAM–CD6 engagement sustains DC-induced T-cell proliferation and γδ T cell activation. ALCAM is dynamically regulated at the cell surface: it undergoes clathrin-mediated endocytosis with recycling back to the surface, clathrin-independent endocytosis via endophilin-A3 driven by galectin-8, and proteolytic ectodomain shedding by ADAM17 (upregulated by TGF-β and modulated by CD9, which inhibits ADAM17 to increase ALCAM surface levels). Intracellularly, ALCAM couples to the actin cortex through adaptor proteins, stiffening the cortex and strengthening cell adhesion without changing individual bond affinity. ALCAM controls MMP-2 activation by regulating MT1-MMP processing in a cell-density-dependent manner, promotes PI3K/AKT-YAP and AKT-FOXO survival signaling in cancer cells, and mediates osteoblast-HSC homophilic interactions critical for HSC niche function and self-renewal. ALCAM expression is regulated transcriptionally by NF-κB and HIF-1α (in macrophages) and post-translationally by E3 ligases CHIP (ubiquitin-proteasome degradation) and PRMT1. ALCAM also serves as a ligand for ILT3 (LILRB4) and galectin-8, and is required for BBB tight junction integrity, lymphatic network formation, and cardiac/intestinal stem cell niche maintenance."},"narrative":{"mechanistic_narrative":"ALCAM (CD166) is a transmembrane immunoglobulin-superfamily cell adhesion molecule that engages in strong heterophilic binding to CD6 (KD ~0.4–1 µM) through its membrane-distal N-terminal Ig domain and weaker homophilic ALCAM–ALCAM interactions (KD ~29–48 µM) [PMID:7760007, PMID:15048703, PMID:26146185]. Homophilic adhesion is biphasic: the N-terminal Ig domain mediates ligand binding, while membrane-proximal Ig domains drive clustering that controls avidity [PMID:11306570]. Crystal structures of the CD6 SRCR domains bound to the two N-terminal ALCAM Ig domains define the interface, and native mass spectrometry shows that heterophilic CD6–ALCAM and homophilic ALCAM–ALCAM engagement compete [PMID:26146185]. Intracellularly, ALCAM is recruited to cell-cell contacts by α-catenin and couples to the actin cortex; this linkage does not alter individual bond affinity but stiffens the cortex and strengthens overall adhesion to CD6 at the immunological synapse [PMID:10673383, PMID:24496453]. ALCAM surface levels are dynamically controlled by clathrin-mediated endocytosis with recycling, an endophilin-A3/galectin-8-driven clathrin-independent pathway, and ADAM17-mediated ectodomain shedding that is induced by TGF-β and restrained by the tetraspanin CD9 [PMID:15769845, PMID:32193381, PMID:27130882, PMID:24385212, PMID:23052204]. Through CD6–ALCAM engagement at the immunological synapse, ALCAM sustains DC-induced T-cell proliferation and is required for γδ T cell activation [PMID:16352806, PMID:16818742]. In vivo, ALCAM is required for hematopoietic stem cell self-renewal and engraftment, lymphatic network formation and dendritic cell migration, blood-brain barrier tight-junction integrity and leukocyte transmigration, and intestinal stem cell niche maintenance via Wnt signaling [PMID:23280653, PMID:23169771, PMID:28069965, PMID:28462380]. In cancer, ALCAM controls MMP-2 activation through MT1-MMP processing and promotes survival signaling through PI3K/AKT–YAP and AKT–FOXO axes [PMID:16204050, PMID:24482231, PMID:24891117]. ALCAM additionally serves as a binding partner for ILT3 (LILRB4), galectin-8, and S100B, and its expression is regulated transcriptionally by NF-κB and HIF-1α and post-translationally by the E3 ligase CHIP and by PRMT1 [PMID:29263213, PMID:27130882, PMID:23729438, PMID:21572107, PMID:38956900, PMID:28279658, PMID:27175582].","teleology":[{"year":1995,"claim":"Establishing ALCAM's first molecular partner: it was unknown what receptor ALCAM engaged, and reciprocal fusion-protein binding defined a direct heterophilic ALCAM–CD6 interaction, founding the entire CD6–ALCAM axis.","evidence":"COS cell transfection with reciprocal Ig-fusion-protein binding and antibody blocking","pmids":["7760007"],"confidence":"High","gaps":["Did not map the binding site at residue resolution","Did not address homophilic binding or downstream signaling"]},{"year":1997,"claim":"Localizing the CD6-binding determinant: mutagenesis and truncation mapped CD6 binding to specific residues on the N-terminal Ig domain β-sheet and explained cross-species interaction.","evidence":"Truncation constructs, site-directed mutagenesis, and molecular modeling with soluble CD6","pmids":["9209500"],"confidence":"High","gaps":["No atomic structure yet","Did not resolve how homophilic binding uses the same domain"]},{"year":1998,"claim":"Defining a second adhesion mode: whether ALCAM could act independently of CD6 was unknown, and transfection into CD6-negative melanoma showed ALCAM expression is sufficient for homophilic clustering.","evidence":"ALCAM transfection into ALCAM-negative melanoma with FACS aggregation assays","pmids":["9502422"],"confidence":"Medium","gaps":["Affinity and structural basis of homophilic binding not measured","Single-lab gain-of-function"]},{"year":2000,"claim":"Quantifying the adhesion hierarchy and its modularity: biophysics revealed heterophilic CD6 binding is ~100-fold stronger than homophilic binding, and structure-function work showed ligand binding and avidity-controlling clustering are separable modules.","evidence":"SPR/biophysical KD measurements and deletion/dominant-negative adhesion assays","pmids":["15048703","11306570"],"confidence":"High","gaps":["Did not define the clustering interface structurally","Intracellular coupling not addressed"]},{"year":2000,"claim":"Connecting ALCAM to the junctional cytoskeleton: α-catenin was shown to be required to recruit ALCAM to cell-cell contacts alongside E-cadherin.","evidence":"α-N-catenin rescue in α-catenin-null prostate cancer cells with immunofluorescence","pmids":["10673383"],"confidence":"Medium","gaps":["Direct vs indirect coupling to actin not resolved","No biophysical adhesion measurement"]},{"year":2006,"claim":"Defining ALCAM's immune function: CD6–ALCAM engagement was shown not only to initiate but to sustain DC-induced T-cell proliferation and to be required for γδ T cell activation at the immunological synapse.","evidence":"Blocking antibodies, ALCAM-Fc, CD6/CD3 crosslinking, synapse imaging, and gain/loss-of-function in tumor lines","pmids":["16352806","16818742"],"confidence":"High","gaps":["Downstream T-cell signaling from CD6 not dissected","Relative contribution of homophilic vs heterophilic binding unclear"]},{"year":2005,"claim":"Linking ALCAM to proteolytic invasion machinery: ALCAM was shown to control MMP-2 activation in a cell-density-dependent manner by regulating MT1-MMP transcription and processing.","evidence":"Dominant-negative truncation and RNAi with zymography, 3D collagen culture, and xenografts","pmids":["16204050"],"confidence":"High","gaps":["Mechanism linking ALCAM adhesion to MT1-MMP regulation not defined","Direct ALCAM signaling intermediates unidentified"]},{"year":2005,"claim":"Establishing ALCAM surface dynamics: ligand engagement was shown to drive clathrin-mediated internalization with recycling back to the surface.","evidence":"Antibody-induced internalization, clathrin/caveolin colocalization, surface biotinylation, and recycling assays","pmids":["15769845"],"confidence":"High","gaps":["Adaptor proteins for clathrin uptake not identified","Did not address alternative endocytic routes"]},{"year":2012,"claim":"Connecting surface regulation to a tetraspanin: ALCAM was shown to associate with CD9 and ADAM17, with CD9 boosting ALCAM adhesion by promoting clustering and inhibiting sheddase activity.","evidence":"Co-IP, confocal colocalization, ADAM17 activity assays, and adhesion/migration functional readouts","pmids":["23052204"],"confidence":"High","gaps":["Structural basis of the CD9–ALCAM–ADAM17 assembly unknown","How CD9 inhibits ADAM17 not resolved"]},{"year":2014,"claim":"Defining the biophysics of intracellular coupling: actin-cortex linkage of the ALCAM tail was shown to strengthen adhesion by stiffening the cortex rather than altering bond affinity.","evidence":"Single-cell force spectroscopy, TIRF microscopy, and cytoplasmic-tail deletion mutants","pmids":["24496453"],"confidence":"High","gaps":["Identity of the actin-coupling adaptors not defined","Link to α-catinin-mediated recruitment unresolved"]},{"year":2014,"claim":"Establishing ALCAM as a sheddable driver of metastasis: ADAM17 was identified as the TGF-β-induced sheddase, and ALCAM shedding was shown required for prostate cancer bone metastasis.","evidence":"Biochemical shedding assays, ADAM17 inhibitor/siRNA, ALCAM shRNA, and in vivo intratibial bone metastasis model","pmids":["24385212"],"confidence":"High","gaps":["Function of the shed ectodomain vs membrane stub not separated","Signaling triggered by shedding unclear"]},{"year":2014,"claim":"Placing ALCAM in cancer survival signaling: ALCAM/CD166 was shown to drive PI3K/AKT–YAP and AKT–FOXO axes and to stabilize partner proteins, with CD9 facilitating CD166 homophilic interaction.","evidence":"Knockdown/overexpression, AKT rescue epistasis, ubiquitination, fractionation, and reporter assays","pmids":["24482231","24891117","24385212"],"confidence":"Medium","gaps":["Direct molecular link between surface ALCAM and AKT activation not defined","Single-lab dissection of these feedback loops"]},{"year":2015,"claim":"Achieving atomic resolution: crystal structures of CD6 SRCR and ALCAM Ig domains defined the binding interface and showed heterophilic and homophilic interactions compete.","evidence":"X-ray crystallography, native mass spectrometry, and SNP glycosylation analysis","pmids":["26146185"],"confidence":"High","gaps":["Full-length complex in membrane context not resolved","Structural basis of avidity clustering not captured"]},{"year":2016,"claim":"Expanding the ALCAM ligand repertoire: SPR established glycosylation-dependent galectin-8 binding, and CRISPR work defined ILT3 (LILRB4) as a direct partner that inhibits tumor growth via p70S6K.","evidence":"SPR with recombinant and endogenous ALCAM, mass spectrometry, CRISPR-Cas9 KO, and p70S6K signaling assays","pmids":["27130882","29263213"],"confidence":"High","gaps":["Physiological context of ILT3–ALCAM signaling beyond tumors unclear","Whether galectin-8 binding competes with CD6 not tested"]},{"year":2013,"claim":"Establishing ALCAM's stem-cell-niche and vascular-barrier roles: knockout mice revealed requirements for HSC self-renewal, lymphatic network formation, and DC migration.","evidence":"Alcam knockout mice with serial transplantation, LEC adhesion/tube assays, and in vivo DC migration","pmids":["23280653","23169771"],"confidence":"High","gaps":["Whether niche function is homophilic or heterophilic not resolved","Molecular signaling downstream of niche adhesion unidentified"]},{"year":2017,"claim":"Defining ALCAM in barrier and epithelial niches: knockouts showed ALCAM is required for BBB tight-junction integrity and Th1/monocyte transmigration, and for intestinal stem cell maintenance via Wnt3/β-catenin.","evidence":"ALCAM KO mice, EAE and passive-transfer models, BBB permeability and transmigration assays, and enteroid/ISC analysis","pmids":["28069965","28273717","28462380"],"confidence":"High","gaps":["How ALCAM mechanistically promotes tight-junction assembly unknown","Link between ALCAM adhesion and Wnt ligand expression not defined"]},{"year":2020,"claim":"Identifying a selective clathrin-independent endocytic route: endophilin-A3 and galectin-8 were shown to drive ALCAM internalization that tunes surface abundance and migration.","evidence":"Isoform-specific endophilin knockdown, galectin-8 perturbation, live-cell carrier imaging, and co-IP","pmids":["32193381"],"confidence":"High","gaps":["How cells choose between clathrin-dependent and -independent routes unknown","Fate of internalized ALCAM not fully tracked"]},{"year":2022,"claim":"Linking the CD6–ALCAM axis to disease and exhaustion: antibody blockade implicated ALCAM in lupus nephritis, and HIF-1α-driven ALCAMhigh macrophages were shown to promote CD8 T cell exhaustion.","evidence":"Anti-CD6 blockade in lupus models, patient uALCAM ELISA, and spatial/single-cell transcriptomics with HIF-1α ChIP and inhibition","pmids":["34981775","38956900"],"confidence":"Medium","gaps":["Direct ALCAM ligand mediating exhaustion in this context not defined","Causality of macrophage ALCAM in human tumors not established"]},{"year":null,"claim":"The identity of the cytoplasmic adaptors that physically couple the ALCAM tail to the actin cortex, and the unified signaling output that links surface adhesion to AKT/YAP/FOXO regulation, remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct ALCAM intracellular signaling effector identified","Mechanism converting adhesion into survival signaling unknown","Structure of full-length clustered ALCAM in membranes unsolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0,2,5,22]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[20,23,27]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,9,15,35]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[9,35]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[4,22]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7,10,29,39]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,5,22]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[23,25,27]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[9,35]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[21,28,39]}],"complexes":["immunological synapse"],"partners":["CD6","CD9","ADAM17","LILRB4","LGALS8","S100B","SOSTDC1","CTNNA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13740","full_name":"CD166 antigen","aliases":["Activated leukocyte cell adhesion molecule"],"length_aa":583,"mass_kda":65.1,"function":"Cell adhesion molecule that mediates both heterotypic cell-cell contacts via its interaction with CD6, as well as homotypic cell-cell contacts (PubMed:15048703, PubMed:15496415, PubMed:16352806, PubMed:23169771, PubMed:24945728, PubMed:7760007). Promotes T-cell activation and proliferation via its interactions with CD6 (PubMed:15048703, PubMed:16352806, PubMed:24945728). Contributes to the formation and maturation of the immunological synapse via its interactions with CD6 (PubMed:15294938, PubMed:16352806). Mediates homotypic interactions with cells that express ALCAM (PubMed:15496415, PubMed:16352806). Acts as a ligand for the LILRB4 receptor, enhancing LILRB4-mediated inhibition of T cell proliferation (PubMed:29263213). Required for normal hematopoietic stem cell engraftment in the bone marrow (PubMed:24740813). Mediates attachment of dendritic cells onto endothelial cells via homotypic interaction (PubMed:23169771). Inhibits endothelial cell migration and promotes endothelial tube formation via homotypic interactions (PubMed:15496415, PubMed:23169771). Required for normal organization of the lymph vessel network. Required for normal hematopoietic stem cell engraftment in the bone marrow. Plays a role in hematopoiesis; required for normal numbers of hematopoietic stem cells in bone marrow. Promotes in vitro osteoblast proliferation and differentiation (By similarity). Promotes neurite extension, axon growth and axon guidance; axons grow preferentially on surfaces that contain ALCAM. Mediates outgrowth and pathfinding for retinal ganglion cell axons (By similarity) Inhibits activities of membrane-bound isoforms by competing for the same interaction partners. Inhibits cell attachment via homotypic interactions. Promotes endothelial cell migration. Inhibits endothelial cell tube formation","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q13740/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALCAM","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000170017","cell_line_id":"CID000884","localizations":[{"compartment":"cell_contact","grade":3},{"compartment":"membrane","grade":3}],"interactors":[{"gene":"LAMB1","stoichiometry":0.2},{"gene":"ABCC1","stoichiometry":0.2},{"gene":"CANX","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000884","total_profiled":1310},"omim":[{"mim_id":"611735","title":"CUB DOMAIN-CONTAINING PROTEIN 1; CDCP1","url":"https://www.omim.org/entry/611735"},{"mim_id":"601662","title":"ACTIVATED LEUKOCYTE CELL ADHESION MOLECULE; ALCAM","url":"https://www.omim.org/entry/601662"},{"mim_id":"186720","title":"CD6 ANTIGEN; CD6","url":"https://www.omim.org/entry/186720"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Vesicles","reliability":"Uncertain"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"parathyroid gland","ntpm":412.8}],"url":"https://www.proteinatlas.org/search/ALCAM"},"hgnc":{"alias_symbol":["CD166","MEMD"],"prev_symbol":[]},"alphafold":{"accession":"Q13740","domains":[{"cath_id":"2.60.40.10","chopping":"31-132","consensus_level":"high","plddt":94.0663,"start":31,"end":132},{"cath_id":"2.60.40.10","chopping":"139-244","consensus_level":"high","plddt":96.3066,"start":139,"end":244},{"cath_id":"2.60.40.10","chopping":"250-329","consensus_level":"high","plddt":95.2166,"start":250,"end":329},{"cath_id":"2.60.40.10","chopping":"333-412","consensus_level":"medium","plddt":93.6196,"start":333,"end":412},{"cath_id":"2.60.40.10","chopping":"415-505","consensus_level":"medium","plddt":86.7821,"start":415,"end":505}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13740","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13740-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13740-F1-predicted_aligned_error_v6.png","plddt_mean":85.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALCAM","jax_strain_url":"https://www.jax.org/strain/search?query=ALCAM"},"sequence":{"accession":"Q13740","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13740.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13740/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13740"}},"corpus_meta":[{"pmid":"7760007","id":"PMC_7760007","title":"Cloning, 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protein binding assay, antibody-blocking studies\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reciprocal binding demonstrated with soluble fusion proteins and blocking antibodies; foundational paper independently replicated across multiple subsequent labs.\",\n      \"pmids\": [\"7760007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The CD6-binding site of ALCAM maps to the N-terminal Ig-like domain, and mutagenesis of hALCAM identified residues critical for CD6 binding on the predicted A'GFCC'C\\\" β-sheet of this domain; all critical residues are conserved in mouse ALCAM, explaining cross-species CD6/ALCAM interaction.\",\n      \"method\": \"Truncation constructs of ALCAM extracellular region, binding assays with soluble CD6, site-directed mutagenesis, molecular modeling\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis plus truncation binding assays in a single study, findings replicated by subsequent structural work.\",\n      \"pmids\": [\"9209500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"ALCAM (MEMD) mediates homophilic (ALCAM–ALCAM) cell-cell clustering in CD6-negative melanoma cells; transfection of ALCAM into ALCAM-negative melanoma cells restored cell-cell interaction, demonstrating that ALCAM expression is sufficient for homophilic adhesion in this context.\",\n      \"method\": \"Transfection experiments, FACS-based aggregation assays\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function transfection with functional adhesion readout in a single lab; supported by correlation data.\",\n      \"pmids\": [\"9502422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CD6–CD166 heterophilic interaction has a KD of ~0.4–1.0 µM with fast off-rate (Koff ≥0.4 s⁻¹); homophilic ALCAM–ALCAM interaction is ~100-fold weaker (KD ~29–48 µM, Koff ≥5.3 s⁻¹), demonstrating that heterophilic binding is substantially stronger than homophilic binding.\",\n      \"method\": \"In vitro binding assays with soluble recombinant proteins (surface plasmon resonance / biophysical characterization)\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — quantitative biophysical measurement with soluble recombinant proteins; replicated in the 2015 structural study.\",\n      \"pmids\": [\"15048703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"α-catenin is required to recruit ALCAM to cell–cell contacts; prostate cancer cell lines lacking α-catenin show cytoplasmic ALCAM staining, whereas transfection of α-N-catenin restores ALCAM localization to cell-cell junctions alongside E-cadherin.\",\n      \"method\": \"α-catenin transfection into α-catenin-null cell lines, immunofluorescence localization\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function rescue experiment with defined subcellular localization readout; single lab.\",\n      \"pmids\": [\"10673383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Homophilic ALCAM–ALCAM cell adhesion requires two structurally and functionally distinct modules: (1) ligand binding mediated by the membrane-distal N-terminal Ig domain, and (2) avidity control through ALCAM clustering involving membrane-proximal Ig domains. A transmembrane deletion mutant lacking the ligand-binding domain inhibited cell-cell adhesion by interfering with ALCAM avidity without affecting soluble homophilic ligand binding.\",\n      \"method\": \"Amino-terminally deleted ALCAM constructs, monoclonal antibody blocking, co-expression of dominant-negative mutant, cell adhesion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure-function analysis with multiple deletion constructs, dominant-negative interference, and blocking antibodies; mechanistically rigorous single-lab study.\",\n      \"pmids\": [\"11306570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ALCAM expression on yolk sac endothelium supports hematopoietic progenitor cell development; ALCAM-transfected adult endothelial cells (EOMA) supported hematopoietic progenitor development compared to vector controls, and ALCAM was found to be involved in capillary tube formation and hemangioblast differentiation.\",\n      \"method\": \"ALCAM transfection into EOMA endothelial cells, hematopoietic progenitor co-culture assay, immunohistochemistry\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function transfection with functional hematopoietic readout; single lab.\",\n      \"pmids\": [\"11568000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD6–ALCAM interactions are required not only for establishing initial DC–T-cell contact but also for sustaining T-cell proliferation; ALCAM-blocking antibodies and recombinant ALCAM-Fc proteins strongly and sustainably inhibited DC-induced T-cell proliferation, and simultaneous crosslinking of CD6 and CD3 induced proliferation comparable to CD3+CD28 co-stimulation.\",\n      \"method\": \"Antibody-blocking assays, ALCAM-Fc recombinant protein inhibition, CD6/CD3 co-crosslinking, T-cell proliferation assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (blocking antibody, soluble Fc protein, crosslinking) with defined proliferative readout; replicated conceptually across multiple studies.\",\n      \"pmids\": [\"16352806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ALCAM controls MMP-2 activation in melanoma: truncation of ALCAM (dominant-negative) severely impaired pro-MMP-2 activation by reducing MT1-MMP transcript levels and impairing MT1-MMP processing. ALCAM depletion by RNAi recapitulated this failure of the proteolytic cascade mainly through incomplete MT1-MMP processing. Extensive cell–cell contacts, wild-type ALCAM, and cell–matrix interactions were all required for efficient MMP-2 activation.\",\n      \"method\": \"Dominant-negative ALCAM truncation mutant, RNA interference (RNAi), nude mouse xenograft model, 2D and 3D collagen-gel cultures, zymography/MMP-2 activation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — two independent loss-of-function approaches (truncation mutant + RNAi) with in vitro and in vivo validation in a single rigorous study.\",\n      \"pmids\": [\"16204050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ALCAM undergoes ligand engagement-induced internalization via a clathrin-mediated pathway (colocalizing with clathrin but not caveolin) and recycles back to the cell surface, as shown by surface biotinylation and recycling assays. This endocytic pathway enables intracellular delivery of ALCAM-targeted immunotoxins.\",\n      \"method\": \"Phage display antibody-induced internalization, immunofluorescence colocalization with clathrin/caveolin, surface biotinylation, recycling assay, immunotoxin kill assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (colocalization, biochemical recycling assay, functional immunotoxin delivery) in a single study.\",\n      \"pmids\": [\"15769845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD6 and CD166 are recruited together to the center of the immunological synapse between γδ T cells and antigen-loaded tumor cells, colocalizing with γδ TCR/CD3. CD166 transfection into a CD166-negative tumor line markedly enhanced γδ T cell activation, while shRNA-mediated CD166 knockdown reduced it, demonstrating that CD6–CD166 engagement at the synapse is required for γδ T cell activation by nonpeptide antigen-presenting tumor cells.\",\n      \"method\": \"CD166 cDNA transfection, shRNA knockdown, immunofluorescence of immunological synapse, T-cell activation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain- and loss-of-function with defined synapse localization readout; multiple orthogonal approaches.\",\n      \"pmids\": [\"16818742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ALCAM gene silencing in breast cancer cells (MCF-7) reduced BCL-2 protein levels and triggered apoptosis (caspase-7 activation, PARP cleavage) and autophagy (MAP1LC3, Beclin1 upregulation), indicating ALCAM supports cell survival downstream of BCL-2.\",\n      \"method\": \"ALCAM gene silencing (siRNA), laser scanning cytometry, Western blotting for apoptosis/autophagy markers\",\n      \"journal\": \"Medical science monitor\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, loss-of-function with defined molecular markers but no mechanistic pathway reconstitution.\",\n      \"pmids\": [\"16865058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NDRG2 expressed in dendritic cells prevents down-regulation of ALCAM during monocyte-to-DC differentiation; NDRG2 siRNA knockdown specifically reduced ALCAM expression in differentiating DCs and diminished their ability to induce T cell proliferation, while NDRG2 overexpression in U937 cells conferred resistance to GM-CSF/IL-4-induced ALCAM reduction.\",\n      \"method\": \"RNA interference of NDRG2, NDRG2 overexpression in U937, flow cytometry for ALCAM, T-cell proliferation assay\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with defined ALCAM expression and T-cell functional readout; single lab.\",\n      \"pmids\": [\"17911180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis in Xenopus laevis; loss-of-function reduced expression of first-heart-field markers (Tbx20, TnIc) but not second-heart-field markers (Isl-1, BMP-4), caused defective cell adhesion and cardiac morphogenesis, and DM-GRASP expression rescued the phenotype caused by loss of non-canonical Wnt11-R signaling, demonstrating functional coupling between ALCAM and Wnt11-R during cardiac development.\",\n      \"method\": \"Xenopus laevis loss-of-function (morpholino knockdown), rescue experiments with DM-GRASP expression, in situ hybridization for cardiac marker genes\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (rescue experiment) with orthogonal molecular readouts in a developmental model organism; single lab.\",\n      \"pmids\": [\"18598690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NF-κB P50/P65 heterodimer activates both CD166/ALCAM and miR-9-1 transcription after serum deprivation. miR-9, induced with a delay, represses ALCAM protein translation via its 3'-UTR, creating a negative auto-regulatory loop. miR-9 also promotes cell migration partly via inhibition of CD166.\",\n      \"method\": \"Luciferase reporter assay, NF-κB inhibition/knockdown, miRNA overexpression and inhibitor experiments, Western blotting, qRT-PCR\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assays plus miRNA gain/loss-of-function with defined molecular endpoints; single lab.\",\n      \"pmids\": [\"21572107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALCAM directly associates with the tetraspanin CD9 and ADAM17/TACE on the leukocyte surface; CD9 upregulates both homophilic and heterophilic ALCAM-mediated adhesion by (1) promoting ALCAM clustering and (2) inhibiting ADAM17 sheddase activity to increase ALCAM surface expression.\",\n      \"method\": \"Confocal microscopy colocalization, co-immunoprecipitation, cell adhesion and migration/proliferation functional assays, ADAM17 activity assays\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, colocalization, and multiple functional assays demonstrating dual mechanism; single lab with multiple orthogonal methods.\",\n      \"pmids\": [\"23052204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALCAM regulates motility, invasiveness, and adherens junction formation in uveal melanoma; shRNA knockdown of ALCAM reduced cell motility and invasion and disrupted adherens junction formation, while ALCAM overexpression enhanced recruitment of β-catenin and N-cadherin to adherens junctions. ALCAM is necessary but not sufficient to promote metastasis-associated behaviors.\",\n      \"method\": \"shRNA stable knockdown, stable overexpression, gap-closure motility assay, transwell invasion assay, immunostaining for adherens junction components\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with multiple functional readouts; single lab.\",\n      \"pmids\": [\"22745734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALCAM mRNA is locally translated in retinal ganglion cell axonal growth cones, regulated by the 3'-UTR and dependent on ERK and TOR kinase activity. Local growth cone translation of ALCAM is required for enhanced axon elongation on ALCAM substrate, rapid compensation for experimentally induced ALCAM internalization, and axonal preference for ALCAM-containing lanes.\",\n      \"method\": \"Isolated growth cone translation assay, 3'-UTR reporter constructs, kinase inhibitors (ERK, TOR), ALCAM internalization assay, axon choice assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution of local translation in isolated growth cones plus multiple functional assays; mechanistically rigorous single-lab study.\",\n      \"pmids\": [\"22421359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALCAM regulates long-term hematopoietic stem cell (HSC) self-renewal and engraftment; Alcam-/- mice show reduced long-term repopulating capacity and engraftment efficiency, age-associated expansion of CD150hi LT-HSCs with myeloid-biased output, and premature elevation of age-associated genes (Selp, Clu, Cdc42, Foxo3).\",\n      \"method\": \"Alcam knockout mouse model, serial transplantation assays, in vitro replating, gene expression analysis\",\n      \"journal\": \"Stem cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined functional HSC phenotype across multiple in vivo readouts; single lab.\",\n      \"pmids\": [\"23280653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ALCAM mediates adhesion, migration, and tube formation in lymphatic endothelial cells (LECs) and supports dendritic cell adhesion to lymphatic endothelium. ALCAM knockout mice have reduced LEC numbers, defects in organized lymphatic vessel network formation, and compromised DC migration from lung to draining lymph nodes.\",\n      \"method\": \"ALCAM knockout mouse, in vitro LEC adhesion/migration/tube formation assays, DC migration assay in vivo and in vitro\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with multiple in vitro and in vivo functional readouts; multiple orthogonal methods.\",\n      \"pmids\": [\"23169771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"S100B binds CD166/ALCAM and induces dose- and time-dependent NF-κB activation in endothelial cells. siRNA knockdown of CD166/ALCAM completely inhibited S100B-induced NF-κB activation in RAGE-/- cells. In vivo, ALCAM siRNA attenuated delayed-type hypersensitivity (DTH) by ~40–50%; ALCAM-/- mice showed compensatory RAGE upregulation.\",\n      \"method\": \"siRNA knockdown, NF-κB reporter/activation assay, ALCAM-/- and RAGE-/- mouse DTH model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function in vitro and in vivo with defined signaling readout; genetic knockout with compensatory cross-regulation revealed.\",\n      \"pmids\": [\"23729438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ALCAM is shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β signaling, and this ectodomain shedding is required for effective bone metastasis; shRNA knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished skeletal dissemination and tumor growth in bone, associated with increased apoptosis and decreased proliferation.\",\n      \"method\": \"Biochemical shedding assays, ADAM17 identification by inhibitor and siRNA, ALCAM shRNA knockdown, in vivo bone metastasis model (intratibial engraftment), IHC for caspase-3 and Ki67\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical identification of ADAM17 as sheddase plus in vivo loss-of-function with multiple mechanistic readouts; single lab, multiple orthogonal methods.\",\n      \"pmids\": [\"24385212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ALCAM intracellular domain coupling to the actin cortex does not affect the affinity of individual ALCAM–CD6 bonds, but does control ALCAM recruitment to adhesion sites and membrane tether formation. Linking ALCAM to the actin cortex stiffens the cortex and strengthens overall cell adhesion to CD6 at the immunological synapse.\",\n      \"method\": \"Single-cell force spectroscopy (SCFS), TIRF microscopy, ALCAM cytoplasmic tail deletion mutants, actin cytoskeleton perturbation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative biophysical measurement (SCFS) combined with defined ALCAM mutants and imaging; mechanistically rigorous.\",\n      \"pmids\": [\"24496453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD166 promotes anti-apoptotic signaling in liver cancer via PI3K/AKT: AKT upregulates CD166 expression post-transcriptionally, and CD166 in turn promotes AKT expression and activity (positive feedback). CD166 also activates YAP through transcriptional regulation via CREB and post-transcriptional stabilization via AMOT130 inhibition. CD9 enhances CD166-mediated YAP regulation by facilitating CD166–CD166 homophilic interaction.\",\n      \"method\": \"CD166 siRNA knockdown, AKT overexpression rescue, subcellular fractionation, co-immunoprecipitation, luciferase reporter, tissue microarray\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple loss/gain-of-function approaches with defined signaling readouts; single lab.\",\n      \"pmids\": [\"24482231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD166 regulates MCAM protein stability by suppressing the ubiquitin E3 ligases βTrCP and Smurf1 through PI3K/AKT and c-Raf/MEK/ERK signaling, thereby protecting MCAM from proteasomal degradation.\",\n      \"method\": \"CD166 knockdown/overexpression, ubiquitination assays, E3 ligase overexpression, pathway inhibitors (PI3K, MEK), co-immunoprecipitation, tissue microarray\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway dissection with multiple loss/gain-of-function and ubiquitination readouts; single lab.\",\n      \"pmids\": [\"26004137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD166 regulates FOXO protein stability and subcellular localization through AKT: CD166 overexpression accelerates FOXO ubiquitination and degradation and shifts FOXO from nucleus to cytoplasm, while CD166 knockdown reduces FOXO phosphorylation. AKT overexpression rescues CD166 knockdown-induced FOXO dephosphorylation and anti-carcinogenic effects, placing AKT between CD166 and FOXO.\",\n      \"method\": \"CD166 knockdown/overexpression, subcellular fractionation, ubiquitination assay, AKT overexpression rescue, in vitro and in vivo tumor assays\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis rescue (AKT overexpression) with biochemical and localization readouts; single lab.\",\n      \"pmids\": [\"24891117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structures of the three SRCR domains of CD6 and the two N-terminal Ig domains of CD166 were solved by X-ray crystallography. Structural analysis revealed the CD6/CD166 binding interface, showed that a SNP in CD6 introduces glycosylation that sterically hinders the CD6/CD166 interaction, and native mass spectrometry demonstrated competition between heterophilic CD6-CD166 and homophilic CD166-CD166 interactions.\",\n      \"method\": \"X-ray crystallography, native mass spectrometry, SNP glycosylation analysis\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structures of both binding partners with functional validation by native MS; definitive structural evidence.\",\n      \"pmids\": [\"26146185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ILT3 (LILRB4) binds CD166/ALCAM directly; CRISPR-Cas9 knockout of CD166 abolished ILT3.Fc binding and its tumor-inhibitory effect. ILT3.Fc binding to CD166 inhibits tumor cell growth through inactivation of the p70 S6 kinase (p70S6K) signaling pathway.\",\n      \"method\": \"Flow cytometry, mass spectrometry, Biacore (SPR), CD166 knockdown by nucleofection and CRISPR-Cas9 KO, p70S6K signaling assay, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding confirmed by SPR, CRISPR KO abolishes effect, signaling mechanism identified; multiple orthogonal methods.\",\n      \"pmids\": [\"29263213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CD166 in multiple myeloma cells inhibits osteoblastogenesis by suppressing Runx2 gene expression in osteoblast progenitors, and promotes osteoclastogenesis by activating TRAF6-dependent signaling in osteoclast progenitors; CD166 silencing reduced skeletal dissemination and osteolytic lesion formation in vivo.\",\n      \"method\": \"CD166 siRNA/shRNA knockdown in MM cell lines, intratibial engraftment model, ex vivo bone organ culture, osteoblast/osteoclast differentiation assays, Runx2 and TRAF6 signaling analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with defined in vitro and in vivo mechanistic readouts across multiple cell types; single lab, multiple orthogonal methods.\",\n      \"pmids\": [\"27634757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALCAM knockout mice develop more severe EAE (experimental autoimmune encephalomyelitis) with increased CNS-infiltrating leukocytes; passive transfer experiments linked this to absence of ALCAM on blood-brain barrier endothelial cells. ALCAM KO mice also show reduced expression of BBB tight junction proteins and increased CNS blood vessel permeability, establishing ALCAM as a component required for tight junction assembly and BBB integrity.\",\n      \"method\": \"ALCAM knockout mouse, active and passive EAE model, phenotypic characterization, tight junction protein expression, in vitro BBB permeability assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with in vivo disease model, passive transfer epistasis, and molecular characterization of junction proteins; multiple orthogonal methods.\",\n      \"pmids\": [\"28069965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ALCAM mediates preferential diapedesis of CD4+ Th1 cells (but not Th17 cells) across the human BBB in vitro; antibody-mediated ALCAM inhibition reduced Th1 but not Th17 diapedesis under static conditions. ALCAM also contributes to rolling, adhesion, and diapedesis of CD14+ monocytes across the human BBB under flow and static conditions.\",\n      \"method\": \"Anti-ALCAM antibody blocking, ALCAM-/- in vitro BBB model, leukocyte transmigration assays under static and flow conditions\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antibody blocking plus ALCAM-/- BBB model with defined cell-type-specific readouts; single lab.\",\n      \"pmids\": [\"28273717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD166 loss in intestinal crypts reduces active-cycling Lgr5+ ISC numbers; homeostasis is maintained by transit-amplifying compartment expansion (not slow-cycling Bmi1+ ISC stimulation). Loss of active-cycling ISCs is coupled to defective Paneth cell terminal differentiation linked to reduced Wnt3 ligand expression and depleted nuclear β-catenin in CD166-/- Paneth cells.\",\n      \"method\": \"CD166-/- mouse, immunohistochemistry, flow cytometry, gene expression analysis, enteroid culture\",\n      \"journal\": \"Cellular and molecular gastroenterology and hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with multiple orthogonal methods revealing ISC niche signaling mechanism; Wnt pathway placement established.\",\n      \"pmids\": [\"28462380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALCAM mediates DC migration through afferent lymphatics and promotes allospecific immune reactions; anti-murine ALCAM blocking antibody reduced DC transmigration across lymphatic endothelial monolayers, DC emigration from human skin explants, lymphangiogenic processes in vitro, and prevented corneal allograft rejection by retaining DCs in the cornea.\",\n      \"method\": \"Blocking monoclonal antibody, in vitro DC transmigration assay, human skin explant emigration, in vivo corneal allograft model, developmental lymphangiogenesis assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple in vitro and in vivo functional assays with defined blocking reagent; multiple orthogonal methods in a single rigorous study.\",\n      \"pmids\": [\"31031759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"E3 ubiquitin ligase CHIP directly regulates ALCAM protein stability through the ubiquitin proteasome system; CHIP negatively correlates with CD166 in head and neck cancer samples, and CHIP expression represses cancer stem-like cell characteristics via targeting CD166 for degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin proteasome inhibitor assay, CHIP overexpression/knockdown, Western blotting, tissue microarray\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional ubiquitination assay with defined CSC readout; single lab.\",\n      \"pmids\": [\"28279658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PRMT1 interacts with ALCAM directly (confirmed by co-immunoprecipitation and LC-MS); PRMT1 silencing reduced ALCAM protein levels and suppressed melanoma tumor growth and metastasis, while re-expression of ALCAM in PRMT1-silenced cells restored colony formation and metastatic ability, placing ALCAM downstream of PRMT1.\",\n      \"method\": \"LC-MS/MS, co-immunoprecipitation, PRMT1 shRNA knockdown, ALCAM rescue overexpression, colony formation and migration assays\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical interaction confirmed by co-IP and LC-MS, epistasis rescue experiment; single lab.\",\n      \"pmids\": [\"27175582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD166/ALCAM is internalized via a clathrin-independent endocytic pathway driven by endophilin-A3 (not A1 or A2 isoforms) and extracellular galectin-8. Endophilin-A3 physically interacts with CD166-containing early endocytic carriers. This endocytic modality modulates CD166 surface abundance and regulates adhesive and migratory properties of cancer cells.\",\n      \"method\": \"Endophilin-A isoform-specific knockdown, galectin-8 perturbation, live-cell endocytic carrier imaging, co-immunoprecipitation, cell adhesion and migration assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — isoform-specific knockdown demonstrating selectivity, physical interaction data, and functional consequences on adhesion/migration; multiple orthogonal methods.\",\n      \"pmids\": [\"32193381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SOSTDC1 interacts with ALCAM/CD166 (identified by immunoprecipitation and mass spectrometry, confirmed by confocal microscopy and competition ELISA); this interaction involves the N-terminal region of SOSTDC1, which contains a sequence similar to the CD6-binding motif for ALCAM. ALCAM also interacts with α2β1 and α1β1 integrins. Knockdown of either SOSTDC1 or ALCAM, or antibody blockade, reduces invasion by inhibiting Src and PI3K/AKT signaling.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, confocal microscopy, competition ELISA, SOSTDC1/ALCAM knockdown, invasion assay, Src/PI3K-AKT signaling readout, in vivo liver metastasis model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical interaction confirmed by multiple orthogonal methods (co-IP, MS, ELISA, imaging), pathway placement by loss-of-function, in vivo validation.\",\n      \"pmids\": [\"32801337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Galectin-8 interacts with ALCAM/CD166 in a glycosylation-dependent manner (demonstrated by surface plasmon resonance with recombinant glycosylated ALCAM ectodomain and endogenous ALCAM from breast cancer cells); ALCAM-silenced cells showed reduced binding to Gal-8. Exogenous Gal-8 caused ALCAM surface segregation/trapping at the cell surface.\",\n      \"method\": \"Surface plasmon resonance (SPR) binding assay, ALCAM siRNA knockdown, SPR binding of ALCAM-silenced cells, internalization/surface localization assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biophysical binding measurement (SPR) with glycosylation dependency demonstrated, plus functional silencing; rigorous single-lab study.\",\n      \"pmids\": [\"27130882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALCAM/CD166 is involved in binding and uptake of cancer-derived extracellular vesicles (EVs) by recipient cancer cells; ALCAM participates in EV docking and subsequent uptake, demonstrated in colorectal and ovarian cancer cell systems.\",\n      \"method\": \"ALCAM expression manipulation, EV binding and uptake assays, flow cytometry\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — functional assay with defined molecular readout; single lab, mechanistic detail limited to abstract description.\",\n      \"pmids\": [\"35628559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The CD6/ALCAM pathway promotes lupus nephritis (LN) via T cell-mediated responses; ALCAM is expressed by renal structural cells while CD6 is exclusive to T cells in the LN kidney. Antibody blockade of CD6 in murine lupus and immune-complex glomerulonephritis models significantly decreased immune cell infiltration, inflammatory markers, and disease measures.\",\n      \"method\": \"Immunophenotyping of LN kidney cells, anti-CD6 antibody blockade in spontaneous lupus and immune-complex glomerulonephritis mouse models, uALCAM ELISA in patient cohorts\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo antibody blockade in two mouse models with defined immunological and disease readouts; replicated in two model systems and corroborated by large human cohort data.\",\n      \"pmids\": [\"34981775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Hypoxia promotes ALCAM expression in macrophages via HIF-1α binding to the ALCAM promoter; ALCAMhigh macrophages co-localize with exhausted CD8+ T cells in the tumor spatial microenvironment and promote T cell exhaustion. HIF-1α inhibition reduces ALCAM expression in macrophages and potentiates T cell anti-tumor function.\",\n      \"method\": \"Bulk, single-cell, and spatial transcriptomics integration, HIF-1α ChIP on ALCAM promoter, HIF-1α inhibitor experiments, preclinical immunotherapy models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — HIF-1α promoter binding demonstrated, functional consequence shown with HIF-1α inhibitor in preclinical model; single study.\",\n      \"pmids\": [\"38956900\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALCAM/CD166 is a transmembrane IgSF cell adhesion molecule that engages in strong heterophilic interactions with CD6 (KD ~0.4–1 µM, mediated by its membrane-distal N-terminal Ig domain) and weaker homophilic ALCAM–ALCAM interactions (KD ~30–48 µM, dependent on membrane-proximal domain-driven clustering); its crystal structure with CD6 is solved. At the immunological synapse, ALCAM–CD6 engagement sustains DC-induced T-cell proliferation and γδ T cell activation. ALCAM is dynamically regulated at the cell surface: it undergoes clathrin-mediated endocytosis with recycling back to the surface, clathrin-independent endocytosis via endophilin-A3 driven by galectin-8, and proteolytic ectodomain shedding by ADAM17 (upregulated by TGF-β and modulated by CD9, which inhibits ADAM17 to increase ALCAM surface levels). Intracellularly, ALCAM couples to the actin cortex through adaptor proteins, stiffening the cortex and strengthening cell adhesion without changing individual bond affinity. ALCAM controls MMP-2 activation by regulating MT1-MMP processing in a cell-density-dependent manner, promotes PI3K/AKT-YAP and AKT-FOXO survival signaling in cancer cells, and mediates osteoblast-HSC homophilic interactions critical for HSC niche function and self-renewal. ALCAM expression is regulated transcriptionally by NF-κB and HIF-1α (in macrophages) and post-translationally by E3 ligases CHIP (ubiquitin-proteasome degradation) and PRMT1. ALCAM also serves as a ligand for ILT3 (LILRB4) and galectin-8, and is required for BBB tight junction integrity, lymphatic network formation, and cardiac/intestinal stem cell niche maintenance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALCAM (CD166) is a transmembrane immunoglobulin-superfamily cell adhesion molecule that engages in strong heterophilic binding to CD6 (KD ~0.4–1 µM) through its membrane-distal N-terminal Ig domain and weaker homophilic ALCAM–ALCAM interactions (KD ~29–48 µM) [#0, #3, #26]. Homophilic adhesion is biphasic: the N-terminal Ig domain mediates ligand binding, while membrane-proximal Ig domains drive clustering that controls avidity [#5]. Crystal structures of the CD6 SRCR domains bound to the two N-terminal ALCAM Ig domains define the interface, and native mass spectrometry shows that heterophilic CD6–ALCAM and homophilic ALCAM–ALCAM engagement compete [#26]. Intracellularly, ALCAM is recruited to cell-cell contacts by α-catenin and couples to the actin cortex; this linkage does not alter individual bond affinity but stiffens the cortex and strengthens overall adhesion to CD6 at the immunological synapse [#4, #22]. ALCAM surface levels are dynamically controlled by clathrin-mediated endocytosis with recycling, an endophilin-A3/galectin-8-driven clathrin-independent pathway, and ADAM17-mediated ectodomain shedding that is induced by TGF-β and restrained by the tetraspanin CD9 [#9, #35, #37, #21, #15]. Through CD6–ALCAM engagement at the immunological synapse, ALCAM sustains DC-induced T-cell proliferation and is required for γδ T cell activation [#7, #10]. In vivo, ALCAM is required for hematopoietic stem cell self-renewal and engraftment, lymphatic network formation and dendritic cell migration, blood-brain barrier tight-junction integrity and leukocyte transmigration, and intestinal stem cell niche maintenance via Wnt signaling [#18, #19, #29, #31]. In cancer, ALCAM controls MMP-2 activation through MT1-MMP processing and promotes survival signaling through PI3K/AKT–YAP and AKT–FOXO axes [#8, #23, #25]. ALCAM additionally serves as a binding partner for ILT3 (LILRB4), galectin-8, and S100B, and its expression is regulated transcriptionally by NF-κB and HIF-1α and post-translationally by the E3 ligase CHIP and by PRMT1 [#27, #37, #20, #14, #40, #33, #34].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing ALCAM's first molecular partner: it was unknown what receptor ALCAM engaged, and reciprocal fusion-protein binding defined a direct heterophilic ALCAM–CD6 interaction, founding the entire CD6–ALCAM axis.\",\n      \"evidence\": \"COS cell transfection with reciprocal Ig-fusion-protein binding and antibody blocking\",\n      \"pmids\": [\"7760007\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the binding site at residue resolution\", \"Did not address homophilic binding or downstream signaling\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Localizing the CD6-binding determinant: mutagenesis and truncation mapped CD6 binding to specific residues on the N-terminal Ig domain β-sheet and explained cross-species interaction.\",\n      \"evidence\": \"Truncation constructs, site-directed mutagenesis, and molecular modeling with soluble CD6\",\n      \"pmids\": [\"9209500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic structure yet\", \"Did not resolve how homophilic binding uses the same domain\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defining a second adhesion mode: whether ALCAM could act independently of CD6 was unknown, and transfection into CD6-negative melanoma showed ALCAM expression is sufficient for homophilic clustering.\",\n      \"evidence\": \"ALCAM transfection into ALCAM-negative melanoma with FACS aggregation assays\",\n      \"pmids\": [\"9502422\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Affinity and structural basis of homophilic binding not measured\", \"Single-lab gain-of-function\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Quantifying the adhesion hierarchy and its modularity: biophysics revealed heterophilic CD6 binding is ~100-fold stronger than homophilic binding, and structure-function work showed ligand binding and avidity-controlling clustering are separable modules.\",\n      \"evidence\": \"SPR/biophysical KD measurements and deletion/dominant-negative adhesion assays\",\n      \"pmids\": [\"15048703\", \"11306570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the clustering interface structurally\", \"Intracellular coupling not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Connecting ALCAM to the junctional cytoskeleton: α-catenin was shown to be required to recruit ALCAM to cell-cell contacts alongside E-cadherin.\",\n      \"evidence\": \"α-N-catenin rescue in α-catenin-null prostate cancer cells with immunofluorescence\",\n      \"pmids\": [\"10673383\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect coupling to actin not resolved\", \"No biophysical adhesion measurement\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defining ALCAM's immune function: CD6–ALCAM engagement was shown not only to initiate but to sustain DC-induced T-cell proliferation and to be required for γδ T cell activation at the immunological synapse.\",\n      \"evidence\": \"Blocking antibodies, ALCAM-Fc, CD6/CD3 crosslinking, synapse imaging, and gain/loss-of-function in tumor lines\",\n      \"pmids\": [\"16352806\", \"16818742\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream T-cell signaling from CD6 not dissected\", \"Relative contribution of homophilic vs heterophilic binding unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linking ALCAM to proteolytic invasion machinery: ALCAM was shown to control MMP-2 activation in a cell-density-dependent manner by regulating MT1-MMP transcription and processing.\",\n      \"evidence\": \"Dominant-negative truncation and RNAi with zymography, 3D collagen culture, and xenografts\",\n      \"pmids\": [\"16204050\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking ALCAM adhesion to MT1-MMP regulation not defined\", \"Direct ALCAM signaling intermediates unidentified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing ALCAM surface dynamics: ligand engagement was shown to drive clathrin-mediated internalization with recycling back to the surface.\",\n      \"evidence\": \"Antibody-induced internalization, clathrin/caveolin colocalization, surface biotinylation, and recycling assays\",\n      \"pmids\": [\"15769845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Adaptor proteins for clathrin uptake not identified\", \"Did not address alternative endocytic routes\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connecting surface regulation to a tetraspanin: ALCAM was shown to associate with CD9 and ADAM17, with CD9 boosting ALCAM adhesion by promoting clustering and inhibiting sheddase activity.\",\n      \"evidence\": \"Co-IP, confocal colocalization, ADAM17 activity assays, and adhesion/migration functional readouts\",\n      \"pmids\": [\"23052204\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the CD9–ALCAM–ADAM17 assembly unknown\", \"How CD9 inhibits ADAM17 not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defining the biophysics of intracellular coupling: actin-cortex linkage of the ALCAM tail was shown to strengthen adhesion by stiffening the cortex rather than altering bond affinity.\",\n      \"evidence\": \"Single-cell force spectroscopy, TIRF microscopy, and cytoplasmic-tail deletion mutants\",\n      \"pmids\": [\"24496453\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the actin-coupling adaptors not defined\", \"Link to α-catinin-mediated recruitment unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Establishing ALCAM as a sheddable driver of metastasis: ADAM17 was identified as the TGF-β-induced sheddase, and ALCAM shedding was shown required for prostate cancer bone metastasis.\",\n      \"evidence\": \"Biochemical shedding assays, ADAM17 inhibitor/siRNA, ALCAM shRNA, and in vivo intratibial bone metastasis model\",\n      \"pmids\": [\"24385212\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Function of the shed ectodomain vs membrane stub not separated\", \"Signaling triggered by shedding unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placing ALCAM in cancer survival signaling: ALCAM/CD166 was shown to drive PI3K/AKT–YAP and AKT–FOXO axes and to stabilize partner proteins, with CD9 facilitating CD166 homophilic interaction.\",\n      \"evidence\": \"Knockdown/overexpression, AKT rescue epistasis, ubiquitination, fractionation, and reporter assays\",\n      \"pmids\": [\"24482231\", \"24891117\", \"24385212\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between surface ALCAM and AKT activation not defined\", \"Single-lab dissection of these feedback loops\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Achieving atomic resolution: crystal structures of CD6 SRCR and ALCAM Ig domains defined the binding interface and showed heterophilic and homophilic interactions compete.\",\n      \"evidence\": \"X-ray crystallography, native mass spectrometry, and SNP glycosylation analysis\",\n      \"pmids\": [\"26146185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length complex in membrane context not resolved\", \"Structural basis of avidity clustering not captured\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanding the ALCAM ligand repertoire: SPR established glycosylation-dependent galectin-8 binding, and CRISPR work defined ILT3 (LILRB4) as a direct partner that inhibits tumor growth via p70S6K.\",\n      \"evidence\": \"SPR with recombinant and endogenous ALCAM, mass spectrometry, CRISPR-Cas9 KO, and p70S6K signaling assays\",\n      \"pmids\": [\"27130882\", \"29263213\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context of ILT3–ALCAM signaling beyond tumors unclear\", \"Whether galectin-8 binding competes with CD6 not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Establishing ALCAM's stem-cell-niche and vascular-barrier roles: knockout mice revealed requirements for HSC self-renewal, lymphatic network formation, and DC migration.\",\n      \"evidence\": \"Alcam knockout mice with serial transplantation, LEC adhesion/tube assays, and in vivo DC migration\",\n      \"pmids\": [\"23280653\", \"23169771\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether niche function is homophilic or heterophilic not resolved\", \"Molecular signaling downstream of niche adhesion unidentified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defining ALCAM in barrier and epithelial niches: knockouts showed ALCAM is required for BBB tight-junction integrity and Th1/monocyte transmigration, and for intestinal stem cell maintenance via Wnt3/β-catenin.\",\n      \"evidence\": \"ALCAM KO mice, EAE and passive-transfer models, BBB permeability and transmigration assays, and enteroid/ISC analysis\",\n      \"pmids\": [\"28069965\", \"28273717\", \"28462380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ALCAM mechanistically promotes tight-junction assembly unknown\", \"Link between ALCAM adhesion and Wnt ligand expression not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying a selective clathrin-independent endocytic route: endophilin-A3 and galectin-8 were shown to drive ALCAM internalization that tunes surface abundance and migration.\",\n      \"evidence\": \"Isoform-specific endophilin knockdown, galectin-8 perturbation, live-cell carrier imaging, and co-IP\",\n      \"pmids\": [\"32193381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How cells choose between clathrin-dependent and -independent routes unknown\", \"Fate of internalized ALCAM not fully tracked\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linking the CD6–ALCAM axis to disease and exhaustion: antibody blockade implicated ALCAM in lupus nephritis, and HIF-1α-driven ALCAMhigh macrophages were shown to promote CD8 T cell exhaustion.\",\n      \"evidence\": \"Anti-CD6 blockade in lupus models, patient uALCAM ELISA, and spatial/single-cell transcriptomics with HIF-1α ChIP and inhibition\",\n      \"pmids\": [\"34981775\", \"38956900\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ALCAM ligand mediating exhaustion in this context not defined\", \"Causality of macrophage ALCAM in human tumors not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the cytoplasmic adaptors that physically couple the ALCAM tail to the actin cortex, and the unified signaling output that links surface adhesion to AKT/YAP/FOXO regulation, remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct ALCAM intracellular signaling effector identified\", \"Mechanism converting adhesion into survival signaling unknown\", \"Structure of full-length clustered ALCAM in membranes unsolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 2, 5, 22]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [20, 23, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 9, 15, 35]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [9, 35]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7, 10, 29, 39]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 5, 22]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [23, 25, 27]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [9, 35]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [21, 28, 39]}\n    ],\n    \"complexes\": [\"immunological synapse\"],\n    \"partners\": [\"CD6\", \"CD9\", \"ADAM17\", \"LILRB4\", \"LGALS8\", \"S100B\", \"SOSTDC1\", \"CTNNA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}