Cleavage of p75 Neurotrophin Receptor by α-Secretase and γ-Secretase Requires Specific Receptor Domains

The p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an α-secretase-mediated release of the extracellular domain followed by a γ-secretase-mediated intramembrane cleavage. However, the specific determinants of p75NTR cleavage events are unknown. Many other substrates of γ-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by γ-secretase. Using a series of deletion mutations and chimeric receptors of p75NTR and the related Fas receptor, we have identified domains that are essential for p75NTR proteolysis. The initial α-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75NTR, however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75NTR. Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75NTR displaying only 15 extracellular amino acids in the stalk region. The p75 neurotrophin receptor (p75NTR), a member of the tumor necrosis factor superfamily of receptors, undergoes multiple proteolytic cleavage events. These events are initiated by an α-secretase-mediated release of the extracellular domain followed by a γ-secretase-mediated intramembrane cleavage. However, the specific determinants of p75NTR cleavage events are unknown. Many other substrates of γ-secretase cleavage have been identified, including Notch, amyloid precursor protein, and ErbB4, indicating there is broad substrate recognition by γ-secretase. Using a series of deletion mutations and chimeric receptors of p75NTR and the related Fas receptor, we have identified domains that are essential for p75NTR proteolysis. The initial α-secretase cleavage was extracellular to the transmembrane domain. Unfortunately, deletion mutants were not capable of defining the requirements of ectodomain shedding. Although this cleavage is promiscuous with respect to amino acid sequence, its position with respect to the transmembrane domain is invariant. The generation of chimeric receptors exchanging different domains of noncleavable Fas receptor with p75NTR, however, revealed that a discrete domain above the membrane is sufficient for efficient cleavage of p75NTR. Mass spectrometric analysis confirmed the cleavage can occur with a truncated p75NTR displaying only 15 extracellular amino acids in the stalk region. The p75 neurotrophin receptor, p75NTR, is a member of the TNF 1The abbreviations used are: TNF, tumor necrosis factor; FasR, Fas receptor; CTF, C-terminal fragment; PMA, phorbol-12 myristate 13-acetate; HA, hemagglutinin; MALDI Q-TOF, matrix-assisted laser desorption ionization quadrupole time-of-flight; ICD, intracellular domain. receptor superfamily that includes the Fas (CD95) antigen, CD30, and CD40 (1Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1839) Google Scholar). This family of receptors is distinguished with multiple cysteine-rich domains for ligand binding, a single transmembrane sequence, and a noncatalytic cytoplasmic domain (2Locksley R. Killeen N. Lenardo M. Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3038) Google Scholar). X-ray crystallographic analyses show a great deal of similarity between the cysteine-rich repeats of p75NTR and the p55 TNF receptor (3Banner D.W. D'Arcy A. Janes W. Gentz R. Schoenfeld H-J. Broger C. Loetscher H. Lesslauer W. Cell. 1993; 73: 431-445Abstract Full Text PDF PubMed Scopus (990) Google Scholar, 4He X.-L. Garcia K. Science. 2004; 304: 870-875Crossref PubMed Scopus (242) Google Scholar). The intracellular region of several receptors, including the p55 TNF receptor, Fas receptor (FasR), and p75NTR, contains a death domain sequence (5Feinstein E. Kimchi A. Wallach D. Boldin M. Varfolomeev E. Trends Biochem. Sci. 1995; 20: 342-344Abstract Full Text PDF PubMed Scopus (271) Google Scholar). The death domain serves as a protein-protein docking site and is required for initiating TNF- and Fas-dependent cytotoxicity. Activation of TNF receptor members leads to the recruitment of proteins including TNF-associated factors, death domain-containing proteins TRADD (6Hsu H. Shu H-B. Pan M.-P. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar), FADD (7Chinnaiyan A.M. Tepper C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.E. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar), and RIP (8Stanger B.Z. Leder P. Lee T-H. Kim E. Seed B. Cell. 1995; 81: 513-524Abstract Full Text PDF PubMed Scopus (869) Google Scholar). p75NTR is recognized by all the neurotrophins (nerve growth factor, brain-derived nerve factor (BDNF), NT-3, and NT-4) that promote differentiation, growth, and survival of diverse cell types in the nervous system (9Huang E. Reichardt L. Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3398) Google Scholar, 10Chao M.V. Nat. Rev. Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1768) Google Scholar). In addition, neurotrophins also initiate signaling through tropomyosin-related kinase tyrosine kinase receptors, which are capable of forming high affinity binding sites with p75NTR that potentiate responses to low concentrations of neurotrophins (11Chao M.V. Hempstead B.L. Trends Neurosci. 1995; 19: 321-326Abstract Full Text PDF Scopus (778) Google Scholar). In the absence of tropomyosin-related kinase receptors, p75NTR is capable of independent signaling that activates NF-κB or c-Jun N-terminal kinase activity (12Hempstead B. Curr. Opin. Neurobiol. 2002; 12: 260-267Crossref PubMed Scopus (274) Google Scholar, 13Roux P. Barker P. Prog. Neurobiol. 2002; 67: 203-233Crossref PubMed Scopus (596) Google Scholar). In selected cell types, p75NTR can initiate cell death (14Frade J.M. Rodriguez-Tebar A. Barde Y-A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (668) Google Scholar, 15Casaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (719) Google Scholar, 16Bredesen D.E. Rabizadeh S. Trends Neurosci. 1997; 20: 287-291Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 17Bamji S. Majdan M. Pozniak C.D. Belliveau D.J. Aloyz R. Cohn J. Causing C.G. Miller F.D. J. Cell Biol. 1998; 140: 911-923Crossref PubMed Scopus (443) Google Scholar). Alternatively, p75NTR can serve as a co-receptor with Nogo receptor in blocking regeneration in the central nervous system (18Wang K. Kim J. Sivasankaran R. Segal R. He Z. Nature. 2002; 420: 74-78Crossref PubMed Scopus (719) Google Scholar, 19Wong S. Henley J. Kanning K. Huang K. Bothwell M. Poo M. Nat. Neurosci. 2002; 5: 1302-1308Crossref PubMed Scopus (398) Google Scholar, 20Bandtlow C. Dechant G. Sci. STKE 2004. 2004; 235: pe24Google Scholar). Intramembrane cleavage events have been described recently for p75NTR (21Kanning K. Hudson M. Amieux P. Wiley J. Bothwell M. Schecterson L. J. Neurosci. 2003; 23: 5425-5436Crossref PubMed Google Scholar, 22Jung K-M. Landman N. Tan S. Petrova K. Murray S. Lewis R. Kim P. Kim D. Ryu S. Chao M. Kim T.-W. J. Biol. Chem. 2003; 278: 42161-42169Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Proteolysis through regulated intramembrane proteolysis has emerged as a highly conserved mechanism in receptor signaling (23Fortini M. Nat. Rev. Mol. Cell Biol. 2002; 3: 673-684Crossref PubMed Scopus (345) Google Scholar, 24Ebinu J. Yankner B. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 25Wolfe M. Kopan R. Science. 2004; 305: 1119-1123Crossref PubMed Scopus (310) Google Scholar). Presenilin-dependent γ-secretase activity is responsible for the intramembrane proteolysis of an increasing number of membrane proteins, including Notch, ErbB4 tyrosine kinase receptors, CD44, low density lipoprotein, and β-amyloid precursor protein (24Ebinu J. Yankner B. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). γ-Secretase is a large protein complex with an unusual aspartyl protease activity that cleaves substrates within the transmembrane domain and requires presenilins and other components, such as nicastrin, Pen-2, and Aph-1 (26Iwatsubo T. Curr. Opin. Neurobiol. 2004; 14: 379-383Crossref PubMed Scopus (153) Google Scholar). Prior to γ-secretase cleavage, p75NTR undergoes extracellular cleavage events that release the ectodomain of the receptor. Previous studies indicated that the γ-secretase cleavage occurs in the middle of the transmembrane domain (22Jung K-M. Landman N. Tan S. Petrova K. Murray S. Lewis R. Kim P. Kim D. Ryu S. Chao M. Kim T.-W. J. Biol. Chem. 2003; 278: 42161-42169Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). However, the requirements for the initial α-secretase events remain unknown. In this study we defined the specific domains in p75NTR that are susceptible to α-secretase and subsequent γ-secretase cleavage. Analyses of a series of chimeric receptors identified a 15-amino acid region of p75NTR that is sufficient for inducing α-secretase cleavage. We also found that the initial α-secretase cleavage is required for the subsequent γ-secretase cleavage. Therefore, susceptibility to proteolytic cleavage is dependent upon specific regions and structural features of p75NTR. Materials—Phorbol-12 myristate 13-acetate (PMA) was purchased from Sigma. TAPI-1, GM 6001, clasto-lactacystin β-lactone (27Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar), and compound E (γ-secretase inhibitor XVIII) were purchased from Calbiochem. Plasmids—Deletion constructs Δ204–209, Δ210–215, and Δ216–221 in p75NTR were produced by site-directed mutagenesis using rat pCDNA3-p75NTR as a template. Deletion constructs of human pIRES-p75NTRΔ168–218 and Δ187–218 were from A. Le Bivic (Faculté des Sciences de Luminy, Marseille, France). The construction of p75NTR-Δ14 was described previously (22Jung K-M. Landman N. Tan S. Petrova K. Murray S. Lewis R. Kim P. Kim D. Ryu S. Chao M. Kim T.-W. J. Biol. Chem. 2003; 278: 42161-42169Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The deletion construct HA p75NTR-Δ15 lacking the entire extracellular domain except 15 amino acids proximal to the membrane was generated using a PCR strategy. Rat pCDNA3-p75NTR was used as a template, and the insert was cloned into pCDNA3.1 using XbaI and KpnI sites. The construction of the P1 and P2 chimeric receptors of p75NTR and the Fas receptor (called B9 and A7) was described previously (28Kong H. Kim A.H. Orlinick J.R. Chao M.V. Cell Death Differ. 1999; 6: 1133-1142Crossref PubMed Scopus (37) Google Scholar). Additional chimeras in this study were produced using a similar hybrid PCR-based strategy and appropriate pairs of primers. All of the constructs were verified by automated DNA sequencing. Cell Culture Treatments and Transfection—PC12 cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 10% horse serum supplemented with 2 mm glutamine. HEK293 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For transient transfection, Lipofectamine 2000 (Invitrogen) reagents were used according to the manufacturer's protocol. In cleavage experiments cells were treated for 45 min with 100 ng/ml PMA and overnight with 1 μm compound E, 20 μm TAPI-1, 10 μm GM 6001, and 5 μmclasto-lactacystin β-lactone. Cells were harvested 48 h after transfection, washed once with cold phosphate-buffered saline on ice, and lysed in radioimmune precipitation assay III buffer (10 mm Tris, pH 8, 1 mm EDTA, 150 mm NaCl, 2 1 and concentrations were by Cell cells were from rat J. K. M. PubMed Scopus Google Scholar). Cells were maintained in the of growth in Dulbecco's modified Eagle's medium supplemented with 10% fetal serum 2 mm 100 and 2 μm were as described previously N. P. R. B. of Scholar). were on using in the of medium modified Eagle's medium with 10% fetal bovine ng/ml nerve growth factor for 2 mm and mm The the were treated with medium medium and The were or medium for to and analysis were on or and protein to the were used in the intracellular domain of p75NTR and the Fas receptor the intracellular domain of of p75 cells were with using Lipofectamine 2000 (Invitrogen) and in Dulbecco's modified Eagle's the medium was by and overnight with 1 of affinity The affinity was by and washed with 20 of buffer mm Tris, pH NaCl, mm EDTA, and The protein was with pH The affinity proteins were by the using The for HA was by MALDI MALDI Mass was and in 5 of acid in The was using a The in was and in of MALDI The was by of in 1 of followed by the of 100 mm in a of The was the MALDI and MALDI were with a MALDI The was in with a of which the of after Mass were and using with of laser were the and the using appropriate for the has been for that p75NTR undergoes ectodomain P. D. PubMed Scopus Google Scholar, P. D. C. J. J. Neurosci. 1993; PubMed Google Scholar). cleavage of p75NTR the extracellular domain from an C-terminal The is by γ-secretase to to the p75NTR intracellular (21Kanning K. Hudson M. Amieux P. Wiley J. Bothwell M. Schecterson L. J. Neurosci. 2003; 23: 5425-5436Crossref PubMed Google Scholar, 22Jung K-M. Landman N. Tan S. Petrova K. Murray S. Lewis R. Kim P. Kim D. Ryu S. Chao M. Kim T.-W. J. Biol. Chem. 2003; 278: 42161-42169Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The cleavage of p75NTR has been followed in p75NTR is we cells from rat The receptor is in cells G. Chao M.V. PubMed Google Scholar, P. E. J. Neurosci. PubMed Google Scholar). with an the cytoplasmic domain of p75NTR revealed an 45 and with the p75NTR protein with a γ-secretase cleavage was also We were to of γ-secretase. γ-secretase can the intramembrane cleavage and an of the C-terminal of p75NTR. of the cells with the γ-secretase inhibitor compound E D. J. C. D. J. A. L. S. J. S. A. R. S. P. D. B. A. R. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in the of p75NTR C-terminal and the of the a between protein TAPI-1, a inhibitor of B. L. C. Biochem. J. 2001; PubMed Scopus Google Scholar), extracellular domain and γ-secretase cleavage, with such as PMA, of ectodomain of several receptors C. Nat. Rev. Mol. Cell Biol. 6: PubMed Scopus Google Scholar), of These experiments that the of the of the extracellular region is a to intramembrane proteolysis by γ-secretase. of the with clasto-lactacystin β-lactone to an of the by blocking its This that the is the of this cleavage cells and were for the proteolytic of p75NTR. similar of p75NTR was in and in cells with PMA and compound E the of of the and indicating that cleavage of p75NTR occurs in and cell the and the cleavage sites we an N-terminal p75NTR in HEK293 with PMA and compound E, p75NTR proteolytic were the the were with the the of the receptor not with the N-terminal HA The in and in the of PMA by with GM 6001, a broad inhibitor R. D. H. L. 1994; Google Scholar), that the of the extracellular domain is for intramembrane proteolysis by γ-secretase the α-secretase cleavage we produced a series of of the stalk domain of p75NTR that from the transmembrane domain to the cysteine-rich repeats of the extracellular domain. This region of amino acids and is in and of p75NTR on occurs in this domain C. A. A. L. A. E. J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar). with the N-terminal for of the of the receptor. We on the stalk domain for a number of according to the of the CTF, the cleavage is to occur in the of the transmembrane domain. the stalk domain proximal to the transmembrane domain is highly conserved proteolytic cleavage events by have been identified in the stalk domain of other We produced of amino acids Δ210–215, and that this conserved region. We also and All the mutants in HEK293 cells produced the as by a specific p75NTR a deletion that of the stalk domain except for amino acids was is to that the generated by the deletion constructs all with the that the site is the C-terminal deletion study found that the initial sequence of the transmembrane domain is also not for α-secretase cleavage G. J. L. J. Kim T. P. D. G. C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Alternatively, the cleavage site or the recognition site for α-secretase not by sequence by a to a from the The recognition site to the cleavage This mechanism is has been for several transmembrane proteins extracellular domain by such as amyloid precursor protein and J. C. J. Cell Sci. 1999; PubMed Google Scholar, M. M. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Mass the site by an independent we the truncated in the cell of of p75NTR in the extracellular was not to to the of the receptor in the We generated a p75NTR construct that all cysteine-rich repeats and a large of the HA was the of of the p75NTR construct in HEK293 cells produced a and with the of the and ICD, as by from cells were and using an HA affinity The were by and by MALDI The MALDI analysis different with of and to the of and The to the sequence of The were also with which confirmed the by MALDI was previously in deletion mutants and that in the stalk domain The cleavage sites that were in the that are different from the cleavage site by of the cleavage of p75NTR that cleavage after the amino acid sequence G. J. L. J. Kim T. P. D. G. C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), which is with is that the cleavage site is not by a specific sequence occurs an to the In this of cleavage by from the cleavage which domains are responsible for the initial cleavage of p75NTR, we generated a series of receptors between p75NTR and the FasR, a related member of the TNF receptor The is also a transmembrane receptor containing cysteine-rich domains followed by a stalk region and a single domain. In addition, the contains a death domain in the cytoplasmic region. We is in a similar to p75NTR The was in HEK293 cells and with an the intracellular domain of The Fas receptor however, other were after PMA or compound E These that is not an efficient substrate for α-secretase and γ-secretase proteolytic receptors were generated between the extracellular of the and the intracellular of p75NTR. The initial chimeric receptors the p75NTR transmembrane and cytoplasmic domains or only the cytoplasmic domain of p75NTR These chimeric receptors were in HEK293 cells with p75NTR. chimeric receptors were not as p75NTR The of by chimeras and was not to with not in the of not the extracellular and transmembrane domains of p75NTR were in α-secretase cleavage, domain was into the in the of a Fas containing the p75NTR extracellular domain a proteolytic of the of a produced from The is a Fas chimeric receptor the extracellular and transmembrane domains of p75NTR was a to γ-secretase cleavage was also and its was by compound E These that the extracellular and the transmembrane domains of p75NTR are and sufficient for and γ-secretase of the receptor. the domain of p75NTR that is susceptible to and γ-secretase we generated a chimeric receptor in which the transmembrane domain of the was into p75NTR We a high of produced from the receptor The of was by PMA not by compound E. The absence of an from the chimeric receptor is with the of the p75NTR transmembrane domain in the by γ-secretase. of was construct in which the p75NTR transmembrane domain was into produced cleavage indicating that the p75 transmembrane sequence was not sufficient to α-secretase cleavage we the stalk domains between the p75NTR the stalk domain of was and to PMA and compound E the other the stalk domain of p75NTR was in the the was The was also in other chimeras the p75NTR extracellular domain the sequence required for cleavage, we the 15 amino acids of p75NTR proximal to the transmembrane region to the region of the The was in the chimeric receptor The of the p75NTR was by GM indicating that a activity this the analysis of the chimeric receptors with the spectrometric analysis that the 15 amino acids of the stalk domain of p75NTR are and sufficient for inducing the extracellular of the receptor. this is required for of p75NTR by γ-secretase. intramembrane proteolysis is a conserved mechanism that has been to intracellular signaling events. transmembrane protein substrates for γ-secretase cleavage have been identified, an of the by which intramembrane and proteins is Previous studies that ectodomain is responsible for transmembrane cleavage G. A. Mol. Cell. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the substrate requirements for diverse membrane proteins are not The from this study that p75NTR lacking the of the extracellular domain serve as efficient substrates for γ-secretase is that a p75 that the cysteine-rich is also by α-secretase activity (21Kanning K. Hudson M. Amieux P. Wiley J. Bothwell M. Schecterson L. J. Neurosci. 2003; 23: 5425-5436Crossref PubMed Google Scholar). The extracellular domain of is and not have with p75NTR. is a of not only α-secretase also γ-secretase is that the of α-secretase not to upon a specific amino acid in Deletion mutants in p75NTR that α-secretase is in the receptor large of the stalk domain are G. J. L. J. Kim T. P. D. G. C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We have confirmed with in above the transmembrane domain. we found that the of the and the produced by deletion are indicating that the α-secretase is a structural of the receptor for its proteolytic However, the structural requirements for α-secretase and γ-secretase of sequence related TNF receptor such as the Fas receptor, not cleavage p75NTR, the similarity in structural features between the of chimeric receptors indicated that the stalk and transmembrane domains of the receptors in α-secretase and γ-secretase cleavage. 15-amino acid sequence in the stalk domain of p75NTR was sufficient to ectodomain of the the transmembrane sequence of p75NTR as a substrate for γ-secretase the transmembrane of the stalk and transmembrane p75NTR to cleavage. of chimeric receptors with indicated that not for the of p75NTR and a similar chimeric receptor experiments there are also sequence requirements for efficient cleavage. of p75NTR by regulated intramembrane proteolysis is of for a number of of the cleavage serve multiple The release of the extracellular domain a binding protein for including and the P. Barker P. Prog. Neurobiol. 2002; 67: 203-233Crossref PubMed Scopus (596) Google Scholar, G. Barde Y-A. Nat. Neurosci. 2002; 5: PubMed Scopus Google Scholar). the domain of p75NTR has the of binding intracellular proteins, including TNF-associated factor and (12Hempstead B. Curr. Opin. Neurobiol. 2002; 12: 260-267Crossref PubMed Scopus (274) Google Scholar, P. Neuron. 2004; Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The p75NTR cytoplasmic domain proteins to in different The cleavage of the also to a the of which is that is to the generated from amyloid precursor that p75NTR cleavage is is that cell types p75 receptors or For after there is of p75NTR in P. M. Barker P. T. J. Neurosci. 1999; 19: PubMed Google Scholar). of p75NTR has been in cell types, including and cells M.V. Nat. Rev. Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1768) Google Scholar, P. Neuron. 2004; Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The and cleavage of p75NTR in that are to γ-secretase cleavage of amyloid precursor protein (24Ebinu J. Yankner B. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google that p75NTR cleavage to other events is that neurotrophin of p75NTR have a upon the cleavage or upon the of γ-secretase W. In addition, the responsible for the α-secretase-mediated cleavage of p75NTR have not been members of the and family are G. J. L. J. Kim T. P. D. G. C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. P. K. S. Lee D. W. B. R. J. R. N. C. M. C. D. R. C. R. Science. 1998; PubMed Scopus Google Scholar). The number of substrates cleavage the of are recognized by γ-secretase. the and the cleavage events of p75NTR using receptor to proteolysis an to defining the of events. We for deletion mutants of p75NTR and and for with