The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and ␥-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require ␥-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n ‫؍‬ 3) or in the presence of urea (n ‫؍‬ 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.The development of multicellular organisms is orchestrated by a limited number of highly conserved signaling pathways. One such pathway involves NOTCH receptors and downstream mediators, which can variously regulate the specification of cell fate, proliferation, self-renewal, survival, and apoptosis in a dose-and context-dependent fashion (3,47).Like other members of the NOTCH receptor family, mammalian NOTCH1 is a large multimodular type I transmembrane glycoprotein (Fig. 1A). During maturation, NOTCH1 undergoes proteolytic processing by furin at a site termed S1 that lies ϳ70 amino acids external to the transmembrane domain (25), yielding two noncovalently associated extracellular (N EC ) and transmembrane (N TM ) subunits (6,25,37). N EC contains 36 N-terminal epidermal growth factor (EGF)-like repeats that participate in binding to ligands (23, 39, 51) and three iterated LIN-12/NOTCH repeats (LNR), which help to maintain NOTCH receptors in the "off" state prior to ligand binding (13,24,40). The association of N EC and N TM is mediated by sequences lying immediately N terminal (HD-N) and C terminal (HD-C) of site S1; together, these sequences constitute the NOTCH subunit association, or "heterodimerization" (HD) domain (40).Binding of ligands to N EC triggers two sequential proteolytic events within the N TM subunit at sites S2 and S3. S2 cleavage occurs just...

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BackgroundNotch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.Principal FindingsThe X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.Conclusions/SignificanceS1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or T-ALL-associated mutations lead to conformational changes of the NRR that permit metalloprotease cleavage.