Abstract
Notch signaling is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis. This unique, highly conserved signaling pathway relies on cell-to-cell contact, which triggers the proteolytic release of the cytoplasmic domain of the membrane-anchored transcription factor Notch from the membrane. A disintegrin and metalloproteinase (ADAM) proteins are crucial for Notch activation by processing its S2 site. While ADAM10 cleaves Notch1 under physiological, ligand-dependent conditions, ADAM17 mainly cleaves Notch1 under ligand-independent conditions. However, the mechanism(s) that regulate the distinct contributions of these ADAMs in Notch processing remain unclear. Using cell-based assays in mouse embryonic fibroblasts (mEFs) lacking ADAM10 and/or ADAM17, we aimed to clarify what determines the relative contributions of ADAM10 and ADAM17 to ligand-dependent or ligand-independent Notch processing. We found that EDTA-stimulated ADAM17-dependent Notch1 processing is rapid and requires the ADAM17-regulators iRhom1 and iRhom2, whereas the Delta-like 4-induced ligand-dependent Notch1 processing is slower and requires ADAM10. The selectivity of ADAM17 for EDTA-induced Notch1 processing can most likely be explained by a preference for ADAM17 over ADAM10 for the Notch1 cleavage site and by the stronger inhibition of ADAM10 by EDTA. The physiological ADAM10-dependent processing of Notch1 cannot be compensated for by ADAM17 in Adam10-/- mEFs, or by other ADAMs shown here to be able to cleave the Notch1 cleavage site, such as ADAMs9, 12, and 19. Collectively, these results provide new insights into the mechanisms underlying the substrate selectivity of ADAM10 and ADAM17 towards Notch1.
Highlights
The Notch signaling pathway is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis [1,2,3]
Following Notch receptor-ligand binding, two sequential cleavage events lead to the release of the Notch intracellular domain (NICD), allowing it to move into the nucleus, where it binds to the transcription factor CSL/RBPJ (CBF-1, Suppressor of Hairless, Lag-2/Recombination signal binding protein for immunoglobulin kappa J region) and regulates the transcription of Notch target genes [10,11]
WT mouse embryonic fibroblasts (mEFs) treated with the phorbol ester phorbol myristate-12-acetate (PMA), which strongly stimulates ADAM17 [43,44,45], or with the ionophore ionomycin, which strongly activates both ADAM10 and ADAM17 [46,47,48,49,50,51], did not yield detectable levels of site 2 (S2) product, with ethylenediaminetetraacetic acid (EDTA) stimulation serving as a positive control (Figure 4). These results demonstrate that the stimulation of ADAM10 and ADAM17 is not sufficient to induce processing at the endogenous Notch1 S2 site, so long as this site is protected by the negative regulatory region (NRR) domain
Summary
The Notch signaling pathway is critical for controlling a variety of cell fate decisions during metazoan development and homeostasis [1,2,3] It is a unique, highly conserved signaling pathway that relies on cell-to-cell engagement and on the proteolytic release of the intracellular domain of Notch [4,5]. During transport to the cell surface, Notch receptors are processed by furin-type pro-protein convertases in the trans Golgi network [6,7] This initial, constitutive cleavage at a position referred to as site 1 (S1) gives rise to the mature, functional Notch receptors that exist as heterodimers at the cell surface [6,7]. Foundational work in the field showed that the processing of the Notch receptor in its transmembrane domain at a 3rd site (S3), between G1743 and V1744 in mammalian Notch, by gamma-secretase was required for releasing the NICD from the cell membrane—an essential step in Notch signaling [11,17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
