ARHGDIB (Rho GDP-dissociation inhibitor 2, RhoGDI2, also called Ly-GDI or D4-GDI) is a 201-residue cytosolic member of the Rho GDI family that is expressed mainly in hematopoietic cells, especially lymphocytes and neutrophils. It binds GDP-bound Rho family GTPases (RAC1, RAC2, RHOA, CDC42, RHOG), slows dissociation of GDP and so keeps them inactive, and through an immunoglobulin-like domain that shields their geranylgeranyl tail it extracts them from membranes and holds them as a soluble cytosolic pool. The crystal structure with GDP-Rac2 shows that the N-terminal regulatory arm contacts the switch regions and stabilizes the Mg2+ ion in the nucleotide-binding pocket. By restraining Rho/Rac activation, RhoGDI2 modulates actin cytoskeleton organization and cell migration; over-expression causes loss of stress fibres, and it limits migration of trophoblast and bladder cancer cells, where it acts as a metastasis suppressor. Ly-GDI-deficient mice have largely normal lymphoid development, consistent with partial redundancy with RhoGDI1 (ARHGDIA). RhoGDI2 is cleaved by caspase-3 during apoptosis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003924 GTPase activity | IMP PMID:24554735 RhoGDI2 is expressed in human trophoblasts and involved in t... | MODIFY | Summary: CACAO IMP annotation of GTPase activity based on RhoGDI2 over-expression and siRNA knockdown in HTR-8/SVneo trophoblast cells, which lowered or raised RAC1-GTP levels respectively. ARHGDIB is a Rho GDI with no P-loop GTPase fold; the read-out is inhibition of Rac1 activation, not GTP hydrolysis by RhoGDI2. Reason: GO:0003924 GTPase activity is defined as catalysis of GTP + H2O = GDP + H+ + phosphate, i.e. the activity of the GTPase itself. ARHGDIB belongs to the Rho GDI family (Pfam Rho_GDI, InterPro IPR000406) and has no nucleotide-binding or hydrolase domain. The cited study (abstract only cached) reports reduced RAC1-GTP on RhoGDI2 over-expression and increased RAC1-GTP on knockdown, which is what a GDP-dissociation inhibitor does to its target: it keeps Rac1 in the GDP-bound state. Nothing in the paper measures GTP hydrolysis by RhoGDI2. The essence of the annotation (RhoGDI2 acts on the Rac1 nucleotide cycle) is sound, so the correct term is the paralog-wide, experimentally established Rho GDP-dissociation inhibitor activity (GO:0005094), which this gene already carries by IDA and IBA. This row was flagged by rule FRR:0010. Proposed replacements: Rho GDP-dissociation inhibitor activity Supporting Evidence: PMID:24554735 Overexpression of RhoGDI2 in HTR-8/SVneo cells was associated with reduced RAC1-guanosine triphosphate (GTP) levels and inhibited cell migration. PMID:24554735 small interfering RNA-mediated downregulation of RhoGDI2 resulted in significantly increased RAC1-GTP levels PMID:8356058 The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. |
| GO:0005094 Rho GDP-dissociation inhibitor activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of Rho GDI activity across the Rho GDI family (PTN000104723); ARHGDIB's own experimental annotation is among the donors. Reason: Rho GDP-dissociation inhibitor activity is the defining, conserved activity of the Rho GDI family, and ARHGDIB has direct biochemical evidence for it against RhoA, CDC42 and Rac1. The presence of P52566 in WITH/FROM reflects its own experimental grounding. Core function. Supporting Evidence: file:human/ARHGDIB/ARHGDIB-uniprot.txt Belongs to the Rho GDI family. PMID:7512369 D4 has GDI activity against the CDC42Hs and rac I proteins |
| GO:0005094 Rho GDP-dissociation inhibitor activity | IDA PMID:7512369 Identification of a novel protein with GDP dissociation inhi... | ACCEPT | Summary: Adra et al. cloned D4 (ARHGDIB) and showed with recombinant human protein that it has GDI activity toward CDC42Hs and Rac1, with lower affinity than RhoGDI1. Reason: Direct in vitro demonstration of GDP-dissociation inhibitor activity on Rho-family GTPases. Core function. Supporting Evidence: PMID:7512369 Functional studies with the human D4 protein demonstrate that D4 has GDI activity against the CDC42Hs and rac I proteins, but binds to these proteins with a significantly weaker affinity than does the rho-subtype GDI. |
| GO:0005094 Rho GDP-dissociation inhibitor activity | IDA PMID:8356058 Ly-GDI, a GDP-dissociation inhibitor of the RhoA GTP-binding... | ACCEPT | Summary: Scherle et al. isolated Ly-GDI from lymphocytes and showed that it binds RhoA and inhibits GDP dissociation from RhoA. Reason: Direct biochemical evidence of Rho GDI activity toward RhoA. Core function. Supporting Evidence: PMID:8356058 The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. |
| GO:0005094 Rho GDP-dissociation inhibitor activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from the Rho GDI family signature (IPR000406). Reason: The Rho_GDI family mapping is accurate for ARHGDIB and agrees with direct experimental evidence. Supporting Evidence: file:human/ARHGDIB/ARHGDIB-uniprot.txt InterPro; IPR000406; Rho_GDI. |
| GO:0005094 Rho GDP-dissociation inhibitor activity | TAS PMID:8262133 Identification of two human Rho GDP dissociation inhibitor p... | ACCEPT | Summary: Leffers et al. identified two human Rho GDIs (IEF 8118 = RhoGDI1 and IEF 8120 = RhoGDI2) and showed that their over-expression in keratinocytes disrupts the actin cytoskeleton, mimicking inactivation of Rho. Reason: Consistent with the established GDI activity; the over-expression phenotype is the expected consequence of Rho inhibition. Supporting Evidence: PMID:8262133 the results imply that Rho GDI's play a role in modulating the activity of the Rho proteins as their overexpression mimics phenotypic changes associated with the inactivation of these proteins |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: High-throughput yeast two-hybrid interaction with FOS from a neurodegenerative disease interactome screen. Reason: Bare protein binding is uninformative under the protein-binding policy. The interaction comes from a large-scale Y2H screen with no functional follow-up for ARHGDIB; removal does not assert that the interaction is false. Supporting Evidence: PMID:32814053 generated by systematic yeast two-hybrid interaction screening of βΌ500 ND-related proteins and integration of literature interactions |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: High-throughput yeast two-hybrid interaction with UQCRC1. Reason: Bare protein binding is uninformative; interaction with a mitochondrial inner-membrane complex III subunit from a Y2H screen has no established functional relevance for cytosolic RhoGDI2. Removal does not assert the interaction is false. Supporting Evidence: PMID:32814053 generated by systematic yeast two-hybrid interaction screening of βΌ500 ND-related proteins and integration of literature interactions |
| GO:0005515 protein binding | IPI PMID:32814053 Interactome Mapping Provides a Network of Neurodegenerative ... | REMOVE | Summary: High-throughput yeast two-hybrid interaction with GRB2. Reason: Bare protein binding is uninformative and the screen gives no functional context. Removal does not assert the interaction is false. Supporting Evidence: PMID:32814053 generated by systematic yeast two-hybrid interaction screening of βΌ500 ND-related proteins and integration of literature interactions |
| GO:0005515 protein binding | IPI PMID:7512369 Identification of a novel protein with GDP dissociation inhi... | MODIFY | Summary: Binding of D4 (ARHGDIB) to CDC42 in the GDI assays of Adra et al. Reason: The interaction with CDC42 is the substrate binding of a Rho GDI and is better captured by small GTPase binding (GO:0031267), which ARHGDIB already carries from the same paper, alongside Rho GDP-dissociation inhibitor activity. Proposed replacements: small GTPase binding Supporting Evidence: PMID:7512369 D4 has GDI activity against the CDC42Hs and rac I proteins, but binds to these proteins with a significantly weaker affinity than does the rho-subtype GDI. |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping placing Rho GDI family members in the cytoplasm. Reason: Correct but less specific than the evidence supports. RhoGDI2 is a soluble cytosolic protein (UniProt, IDA and IBA cytosol rows in this review); the specific descendant cytosol (GO:0005829) is preferred. Proposed replacements: cytosol Supporting Evidence: file:human/ARHGDIB/ARHGDIB-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cytosol {ECO:0000269|PubMed:10802295}. |
| GO:0005737 cytoplasm | TAS PMID:16130169 Proteomics of human umbilical vein endothelial cells applied... | MODIFY | Summary: TAS from a HUVEC two-dimensional gel proteome; the paper inventories proteins and does not study ARHGDIB localization. Reason: The cytoplasmic location is correct but the specific supported location is cytosol (GO:0005829). The cited proteome study itself does not address localization, so support comes from the cytosol evidence elsewhere in this review. Proposed replacements: cytosol Supporting Evidence: PMID:9113980 A similar construction with GFP tagged to the amino terminus of GDI/D4 or RhoGDI showed only a diffuse distribution in the cytoplasm without any of the perinuclear aggregates seen with RhoGDIΞ³ (figure not shown). |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference that Rho GDI family members act in the cytosol. Reason: RhoGDIs sequester prenylated Rho GTPases as soluble cytosolic complexes; the cytosol is where ARHGDIB holds its targets. Core location. Supporting Evidence: PMID:10655614 release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments |
| GO:0005829 cytosol | IDA GO_REF:0000052 | ACCEPT | Summary: Human Protein Atlas immunofluorescence localization to the cytosol. Reason: Consistent with all other evidence for a soluble cytosolic GDI. Supporting Evidence: file:human/ARHGDIB/ARHGDIB-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cytosol {ECO:0000269|PubMed:10802295}. |
| GO:0005829 cytosol | IDA PMID:10802295 Cloning and characterization of bovine low molecular weight ... | ACCEPT | Summary: D4-GDI detected as the predominant GDI in (bovine and human) neutrophil cytosol by comparative Western blotting. Reason: Fractionation evidence for cytosolic location; matches the UniProt curated location. Supporting Evidence: PMID:10802295 D4-GDI was the predominant GDI species in bovine neutrophil cytosol |
| GO:0005829 cytosol | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping from the UniProt subcellular location "Cytoplasm, cytosol". Reason: Based on a curated, experimentally supported UniProt location. Supporting Evidence: file:human/ARHGDIB/ARHGDIB-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm, cytosol {ECO:0000269|PubMed:10802295}. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9012835 | ACCEPT | Summary: Reactome reaction in which ARHGDIB binds RHOA in the cytosol and blocks its activation. Reason: Consistent with the cytosolic GDI-GTPase complex. Supporting Evidence: Reactome:R-HSA-9012835 ARHGDIB (Watanabe et al. 2012) |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9013147 | ACCEPT | Summary: Reactome reaction in which ARHGDIB binds RAC1 and blocks its activation. Reason: Consistent with the cytosolic GDI-GTPase complex. Supporting Evidence: Reactome:R-HSA-9013147 ARHGDIB (Adra et al. 1993, Zhang et al. 2009) |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9013158 | ACCEPT | Summary: Reactome reaction in which ARHGDIB binds CDC42 and blocks its activation. Reason: Consistent with the cytosolic GDI-GTPase complex. Supporting Evidence: Reactome:R-HSA-9013158 ARHGDIB (Adra et al. 1993) |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9014456 | ACCEPT | Summary: Reactome reaction in which ARHGDIB binds RHOG and blocks its activation. Reason: Consistent with the cytosolic GDI-GTPase complex. Supporting Evidence: Reactome:R-HSA-9014456 ARHGDIB (Vignal et al. 2001) |
| GO:0005829 cytosol | TAS Reactome:R-HSA-9018754 | ACCEPT | Summary: Reactome reaction in which Rho GDIs (including GDIbeta) bind RHOH and sequester it in the cytosol. Reason: Consistent with the cytosolic location of GDI-GTPase complexes. Supporting Evidence: Reactome:R-HSA-9018754 GDIalpha, GDIbeta and GDIgamma bind to RHOH and presumably affect its translocation to the site of activity by sequestering it in the cytosol |
| GO:0005856 cytoskeleton | TAS PMID:16130169 Proteomics of human umbilical vein endothelial cells applied... | REMOVE | Summary: TAS from a HUVEC 2-DE proteome that groups identified proteins into functional categories such as cell motility. The paper does not localize ARHGDIB to the cytoskeleton. Reason: RhoGDI2 regulates the actin cytoskeleton indirectly through Rho GTPases, but it is a soluble cytosolic protein and there is no evidence that it is a component of or located at the cytoskeleton. GFP-tagged GDI/D4 shows only a diffuse cytoplasmic distribution. The cited proteome study is an inventory and does not address localization; the annotation appears to conflate functional category with location. Supporting Evidence: PMID:9113980 A similar construction with GFP tagged to the amino terminus of GDI/D4 or RhoGDI showed only a diffuse distribution in the cytoplasm without any of the perinuclear aggregates seen with RhoGDIΞ³ (figure not shown). PMID:16130169 illustrates various cellular functions more related to cell motility and angiogenesis |
| GO:0007266 Rho protein signal transduction | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference that Rho GDIs take part in Rho GTPase signalling. Reason: RhoGDIs are integral to the Rho GTPase cycle and are conventionally annotated to Rho signalling. Their role is regulatory (restraining activation and controlling membrane cycling), which is captured more precisely by negative regulation of Rho protein signal transduction and by the GDI activity; this broader participation term is kept as non-core. Supporting Evidence: PMID:10655614 representing the cytosolic, resting pool of Rho species to be activated by extracellular signals |
| GO:0007266 Rho protein signal transduction | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the Rho GDI family signature. Reason: Same claim as the IBA row; kept as non-core for the same reasons. Supporting Evidence: file:human/ARHGDIB/ARHGDIB-uniprot.txt InterPro; IPR000406; Rho_GDI. |
| GO:0016020 membrane | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic inference that Rho GDIs act at membranes. Reason: RhoGDIs act transiently at membranes, where they extract prenylated Rho GTPases and from which they deliver them; the steady-state location of ARHGDIB is the cytosol. The membrane term is as precise as the family-level evidence allows (which membrane varies), so it is kept but as non-core. Supporting Evidence: PMID:10655614 release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments |
| GO:0031267 small GTPase binding | IDA PMID:7512369 Identification of a novel protein with GDP dissociation inhi... | ACCEPT | Summary: D4 (ARHGDIB) binds CDC42Hs and Rac1, with weaker affinity than RhoGDI1. Reason: Direct binding to Rho-family small GTPases is the basis of ARHGDIB's GDI function and is also shown structurally for GDP-Rac2. Retained alongside the GDI activity. Supporting Evidence: PMID:7512369 D4 has GDI activity against the CDC42Hs and rac I proteins, but binds to these proteins with a significantly weaker affinity than does the rho-subtype GDI. PMID:10655614 We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2 |
| GO:0031410 cytoplasmic vesicle | TAS PMID:9113980 RhoGDIgamma: a GDP-dissociation inhibitor for Rho proteins w... | REMOVE | Summary: PINC TAS from the paper describing RhoGDIΞ³ (ARHGDIG). The vesicular, perinuclear localization reported there is for RhoGDIΞ³; the same paper states that GFP-tagged GDI/D4 (ARHGDIB) is only diffusely cytoplasmic. Reason: The cited paper explicitly contrasts the vesicle-associated distribution of RhoGDIΞ³, attributed to its unique hydrophobic N-terminus absent from the other two GDIs, with the diffuse cytoplasmic distribution of GDI/D4 and RhoGDI. The annotation is attached to the wrong paralog; there is no evidence that ARHGDIB localizes to cytoplasmic vesicles. Supporting Evidence: PMID:9113980 A similar construction with GFP tagged to the amino terminus of GDI/D4 or RhoGDI showed only a diffuse distribution in the cytoplasm without any of the perinuclear aggregates seen with RhoGDIΞ³ (figure not shown). PMID:9113980 Immunohistochemical studies showed a diffuse punctate distribution of the protein in the cytoplasm with concentration around the nucleus in cytoplasmic vesicles. |
| GO:0035023 regulation of Rho protein signal transduction | IDA PMID:7512369 Identification of a novel protein with GDP dissociation inhi... | MODIFY | Summary: D4 inhibits GDP dissociation from CDC42 and Rac1, keeping them inactive, i.e. it negatively regulates Rho-family signalling. Reason: Correct but less specific than the evidence supports. GDP-dissociation inhibition holds Rho GTPases in the inactive GDP-bound state, so the direction of regulation is negative; the descendant GO:0035024 negative regulation of Rho protein signal transduction is preferred. Proposed replacements: negative regulation of Rho protein signal transduction Supporting Evidence: PMID:7512369 Functional studies with the human D4 protein demonstrate that D4 has GDI activity against the CDC42Hs and rac I proteins |
| GO:0035023 regulation of Rho protein signal transduction | IMP PMID:8262133 Identification of two human Rho GDP dissociation inhibitor p... | MODIFY | Summary: Over-expression of RhoGDI2 (IEF 8120) in keratinocytes causes cell rounding and loss of stress fibres, focal contacts and adhesion belts, mimicking Rho inactivation. Reason: The phenotype is that of Rho inactivation, so the supported regulation is negative; refine to GO:0035024 negative regulation of Rho protein signal transduction. Proposed replacements: negative regulation of Rho protein signal transduction Supporting Evidence: PMID:8262133 overexpression of both GDI proteins leads to rounding up of the cells and loss of stress fibers and focal contact sites |
| GO:0070062 extracellular exosome | HDA PMID:19056867 Large-scale proteomics and phosphoproteomics of urinary exos... | KEEP AS NON CORE | Summary: Detected in the urinary exosome proteome by LC-MS/MS. Reason: High-throughput detection of an abundant cytosolic protein in exosomes; plausible passive cargo with no known function there. Supporting Evidence: PMID:19056867 we used LC-MS/MS to profile the proteome of human urinary exosomes |
| GO:0070062 extracellular exosome | HDA PMID:20458337 MHC class II-associated proteins in B-cell exosomes and pote... | KEEP AS NON CORE | Summary: Detected in the proteome of B-cell-derived exosomes. Reason: ARHGDIB is abundant in lymphocytes, so its presence in B-cell exosomes is expected as cytosolic cargo; no exosome-specific function is known. Supporting Evidence: PMID:20458337 we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins |
| GO:0071461 cellular response to redox state | IEA GO_REF:0000107 | MARK AS OVER ANNOTATED | Summary: Ensembl Compara transfer from a rat ortholog annotation. Reason: No human evidence links ARHGDIB to a response to redox state, and nothing in the known mechanism of a Rho GDI implies one. The term is a peripheral finding projected from a single rodent annotation and does not describe ARHGDIB's function; the source experiment could not be inspected. |
| GO:1901164 negative regulation of trophoblast cell migration | IDA PMID:24554735 RhoGDI2 is expressed in human trophoblasts and involved in t... | KEEP AS NON CORE | Summary: RhoGDI2 over-expression in HTR-8/SVneo trophoblast cells inhibited migration and lowered RAC1-GTP. Reason: Supported by the cited study and mechanistically consistent with RhoGDI2 restraining Rac1 activation, but this is one cell-type-specific consequence of the core GDI function (single study, abstract only cached). Supporting Evidence: PMID:24554735 In conclusion, RhoGDI2 inhibits trophoblast cell migration, and this function may involve suppression of RAC1 activation. |
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Download this section (compressed HTML)Q: Which Rho GTPases are the physiological targets of RhoGDI2 in lymphocytes and neutrophils, given its lower in vitro affinity than RhoGDI1 and its abundance alongside RAC2?
Q: Does caspase-3 cleavage of RhoGDI2 during apoptosis alter its GDI activity or target selectivity, and does the cleaved fragment have a distinct function?
Experiment: Quantify RhoGDI2 binding affinity and GDP-dissociation inhibition for RAC1, RAC2, RHOA, CDC42 and RHOG in parallel with RhoGDI1, using prenylated GTPases, and compare GDI-bound pools in ARHGDIB-knockout versus wild-type primary leukocytes.
Experiment: Express caspase-resistant and caspase-cleaved RhoGDI2 in lymphoid cells and measure Rho GTPase activation, membrane extraction and apoptosis.
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