Introduction We recently described neurotrophin receptor alike death domain protein (NRADD; PLAIDD/NRH2) as a death-receptor-like protein that causes apoptosis upon expression in neuronal cells (Wang et al., 2003). This protein is highly conserved across mammalian species and has extensive homology to the cytoplasmic regions of p75 NTR but retains a unique N-terminal ectodomain. This ectodomain is N-glycosylated and its deletion leads to a dominant negative form that protects cells from death induced by a range of endoplasmic reticulum (ER) stressors. Our studies showed that the NRADD ectodomain is required for subcellular localization and apoptotic function. Whether the short ectodomain creates enough of a surface to bind a specific ligand is not currently known. Alternate mechanisms of NRADD activation (such as proteolytic processing or association with other ligand-binding receptors) could also be operating. Further, the lack of homotypic interaction partners for p75 NTR -like death domains also suggest an intracellular signaling cascade differing from that of other death receptors (reviewed by Hempstead, 2002). Regulated intramembrane proteolysis (RIP) is one way by which transmembrane proteins are cleaved to release cytosolic domains from the membrane that are biologically active (Brown et al., 2000). Two sequential cleavages occur during RIP. The first regulated hydrolysis sheds the ectodomain and results in a substrate that is then processed by the second cleavage within the transmembrane domain. An intracellular domain is released that, in many instances, transduces nuclear signals (Cao and Sudhof, 2001) (reviewed by Ebinu and Yankner, 2002). Intramembrane cleaving proteases (I-CLiPs) have catalytic site motifs embedded in predicted transmembrane regions. Presenilins (PSs), a component of the γ-secretase, are I-CLiPs that were initially shown to cleave APP and Notch (De Strooper et al., 1999; De Strooper et al., 1998). Subsequently, other type-I transmembrane proteins have been found to be RIP substrates, including Notch 1-4, the Notch ligands Jagged and Delta, APP, APLP1, APLP2, ErbB- 4, E-Cadherin, N-Cadherin, CD44, α-Nectin, LRP (Martoglio and Golde, 2003) and, more recently, DCC (Taniguchi et al., 2003), ApoER2 (May et al., 2003), p75 NTR (Kanning et al., 2003) and Syndecan 3 (Schulz et al., 2003). The cytoplasmic domains released from these substrates do not contain a common signaling motif and only p75 NTR has a death domain. Among these substrates, only RIP of ErB-4 has been shown to contribute directly to cell death (Ni et al., 2003). Additional substrates are likely to exist because the γ-secretase cleavage requires only an ectodomain stump of 50 amino acids or less but no consensus sequences (Struhl and Adachi, 2000). Mutations in the human genes encoding presenilins 1 and 2 (PS1 and PS2) are the main cause of familial early-onset Alzheimer’s disease (FAD). PSs are components of γ-secretase and have been shown to be tightly associated with Nicastrin (Yu et al., 2000), APH-1 (Goutte et al., 2002) and Pen-2 (Francis et al., 2002). Together, these form the currently known minimal essential components of the active γ-secretase complex (Edbauer et al., 2003; Hu and Fortini, 2003; Kimberly 4099 Neurotrophin receptor alike death domain protein (NRADD) is a death-receptor-like protein with a unique ectodomain and an intracellular domain homologous to p75 NTR . Expression of NRADD results in apoptosis, but only in certain cell types. This paper characterizes the expression and proteolytic processing of the mature 55 kDa glycoprotein. N-terminally truncated NRADD is processed by a γ-secretase activity that requires presenilins and has the same susceptibility to γ-secretase inhibitors as the secretion of amyloid β (Aβ). The ectodomain of endogenous NRADD is shed by activation of metalloproteinases. Inhibitor studies provide evidence that NRADD is cleaved in two steps typical of regulated intramembrane proteolysis (RIP). Inhibition of γ-secretase abrogates both the production of the soluble intracellular domain of NRADD and the appearance of NRADD in subnuclear structures. Thus, solubilized death domains with close homology to p75 NTR might have a nuclear function. Furthermore, presenilin deficiency leads to abnormally glycosylated NRADD and overexpression of presenilin 2 inhibits NRADD maturation, which is dependent on the putative active site residue D366 but not on γ-secretase activity. Our results demonstrate that NRADD is an additional γ- secretase substrate and suggest that drugs against Alzheimer’s disease will need to target γ-secretase in a substrate-specific manner. Key words: γ-Secretase, Death domain, Metalloproteinases, NRADD, Presenilin Summary Release of a membrane-bound death domain by γ-secretase processing of the p75 NTR homolog NRADD Kavitha Gowrishankar 1 , Michael G. Zeidler 1 and Claudius Vincenz 2, * 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 *Author for correspondence (e-mail: vincenz@umich.edu) Accepted 7 April 2004 Journal of Cell Science 117, 4099-4111 Published by The Company of Biologists 2004 doi:10.1242/jcs.01263 Research Article