Ectodomain Shedding and Intramembrane Cleavage of Mammalian Notch Proteins Are Not Regulated through Oligomerization

Abstract

Intramembrane cleaving proteases such as site 2 protease, ␥-secretase, and signal peptide peptidase hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. It has been proposed that removing the extracellular domain allows dissociation of substrate TMD, held together by the extracellular domain or loop. Using ␥-secretase as a model intramembrane cleaving protease and Notch as a model substrate, we investigated whether activating and inactivating mutations in Notch modulate ␥-secretase cleavage through changes in oligomerization. We find that although the Notch epidermal growth factor repeats can promote dimer formation, most surface Notch molecules in mammalian cells are monomeric as are constitutively active or inactive Notch1 proteins. Using a bacterial assay for TM dimerization, we find that the isolated TMD of Notch and amyloid precursor protein self-associate and that mutations affecting Notch cleavage by ␥-secretase cleavage do not alter TMD dimerization. Our results indicate that ligand-induced reversal of controlled TMD dimerization by the Notch extracellular domain is unlikely to underlie the regulatory mechanism of intramembranous cleavage.

Notch-1 associates with IKKα and regulates IKK activity in cervical cancer cells

“…Annexin-V flow cytometry ( Figure 6c) indicated that cytotoxicity was at least in part due to apoptosis. Either Notch-1 or IKKa siRNAs dramatically inhibited both NF-kB and CBF-1 reporter activity in the presence or absence of 10 mM cisplatin (Figures 6d and e), consistent with the hypothesis that NF-kB and CBF-1 activities are (Vooijs et al, 2004). Forty-eight hours after transfection, cells were treated for 30 min with vehicle or TNF-a (10 ng/ml).…”

“…Both direct and reverse immunoprecipitation experiments detected a Notch-1/IKKa-containing complex in normal keratinocytes as well (Figure 3e), indicating that this complex is not exclusive to CaSki and T-ALL cell lines. To confirm the interaction, we used a quantitative immunoprecipitation assay described by Vooijs et al (2004). This assay uses a full-length Notch-1 construct tagged at the C terminus with Renilla luciferase.…”

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

“…Similar to APP, numerous other γ-secretase substrates dimerize via their TMS, e.g., E-cadherin (23), ErbB4 (24), or Notch (25), and it is reasonable to speculate that certain GSMs may similarly modulate processing of those substrates. Indeed, sulindac sulfide and indomethacin decreased the relative level of released Notch-1 Aβ-like peptide species, i.e., Nβ25, without changing the total Nβ level (26).…”

Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes

“…Models involving receptor oligomerization have been proposed, but recent data suggest that both metalloprotease-sensitive and -resistant forms of NOTCH receptors exist primarily as monomers (46). An alternative mechanism supposes that NOTCH1 is rendered sensitive to S2 cleavage by a change in conformation (46), which could occur in response to mechanical forces generated by the endocytic machinery of the ligand-expressing cell (1,22,30,43).…”