Both wild-type Notch 3 and Notch 3^R142C receptors respond to Notch ligands. HEK 293 stable cell lines expressing either the wild-type mNotch 3 (Notch 3) or the mNotch 3^R142C receptor were cocultured with 3T3 cells expressing either Jagged 1 (3T3-hJagged 1, black), HEK 293 cells expressing Delta 1 (293-hDelta 1, light gray), or the respective control line (Ctrl-3T3, white; Ctrl-293, white) and analyzed for 6× RBP–luciferase reporter gene activity. Similar levels of receptor activation were observed for the mNotch 3- and mNotch 3^R142C-expressing cells. All transfections were performed in triplicate, and the experiment was repeated twice.

Reduced amounts of Notch 3^R142C at the cell surface. Cell surface proteins of HEK 293 stable cell lines expressing either the mNotch 3 or the mNotch 3^R142C receptor were biotinylated. Five percent of the protein extract was removed before the pull down with magnetic beads coupled to streptavidin. The remaining 95% of the extracted proteins was subjected to the streptavidin pull down (B) and followed by SDS/PAGE and Western blot analysis by using the 5E1 antibody along with 5% of total protein extract (T). Note that the ratio between the pulled-down receptor (B) and the total amount of expressed receptor (T) is lower for mNotch 3^R142C compared with wild-type mNotch 3. The 210-kDa immunoreactive band that is present in all pulled-down fractions suggests that both the vast majority of wild-type and mNotch 3^R142C receptors are expressed as S1-cleaved bipartite proteins at the cell surface. The Western blot is from one representative experiment, whereas the 210:280-kDa ratios are mean ± SD from three independent experiments.

Introduction of the R142C mutation enhances the formation of cytoplasmic aggregates. The subcellular localization of the mNotch 3 receptors in stable HEK 293 cells was assayed by immunocytochemistry. mNotch 3^R142C-GVP (R142C) expressing cells contain a single large cytoplasmic aggregate in 75% of the cells (A). In contrast, mNotch 3-GVP in most cells is distributed in patches at the cell surface, and the large cytoplasmic aggregate is found in only 25% of the cells (B). Brefeldin A treatment of the mNotch 3-GVP cells, however, increases the proportion of aggregate-containing cells (C). Costaining of the mNotch 3-containing aggregates with calnexin demonstrates that the aggregates do not colocalize with ER (D). Costaining with the Golgi-marker CTR433 revealed that the aggregates were located close to the Golgi, but with no apparent overlap (E). Similarly, no colocalization with the intermediate compartment was observed, as visualized by double staining with the p58 antibody (F). To investigate the relationship with aggresomes in more detail, double immunocytochemistry with antibodies recognizing pericentrin (G), vimentin (H), and ubiquitin (I) was performed. The aggregates are located close to the centrosomes (G) but are not caged with vimentin (H) nor do they contain ubiquitin (I). Coexpression of GFP-CFTRΔF508 (CFTR) in stable mNotch 3^R142C-GVP cell lines shows that mNotch 3- and CFTR-containing aggregates are close to each other but form separate clusters (K). Single large cytoplasmic aggregates were detected at low frequency also in mNotch 3^R142C cells (L). mNotch 3-containing proteins were detected by anti(α)-Gal4 antibodies (A–C and G), −VP16 antibodies (D–F and H), or with the M20 antibody raised against the intracellular domain of Notch 3 (L).

Reduced expression of S1-cleaved Notch 3^R142C receptor. (A) Schematic depiction of presumed processing events of the Notch 3 receptor. The full length (FL) Notch 3 receptor is processed at site 1 (S1) by a furin-like convertase during intracellular trafficking and is presented at the plasma membrane as a bipartite protein consisting of the EC and the transmembrane + intracellular domain (TMIC). The corresponding proteins migrate as 280- (FL), 210- (EC), and 97-kDa (TMIC) proteins. As a result of ligand activation, the receptor undergoes two consecutive cleavages (S2 and S3). The CADASIL-causing R142C mutation (R142C) is located in the third EGF repeat in the EC. (B) Total protein extracts from three different HEK 293 stable cell lines expressing wild-type mNotch 3 (clones 6, 8, and 17) and three lines expressing mNotch 3^R142C (clones 8, 9, and 12) receptor were analyzed by Western blot using the α-Notch 3 EC antibody 5 E1. Note the increased levels of the 280-kDa band (FL receptor) in cells expressing the mNotch 3^R142C receptor compared with wild-type mNotch 3-expressing cells. The Western blot is from one representative experiment, whereas the 210:280-kDa ratios are mean ± SD from three independent experiments.

The Notch 3^R142C-GVP receptor is impaired in S1-processing and appears in reduced amounts at the cell surface but retains signaling. (A) A GVP domain was inserted immediately C-terminal to the mNotch 3 S3-cleavage site of different mNotch 3 derivatives to enable specific recording of the engineered receptors from a UAS–luciferase reporter gene construct. (i) N3IC-GVP, the Notch 3 IC with GVP, ligand- and S3 cleavage-independent; (ii) N3 ΔE-GVP, an N-terminally truncated Notch 3 receptor, ligand-independent but S3-cleavage dependent; (iii) N3-GVP, ligand- and S3-cleavage dependent; (iv) N3^R142C-GVP, the same as N3-GVP but with the R142C mutation. (B) Total protein (T) and cell surface biotinylated extracts (B) of HEK 293 stable cell lines expressing either mNotch 3-GVP or mNotch 3^R142C-GVP were analyzed by Western blot by using the 5E1 antibody. As for the mNotch 3 receptors with a wild-type backbone (Fig. 2), we observed an impaired S1 cleavage as well as a decreased cell surface expression of mNotch 3^R142C-GVP as compared with the corresponding mNotch 3-GVP receptors. The Western blot is from one representative experiment, and the B:T ratios are mean values from three independent experiments. *, Postlysis degradation product. The mNotch 3-GVP expressing cells were mixed with either 3T3-hJagged 1 cells (C), 293-hDelta 1 cells (D), or control (Ctrl) cells, and cultured in the presence or absence of the γ-secretase inhibitor MW167 (100 μM). The N3 IC-GVP and N3 ΔE-GVP transfected cells were included as controls for S3 cleavage and ligand-dependent activation, respectively. The experiment was performed in triplicate and repeated at least three times. *, P < 0,05; ***, P < 0.001 vs. Ctrl + Jagged 1 Delta 1; #, P < 0.05 vs. Ctrl.