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First published online 27 July 2004
doi: 10.1242/jcs.01263

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Release of a membrane-bound death domain by -secretase processing of the p75^NTR homolog NRADD

¹ Department of Pathology, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA
² Howard Hughes Medical Institute, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA

View larger version (20K): [in a new window]   Fig. 1. Mature NRADD is a 55 kDa glycoprotein. (A) Pulse chase. Full-length NRADD was transiently transfected into 293 cells labeled 24 hours later with 35S-methionine and [35S]cysteine for 15 minutes and chased with cold medium as indicated. Cell lysates were immunoprecipitated with anti-NRADD antibody and analysed by SDS-PAGE and fluorography. `TUN.' indicates sample derived from cells labeled and chased in the presence of tunicamycin (10 µg ml-1). (B) In vitro deglycosylation. Lysates from 293 cells transfected with NRADD were immunoprecipitated as in (A) and subjected to endoglycosidase digestions as indicated. dglNR, deglycosylated NRADD; iNR, immature NRADD; mNR, mature NRADD.

View larger version (53K): [in a new window]   Fig. 2. NRADD expression in mouse tissues. (A) Characterization of an anti-NRADD antibody. 293 cells were transfected with full length p75NTR and NRADD FLAG-tagged at the N-terminus. Cell lysates from N2a or transfected 293 cells were analysed by three identical western blots probed with anti-NRADD, antigen blocked anti-NRADD and anti-FLAG antibodies. The N2a and 293 lanes were exposed to film for 5 minutes and the transfected 293 lanes were exposed for 30 seconds. (B) Mouse tissues were homogenized and separated into membrane (M) and soluble (S100) fractions. An aliquot of each fraction was deglycosylated in vitro with PNG-F or Endo H. Deglycosylation converted the fuzzy mNR band into a darker dglNR band. sNR indicates the small NRADD isoform. N.S. indicates a non-specific band in the brain lysates that was not blocked by preincubation of the antibody with antigen.

View larger version (54K): [in a new window]   Fig. 8. Endogenous NRADD is a RIP substrate. (A) NRADD processing in vivo. NIH3T3 cells were scraped from the tissue culture dish and incubated at 37°C for 3 hours in the presence of the indicated inhibitors. The first lane (4°C) shows cells kept at 4°C after scraping and lysed immediately. Cell lysates were analysed by anti-NRADD western blot. -Secretase substrate (NpNR) and product (-PNR) are indicated. (B) NRADD processing in vitro. N2a cell membranes were isolated and incubated at 37°C for 1 hour with the indicated inhibitors. (C) Subcellular fractionation. N2a cells were treated for 40 minutes with APMA or left untreated, then washed and incubated for 3 hours in the presence of lactacystin (10 µM). Cells were harvested and fractionated as described in Materials and Methods. N, Nuclear fraction; S100SN, Cytosol; X100P, Triton-X-100-insoluble fraction; X100SN, 1% Triton-X-100-soluble membranes (n=3). (D) Detection of ECD in conditioned medium. 293 cells were transfected with NRADD tagged at the N-terminus with AU1 (AU1 NR) or vector (Vect.). 18 hours after transfection PMA (100 ng ml-1) was added as indicated and, after 70 hours, medium was harvested and cell lysates prepared. Lysates and conditioned medium were PNG-F treated. Conditioned media were immunoprecipitated with anti-AU1 antibody and analysed by anti-AU1 western blot of a 16% Tricine gel. *NRADD truncated at the C-terminus.

View larger version (96K): [in a new window]   Fig. 3. Subcellular localization of NRADD. Cells were grown on gelatin-coated coverslips, stained with Hoechst dye 33342 (blue) and processed for immunocytochemistry with anti-NRADD antibody (green) and visualized by confocal microscopy. MEFs are mouse embryonic fibroblasts from day 16.5 embryos. Notice the common perinuclear staining (open arrowhead) in MEFs. The edge of the cells are also stained more prominently, particularly structures resembling lamellipodia (closed arrowhead). In the fibroblast line NIH3T3 and the neuroblastoma line N2a, additional staining of nuclear substructure is observed (arrows). All samples were stained in parallel with antigen-blocked antibody. Notice the complete lack of background in the nuclei.

View larger version (97K): [in a new window]   Fig. 9. Nuclear staining of NRADD requires -secretase. (A) N2a cells were treated with APMA or PMA for 40 minutes, washed and incubated for 3 hours in lactacystin and DAPT where indicated. Cells were fixed, immunostained with anti-NRADD antibody and visualized by confocal microscopy. Notice the diffuse nuclear staining in the presence of lactacystin alone. NRADD containing nuclear substructures are induced by treatment with APMA or PMA (arrows). Co-incubation with the -secretase inhibitor DAPT prevents the formation of these structures. (B) A series of confocal pictures show that the nuclear substructures localize within the DAPI-stained nuclei but do not localize with high-intensity DAPI subnuclear structures.

View larger version (60K): [in a new window]   Fig. 4. NRADD is proteolytically processed. NRADD-MycHis was transfected into CHO lines. (A) Cells were incubated for 24 hours with the compounds as indicated. Cell lysates were analysed by western blot with anti-NRADD antibody. (B) The same as (A) except that the indicated lysates were also digested with PNG-F. NpNR, N-terminally processed NRADD.

View larger version (31K): [in a new window]   Fig. 5. Truncated NRADD is a -secretase substrate. (A) The GV constructs. The amino acid numbering refers to wild-type NRADD. The two glycosylation sites are indicated in the extracellular domain (ECD) of FL-NRGV. (B) Expression of the GV constructs in 293 cells. The western blot was probed with anti-NRADD antibody. (C) Luciferase assay in N2a cells. Cells were co-transfected with GV constructs, Gal4 luciferase reporter and renilla luciferase-TK reporter to normalize for transfection efficiency. The -secretase inhibitor L-685,458 (10 µM) was added 5 hours after transfection. Cells were lysed 48 hours after transfection. Dual luciferase assays were performed and the fold induction calculated. Error bars represent 95% confidence limit. (D) The same lysates were analysed by western blot either before or after N-glycanase digestion. End., endogenous NRADD; Exo., transfected NRADD.

View larger version (25K): [in a new window]   Fig. 6. Genetic and chemical inhibition of NRADD processing by -secretase. (A) Wild-type (+/+) and PS1,2 double knockout (-/-) cells were transfected with the three NRGV constructs. Luciferase assays were performed as follows. Cells were co-transfected with GV constructs, Gal4 luciferase reporter and renilla luciferase-TK reporter to normalize for transfection efficiency. The -secretase inhibitor L-685,458 (10 µM) was added 5 hours after transfection. Cells were lysed 48 hours after transfection. Dual luciferase assays were performed and the fold induction calculated. (B) Cell lysates were analysed by western blot with anti-NRADD antibody. (C) N2a cells were transfected with NR45GV and incubated with various concentrations of the -secretase inhibitors DAPT or compound E. Luciferase activities were normalized to the activation obtained without inhibitor. Expression of NR45GV was monitored by western blot of the cell lysates (bottom). All error bars represent 95% confidence limit.

View larger version (72K): [in a new window]   Fig. 7. Presenilins affect NRADD maturation. (A) Lysates from wild-type (+/+) or PS1,2 double knockout (-/-) cells were analysed by western blot with anti-NRADD antibody. Protein loading was determined by probing with anti-GAPDH. m+NR, mature NRADD of higher molecular weight. One of four experiments shown. The independently blastocyst derived clonal lines are designated as in Sato et al. (Sato et al., 2000). NRADD and PS2 were co-transfected into 293 cells and pulse-chase experiment was performed as in Fig. 1A (one of three experiments shown). mglNR indicates an NRADD species migrating with N4Q single glycosylation mutant (Wang et al., 2003). (C) In vitro deglycosylation. FLAG-NRADD was transfected alone or together with PS2 in 293 cells. Cell lysates (lanes 1-4) were immunoprecipitated with FLAG beads and either mock treated (lanes 1,3) or treated with PNG-F (lanes 2,4) before western-blot analysis. Lanes 5, 6 were loaded with cell lysates produced in the absence or presence of tunicamycin, respectively (n=4). (D) NRADD was co-transfected with the indicated presenilin constructs and cell lysates prepared 48 hours later. Lysates were analysed on anti-NRADD, anti-PS2 and anti-PS1 western blots. +, * and -indicate aggregated, full-length and processed forms of PS, respectively (n=5).