RHBDL2

RHBDL2 is an intramembrane serine protease that sheds the ectodomains of diverse single-pass transmembrane substrates, positioning it as a broad regulator of receptor signaling, tissue repair, and cell survival (PMID:21833011, PMID:28779096). It cleaves substrates within or just outside the transmembrane domain, with cleavage selectivity dictated by the luminal face of the substrate TM domain, as established for ephrinB3 and for the IL-11 receptor, where the precise scissile bond (Ala-370/Ser-371) was mapped and a human A370V variant abolishes cleavage without affecting classic signaling (PMID:15047175, PMID:33566379). Its validated substrate repertoire includes EGF—whose cleavage drives EGF secretion and EGFR activation (PMID:21494248)—thrombomodulin, whose shedding from keratinocytes is required for wound healing (PMID:21833011), CLEC14A, whose shed ectodomain restrains sprouting angiogenesis (PMID:26939791), and an endogenously confirmed set comprising IL6R, Spint-1, DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, and BCAM (PMID:28779096). Through EGFR-coupled signaling RHBDL2 promotes proliferation, reduces adhesion, and confers anoikis resistance (PMID:24977233), and it activates Notch signaling by cleaving Notch1 to release N1ICD while collaborating with the deubiquitinase OTUD7B to stabilize N1ICD against proteasomal degradation (PMID:36351890). Beyond proteolysis, RHBDL2 acts as a non-proteolytic scaffold: a Val245-mediated hydrophobic interaction stabilizes the deubiquitinase USP3 independently of catalytic activity, driving a USP3–PPT1–FASN axis of de novo lipogenesis (PMID:42031733). In zebrafish, loss of Rhbdl2 enhances regenerative growth and macrophage recruitment through elevated Rac2, placing it upstream of Rac2-dependent immune signaling during injury (PMID:41726946).

1. 2004 Medium

   Established RHBDL2 as a functional intramembrane protease and defined the substrate determinant of its activity—the luminal face of the substrate TM domain controls cleavage susceptibility.

   Evidence Co-expression cleavage of ephrinB3 in mammalian cells with substrate domain-swap mutagenesis

   PMID:15047175

   Open questions at the time

   • No physiological readout for ephrinB3 cleavage shown
   • Endogenous RHBDL2 activity not yet demonstrated
   • Single lab
2. 2011 High

   Connected RHBDL2 cleavage to defined signaling and physiological outputs—EGF shedding to drive EGFR activation, and thrombomodulin shedding required for wound healing.

   Evidence Cleavage assays with EGFR activation readout in tumour cell lines; shRNA/DCI inhibition with ex vivo skin and in vivo mouse wound models, rescued by recombinant sTM

   PMID:21494248 PMID:21833011

   Open questions at the time

   • Structural basis of substrate selection not resolved
   • Relative contribution of multiple substrates to wound phenotype unclear
3. 2014 Medium

   Placed RHBDL2 upstream of EGFR-mediated anoikis resistance, linking its protease activity to cell survival and adhesion programs.

   Evidence Gain/loss-of-function with caspase-3 and FAK readouts and EGFR-inhibitor epistasis in suspension culture

   PMID:24977233

   Open questions at the time

   • Direct substrate driving anoikis resistance not identified
   • Single lab
4. 2016 High

   Demonstrated rhomboid-family selectivity (RHBDL2 but not RHBDL1/3) and mapped a precise CLEC14A cleavage site, linking shedding to suppression of angiogenesis.

   Evidence Site-directed mutagenesis, siRNA knockdown of endogenous RHBDL2, in vitro and in vivo angiogenesis assays

   PMID:26939791

   Open questions at the time

   • In vivo contribution of endogenous RHBDL2 to vascular biology not established
5. 2017 High

   Defined the endogenous substrate repertoire systematically, distinguishing RHBDL2 shedding from metalloprotease-mediated shedding.

   Evidence SILAC quantitative proteomics with endogenous RHBDL2-dependent shedding validation

   PMID:28779096

   Open questions at the time

   • Physiological consequence of most identified substrates not characterized
   • Cleavage sites for most substrates not mapped
6. 2021 Medium

   Extended RHBDL2 into cytokine trans-signaling and to atypical conformational substrate sensing—generating soluble IL-11R for trans-signaling, and surveying activated Orai1/CRAC channels.

   Evidence IL-11R cleavage site mapping, TM mutagenesis, subcellular localization, trans-signaling assay and disease variant analysis; CRAC channel surveillance described via Molecular Cell commentary

   PMID:33566379 PMID:34861185

   Open questions at the time

   • CRAC channel surveillance is a commentary without primary experimental detail here
   • In vivo relevance of IL-11R shedding not established
7. 2022 Medium

   Showed RHBDL2 activates Notch signaling not only by cleaving Notch1 but by co-stabilizing the released N1ICD via OTUD7B-dependent deubiquitination, revealing a combined proteolytic and stabilization mechanism in cancer.

   Evidence Co-IP, cleavage and ubiquitination assays, RNA-seq, in vitro/in vivo gain/loss-of-function with Notch-inhibitor rescue in pancreatic cancer

   PMID:36351890

   Open questions at the time

   • Direct vs indirect role in N1ICD ubiquitin regulation not fully separated
   • Single lab
8. 2026 Medium

   Revealed a protease-independent scaffolding function—Val245-mediated stabilization of USP3—coupling RHBDL2 to a USP3–PPT1–FASN lipogenic axis.

   Evidence Structural analysis, Val245 mutagenesis, Co-IP, ubiquitination assay, EGCG disruption and in vivo osteosarcoma model

   PMID:42031733

   Open questions at the time

   • Structural model of the RHBDL2–USP3 interface not fully resolved
   • Generality of scaffold function beyond osteosarcoma unknown
9. 2026 Medium

   Placed Rhbdl2 as a brake on regenerative growth and macrophage recruitment via Rac2 in an in vivo vertebrate injury model.

   Evidence Zebrafish CRISPR-Cas9 knockout, proteomics, Rac2 morpholino epistasis and live imaging (preprint)

   PMID:41726946

   Open questions at the time

   • Preprint, not yet peer-reviewed
   • Substrate linking Rhbdl2 to Rac2 levels unidentified
   • Mammalian relevance untested

• How RHBDL2 substrate selection, subcellular site of cleavage, and the switch between proteolytic and scaffolding modes are regulated in vivo remains unresolved.
• No structure of RHBDL2 with a substrate or with USP3
• Determinants governing proteolytic vs non-proteolytic activity unknown
• In vivo physiological functions in mammals largely uncharacterized