B0L3A2 is an 85-amino-acid single-pass membrane protein, named DEspR (dual endothelin-1/VEGF signal peptide receptor), encoded on the locus that HGNC classifies as the long non-coding RNA FBXW7-AS1, antisense to exon 5 of FBXW7 on chromosome 4q31.3. In the reference genome the reading frame is interrupted at codon 14 by a TGA; expression of the full-length protein is reported to depend on ADAR1-mediated adenosine-to-inosine editing of that codon to tryptophan. The protein has a short extracellular N terminus, one transmembrane helix and a cytoplasmic C-terminal tail, is N-glycosylated, and is detected at the plasma membrane of tumour cells, cancer stem-like cells, endothelial cells and a subset of circulating neutrophils, where it is internalised after antibody binding. It is reported to bind endothelin-1 and the signal peptide of VEGF-A - but not mature VEGF-A - and to trigger intracellular phosphorylation events overlapping only partly with VEGFR2 signalling; the rat orthologue was originally described as also binding angiotensin II, an activity not found in mouse or human. Mice lacking the gene die in mid-gestation with impaired angiogenesis and disordered neuroepithelial development. Protein-level detection rests on monoclonal antibodies rather than mass spectrometry, and the receptor pharmacology has not been taken up independently of the originating laboratory, so both the existence and the ligand specificity of the protein remain contested.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004962 endothelin receptor activity | IDA PMID:24465725 DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC... | MODIFY | Summary: Anti-DEspR monoclonal antibody binding to DEspR-expressing Cos1 transfectants and to Panc1 cancer stem-like cells is displaced by endothelin-1, and ET-1 addition elicits a distinct intracellular phosphorylation signature, supporting an ET-1-binding, signal-transmitting surface protein. Reason: The experiments support ET-1 binding and ligand-initiated transmembrane signalling, and that claim is kept. The term itself, however, is defined as "Combining with endothelin and transmitting the signal across the membrane by activating an associated G-protein; promotes the exchange of GDP for GTP on the alpha subunit of a heterotrimeric G-protein complex". No G-protein coupling has been demonstrated for this 85-residue single-pass protein: the original rat study showed Ca2+ mobilisation (PMID:9508787) and the human study phosphoproteomics (PMID:24465725), neither a GDP/GTP exchange assay. The endothelin field also does not list DEspR among endothelin receptors - the IUPHAR review states that only ETA and ETB are recognised (PMID:26956245), and two further reviews note that the pharmacology of this receptor has not been developed and that the data come from a single laboratory (PMID:19907001, PMID:21081213). Rather than remove an experimental annotation whose full text was read by the curator, the claim is restated with terms that do not assert G-protein coupling. Proposed replacements: transmembrane signaling receptor activity peptide hormone binding Supporting Evidence: PMID:24465725 7c5b2-immunostaining of DEspR+Cos1 cell transfectants was effectively blocked by the antigenic peptide and by ET1 PMID:24465725 Collectively, these findings support the functionality of VEGFsp- and ET1-binding to DEspR. PMID:26956245 Current evidence only supports the existence of two subtypes, ETA and ETB, according to NC-IUPHAR nomenclature PMID:21081213 All these data come from one group of scientists, and the physiology and pharmacology of DEAR has not been studied in depth. |
| GO:0005515 protein binding | IPI PMID:17446437 Association of ATP1A1 and dear single-nucleotide polymorphis... | REMOVE | Summary: Bare protein binding, with endothelin-1 (P05305) and VEGF-A (P15692) given as partners, cited to a single-nucleotide-polymorphism haplotype association study of essential hypertension. Reason: Generic protein binding carries no functional information, and no more informative molecular function can be built from this row: the two partners it names are the two reported ligands, which are already covered by the ligand-binding and receptor rows of this entry. The cited reference is a human genetic association study of ATP1A1 and DEAR haplotypes with hypertension, with no interaction assay in its abstract. Removal does not assert that the reported interactions are false. Supporting Evidence: PMID:17446437 Here we report the association of human ATP1A1 (P<0.000005) and Dear (P<0.03) |
| GO:0005886 plasma membrane | IDA PMID:24465725 DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC... | ACCEPT | Summary: Immunofluorescence of DEspR-expressing Cos1 transfectants, HUVECs and cancer stem-like cells, and competition FACS on intact Panc1 cancer stem-like cells, place the protein at the cell surface. Reason: Surface localisation is the best-supported claim about this entry and is consistent across cell types, laboratories and assay formats (PMID:27301377, PMID:33853558, PMID:34789851). It is also what the single predicted transmembrane helix and the extracellular N-terminal epitopes used for all the antibodies predict. Supporting Evidence: PMID:24465725 7c5b2-immunostaining of DEspR+Cos1 cell transfectants was effectively blocked by the antigenic peptide and by ET1 |
| GO:0005886 plasma membrane | IDA PMID:27301377 Confirmation of translatability and functionality certifies ... | ACCEPT | Summary: Anti-DEspR monoclonal antibody pull-down from isolated cell membranes of glioblastoma cancer stem-like cells and DEspR-positive Cos1 transfectants yields a 17.5 kDa glycosylated protein that shrinks after PNGase F treatment, together with live-cell imaging of antibody-triggered receptor internalisation. Reason: The protein was recovered from the membrane fraction and is N-glycosylated and internalisable, all consistent with a plasma-membrane protein. The paper is candid that mass spectrometry of the same pull-downs did not detect DEspR peptides, so the evidence is antibody-dependent; that limitation bears on whether the protein exists, not on where it is when detected, and the location term is retained. Supporting Evidence: PMID:27301377 PNGase-F digest demonstrates post-translational glycosylation, concordant with PMID:27301377 We note that although DEspR was detected consistently in denatured conditions via Western blot analysis of membrane protein pull-down products using 5g12e8 and 6g8g7 anti-DEspR mAbs, MS did not detect DEspR-specific peptides. |
| GO:0005886 plasma membrane | IDA PMID:33853558 Humanized anti-DEspR IgG4(S228P) antibody increases overall ... | ACCEPT | Summary: Murine and humanised anti-DEspR antibodies bind the surface of pancreatic tumour cells and cancer stem-like cells and are then internalised, in a manner abolished by ADAR1 knockout. Reason: Independent confirmation, in a third cell system, of surface presentation; the ADAR1-knockout control additionally ties the detected signal to the edited transcript of this locus rather than to antibody cross-reactivity alone. Supporting Evidence: PMID:33853558 Blocking anti-DEspR murine precursor and humanized antibodies bind cell-surface DEspR, internalize, and co-translocate with gal1/gal3 nuclear shuttling proteins to the nucleus, subsequently inducing apoptosis in vitro and in vivo. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer of the UniProt "Cell membrane" subcellular-location annotation, which is itself grounded in three experimental papers. Reason: Correct and consistent with the three independent IDA rows for the same term. |
| GO:0038084 vascular endothelial growth factor signaling pathway | IDA PMID:24465725 DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC... | MODIFY | Summary: Phosphoproteomic profiling of DEspR-expressing Cos1 transfectants after addition of the VEGF-A signal peptide shows ligand-specific activation of angiogenesis-associated phosphoproteins, partly overlapping and partly distinct from VEGF-VEGFR2 signalling. Reason: The term is defined as "The series of molecular signals initiated by vascular endothelial growth factor (VEGF) binding its receptor on the surface of the target cell". The ligand used here is the cleaved VEGF-A signal peptide, not VEGF-A itself, and UniProt records that the protein does not bind mature VEGF-A; the paper itself describes the resulting signalling as "distinct from VEGF-VEGFR2". The assertion that is actually supported is a cell-surface receptor signalling pathway initiated by an extracellular peptide ligand, so the generic parent is proposed instead. Proposed replacements: cell surface receptor signaling pathway Supporting Evidence: PMID:24465725 DEspR-mediated signaling activated phosphoproteins implicated in angiogenesis with some overlap with VEGF-VEGFR2 signaling pathways (FAK, PKCa, ERK1/2), but also distinct from VEGF-VEGFR2 |
| GO:0038085 vascular endothelial growth factor binding | IDA PMID:24465725 DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC... | MODIFY | Summary: Competition FACS shows anti-DEspR antibody binding to Panc1 cancer stem-like cells being displaced by the VEGF-A signal peptide, and signal-peptide addition triggers DEspR-dependent phosphorylation events. Reason: The term means "Binding to a vascular endothelial growth factor", i.e. the mature growth factor. What was assayed is binding of the VEGF-A signal peptide (VEGFsp); UniProt states explicitly that this protein does not bind the mature VEGFA protein, and the mouse study likewise reports binding of ET-1 and the VEGF signal peptide rather than VEGF (PMID:16293765). The term therefore overstates the experiment. Signal sequence receptor activity, with generic peptide binding as the weaker fallback, matches what was shown. Proposed replacements: signal sequence receptor activity peptide binding Supporting Evidence: PMID:24465725 Collectively, these findings support the functionality of VEGFsp- and ET1-binding to DEspR. PMID:16293765 to Dahl R/JRHS rat Dear, but binds ET-1 and vascular endothelial growth factor |
| GO:0086100 endothelin receptor signaling pathway | IDA PMID:24465725 DEspR roles in tumor vasculo-angiogenesis, invasiveness, CSC... | MODIFY | Summary: Endothelin-1 addition to DEspR-expressing transfectants produces a ligand-specific intracellular phosphorylation signature, taken as evidence that the protein initiates ET-1-triggered signalling. Reason: The term is defined as "A G protein-coupled receptor signaling pathway initiated by endothelin binding to its receptor", and no G-protein coupling has been demonstrated for this single-pass 85-residue protein; the IUPHAR review recognises only ETA and ETB as endothelin receptors (PMID:26956245). As with the molecular-function row, the supported claim is kept but restated without the GPCR commitment. Proposed replacements: cell surface receptor signaling pathway Supporting Evidence: PMID:24465725 Collectively, these findings support the functionality of VEGFsp- and ET1-binding to DEspR. PMID:26956245 No further Family A GPCRs have been identified that might bind ET peptides. |
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Download this section (compressed HTML)Q: Should a UniProt entry rated PE1 on antibody evidence alone carry molecular-function annotations when the underlying locus is classified by HGNC as a long non-coding RNA and mass spectrometry of the authors' own pull-downs did not detect the protein?
Q: Is there a GO term, or should there be one, for a non-GPCR single-pass peptide receptor? Both GO:0004962 endothelin receptor activity and GO:0086100 endothelin receptor signaling pathway are defined in terms of heterotrimeric G-protein activation, which leaves no endothelin-specific term available for a protein reported to bind ET-1 without a demonstrated G-protein mechanism.
Q: Can GO express "binds the signal peptide of VEGF-A but not mature VEGF-A"? GO:0038085 vascular endothelial growth factor binding conflates the two, and GO:0005048 signal sequence receptor activity carries intracellular-targeting connotations from the SRP receptor literature.
Experiment: Targeted mass spectrometry (parallel reaction monitoring with heavy-labelled synthetic peptides for the tryptic peptides downstream of codon 14) on membrane fractions of DEspR-high cells, with ADAR1 knockout cells as the negative control, to establish protein existence independently of antibodies.
Experiment: Radioligand binding on membranes from cells expressing an epitope-tagged construct, giving Kd and Bmax for 125I-ET-1 and for synthetic VEGF-A signal peptide, with competition by BQ123 and BQ788 to test whether the sites are pharmacologically distinct from ETA and ETB.
Experiment: A mechanism test for signal transmission: BRET or NanoBiT assays for G-protein recruitment and beta-arrestin recruitment on ligand addition, alongside a search for a transducing partner by proximity labelling of the 48-residue cytoplasmic tail, to determine how an 85-residue single-pass protein could transmit a ligand signal.
Experiment: Independent replication, in a laboratory with no stake in the receptor claim, of the ligand-displacement FACS and of the ET-1-induced phosphorylation signature, using gene knockout rather than only antibody blockade as the specificity control.
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