Selective Use of ADAM10 and ADAM17 in Activation of Notch1 Signaling

Notch signaling requires a series of proteolytic cleavage events to release the Notch intracellular domain (NICD) that functions directly in signal transduction. The Notch receptor is locked down in a protease-resistant state by a negative regulatory region (NRR) that protects an ADAM (a disintegrin and metalloprotease) cleavage site. Engagement with ligand-bearing cells induces global conformational movements in Notch that unfold the NRR structure to expose the ADAM cleavage site and initiate proteolytic activation. Although both ADAM10 and ADAM17 have been reported to cleave Notch to facilitate NICD release by γ-secretase, the relevant ADAM has remained controversial. Our study provides new insight into this conflict, as we find that although Notch1 (N1) is a substrate for both ADAM10 and ADAM17, the particular ADAM required for receptor activation is context dependent. Specifically, ADAM10 was absolutely required for N1 signaling induced by ligands, while signaling independent of ligands required ADAM17. In contrast to the strict and differential use of ADAM10 and ADAM17 in normal and dysregulated signaling, respectively, both proteases participated in signaling intrinsic to N1 mutations associated with leukemia. We propose that in addition to exposing the ADAM cleavage site, activating N1 conformational changes facilitate selective cleavage by specific proteases.Tight control over Notch signaling is critical to normal development as both gains and losses in signaling produce developmental defects (1, 2). Signaling from the Notch receptor is dependent on, and highly regulated by, three distinct types of proteases that cleave Notch at defined sites (35). The first cleavage takes place at a site in the ectodomain (S1) by a furin-like convertase (28) to generate an intramolecular, heterodimeric cell surface receptor that exhibits a protease-resistant conformation in the absence of ligand (10). Interactions with Notch ligands override the autoinhibitory state to allow additional proteolysis at a second site (S2) within the Notch extracellular juxtamembrane region by ADAM proteases. S2 cleavage allows subsequent proteolysis within the membrane-spanning region at additional sites (S3 and S4) by the aspartyl protease γ-secretase. Completion of this proteolytic cascade allows Notch intracellular domain (NICD) membrane release and nuclear translocation resulting in transcriptional complex formation with the CSL [CBF-1/Su(H)/Lag-1] DNA binding protein to directly activate target gene expression (2, 35).Numerous functional studies have identified a negative regulatory region (NRR) located within the ectodomain that prevents Notch proteolytic activation (13, 20, 25). Recent structural studies have provided mechanistic insight into how the Notch NRR conformation contributes to the protease-resistant state and, importantly, how ligands might override this to activate Notch signaling (10). Specifically, the ADAM cleavage site is buried deep within the NRR structure, and extensive noncovalent interactions within the NRR function to stabilize the heterodimer and occlude the S2 site in the absence of ligand. Moreover, based on ADAM17 structure data, the S2 site might need to be completely unstructured to gain access to the metalloprotease catalytic site (32). Thus, it has been suggested that ligand binding must produce substantial movement within the Notch heterodimer to destabilize the NRR conformation and allow full exposure of the S2 cleavage site (11, 12). In this regard, studies of flies and mammalian cells have identified a strict requirement for ligand endocytosis in Notch signaling (23, 39), and we have found that ligand endocytosis is required to both dissociate the ligand-bound heterodimer and promote Notch proteolysis for downstream signaling (38). Importantly, our studies indicate that ligand binding alone is not sufficient to dissociate the Notch1 (N1) heterodimer and activate signaling. Accordingly, we have proposed that ligand endocytosis generates a mechanical force to destabilize the NRR and completely dissociate the heterodimer, events that would clearly expose the S2 site and allow ADAM cleavage to initiate the activation of proteolysis.Although it is clear that prior S2 cleavage is required for efficient γ-secretase cleavage to generate the active NICD, it is less clear which ADAM family members actually cleave Notch. Biochemical studies of flies have indicated that ADAM10 interacts with and cleaves Notch to activate signaling, and genetic manipulations that result in losses in protease activity lead to developmental defects similar to those described for defects in Notch signaling (26, 41, 48, 53). Together, these data provide strong support for the idea that ADAM10 directly participates in the proteolytic activation of Notch to regulate signaling. However, genetic studies of Caenorhabditis elegans have identified functional redundancy for the orthologs of ADAM10 and ADAM17, SUP-17 and ADM-4, respectively, for a subset of developmental decisions mediated by the Notch-related LIN-12 receptor (19). Further complicating these issues are the in vitro studies of mammalian cells that have both identified ADAM17 as the relevant metalloprotease in S2 cleavage and excluded a role for ADAM10 in this event (3, 36). However, a role for ADAM17 in the proteolytic activation of N1 is difficult to reconcile with genetic studies demonstrating that ADAM10 knockout mice display classic Notch loss-of-function phenotypes (4, 14, 56), while ADAM17 mutant mice clearly do not (42).Despite a large body of evidence supporting a role for ADAM10 in Notch cleavage and activation of signaling, ADAM17 is often considered in the literature as the relevant ADAM responsible for activating Notch proteolysis. We reasoned that this discrepancy is due to the fact that the studies implicating ADAM17 in S2 cleavage employed forms of N1 that either lacked sequences required to form the NRR or encoded destabilizing NRR mutations (3, 36), which, based on structure data, likely expose the S2 site to proteolysis independent of ligand. Given the strong dependency on Notch ligands to expose the S2 site and release autoinhibition, we reevaluated the requirement of these ADAMs in Notch signaling by using cell coculture assays that rely on direct interactions between Notch ligands and receptor-expressing cells to activate N1 proteolysis and downstream signaling. Our findings indicate that N1 proteolytic processing by ADAM10 is strictly ligand dependent and, importantly, ADAM17 cannot functionally replace ADAM10 activity, suggesting an exclusive role for this protease in ligand-induced signaling. Interestingly, while ADAM17 cannot activate N1 in response to ligand, it can nonetheless activate signaling in a ligand-independent manner.