RHBDL2

RHBDL2 is a mammalian intramembrane serine protease that sheds the ectodomains of diverse type I transmembrane substrates—including EGF, thrombomodulin, ephrinB3, CLEC14A, IL-6R, IL-11R, DDR1, N-cadherin, and Notch1—by cleaving within or near their transmembrane domains, with substrate recognition governed by the amino acid sequence at the luminal face of the transmembrane helix (PMID:15047175, PMID:28779096, PMID:33566379). Cleavage can occur both at the plasma membrane and in the early secretory pathway, and the released ectodomains activate downstream signaling cascades including EGFR-mediated survival signaling, IL-11 trans-signaling, Notch pathway activation, and anti-angiogenic signaling via shed CLEC14A (PMID:21494248, PMID:33566379, PMID:36351890, PMID:26939791). RHBDL2-dependent EGF shedding promotes anoikis resistance in epithelial cancer cells, thrombomodulin shedding by RHBDL2 in keratinocytes promotes wound healing, and Notch1 cleavage cooperates with OTUD7B-mediated deubiquitination to drive pancreatic cancer cell proliferation (PMID:24977233, PMID:21833011, PMID:36351890). Beyond its proteolytic function, RHBDL2 acts as a non-proteolytic scaffold that stabilizes the deubiquitinase USP3 through a Val245-anchored hydrophobic interface, thereby promoting USP3-dependent deubiquitination of PPT1 and FASN-dependent de novo lipogenesis in osteosarcoma (PMID:42031733).

1. 2004 Medium

   Establishing RHBDL2 as a functional intramembrane protease in mammalian cells resolved whether mammalian rhomboid family members possessed catalytic activity and revealed that substrate selectivity is encoded in the luminal-face transmembrane sequence of substrates.

   Evidence Co-expression cleavage assay with ephrinB3 and site-directed mutagenesis of substrate transmembrane domain in mammalian cells

   PMID:15047175

   Open questions at the time

   • Only one substrate (ephrinB3) tested; breadth of substrate repertoire unknown
   • Catalytic mechanism and active-site residue requirements not characterized
   • No endogenous activity demonstrated
2. 2011 High

   Identification of EGF and thrombomodulin as endogenous RHBDL2 substrates established that this protease functions as an ectodomain sheddase with physiological consequences—EGFR activation in tumour cells and wound healing in keratinocytes—broadening its role beyond intramembrane cleavage of ephrinB3.

   Evidence Cell-based cleavage assays with endogenous activity in tumour lines (EGF/EGFR); shRNA knockdown, conditioned-medium rescue, ex vivo skin culture, and in vivo mouse wound model (thrombomodulin)

   PMID:21494248 PMID:21833011

   Open questions at the time

   • Full substrate repertoire still undefined
   • Whether RHBDL2 cleaves within or just outside the transmembrane domain was not yet precisely mapped
   • Regulation of RHBDL2 catalytic activity in vivo unknown
3. 2014 Medium

   Demonstrating that RHBDL2-mediated EGF shedding confers anoikis resistance through EGFR and FAK signaling placed the protease in a pro-survival oncogenic circuit, explaining its potential role in tumour progression.

   Evidence Overexpression/shRNA knockdown with rhomboid inhibitor, EGFR inhibitor epistasis, caspase-3 cleavage in suspension culture

   PMID:24977233

   Open questions at the time

   • Single-lab study; independent replication in additional cancer models needed
   • In vivo tumourigenesis data not provided
   • Whether other RHBDL2 substrates contribute to anoikis resistance unknown
4. 2016 High

   Identification of CLEC14A as a specific RHBDL2 substrate with anti-angiogenic function upon shedding revealed a selective role among rhomboid family members (RHBDL1/3 do not cleave CLEC14A) and extended RHBDL2 biology to vascular biology.

   Evidence Co-expression cleavage, site-directed mutagenesis of cleavage site, siRNA knockdown, in vitro sprouting assay, in vivo sponge implant angiogenesis model

   PMID:26939791

   Open questions at the time

   • Mechanism of RHBDL2 selectivity over RHBDL1/RHBDL3 not structurally resolved
   • Whether CLEC14A shedding occurs in pathological angiogenesis settings not tested
5. 2017 High

   Unbiased quantitative proteomics greatly expanded the RHBDL2 substrate repertoire (IL-6R, DDR1, N-cadherin, KIRREL, BCAM, HAI-1, DCBLD2), demonstrating that RHBDL2 functions as a broad ectodomain sheddase complementary to—but partially non-overlapping with—metalloprotease-mediated shedding.

   Evidence SILAC-based quantitative proteomics in human cells, endogenous RHBDL2 shedding validation, metalloprotease inhibitor comparison

   PMID:28779096

   Open questions at the time

   • Cleavage sites not mapped for most new substrates
   • Physiological significance of each shedding event not individually addressed
   • Redundancy with other rhomboid proteases not fully explored
6. 2021 High

   Precise mapping of the IL-11R cleavage site (Ala370-Ser371) and demonstration that cleavage generates trans-signaling-competent soluble IL-11R—even in the early secretory pathway—revealed that RHBDL2 activity is not confined to the plasma membrane and has direct implications for cytokine signaling; the disease-associated IL-11R-A370V mutation blocks this cleavage.

   Evidence Cleavage site mapping, substrate TM-helix mutagenesis, subcellular fractionation, IL-11 trans-signaling bioassay, disease mutation analysis

   PMID:33566379

   Open questions at the time

   • How RHBDL2 accesses substrates in the ER/Golgi is mechanistically unclear
   • In vivo consequences of impaired IL-11R shedding (A370V) not demonstrated
   • Whether early secretory cleavage applies to other substrates not tested
7. 2022 Medium

   Discovery that RHBDL2 cleaves Notch1 to release N1ICD and cooperates with the deubiquitinase OTUD7B to stabilize N1ICD expanded RHBDL2 into the Notch signaling pathway and demonstrated functional collaboration between a rhomboid protease and a DUB.

   Evidence Co-immunoprecipitation, RNA-seq, ubiquitination assay, Notch inhibitor (IMR-1) epistasis, in vivo xenograft in pancreatic cancer

   PMID:36351890

   Open questions at the time

   • Notch1 cleavage site not mapped
   • Relationship to canonical γ-secretase-mediated Notch processing unclear
   • Single-lab study in one cancer type
8. 2026 Medium

   Identification of a protease-independent scaffold function of RHBDL2 that stabilizes USP3 via a Val245-mediated hydrophobic interface revealed a second molecular activity for the protein, linking it to deubiquitination of PPT1 and FASN-dependent lipogenesis in osteosarcoma.

   Evidence Multi-omics, structural analysis, Val245 mutagenesis, Co-IP, ubiquitination assay, in vivo tumour model, EGCG pharmacological inhibition

   PMID:42031733

   Open questions at the time

   • Scaffold function demonstrated in a single cancer context; generality unknown
   • No reconstituted biochemical assay for direct RHBDL2–USP3 stabilization
   • Structural basis of the Val245 interface resolved only computationally

• Key unresolved questions include how RHBDL2 activity is regulated (transcriptionally, post-translationally, or by lipid environment), the structural basis for substrate selectivity among rhomboid family members, whether the scaffold function extends to additional partners, and the in vivo physiological consequences of RHBDL2 loss in mammals.
• No mammalian RHBDL2 knockout phenotype reported
• No high-resolution structure of RHBDL2
• Regulatory mechanisms (transcriptional or post-translational) of RHBDL2 expression and activity unknown
• Relative contribution of RHBDL2 versus metalloproteases for shared substrates in vivo not established