Characterization of the H1545P mutation in LNR-C. (A) Structure of the region surrounding H1545. Calcium coordinating residues are shown in ball-and-stick representation and are colored by atom type (gray indicates carbon; red: oxygen; and blue: nitrogen). Disulfide bonds are yellow, and the calcium ion is a green sphere. (B) Reporter gene assay for ligand-independent Notch1 activation. H1545P and several other well-characterized T-ALL–associated mutations were tested for their ability to induce reporter gene transcription in the context of a Notch1 polypeptide lacking the EGF-like ligand binding repeats (ΔEGF). The ΔEGF forms of the receptors were transiently transfected into U2OS cells together with a plasmid encoding a luciferase reporter gene under control of 4 iterated CSL binding sites, and an internal control plasmid expressing Renilla luciferase. Firefly luciferase activities were normalized to the internal Renilla control and expressed relative to the activity produced by the unmutated ΔEGF form of the receptor, which was assigned a relative value of 1. Error bars represent the SE of the 3 replicate measurements made for each experimental condition. (C) The H1545P mutation confers ligand-independent proteolytic sensitivity. U2OS cells were transfected with plasmids encoding the indicated Notch1 receptor variants. Western blots with antibodies specific for the intracellular portion of human Notch1 (top panel) and for the S3-cleaved product (bottom panel) are shown. (D) Effect of the H1545P mutation on sensitivity to urea-induced subunit dissociation. Conditioned media from HEK 293T cells expressing epitope-tagged Notch1 NRR minireceptors were immunoprecipitated with α-HA coupled beads followed by incubation in buffer containing different concentrations of urea for 30 minutes (0 to 3.5 M). Subunit dissociation was evaluated by SDS–polyacrylamide gel electrophoresis (PAGE) followed by Western blot analysis. The N-terminal and C-terminal subunits were detected with α-FLAG and α-HA antibodies, respectively.