Regulated Intramembrane Proteolysis of the p75 Neurotrophin Receptor Modulates Its Association with the TrkA Receptor

Abstract

The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent γ-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including β-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for γ-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that γ-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid β-peptide 40 (Aβ40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that γ-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that γ-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction. The generation of biologically active proteins by regulated intramembrane proteolysis is a highly conserved mechanism in cell signaling. Presenilin-dependent γ-secretase activity is responsible for the intramembrane proteolysis of selected type I membrane proteins, including β-amyloid precursor protein (APP) and Notch. A small fraction of intracellular domains derived from both APP and Notch translocates to and appears to function in the nucleus, suggesting a generic role for γ-secretase cleavage in nuclear signaling. Here we show that the p75 neurotrophin receptor (p75NTR) undergoes presenilin-dependent intramembrane proteolysis to yield the soluble p75-intracellular domain. The p75NTR is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by forming a heteromeric co-receptor complex with the Trk receptors. Mass spectrometric analysis revealed that γ-secretase-mediated cleavage of p75NTR occurs at a position located in the middle of the transmembrane (TM) domain, which is reminiscent of the amyloid β-peptide 40 (Aβ40) cleavage of APP and is topologically distinct from the major TM cleavage site of Notch 1. Size exclusion chromatography and co-immunoprecipitation analyses revealed that TrkA forms a molecular complex together with either full-length p75 or membrane-tethered C-terminal fragments. The p75-ICD was not recruited into the TrkA-containing high molecular weight complex, indicating that γ-secretase-mediated removal of the p75 TM domain may perturb the interaction with TrkA. Independent of the possible nuclear function, our studies suggest that γ-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of the p75-TrkA receptor complex by regulating the availability of the p75 TM domain that is required for this interaction. The p75 neurotrophin receptor (p75NTR) 1The abbreviations used are: p75NTR, p75 neurotrophin receptor; APP, β-amyloid precursor protein; Aβ, amyloid β-peptide; TM, transmembrane; ICD, intracellular domain; PS, presenilins; NGF, nerve growth factor; HA, hemagglutinin; PBS, phosphate-buffered saline;MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TPA, 12-O-tetradecanoylphorbol-13-acetate. is a multifunctional type I membrane protein that promotes neurotrophin-induced neuronal survival and differentiation by regulating the specificity and affinity of the Trk receptors (for review, see Refs. 1Chao M.V. Nat. Rev. Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1768) Google Scholar, 2Huang E.J. Reichardt L.F. Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3398) Google Scholar, 3Hempstead B.L. Curr. Opin. Neurobiol. 2002; 12: 260-267Crossref PubMed Scopus (274) Google Scholar, 4Dechant G. Barde Y.-A. Nat. Neurosci. 2002; 5: 1131-1136Crossref PubMed Scopus (482) Google Scholar). Recent studies demonstrate that the p75NTR participates in more diverse biological events including cell death, migration, and axonal elongation. Given a widely postulated role in neuronal cell survival, the p75NTR is also thought to be associated with many neurodegenerative diseases, including Alzheimer's disease (reviewed in Ref. 5Sofroniew M.V. Howe C.L. Mobley W.C. Annu. Rev. Neurosci. 2001; 24: 1217-1281Crossref PubMed Scopus (1107) Google Scholar). For instance, the p75NTR is a marker for basal forebrain cholinergic neurons that undergo selective and severe degeneration in Alzheimer's disease (6Coyle J.T. Price D.L. DeLong M.R. Science. 1983; 219: 1184-1190Crossref PubMed Scopus (2618) Google Scholar, 7Auld D.S. Kornecook T.J. Bastianetto S. Quirion R. Prog. Neurobiol. 2002; 68: 209-245Crossref PubMed Scopus (591) Google Scholar). The precise mechanism as to how the p75NTR transmits diversified signaling in the normal or diseased nervous system, remains elusive. γ-Secretase is an unusual aspartyl protease that cleaves a growing number of substrates (8De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1808) Google Scholar, 9Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 10Lee H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 11Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Baki L. Wen P. Efthimiopoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (626) Google Scholar, 12Kim D.Y. Ingano L.A. Kovacs D.M. J. Biol. Chem. 2002; 277: 49976-49981Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), including APP, Notch, and ErbB4, within the predicted transmembrane domain (reviewed in Refs. 13Ebinu J.O. Yankner B.A. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 14Fortini M.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 673-684Crossref PubMed Scopus (345) Google Scholar, 15Strooper B.D. Annaert W. Nat. Cell Biol. 2001; 3: E221-E225Crossref PubMed Scopus (48) Google Scholar, 16Haass C. Steiner H. Trends Cell Biol. 2002; 12: 556-562Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 17Selkoe D. Kopan R. Annu. Rev. Neurosci. 2003; 26: 565-597Crossref PubMed Scopus (564) Google Scholar). Limited homology among the putative cleavage sites of known γ-secretase substrates indicates that the intramembrane cleaving γ-secretase activity is not dependent on a specific amino acid target sequence immediately adjacent to the cleavage site. The presenilins 1 (PS1) or PS2 are required for its activity (18De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar) and appear to be essential catalytic components in the heteromultimeric γ-secretase complex (19Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (781) Google Scholar, 20Kimberly W.T. LaVoie M.J. Ostaszewski B.L. Ye W. Wolfe M.S. Selkoe D.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6382-6387Crossref PubMed Scopus (684) Google Scholar, 21Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar). γ-Secretase-mediated intramembrane proteolysis recently emerged as a highly conserved mechanism in receptor signaling (13Ebinu J.O. Yankner B.A. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 14Fortini M.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 673-684Crossref PubMed Scopus (345) Google Scholar). A small fraction of intracellular domains (ICD) derived from APP, Notch, and ErbB4 translocates and appears to function in the nucleus, suggesting a generic role for γ-secretase cleavage in nuclear signaling (9Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 10Lee H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 22Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar, 23Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar, 24Baek S.H. Ohgi K.A. Rose D.W. Koo E.H. Glass C.K. Rosenfeld M.G. Cell. 2002; 110: 55-67Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 25Scheinfeld M.H. Ghersi E. Laky K. Fowlkes B.J. D'Adamio L. J. Biol. Chem. 2002; 277: 44195-44201Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, it is unclear whether γ-secretase cleavage plays a role in non-nuclear functions. Although γ-secretase is incapable of processing the full-length form of its substrates, it efficiently cleaves membrane-anchored truncated C-terminal derivatives produced by ectodomain shedding (26Struhl G. Adachi A. Mol. Cell. 2000; 6: 625-636Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Early reports showed that the truncated p75 extracellular domain that contains the NGF-binding region is released into the culture medium and biological fluids, indicating that the p75NTR undergoes ectodomain shedding (27DiStefano P.S. Johnson Jr., E.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 270-274Crossref PubMed Scopus (109) Google Scholar, 28Zupan A.A. Osborne P.A. Smith C.E. Siegel N.R. Leimgruber R.M. Johnson Jr., E.M. J. Biol. Chem. 1989; 264: 11714-11720Abstract Full Text PDF PubMed Google Scholar, 29Barker P.A. Miller F.D. Large T.H. Murphy R.A. J. Biol. Chem. 1991; 266: 19113-19119Abstract Full Text PDF PubMed Google Scholar, 30Zupan A.A. Johnson Jr., E.M. J. Biol. Chem. 1991; 266: 15384-15390Abstract Full Text PDF PubMed Google Scholar, 31DiStefano P.S. Chelsea D.M. Schick C.M. McKelvy J.F. J. Neurosci. 1993; 13: 2405-2414Crossref PubMed Google Scholar). We predicted that there may be cell-associated C-terminal proteolytic fragments resulting from ectodomain shedding of the full-length p75 receptor in addition to the corresponding extracellular domain that is released from the cell. Because extracellular cleavage of type I membrane proteins is a pre-requisite for their subsequent γ-secretase-mediated cleavage (26Struhl G. Adachi A. Mol. Cell. 2000; 6: 625-636Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), we set out to examine whether the p75NTR could be subjected to γ-secretase-mediated proteolysis. Here we report that p75NTR is a novel substrate of the presenilin-dependent γ-secretase and that intramembrane proteolysis of the p75NTR may play a role in the formation or disassembly of the neurotrophin receptor complex containing the p75NTR and Trk receptors. Plasmids—To generate constructs encoding C-terminal V5/His-tagged p75NTR and Trk receptors, coding sequences from the pCMV constructs (32Perez P. Coll P.M. Hempstead B.L. Martin-Zanca D. Chao M.V. Mol. Cell. Neurosci. 1995; 6: 97-105Crossref PubMed Scopus (89) Google Scholar) coding for the full-length human p75NTR, rat TrkA, rat TrkB, or rat TrkC were amplified by PCR using High Fidelity PCR Master (Roche Applied Science) and subcloned into pEF-V5/His vector using TOPO cloning kit (Invitrogen). To generate constructs encoding truncated p75 lacking the entire ectodomain (p75-ΔE14), the following primers were used: a 5′ primer consisting of the KpnI restriction site fused to Kozak and start codons followed by the preprotrypsin signal peptide, HA recognition sequence, and the 5′ region of p75 consisting of the first extracellular amino acid The primer of the region of p75 of the followed by an site The encoding full-length rat p75 with the signal followed by the and HA was by PCR using signal and full-length rat p75 as The primers used were and the sequence PCR were with and subcloned into signal and the full-length rat p75 constructs were by Cell and were in medium with and (Invitrogen). For or were used to the cell were by with the or and by using The was by with and that a at the C-terminal of TrkA. and were by using and was and by T. A. and C. were using with the protease (Roche Applied or and analyses were out as H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). were used at the following and of p75 from p75 or were subjected to affinity using as by the were with for at and with proteins were with were using in and subjected to and of p75-ICD and Mass membrane and generation of the p75-ICD were as H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). from of with were at the soluble p75-ICD was by of the at for The were a with at a of 1 proteins were with a of to and were by using an containing p75-ICD were and by using affinity to the the affinity proteins by analysis and using kit were and on The protein corresponding to p75-ICD was subjected to by and by as Kim Kim Y. S.H. J.S. G. S.H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of by was with of p75 fragments. were for at with in PBS, with PBS, and for at with normal in were at with the following in normal in in normal in or were used for were and were using an with of were with and or with protease The were at for and the were with of protein at The were with and of protein at The were with with and by Size were with and with a protease The were at for and the were and using a that was with were and by The protein were as and shedding of many cell receptors to be by with as J. J. Cell Sci. 1999; PubMed Google Scholar). We first whether ectodomain shedding of the full-length p75NTR truncated C-terminal fragments that may as putative substrate fragments of of the with in p75NTR C-terminal fragments However, not of fragments. Cell studies that fragments are associated with the membrane not To the soluble p75 extracellular domain, were with constructs encoding the p75NTR with an affinity released p75 ectodomain was in the medium and by of the soluble p75 ectodomain was efficiently by a indicating that a as is responsible for p75 ectodomain the p75-ICD to fragments in the medium not We whether the C-terminal fragments derived from ectodomain shedding are to γ-secretase that γ-secretase of substrate fragments by γ-secretase-mediated (9Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (759) Google Scholar, 10Lee H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar). with a γ-secretase D. C.M. F. Jr., L.A. R.A. D.W. B. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), the of the p75 C-terminal fragments that were by The of full-length p75NTR were not by the γ-secretase of a of p75 C-terminal fragments in the of suggesting that both ectodomain shedding and subsequent γ-secretase cleavage in the γ-secretase M.S. D. T. P. A. Smith G. 2000; PubMed Scopus Google Scholar), to not However, γ-secretase not on C-terminal in with constructs encoding TrkA, TrkB, or TrkC Although γ-secretase to the of putative substrate fragments of the predicted intracellular domain derived from p75NTR was not by suggesting that the p75-ICD is in the We to the p75-ICD by a that to of Notch intracellular domain E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar, C. J. A. E. M. U. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of with the of fragments of p75 fragments could be efficiently by with indicating that this the including and the of the not on p75-ICD The of and on the p75-ICD were at 1 not that p75-ICD is To demonstrate a generation of p75-ICD from p75 C-terminal fragments we a which a truncated p75 protein that the entire ectodomain contains the entire and transmembrane domains of the p75NTR immediately following the signal sequence the proteins are predicted to undergo cleavage by γ-secretase in the of ectodomain to the Notch constructs E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar). The p75-ICD was in with constructs the in indicating that the p75-ICD is from the truncated To demonstrate γ-secretase-mediated cleavage of p75NTR, we cholinergic A. Lee J. Neurosci. 12: PubMed Google Scholar). in the p75-ICD was and in the of the of p75 C-terminal fragments and subsequent generation of p75-ICD in show that the p75-ICD is produced in We whether presenilins are required for γ-secretase cleavage of the For this we first the of the p75 C-terminal fragments in the the forms of and PS2 H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The in conserved and the biological of and including γ-secretase cleavage M.S. W. Ostaszewski B.L. W.T. Selkoe D.J. Nature. 1999; 398: PubMed Scopus Google Scholar, H. K. A. H. M.G. S. J. X. M. K. M. Kopan R. Pesold B. S. M. Tomita T. Iwatsubo T. R. Haass C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the p75 C-terminal fragments was as with cell of forms of and PS2 the of p75 C-terminal fragments in indicating that γ-secretase-mediated of the p75 C-terminal fragments presenilins in the of the p75 C-terminal fragments were also in lacking both and PS2 not To demonstrate presenilin-dependent generation of we the generation of p75-ICD from membrane from either or presenilins by at in the or of E. The generation of p75-ICD was in the membrane from and by the cell membrane with in that presenilin-dependent γ-secretase cleavage is responsible for the generation of p75-ICD and of this cleavage to of p75 C-terminal substrate fragments. Because the p75-ICD is in to of the p75-ICD for the major γ-secretase cleavage we a γ-secretase H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) using membrane from the p75NTR with C-terminal and affinity were on a and subjected to in γ-secretase cleavage The p75-ICD fragments containing were in a γ-secretase and a of the p75-ICD was in the soluble fraction The from and in on indicating that the p75-ICD in the soluble fraction is to an to the p75-ICD in The soluble fraction was and subjected to using and for the p75-ICD were on and The corresponding to the p75-ICD were subjected to and analysis Mass analysis revealed a major of molecular of as as many derived from the p75-ICD were in our analysis The major and a were at and which to the sequences of and were for and for start at of the human p75NTR and indicating that the p75-ICD is as a of proteolysis amino and the major cleavage site of the p75NTR is located immediately the amino acid sequence to the cleavage site that immediately following an sequence that the major intramembrane cleavage site of the p75NTR is located in the middle of the transmembrane domain that is reminiscent of the cleavage of APP and is topologically distinct from the major transmembrane cleavage site of Notch 1 E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1365) Google Scholar) or from the site of APP Y. H. T. N. K. Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Steiner H. K. A. G. Haass C. EMBO 2001; PubMed Scopus Google Scholar, A. S. M. K. G. C.L. K. G. 2002; PubMed Scopus Google sequence of the by are in the amino acid in full-length of not are in the amino acid in full-length not in a of the of the p75NTR is to as a co-receptor for by forming a high affinity heteromeric receptor complex with Trk receptors D. P. R.M. P. Chao M.V. Hempstead B.L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the transmembrane domain and of the p75NTR appear to be the essential for its interaction with the Trk receptor M. E. Barde EMBO J. 1999; PubMed Scopus Google Scholar) as as for forming a high affinity receptor D. P. R.M. P. Chao M.V. Hempstead B.L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we postulated that the extracellular intramembrane cleavage of the p75NTR subsequent domain may its interaction with Trk We first whether TrkA with truncated p75NTR produced by constructs were with TrkA constructs C-terminal with constructs encoding vector not p75 the TM and domains not the extracellular domain; and p75-ICD predicted γ-secretase revealed that both full-length p75NTR and truncated are in and an with TrkA the p75-ICD was in the cell and appears to be in the as by nuclear The of p75-ICD was from that of TrkA. with to the by the of of TrkA and to the as as the membrane We whether TrkA with truncated p75NTR by constructs were with TrkA constructs with the p75 constructs and A full-length ErbB4 was as a H.-J. Jung K.-M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.-W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The were subjected to co-immunoprecipitation analyses using or for the co-immunoprecipitation full-length forms of TrkA and p75NTR M. E. Barde EMBO J. 1999; PubMed Scopus Google Scholar). full-length and were by indicating that TrkA with both p75NTR full-length and the predicted C-terminal fragments that the p75NTR C-terminal fragments may be with TrkA and are predicted to as a high affinity receptor D. P. R.M. P. Chao M.V. Hempstead B.L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). the p75-ICD and ErbB4 to with TrkA, indicating that TrkA with membrane-tethered p75NTR not with the suggest that γ-secretase cleavage the TM domain that is required for the of p75NTR with the Trk To the molecular interaction TrkA and p75 proteolytic we exclusion analyses using the from TrkA and full-length To the generation and of the p75 C-terminal fragments and p75-ICD from the full-length the were with and in the or of E. The full-length p75NTR and TrkA in exclusion chromatography and the p75 C-terminal fragments an with TrkA A and The molecular of the high molecular weight TrkA and p75 full-length or C-terminal fragments was as the and p75-ICD as a molecular weight indicating that the p75-ICD is not recruited into the high molecular weight the with TPA, or not the of TrkA and full-length p75NTR in the exclusion chromatography not We showed that p75NTR is the γ-secretase substrate that undergoes intramembrane proteolysis at the site corresponding to the Although it is possible that the γ-secretase in that were not in our our studies that the cleavage in the middle of the TM domain was to the of the a for cleavage the of the as or Notch cleavage Given a widely postulated role for neurotrophin receptors in cholinergic neuronal survival, our studies the APP, proteolytic processing of p75NTR may to the selective degeneration of neurons in Alzheimer's disease basal forebrain cholinergic (6Coyle J.T. Price D.L. DeLong M.R. Science. 1983; 219: 1184-1190Crossref PubMed Scopus (2618) Google Scholar, 7Auld D.S. Kornecook T.J. Bastianetto S. Quirion R. Prog. Neurobiol. 2002; 68: 209-245Crossref PubMed Scopus (591) Google Scholar). it is that on of γ-secretase activity may p75 signaling and cholinergic neuronal survival and The intracellular domains derived from APP and Notch appear to function as nuclear signaling remains to be whether the p75-ICD also function as a nuclear Notch and suggest that the role of the γ-secretase is to the p75 TM domain that is an essential domain for molecular interaction with the Trk receptor γ-Secretase-mediated of p75NTR from membrane-tethered p75 fragments to p75-ICD may the p75 is to TrkA. p75 ectodomain shedding may Trk receptors and cleavage by γ-secretase the membrane-tethered p75 interaction with Trk Independent of the possible nuclear function, our studies suggest that γ-secretase-mediated p75NTR proteolysis plays a role in the formation/disassembly of this receptor complex by regulating the availability of the TM domain that is required for interaction with the Trk receptors. We B. for and B. L. Hempstead for