LRCH4 is a 683-amino-acid membrane protein with nine N-terminal leucine-rich repeats, a C-terminal calponin-homology domain, and a predicted transmembrane helix near its C terminus. Depletion of endogenous LRCH4 in human HEK293 cells engineered to express TLR2 or TLR4/MD-2/CD14 attenuates ligand-induced IL-8 production while sparing the response to TNF-alpha. Mouse macrophage and in vivo studies further connect Lrch4 to signaling by several Toll-like receptors, LPS docking in membrane rafts, raft abundance, and CD14 surface display. Tagged human LRCH4 associates with DOCK6, DOCK7, and DOCK8 in HEK293T cells; in canine MDCK cells, expressed LRCH4 redistributes DOCK8 toward an endoplasmic-reticulum pattern. Human platelets contain LRCH4 RNA and protein, and LRCH4 knockdown alters YAP and TGF-beta/Smad outputs in a colorectal-cancer cell line. The molecular link between LRCH4, Toll-like-receptor signaling, membrane organization, and C-Dock proteins remains unresolved in native human immune cells.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0016605 PML body | IBA GO_REF:0000033 | REMOVE | Summary: Phylogenetic inference assigns LRCH4 activity in a PML body, but the supporting gene-product provenance traces back to an LRCH4 IDA annotation whose cited paper localizes SAP25 rather than LRCH4. Reason: This IBA has no independent evidence for LRCH4 localization. PMID:16449650 characterizes human SAP25 (UniProtKB:Q8TEE9), not LRCH4/O75427, and the target protein correctly appears among the descendant evidence used for the family-node inference. The annotation is removed because that seeding IDA is miscited, not because the target occurs in its own WITH/FROM. Propagation Review Root cause: SOURCE BAD Failure modes: SOURCE MISCITATION Sources checked: PANTHER:PTN002699689 · LRCH4-family PANTHER node SOURCE WEAK OR INFERRED The family-node inference supplies no independent experimental localization. UniProtKB:O75427 · human LRCH4 recipient SOURCE BAD The expected self-source identifies the target IDA used to seed the family node, but that IDA cites the SAP25 paper and is therefore a bad source. Supporting Evidence: PMID:16449650 A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated methods transfer plasma-membrane localization from the mouse Lrch4 ortholog and a UniProt subcellular-location mapping. Reason: LRCH4 is a conserved membrane protein with a predicted C-terminal transmembrane segment, and the single identifiable mouse ortholog supports plasma-membrane localization. The exact topology and distribution of endogenous human LRCH4 remain untested in the current evidence set. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q921G6 · mouse Lrch4 SUPPORTS TRANSFER ensembl:ENSMUSP00000031734 · mouse Lrch4 protein SUPPORTS TRANSFER UniProtKB-SubCell:SL-0039 · cell membrane controlled vocabulary SUPPORTS TRANSFER |
| GO:0005515 protein binding | IPI PMID:16189514 Towards a proteome-scale map of the human protein-protein in... | MARK AS OVER ANNOTATED | Summary: A proteome-scale binary-interaction screen reported an LRCH4-MDFI contact. Reason: The screen-level interaction preserves useful partner provenance but does not establish a specific LRCH4 molecular activity or physiological role; generic protein binding is therefore uninformative. |
| GO:0005515 protein binding | IPI PMID:19060904 An empirical framework for binary interactome mapping. | MARK AS OVER ANNOTATED | Summary: An empirical binary-interactome study also reported an LRCH4-MDFI contact. Reason: This repeated high-throughput edge is compatible with physical association, but it does not define a mechanistic activity for LRCH4 and cannot make the generic protein-binding term informative. |
| GO:0005515 protein binding | IPI PMID:24255178 Protein interaction network of the mammalian Hippo pathway r... | MARK AS OVER ANNOTATED | Summary: A mammalian Hippo-pathway interaction survey recovered LRCH4 with DOCK7 and SUGT1. Reason: The two screen-derived contacts do not establish that LRCH4 is a Hippo pathway component, a DOCK regulator, or a stable complex subunit. No LRCH1, LRCH2, or LRCH3 mechanism should be transferred to LRCH4, and generic protein binding remains uninformative. |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: A proteome-scale binary map reported LRCH4 contacts with KRTAP10-7, KRTAP10-8, KRT40, and NOTCH2NLA. Reason: These heterogeneous high-throughput partners do not converge on a demonstrated LRCH4 activity or cellular mechanism. Retaining the exact partner provenance is useful, but GO:0005515 adds no functional specificity. |
| GO:0005515 protein binding | IPI PMID:32203420 Systems analysis of RhoGEF and RhoGAP regulatory proteins re... | MARK AS OVER ANNOTATED | Summary: A family-wide Rho-regulator study validated LRCH4 association with DOCK8 isoform 3, DOCK6, and DOCK7. Reason: Targeted anti-tag immunoprecipitation and domain mapping make the clustered C-Dock associations informative, but the experiments do not demonstrate that LRCH4 regulates a Rho GTPase, acts as an adaptor, or shares the distinct mechanisms established for other LRCH paralogs. Generic protein binding is therefore not an adequate molecular-function annotation. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: A reference binary-interactome map reported LRCH4 contacts with ELOVL7, CYB5R3, RNASEK, RETREG3, KASH5, and HSD17B11. Reason: The diverse screen-level edges do not establish a coherent biochemical function, endogenous complex, or shared compartment for LRCH4. They should not be generalized into an uninformative protein-binding activity. |
| GO:0001765 membrane raft assembly | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ensembl Compara transfers membrane-raft assembly from the mouse Lrch4 ortholog to human LRCH4. Reason: Mouse Lrch4 loss perturbs raft abundance and LPS docking, and LRCH4 is the identifiable human ortholog with the same domain architecture. The biology therefore supports retaining the transferred process, but its physiological importance has not been tested in human cells and it is not part of the narrower TLR-signaling core. The mechanical transfer source remains miscited, while PMID:30523158 independently supports the mouse raft phenotype. Propagation Review Root cause: SOURCE BAD Failure modes: SOURCE MISCITATION Sources checked: UniProtKB:Q921G6 · mouse Lrch4 SOURCE BAD Live QuickGO returns Q921G6, GO:0001765, IDA, PMID:16449650; PMID:30523158 independently supports the mouse raft biology, but the propagated source row itself is miscited. ensembl:ENSMUSP00000031734 · mouse Lrch4 protein SOURCE WEAK OR INFERRED This is the orthology carrier rather than independent experimental evidence. Supporting Evidence: PMID:30523158 We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. |
| GO:0034123 positive regulation of toll-like receptor signaling pathway | IEA GO_REF:0000107 | ACCEPT | Summary: Ensembl Compara transfers positive regulation of Toll-like-receptor signaling from mouse Lrch4 to human LRCH4; direct human-cell depletion evidence independently supports the transferred process. Reason: Endogenous human LRCH4 depletion in HEK293 cells engineered with TLR2 or TLR4/MD-2/CD14 reduced ligand-induced IL-8 without reducing the TNF-alpha response. Together with the broader mouse macrophage and in vivo evidence, this supports a positive contribution to Toll-like-receptor signaling while leaving its native human cell-type and ligand breadth unresolved. Propagation Review Root cause: SOURCE BAD Failure modes: SOURCE MISCITATION Sources checked: UniProtKB:Q921G6 · mouse Lrch4 SOURCE BAD Live QuickGO returns Q921G6, GO:0034123, IDA, PMID:16449650; PMID:30523158 independently supports the mouse TLR biology, but the propagated source row itself is miscited. ensembl:ENSMUSP00000031734 · mouse Lrch4 protein SOURCE WEAK OR INFERRED This source records one-to-one orthology but does not repair the miscited mouse source annotation. Supporting Evidence: PMID:30523158 HEK293 cells stably expressing either TLR4/MD-2/CD14 or TLR2 were transfected with Lrch4 siRNA or Scr siRNA and then exposed to LPS or Pam3CSK4, respectively. PMID:30523158 As shown in Fig. 2 G , Lrch4 siRNA attenuated IL-8 induction by both ligands in HEK293 cells, providing further support that the shRNA results in macrophages are unlikely to reflect off-target effects. |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | ACCEPT | Summary: Curator-reviewed sequence similarity transfers plasma-membrane localization from mouse Lrch4. Reason: The source is the direct mouse ortholog, and human LRCH4 retains the C-terminal transmembrane segment expected for membrane anchoring. Transfer of the broad plasma-membrane location is reasonable despite the absence of an endogenous human localization assay. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q921G6 · mouse Lrch4 SUPPORTS TRANSFER |
| GO:0001765 membrane raft assembly | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Curator judgment transfers the mouse Lrch4 membrane-raft phenotype to human LRCH4. Reason: The orthologous mouse protein is required for normal raft abundance and LPS docking, and no paralog-specific divergence is evident. That makes the transferred process biologically credible but still secondary to the directly supported TLR-signaling core in human cells. The live mouse GOA source is miscited, while PMID:30523158 independently establishes the mouse phenotype. Propagation Review Root cause: SOURCE BAD Failure modes: SOURCE MISCITATION Sources checked: UniProtKB:Q921G6 · mouse Lrch4 SOURCE BAD Live QuickGO returns Q921G6, GO:0001765, IDA, PMID:16449650; PMID:30523158 independently supports the mouse raft biology, but the propagated source row itself is miscited. Supporting Evidence: PMID:30523158 We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. |
| GO:0034123 positive regulation of toll-like receptor signaling pathway | ISS GO_REF:0000024 | ACCEPT | Summary: Curator judgment transfers the TLR-accessory role of mouse Lrch4 to human LRCH4, consistent with direct depletion experiments in engineered human cells. Reason: Endogenous human LRCH4 depletion in TLR2- or TLR4/MD-2/CD14-expressing HEK293 cells attenuated ligand-induced IL-8, while leaving TNF-alpha-induced IL-8 intact. The human result and broader mouse evidence support this process annotation, with native human immune-cell scope remaining an open question. Propagation Review Root cause: SOURCE BAD Failure modes: SOURCE MISCITATION Sources checked: UniProtKB:Q921G6 · mouse Lrch4 SOURCE BAD Live QuickGO returns Q921G6, GO:0034123, IDA, PMID:16449650; PMID:30523158 independently supports the mouse TLR biology, but the propagated source row itself is miscited. Supporting Evidence: PMID:30523158 HEK293 cells stably expressing either TLR4/MD-2/CD14 or TLR2 were transfected with Lrch4 siRNA or Scr siRNA and then exposed to LPS or Pam3CSK4, respectively. PMID:30523158 Confirming some selectivity for Lrch4 function, Lrch4 silencing did not affect induction of IL-8 by HEK293 cells in response to stimulation with TNFα ( Fig. 2 H ). |
| GO:0016605 PML body | IDA PMID:16449650 Identification and characterization of SAP25, a novel compon... | REMOVE | Summary: The cited study localizes SAP25, not LRCH4, to PML nuclear bodies. Reason: Full-text inspection identifies SAP25/Q8TEE9 as the assayed product and contains no LRCH4, O75427, LRN, or LRRN4 experiment. Because the actual PML-body result belongs to another gene product, this is a verified source miscitation rather than a reason to infer nuclear localization for LRCH4. Supporting Evidence: PMID:16449650 A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. |
| GO:0007399 nervous system development | TAS PMID:9799793 Large-scale sequencing of two regions in human chromosome 7q... | REMOVE | Summary: The chromosome-sequencing paper describes weak sequence similarity to a neuronal leucine-rich protein but does not test nervous-system development. Reason: A descriptive neuronal-protein resemblance or historical name is not evidence that LRCH4 participates in nervous-system development. The cited genomic annotation study provides no developmental phenotype, pathway, or functional assay supporting GO:0007399. Supporting Evidence: PMID:9799793 Two genes showed weak similarity to an insulin-like receptor and a neuronal protein with a leucine-rich amino-terminal domain. |
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Download this section (compressed HTML)Q: Does endogenous LRCH4 support TLR2, TLR4, or additional TLR pathways in primary human innate-immune cells, and is its effect restricted to selected signaling outputs?
Q: Does LRCH4 contact LPS, CD14, MD-2, or a TLR directly, or does it act indirectly by changing membrane-lipid composition or receptor trafficking?
Q: Where is endogenous human LRCH4 located, what is its membrane topology, and does TLR stimulation alter its distribution?
Q: Do DOCK6, DOCK7, or DOCK8 mediate an LRCH4-dependent Rho-GTPase response, and is that module required for the TLR phenotype?
Q: Are any human LRCH4 transcript products physiologically expressed as functionally distinct membrane-bound or soluble forms?
Experiment: Create independent LRCH4 knockouts in primary-cell-derived macrophages or validated human monocyte models, rescue with CRISPR-resistant LRCH4, and measure receptor-proximal phosphorylation, NF-kappa-B and IRF activation, and cytokine output after matched TLR2, TLR4, and control cytokine stimulation.
Hypothesis: LRCH4 selectively amplifies receptor-proximal TLR2 and TLR4 signaling in primary human innate-immune cells.
Type: Genetic perturbation, rescue, and signaling time course
Experiment: Quantify CD14 and TLR4 surface delivery, receptor nanoclustering, detergent-independent membrane-domain partitioning, and lipid composition after LRCH4 loss and rescue; in parallel, test purified LRCH4 extracellular regions for saturable LPS binding with stringent lipid and co-receptor controls.
Hypothesis: LRCH4 affects LPS responsiveness by controlling CD14/TLR4 membrane organization rather than by functioning as a direct LPS receptor.
Type: Surface trafficking, super-resolution imaging, lipidomics, and binding assays
Experiment: Introduce small endogenous tags on both sides of the predicted transmembrane segment, then combine selective permeabilization, extracellular labeling, protease protection, glycosylation-site mapping, and pulse-chase imaging in human immune cells.
Hypothesis: The C-terminal hydrophobic segment anchors LRCH4 with a reproducible topology and controls stimulus-dependent movement between the endoplasmic reticulum and cell surface.
Type: Endogenous tagging and membrane-topology analysis
Experiment: Define interaction-deficient LRCH4 mutants by domain mapping, confirm endogenous binding, and compare wild-type and separation-of-function rescue using DOCK6/7/8 perturbations, RAC1/CDC42/RHOA biosensors, and TLR-induced cytokine readouts.
Hypothesis: A specific LRCH4-C-Dock interaction is required for spatial Rho-GTPase activation and contributes to LRCH4-dependent TLR signaling.
Type: Interaction mapping, epistasis, and live-cell biosensors
Experiment: Perform long-read transcript sequencing, isoform-resolved proteomics, and C-terminal peptide mapping across resting and TLR-stimulated primary human immune-cell types before testing any reproducibly detected products in topology and rescue assays.
Hypothesis: Human immune cells express a regulated LRCH4 transcript or protein form with a distinct membrane association state.
Type: Long-read transcriptomics and isoform-resolved proteomics
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The molecular activity by which LRCH4 augments Toll-like-receptor signaling is unknown: it has not been established whether LRCH4 binds LPS directly, changes membrane-lipid organization, controls CD14 trafficking, or acts through another receptor-proximal component.
OPEN BIOLOGY MF_DARK
What is known: Endogenous LRCH4 depletion selectively reduced TLR2- and TLR4-dependent IL-8 induction in engineered human HEK293 cells, while mouse experiments linked Lrch4 depletion to reduced raft abundance, CD14 surface display, and LPS docking. The LPS pulldown did not distinguish a direct from an indirect association.
Significance: Without a defined molecular activity, the TLR role cannot be represented as a mechanistic molecular-function assertion or separated cleanly from secondary changes in membrane organization.
What would resolve it: Reconstitute LPS, CD14, MD-2, TLR4, and LRCH4 interactions with purified or compositionally defined membrane systems, combined with crosslinking and quantitative lipid analysis, to distinguish direct ligand binding from effects on receptor trafficking or membrane-raft organization.
Gap: It is unknown whether LRCH4 is required for Toll-like-receptor responses in native primary human immune cells and whether the ligand range observed in mouse systems is conserved in humans.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: Direct human evidence is limited to TLR2- or TLR4-engineered HEK293 cells, whereas the broad ligand panel, macrophage mechanism, and in vivo innate-response phenotype were established chiefly in mouse cells and mice.
Significance: Establishing physiological human-cell relevance is necessary to define the native cell types and ligand breadth of the conserved TLR-regulatory role and to decide whether raft assembly is a primary LRCH4 process or a mouse-context phenotype.
What would resolve it: Generate LRCH4 loss-of-function and rescue series in primary human monocytes, macrophages, and dendritic cells, then compare proximal signaling and cytokine responses across a controlled panel of TLR ligands.
Gap: The endogenous subcellular distribution, membrane orientation, and trafficking itinerary of human LRCH4 are unknown.
OPEN BIOLOGY CC_DARK
What is known: Human LRCH4 contains a predicted C-terminal transmembrane helix, transferred mouse annotations place it at the plasma membrane, and overexpressed human LRCH4 has been imaged at the endoplasmic reticulum. None of these observations establishes the topology or steady-state distribution of endogenous human LRCH4.
Significance: Sidedness and compartment determine whether the leucine-rich repeats could contact extracellular ligands, luminal cargo, or cytosolic signaling partners and therefore constrain the possible TLR mechanism.
What would resolve it: Endogenously tag LRCH4 in human immune cells and combine surface biotinylation, protease protection, glycosylation mapping, pulse-chase trafficking, and quantitative microscopy before and after TLR stimulation.
Gap: The physiological significance of LRCH4 binding to DOCK6, DOCK7, and DOCK8 is unknown, including which Rho GTPase output is affected and whether the interaction contributes to Toll-like-receptor or membrane-raft phenotypes.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: Tagged human LRCH4 reproducibly bound the C-Dock proteins in transfected-cell assays and recruited DOCK8 to an endoplasmic-reticulum pattern, but no endogenous stable complex, downstream GTPase, or genetic connection to the TLR phenotype has been shown.
Significance: Resolving this connection would determine whether the C-Dock interactions are a function-defining signaling module or context-dependent binding without a shared physiological output.
What would resolve it: Map the LRCH4-C-Dock interface, test endogenous interaction in immune cells, and use separation-of-function LRCH4 mutants with DOCK6/7/8 perturbation and spatial Rho-GTPase biosensors during TLR stimulation.
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