Human NOTCH2 Is Resistant to Ligand-independent Activation by Metalloprotease Adam17

Abstract

Cell surface receptors of the NOTCH family of proteins are activated by ligand induced intramembrane proteolysis. Unfolding of the extracellular negative regulatory region (NRR), enabling successive proteolysis by the enzymes Adam10 and γ-secretase, is rate-limiting in NOTCH activation. Mutations in the NOTCH1 NRR are associated with ligand-independent activation and frequently found in human T-cell malignancies. In mammals four NOTCH receptors and five Delta/Jagged ligands exist, but mutations in the NRR are only rarely reported for receptors other than NOTCH1. Using biochemical and functional assays, we compared the molecular mechanisms of ligand-independent signaling in NOTCH1 and the highly related NOTCH2 receptor. Both murine Notch1 and Notch2 require the metalloprotease protease Adam17, but not Adam10 during ligand-independent activation. Interestingly, the human NOTCH2 receptor is resistant to ligand-independent activation compared with its human homologs or murine orthologs. Taken together, our data reveal subtle but functionally important differences for the NRR among NOTCH paralogs and homologs.

FIGURE 1.

Calcium chelation activates NOTCH1, but not NOTCH2. A, expression and cleavage of transfected human HA-tagged NOTCH1 and NOTCH2 in U2OS cells after EDTA treatment. B, induction of RBP-jκ/CSL-luciferase reporter gene activity in U2OS cells by EDTA. C, expression and cleavage of endogenous NOTCH1 and NOTCH2 in HEK293 transfected with ectopic Myc-tagged Notch1 and treated with EGTA. D, upper panel, full-length and chimeric NOTCH1/2 receptors with a Gal4 DNA binding domain, replacing the RAM and ANK domain, fused to the NOTCH1 TAD and PEST domain (lower panel). Expression and cleavage of full-length (FL) and ΔEGF Gal4 fusions of NOTCH1 and NOTCH2 (N2L1566P (L>P, ●). E, Gal4 luciferase cleavage assay of N1-Gal4 or N2-Gal4 after Jagged1 stimulation corrected for empty vector-transfected U2OS cells stimulated with ligand. F, Gal4 reporter luciferase assays in U2OS cells showing N1-Gal4 but not N2-Gal4 is stimulated by EDTA. Reporter assays are presented as fold induction by EDTA versus vehicle (PBS) and normalized to empty vector. TMIC, transmembrane and intracellular domain. NEXT, NOTCH extracellular truncation domain. Lamin A/C is used as a loading control. RLU, relative light units. Data are represented as mean ± S.E. of three independent experiments. ev, empty vector *, significant (p < 0.05), and ns, non-significant.

FIGURE 2.

Leukemogenic NOTCH1 mutations do not activate NOTCH2. A, clustal-W2 alignment (28) of human NOTCH1 and 2 and murine Notch2 HD domains, (*) identical residues (:) highly similar and (·) weak similarity (↓) indicate the missence mutations used in this study. B, ligand-independent cleavage of HA-N1-L1594P T-ALL mutant in transfected U2OS cells. NICD1/Val-1744 cleavage and N1EXT accumulation after GSI (DBZ) treatment and (C) RBP-jκ/CSL-luciferase reporter activity inhibited by GSI. D, HA-Immunoblots of U2OS cells transfected with either wild type (WT) or NOTCH2-HD mutants. Note loss of furin-cleaved TMIC in most NOTCH2 HD mutants. E, surface-biotinylation and streptavidin pull-down of U2OS cells transfected with wild type or HD-mutant NOTCH2 receptors. HA immunoblotting of Input (WCL) flow through (FT) and the streptavidin pulldown (IP). Only wild type and V1667I mutant NOTCH2 receptors are furin cleaved and at the cell surface. Biotinylation is specific for surface expressed proteins (i.e. N-cadherin) and does not label cytoplasmic proteins (HIF-1β). F, RBP-jκ/CSL-luciferase reporter gene activation of U2OS cells transfected with wild type or mutant NOTCH2 cultured in the absence of exogenous ligand (H) and (G) after co-culture with OP9-Jag1-expressing cells in presence or absence of GSI (DBZ). I, HA-Immunoblotting of U2OS cells expressing WT or N2-V1667I showing DBZ induces S2/N2EXT accumulation in response to ligand (OP9-Jag1). I, Gal4-luciferase reporter gene activation monitoring cleavage of wild type and N2-L1566P mutated N2ΔEGF-Gal4 proteins. RLU, relative light units. Data are represented as mean ± S.E. of three independent experiments, *, p < 0,05 and ns, non-significant.

FIGURE 3.

Zinc coordinating histidines within the NOTCH2 NRR are not essential for auto-inhibition. A, HA-Immunoblotting of U2OS cells expressing WT or histidine mutant HA-NOTCH2 showing furin/TMIC levels. B, RBP-jκ/CSL-luciferase reporter activation of WT and His-mutant NOTCH2 in U2OS after OP9-Jag1 co-culture in the presence and absence of GSI. C, HA-immunoblots of WT or his-mutant NOTCH2 after OP9-Jag1 showing WT and the His mutants accumulate S2/N2EXT in response to GSI. D, expresion of TMIC and NEXT/S2 in response to EDTA stimulation and GSI treatment in U2OS cells expressing either WT or His-mutant NOTCH2 and (E) RBP-jκ/CSL-luciferase activation of WT and His-mutants after EDTA stimulation. Data are normalized to non-transfected controls and expressed relative to WT transfected cells (set as 1) treated with vehicle (DMSO) only (F) NOTCH transcriptional activation measured by RBP-jκ/CSL-luciferase reporter gene activation in U2OS transfected with WT, H1574R (NOTCH1 mimic) and patient-derived H1638Y MBZL mutant NOTCH2 or empty vector control (EV). RLU, relative light units. Lamin A/C is used as loading control. Data are represented as mean ± S.E. of three independent experiments. *, p < 0,05.

FIGURE 4.

The NOTCH2 NRR is more resistant to unfolding than the NOTCH1 NRR. A, schematic overview of FLAG-HA tagged NRR composed of N and C-terminal HD domains and LNR A, B, and C modules, which are furin (S1) cleaved and secreted into the medium of HEK293 as heterodimers. B and D, HA-immuneprecipitation from HEK293 medium 48 h post-transfection. HA-IP pre-incubated in the absence (B) or presence (D) of EDTA followed by increasing urea and blotted for HA (total IP) and FLAG (eluted fragment). C, stepwise elution with urea (0–5 m) directly from HA-bound NRR1 and NRR2 on beads, followed by anti-FLAG IB from supernatant. Note earlier appearance of FLAG signal with NRR1 than NRR2 and at 5 m urea FLAG-NRR2 is still bound to the HA beads and can be observed in the residual eluted fraction (all). E, RBP-jκ/CSL-luciferase reporter gene activation in NIH3T3 cells transfected with ectodomain truncated NOTCH1 and NOTCH2 ΔE constructs showing a γ-secretase dependent transcriptional activation indicating that NRR domain removal from NOTCH2 is sufficient for constitutive activity. RLU, relative light units. Data are represented as mean ± S.E. of three independent experiments. Urea dissociation experiments are representative of at least three independent experiments.

FIGURE 5.

The mouse Notch2 NRR is sensitive to unfolding and EDTA activation and requires Adam17. A, immunoblotting for endogenous Notch1 and Notch2 after EDTA stimulation of Adam10- or Adam17-deficient MEFs. Cleaved-activated Notch1 (NICD Val1744) is only observed in Adam10-deficient cells and not Adam17-deficient cells. Anti Notch2 IB (upper panel) shows N2ICD and accumulation of N2EXT/S2 only in cells expressing Adam17 (Adam10^−/−) and in a GSI-dependent manner. B, RBP-jκ/CSL-luciferase reporter activation in U2OS cells expressing wild type or murine HD-mutant N2L1566P showing robust ligand-independent, γ-secretase-dependent, transcriptional activation in N2L1566P mutant. C, HA-IP of transfected murine NRR2 48 h post-transfection from medium of HEK293 in the presence or absence of EDTA followed by urea dissociation. Lamin A/C is used as loading control. RLU, relative light units. Data are represented as mean ± S.E. of three independent experiments. Urea dissociation assay with murine NRR2 is representative of at least three independent experiments.