id: A0A0R4IGT8 gene_symbol: wu:fi36a10 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:7955 label: Danio rerio description: 'wu:fi36a10 (ZFIN ZDB-GENE-030131-6139, chromosome 23; NCBI and RefSeq symbol magi3a) is one of two zebrafish co-orthologs of vertebrate MAGI3; PANTHER places the duplication at the teleost genome duplication. Its paralog is magi3b. It encodes a cytoplasmic membrane-associated guanylate kinase with inverted domain order (MAGI): an N-terminal PDZ domain, a guanylate kinase-like (GK) domain that lacks the nucleotide-binding P-loop and serves as a protein-interaction module rather than an enzyme, two WW domains, five further PDZ domains and a long disordered C-terminal tail. MAGI proteins scaffold receptors and signalling proteins at the cytoplasmic face of cell-cell junctions. In a genome-wide yeast two-hybrid screen the zebrafish protein (reported as Magi3a) bound the C-terminal ETTV PDZ-binding motif of the Wnt7 co-receptor Gpr124. The gene is expressed in endothelial cells, and F0 crispants have reduced brain vascularization that phenocopies loss of that motif, as do magi3b crispants. Public RNA-Seq calls cover a broad range of embryonic and adult tissues, as for the single gar magi3 gene, and are more extensive than those of magi3b. Of the two copies it is the closer in sequence to gar and human MAGI3.' existing_annotations: - term: id: GO:0000166 label: nucleotide binding evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: Nucleotide binding from UniRule UR000400325 (MAGUK guanylate kinase-like domain). action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in wu:fi36a10 (as in magi3b, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - &id001 reference_id: PMID:37163606 supporting_text: Unlike other MAGUK GKs, which are composed of three subdomains including guanosine monophosphate (GMP)–binding, LID, and CORE subdomains, MAGI GKs lack the last two subdomains - &id002 reference_id: file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md supporting_text: Neither zebrafish copy, nor gar or human MAGI3, has a Walker A (P-loop) motif at the position UniProt annotates as the ATP-binding site - term: id: GO:0005524 label: ATP binding evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: ATP binding from UniRule UR000400325, based on the predicted P-loop of the GK-like domain. action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in wu:fi36a10 (as in magi3b, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - *id001 - *id002 - term: id: GO:0005634 label: nucleus evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0191 review: summary: Nucleus (UniProt subcellular location; for human MAGI3 this is 'by similarity'). action: KEEP_AS_NON_CORE reason: Human MAGI3 is reported to concentrate in the nucleus as well as at junctions, but only by similarity. There is no zebrafish evidence. Plausible, but not where the scaffold function characterized in zebrafish acts. magi3b lacks this IEA row because of different rule coverage, not because of any known biological difference. - term: id: GO:0005737 label: cytoplasm evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - MGI:MGI:1203522 - MGI:MGI:1354953 - PANTHER:PTN000817101 - RGD:621855 - UniProtKB:Q86UL8 - UniProtKB:Q96QZ7 - ZFIN:ZDB-GENE-060503-301 review: summary: Cytoplasm (IBA, PANTHER PTN000817101 MAGI node). action: ACCEPT reason: MAGI proteins are cytoplasmic scaffolds at the inner face of the plasma membrane. wu:fi36a10 is inside the MAGI clade, keeps all its domains, and nothing argues for loss of the function. Its paralog magi3b has a direct IDA for this location. supported_by: - &id003 reference_id: PMID:10748157 supporting_text: localizes to epithelial cell tight junctions - &id004 reference_id: PMID:35649360 supporting_text: the PDZ proteins membrane-associated guanylate kinase, WW, and PDZ domain containing 3 (Magi3a) and discs large MAGUK scaffold protein 4 (Dlg4a) bound to Gpr124 via its C-terminal ETTV motif. - term: id: GO:0005886 label: plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0039 review: summary: Plasma membrane (UniProt subcellular location, peripheral membrane protein). action: ACCEPT reason: MAGI3 is a peripheral protein on the cytoplasmic face of the plasma membrane at cell-cell junctions. In zebrafish it binds the C terminus of the transmembrane receptor Gpr124. supported_by: - *id003 - *id004 - term: id: GO:0005911 label: cell-cell junction evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - MGI:MGI:1203522 - MGI:MGI:1923484 - PANTHER:PTN002569180 - RGD:621855 - UniProtKB:Q86UL8 - UniProtKB:Q96QZ7 review: summary: Cell-cell junction (IBA, PANTHER PTN002569180). action: ACCEPT reason: Junctional localization is the conserved property of MAGI1-3. wu:fi36a10 keeps the full MAGI domain set, and nothing argues for loss of the function. This is the correct level of specificity for a family-level inference. supported_by: - *id003 - term: id: GO:0005923 label: bicellular tight junction evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0265 review: summary: Bicellular tight junction (UniProt subcellular location). action: ACCEPT reason: Human MAGI3 localizes to epithelial tight junctions. There is no zebrafish test of this, but the domain architecture is conserved. It is a reasonable transfer, more specific than the IBA cell-cell junction. supported_by: - *id003 - term: id: GO:0007165 label: signal transduction evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - MGI:MGI:1354953 - PANTHER:PTN000817101 - RGD:621855 review: summary: Signal transduction (IBA, PANTHER PTN000817101). action: ACCEPT reason: 'Very general, but correct. MAGI3 scaffolds signalling complexes: PTEN and AKT in mammals. In zebrafish this copy (Magi3a) binds the Gpr124 ETTV motif in yeast two-hybrid, and its crispants phenocopy loss of that motif in Wnt7-dependent brain angiogenesis. A more specific process term would need evidence beyond F0 crispants.' supported_by: - reference_id: PMID:10748157 supporting_text: cooperate to modulate the kinase activity of AKT/PKB - *id004 - &id005 reference_id: PMID:35649360 supporting_text: Targeting magi3a, magi3b, or dlg4b, but not dlg4a, partially impaired brain vascularization (Figures 4H and 4I), in agreement with the expression data. - term: id: GO:0016301 label: kinase activity evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: Kinase activity from UniRule UR000400325. action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in wu:fi36a10 (as in magi3b, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - *id001 - *id002 - term: id: GO:0016740 label: transferase activity evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables supporting_entities: - UniRule:UR000400325 review: summary: Transferase activity from UniRule UR000400325 (parent of kinase activity). action: REMOVE reason: 'UniRule UR000400325 transfers the guanylate-kinase chemistry of the GK fold to all MAGUKs. MAGI-family GK domains are degenerate: they lack the LID and CORE subdomains, and in wu:fi36a10 (as in magi3b, gar and human MAGI3) the segment that UniProt labels as the ATP-binding site has no Walker A/P-loop motif. The GK domain of MAGIs is a phosphopeptide-binding module, not a kinase. No kinase or nucleotide-binding activity has been shown for any MAGI protein. This is a family-level IEA overextension to a pseudoenzyme domain.' supported_by: - *id001 - *id002 references: - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000104 title: Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features findings: [] - id: PMID:35649360 title: An integrated model for Gpr124 function in Wnt7a/b signaling among vertebrates. findings: - statement: Magi3a (wu:fi36a10) binds the Gpr124 C-terminal ETTV motif in yeast two-hybrid; its crispants impair brain vascularization. supporting_text: the PDZ proteins membrane-associated guanylate kinase, WW, and PDZ domain containing 3 (Magi3a) and discs large MAGUK scaffold protein 4 (Dlg4a) bound to Gpr124 via its C-terminal ETTV motif. reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text. The paper's magi3a/magi3b names follow NCBI; its probe primers map to wu:fi36a10 and magi3b respectively (see RESULTS.md). - id: PMID:37163606 title: Phosphorylation-dependent recognition of diverse protein targets by the cryptic GK domain of MAGI MAGUKs. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Structural work on MAGI2 GK; states that MAGI GKs lack the LID and CORE subdomains. Family-level background. - id: PMID:10748157 title: Interaction of the tumor suppressor PTEN/MMAC with a PDZ domain of MAGI3, a novel membrane-associated guanylate kinase. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Abstract only; human MAGI3 at tight junctions, binds PTEN. - id: file:DANRE/wu_fi36a10/wu_fi36a10-bioinformatics/RESULTS.md title: magi3b / wu:fi36a10 protein, expression, synteny and primer analyses reference_review: relevance: HIGH correctness: VERIFIED review_notes: Manual audit verified the sidecar maps PMID:35649360 primers to wu:fi36a10 and LOC564220, confirms both MAGI3 copies retain the PDZ/WW/GK-like domain set, and argues against kinase, ATP-binding and nucleotide binding annotations. findings: - statement: Primers of PMID:35649360 identify its magi3a as ZFIN wu:fi36a10. supporting_text: The magi3a primers of PMID:35649360 match wu:fi36a10 with 0 mismatches core_functions: - description: Cytoplasmic PDZ/WW scaffold at cell-cell junctions. It binds the C-terminal ETTV PDZ-binding motif of the Wnt7 co-receptor Gpr124 and, with magi3b, is needed for Gpr124-dependent brain angiogenesis (crispant epistasis). No catalytic activity. directly_involved_in: - id: GO:0007165 label: signal transduction locations: - id: GO:0005911 label: cell-cell junction - id: GO:0005886 label: plasma membrane supported_by: - *id004 - reference_id: PMID:35649360 supporting_text: This observation is compatible with a role for Dlg4 and Magi3 as Gpr124 ETTV adaptor proteins, acting in parallel to Dvl. - *id005 - reference_id: PMID:35649360 supporting_text: dlg4b, magi3a, and magi3b transcripts could readily be amplified, while dlg4a transcripts were not (Figure S2B). suggested_questions: - question: Do stable wu:fi36a10 mutants (ideally RNA-less, to avoid transcriptional adaptation of magi3b) reproduce the brain vascularization defect seen in F0 crispants? - question: Is the broader expression of wu:fi36a10 the ancestral MAGI3 pattern, with magi3b having lost domains? Gar magi3 RNA-Seq calls resemble those of wu:fi36a10. - question: Should the ZFIN symbol wu:fi36a10 be updated to magi3a, the symbol NCBI and RefSeq already use? suggested_experiments: - hypothesis: magi3b and wu:fi36a10 are both required, non-redundantly and dose-dependently, for Gpr124-dependent brain angiogenesis. description: Generate stable single, double and compound-heterozygous mutants and count hindbrain central arteries; test cross-rescue by endothelial (kdrl promoter) expression of each coding sequence. experiment_type: genetic interaction and cross-rescue - hypothesis: The restricted expression of magi3b relative to wu:fi36a10 reflects expression partition, not reduced function. description: Single-cell or FACS-sorted RNA-seq and in situ hybridization for both paralogs in endothelium, retina and kidney, compared with gar magi3. experiment_type: expression profiling