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• W2074701223 abstract "The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts through its cytoplasmic tail with adaptor and scaffold proteins that participate in cellular signaling. Its extracellular domain, like that of the signaling receptor Notch and of amyloid precursor protein (APP), is proteolytically processed at multiple positions. This similarity led us to investigate whether LRP, like APP and Notch, might also be cleaved at a third, intramembranous or cytoplasmic site, resulting in the release of its intracellular domain. Using independent experimental approaches we demonstrate that the cytoplasmic domain is released by a γ-secretase-like activity and that this event is modulated by protein kinase C. Furthermore, cytoplasmic adaptor proteins that bind to the LRP tail affect the subcellular localization of the free intracellular domain and may regulate putative signaling functions. Finally, we show that the degradation of the free tail fragment is mediated by the proteasome. These findings suggest a novel role for the intracellular domain of LRP that may involve the subcellular translocation of preassembled signaling complexes from the plasma membrane. The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts through its cytoplasmic tail with adaptor and scaffold proteins that participate in cellular signaling. Its extracellular domain, like that of the signaling receptor Notch and of amyloid precursor protein (APP), is proteolytically processed at multiple positions. This similarity led us to investigate whether LRP, like APP and Notch, might also be cleaved at a third, intramembranous or cytoplasmic site, resulting in the release of its intracellular domain. Using independent experimental approaches we demonstrate that the cytoplasmic domain is released by a γ-secretase-like activity and that this event is modulated by protein kinase C. Furthermore, cytoplasmic adaptor proteins that bind to the LRP tail affect the subcellular localization of the free intracellular domain and may regulate putative signaling functions. Finally, we show that the degradation of the free tail fragment is mediated by the proteasome. These findings suggest a novel role for the intracellular domain of LRP that may involve the subcellular translocation of preassembled signaling complexes from the plasma membrane. Seven structurally closely related cell surface receptors constitute the core of the low density lipoprotein (LDL) 1The abbreviations used are: LDLlow density lipoproteinLRPLDL receptor-related proteinVLDLvery low density lipoproteinapo-ER2apolipoprotein E receptor 2APPamyloid precursor proteinICDintracellular domainPKCprotein kinase CPMAphorbol 12-myristate 13-acetateDAPTN-[N-(3,5,-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butyl esterVP16viral protein 16JNKc-Jun amino-terminal kinaseDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumMOPS4-morpholinepropanesulfonic acidPS1presenilin-1wtwild typePTBphosphotyrosine binding 1The abbreviations used are: LDLlow density lipoproteinLRPLDL receptor-related proteinVLDLvery low density lipoproteinapo-ER2apolipoprotein E receptor 2APPamyloid precursor proteinICDintracellular domainPKCprotein kinase CPMAphorbol 12-myristate 13-acetateDAPTN-[N-(3,5,-difluorophenacetyl)-l-alanyl]-S-phenylglycine-t-butyl esterVP16viral protein 16JNKc-Jun amino-terminal kinaseDMEMDulbecco's modified Eagle's mediumFCSfetal calf serumMOPS4-morpholinepropanesulfonic acidPS1presenilin-1wtwild typePTBphosphotyrosine binding receptor gene family. They include the LDL receptor, the LDL receptor-related protein (LRP or LRP1), LRP1b, megalin, very low density lipoprotein (VLDL) receptor, apolipoprotein E receptor 2 (apo-ER2), and MEGF7 (multiple epidermal growth factor-like domains containing protein 7) (1Herz J. Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Although the members of this evolutionarily ancient gene family all share the same typical arrangement of protein modules in their extracellular domains, the biological functions of the individual members of the family are highly diverse and include roles in cellular ligand uptake and endocytosis, the transmission of extracellular signals, vitamin and cholesterol homeostasis, brain development, the modulation of neurotransmission, and protection from neurodegeneration (1Herz J. Neuron. 2001; 29: 571-581Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 2Willnow T.E. Nykjaer A. Herz J. Nat. Cell Biol. 1999; 1: E157-E162Crossref PubMed Scopus (185) Google Scholar).Of this entire receptor family, LRP interacts with by far the largest number of proteins (3Herz J. Strickland D.K. J. Clin. Invest. 2001; 108: 779-784Crossref PubMed Scopus (875) Google Scholar). Ligands that bind to its extracellular domain include, for instance, α2-macroglobulin, plasminogen activators, clotting factors, lipases, and the amyloid precursor protein (APP). The cytoplasmic tail of LRP also interacts with an extended set of intracellular adaptor and scaffold proteins, e.g. Dab1, c-Jun amino-terminal kinase interacting proteins, the postsynaptic density protein PSD-95 (4Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), and the trivalent scaffold protein FE65 (5Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). The latter protein contains two phosphotyrosine binding domains, of which the first binds to the LRP tail, whereas the second domain interacts with an NP XY motif in the cytoplasmic tail of APP (6Fiore F. Zambrano N. Minopoli G. Donini V. Duilio A. Russo T. J. Biol. Chem. 1995; 270: 30853-30856Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 7Bressler S.L. Gray M.D. Sopher B.L., Hu, Q. Hearn M.G. Pham D.G. Dinulos M.B. Fukuchi K. Sisodia S.S. Miller M.A. Disteche C.M. Martin G.M. Hum. Mol. Genet. 1996; 5: 1589-1598Crossref PubMed Google Scholar, 8Guenette S.Y. Chen J. Jondro P.D. Tanzi R.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10832-10837Crossref PubMed Scopus (149) Google Scholar, 9Borg J.P. Ooi J. Levy E. Margolis B. Mol. Cell. Biol. 1996; 16: 6229-6241Crossref PubMed Scopus (430) Google Scholar).Mice in which the LRP gene has been disrupted by homologous recombination die early during embryonic development (10Herz J. Clouthier D.E. Hammer R.E. Cell. 1992; 71: 411-421Abstract Full Text PDF PubMed Scopus (506) Google Scholar). The phenotype of the few malformed LRP-deficient embryos that survive until around E9–E10 is complex and difficult to explain by defects in ligand endocytosis alone, suggesting that LRP, like the VLDL receptor and the apo-ER2 (11Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar), could have essential developmental signaling functions that involve interactions of its cytoplasmic tail with the intracellular signal transduction machinery.LRP is a type I integral membrane protein, like the other members of the family. However, only LRP and its closest relative, LRP1b, are cleaved at a site that matches the consensus sequence recognized by furin (12Willnow T.E. Moehring J.M. Inocencio N.M. Moehring T.J. Herz J. Biochem. J. 1996; 313: 71-76Crossref PubMed Scopus (108) Google Scholar, 13Liu C.X., Li, Y. Obermoeller-McCormick L.M. Schwartz A.L. Bu G. J. Biol. Chem. 2001; 276: 28889-28896Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), a processing proteinase that frequently serves to activate or modify cell surface receptors and secreted proteins upon exit from the secretory pathway (14Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Crossref PubMed Scopus (577) Google Scholar). Furin cleavage of the 600-kDa LRP precursor protein produces the mature cell-surface receptor, which consists of a carboxyl-terminal 85-kDa fragment and a non-covalently attached 515-kDa amino-terminal subunit.Notably, furin also cleaves members of the Notch family of cell surface signaling proteins, which, like LRP, contain multiple EGF repeats in their extracellular domain (15Blaumueller C.M., Qi, H. Zagouras P. Artavanis-Tsakonas S. Cell. 1997; 90: 281-291Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 16Logeat F. Bessia C. Brou C. LeBail O. Jarriault S. Seidah N.G. Israel A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8108-8112Crossref PubMed Scopus (572) Google Scholar). Additionally, Notch proteins undergo another metalloproteinase-mediated processing step at the cell surface (17Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israel A. Mol. Cell. 2000; 5: 207-216Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar, 18Mumm J.S. Schroeter E.H. Saxena M.T. Griesemer A. Tian X. Pan D.J. Ray W.J. Kopan R. Mol. Cell. 2000; 5: 197-206Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar), which is followed by the presenilin/γ-secretase-dependent release of their intracellular domains (ICD) (19Lecourtois M. Schweisguth F. Curr. Biol. 1998; 8: 771-774Abstract Full Text Full Text PDF PubMed Google Scholar, 20Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 47De 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 (1787) Google Scholar). Notch-ICD translocates to the nucleus where it stimulates expression of target genes through interaction with transcription factors of the CSL family (21Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4856) Google Scholar).Interestingly, shedding of LRP from the cell surface has also been reported (22Quinn K.A. Grimsley P.G. Dai Y.P. Tapner M. Chesterman C.N. Owensby D.A. J. Biol. Chem. 1997; 272: 23946-23951Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and it, too, involves a metalloproteinase (23Quinn K.A. Pye V.J. Dai Y.P. Chesterman C.N. Owensby D.A. Exp. Cell Res. 1999; 251: 433-441Crossref PubMed Scopus (75) Google Scholar). Furthermore, LRP can form a complex with APP by interactions of both the extracellular (24Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. Cell. 1995; 82: 331-340Abstract Full Text PDF PubMed Scopus (442) Google Scholar) and the intracellular domains (5Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 25Kinoshita A. Whelan C.M. Smith C.J. Mikhailenko I. Rebeck G.W. Strickland D.K. Hyman B.T. J. Neurosci. 2001; 21: 8354-8361Crossref PubMed Google Scholar). APP also undergoes several proteolytic processing events that, as in Notch, lead to the presenilin/γ-secretase-dependent release of the cytoplasmic tail. Recently it was shown that FE65 binding to the APP tail not only greatly enhances nuclear translocation of the complex but that this also serves to recruit transcriptional activators, which could potentially stimulate the expression of target genes that are regulated by APP cleavage events (26Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1045) Google Scholar).The purpose of the current study was to investigate whether LRP, in a manner analogous to Notch and APP, may also be subject to intramembranous or cytoplasmic proteolytic processing that results in the release of its intracellular domain from the membrane. Our results demonstrate that such a cleavage event occurs, that a protease with γ-secretase like properties is involved, and that intracellular proteins such as FE65 under certain conditions can stimulate the translocation of the tail fragment into the nucleus. The amount of biologically active cleaved tail fragment can be regulated by activation of protein kinase C (PKC) by phorbol esters and by inhibition of the proteasome.RESULTSThe γ-secretase-mediated intramembranous cleavage events that in Notch and APP lead to the release of the proteins' intracellular domains into the cytoplasm occur at a site preceding a valine located four or three residues, respectively, from the cytoplasmic face of the membrane (32Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref PubMed Scopus (424) Google Scholar). The transmembrane segment of LRP contains two valine residues at a corresponding position (Fig. 1) raising the possibility that LRP might be cleaved at the same relative position and that this cleavage might also involve γ-secretase.To test whether the cytoplasmic tail of LRP can be released by intramembranous or intracellular proteolysis, we constructed an expression vector encoding a chimeric protein, in which a yeast Gal4 DNA binding domain and a VP16 transactivation domain from herpes simplex virus were fused to the carboxyl-terminal end of the cytoplasmic tail of full-length LRP (pLRP-Gal4/VP16; Fig. 2 A). Proteolytic release of the tail-Gal4/VP16 fusion protein from the membrane was detected by measuring Gal4/VP16-dependent luciferase gene expression from a reporter construct containing multiple Gal4 binding motifs and the adenoviral E1B minimal promoter, followed by the luciferase gene (pG5E1B-Luc). A similar experimental strategy had previously been employed to detect the release of Notch ICD and for identifying the carboxyl-terminal fragment of APP as part of a transcriptionally active complex (19Lecourtois M. Schweisguth F. Curr. Biol. 1998; 8: 771-774Abstract Full Text Full Text PDF PubMed Google Scholar, 20Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 26Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1045) Google Scholar).Figure 2Relative luciferase activity in cells transfected with reporter gene constructs. A, LRP-Gal4/VP16 construct; the Gal4 DNA binding domain (amino acids 1–147) and the VP16 transactivation domain of the herpes simplex virus protein VP16 are fused to the carboxyl-terminal end of full-length LRP.B, reporter gene activity in transfected HEK 293 cells. Cells were transfected with 1.5 μg of reporter gene plasmid pG5E1B-Luc, 0.05 μg of control vector pCMV-β-Gal, and 1 μg of pcDNA3.1-LRP-Gal4/VP16 (expression vector for LRP-Gal4/VP16), pcDNA3.1-LRP (LRP), pMstGV (Gal4/VP16), or pMstGV-LDLR (LDLR-Gal4/VP16), respectively. After 48 h the cells were lysed and luciferase and β-galactosidase activities were determined. Luciferase reporter gene activity was corrected for transfection efficiency by dividing relative light units values by those obtained for galactosidase activity (representative results from more than three independent experiments).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Proteolytic Release of the LRP Cytoplasmic DomainTo determine whether the LRP tail can be proteolytically released from the membrane, we transfected HEK 293 cells with pLRP-Gal4/VP16, pG5E1B-Luc, and with a β-galactosidase expression vector (pCMV-βGal) to control for transfection efficiency. Transcriptional stimulation by a soluble Gal4/VP16 fusion protein or by an LDL receptor-Gal4/VP16 (LDLR-Gal4/VP16) chimeric protein, which is not expected to undergo processing, served as positive and negative controls, respectively. LRP-Gal4/VP16 stimulated luciferase activity by ∼80 fold, whereas LDLR-Gal4/VP16 had no effect (Fig. 2 B), even though expression of the shorter LDLR-Gal4/VP16 fusion protein was considerably higher than that of the almost five times larger LRP-Gal4/VP16 construct (data not shown). These findings indicate that the cytoplasmic tail of LRP can be proteolytically released from the membrane.Reporter Gene Activity in LRP-Gal4/VP16-expressing Cells Is Enhanced by PMA TreatmentWe next sought to determine, whether the rate at which the cytoplasmic tail of LRP is released from the membrane can be modulated. Incubation of LRP-Gal4/VP16-transfected cells with the LRP ligands α2-macroglobulin and thrombospondin, and with a fusion protein of the LRP binding chaperone RAP with the constant region of human IgG (33Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar), which can dimerize receptors, had no effect on the transcriptional activation of the Luc reporter (data not shown), suggesting that binding of ligands to the extracellular domain does not generally activate the cleavage. However, incubation of the transfected cells with the phorbol ester PMA robustly increased reporter gene activity from the LRP-Gal4/VP16 construct by ∼8 fold but not from the APP-Gal4/VP16 or LDLR-Gal4/VP16 control constructs (Fig. 3 A).Figure 3PKC-mediated stimulation of LRP-Gal4/VP16 dependent reporter gene activity. A, LRP-Gal4/VP16-induced reporter gene activity is enhanced by PMA. 293 cells were transfected with 1.5 μg of pG5E1B-Luc, 0.05 μg of pCMV-β-Gal, and 1 μg of pcDNA3.1-LRP-Gal4/VP16 (expression plasmid for LRP-Gal4/VP16), pMstGV-APP (APP-Gal4/VP16), or pMstGV-LDLR (LDLR-Gal4/VP16), respectively. After 24 h the cells were stimulated in the absence or presence of 100 nm phorbol 12-myristate 13-acetate (PMA) for 24 h. Luciferase reporter gene activity was corrected for transfection efficiency as described above. B, PMA-dependent stimulation of reporter activity is blocked by Calphostin C. 293 cells were transfected with 1.5 μg of pG5E1B-Luc, 0.05 μg of pCMV-β-Gal, and 1 μg of pcDNA3.1-LRP-Gal4/VP16. After 24 h the cells were treated with 100 nm PMA, 1 μm Calphostin, or with both for 24 h. Luciferase reporter gene activity was determined as described above.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because phorbol esters are activators of PKC, we determined whether the stimulatory effect of PMA could be blocked by simultaneous treatment of cells with Calphostin C, a non-isozyme-specific PKC inhibitor. Incubation of PMA-treated LRP-Gal4/VP16-transfected cells with 1 μm Calphostin C completely abolished the PMA-mediated increase in reporter gene activity (Fig. 3 B), suggesting that PKC can regulate LRP tail release.The cytoplasmic tail of LRP contains a possible PKC phosphorylation site as part of the NPTYK sequence motif. To test whether this PKC phosphorylation site was directly involved in the regulation of tail release, nuclear import, or stimulation of reporter gene transcription, we mutated the putatively phosphorylated threonine of the PKC consensus site to an alanine residue. This mutation did not significantly affect the ability of PMA to stimulate LRP tail release (Fig. 4 A), suggesting that PKC-mediated phosphorylation of the LRP tail is not the primary mechanism by which PMA increases reporter gene activation. Also, deletion of the carboxyl-terminal 25 or 46 amino acids of the LRP tail did not abolish basal or PMA-induced reporter gene transcription (data not shown).Figure 4PKC augments LRP-Gal4/VP16 reporter gene activity independent of phosphorylation of a PKC consensus site in the LRP tail, but accelerates cellular LRP turnover. A, mutation of the PKC consensus phosphorylation site in the LRP-tail does not alter reporter gene activity. 293 cells were transfected with 1.5 μg of pG5E1B-Luc, 0.05 μg of pCMV-β-Gal, and 1 μg of pcDNA3.1-LRP-Gal4/VP16 (LRP-Gal4/VP16) or pcDNA3.1-LRP-T/A-Gal4/VP16 (LRP-T/A-Gal4/VP16). After 24 h the cells were treated in the presence or absence of 100 nm PMA and luciferase activity was determined and normalized as described. B, endogenous LRP levels are decreased by PMA. Untransfected 293 cells were treated with 100 nm PMA for 24 h. Cell lysates were prepared and subjected to SDS-gel electrophoresis and immunoblotting with an antibody directed against the carboxyl terminus of the receptor.C, 293 cells were transfected with 5 μg of pcDNA3.1-LRP. After 24 h cells were either treated with 100 nm PMA and 1 μm DAPT (lane 1), 100 nm PMA (lane 2), 1 μm DAPT (lane 3), or not treated (lane 4) for 16 h. Cell membranes were then isolated and analyzed by immunoblotting as described in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether accelerated turnover of LRP at the plasma membrane, resulting in increased release of the tail from the membrane, might be the reason for the PMA effect, we incubated untransfected HEK 293 cells in the absence (Fig. 4 B, lane 1) or presence (lane 2) of 100 nm PMA for 24 h and then prepared lysates from the treated and non-treated cells. Immunoblot analysis revealed greatly decreased levels of the 85-kDa LRP fragment and of the 600-kDa precursor after incubation with PMA, whereas a shorter fragment of ∼25kDa that reacted with an antibody directed against the carboxyl-terminal epitope of LRP was increased in the membrane fractions of PMA-treated 293 cells compared with untreated cells (Fig. 4 C, lanes 2 and 4). This truncated carboxyl-terminal fragment of LRP is likely generated by proteolytic processing, or shedding, of the LRP ectodomain (23Quinn K.A. Pye V.J. Dai Y.P. Chesterman C.N. Owensby D.A. Exp. Cell Res. 1999; 251: 433-441Crossref PubMed Scopus (75) Google Scholar). Thus, PMA appears to increase LRP shedding, which would result in increased availability of substrate for the subsequent intramembranous or cytoplasmic cleavage event. γ-Secretase performs such an intramembranous proteolytic cleavage step in Notch and APP and thus is a candidate protease that might mediate the release of the LRP cytoplasmic domain from the 25-kDa precursor. In a preliminary experiment the γ-secretase inhibitor DAPT (27Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S., Wu, J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (790) Google Scholar) did indeed increase the levels of the 25-kDa fragment in the absence (lane 3) or presence (lane 1) of PMA.A γ-Secretase-like Activity Participates in the Release of the LRP Intracellular DomainTo further examine whether a γ-secretase-like activity might be involved in the release of the cytoplasmic tail of LRP, we treated pAPP-Gal4/VP16- or pLRP-Gal4/VP16-transfected cells (Fig. 5 A) with increasing concentrations of the γ-secretase inhibitors DAPT (27Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S., Wu, J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.Y. Little S.P. Mabry T.E. Miller F.D. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (790) Google Scholar) (closed circles and triangles) or l-685,458 (34Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (364) Google Scholar) (open circles) for 24 h. DAPT (closed triangles) and l-685,458 (not shown) almost completely abolished APP cleavage-dependent reporter gene activation, whereas LRP-dependent reporter gene expression (open and closed circles) was partially, but significantly, reduced by addition of both inhibitors.Figure 5γ-Secretase-dependent release of the LRP intracellular domain. A, LRP-Gal4/VP16-induced reporter gene activity is reduced by treatment with the γ-secretase inhibitors DAPT and l-685,458. 293 cells were transfected with 1.5 μg of pG5E1B-Luc, 0.05 μg of pCMV-β-Gal, and 1 μg of pcDNA3.1-LRP-Gal4/VP16 (LRP-Gal4/VP16) or 1 μg pMstGV-APP (APP-Gal4/VP16). After 12 h the cells were treated with increasing amounts of DAPT (LRP, closed circles; APP, closed triangles) or l-685,458 (open circles) for 24 h. Cell lysates were assayed for reporter gene activity as described above. APP-Gal4/VP16 served as a control for the efficacy of DAPT. B, release of the cytoplasmic domain of LRP from membranes in vitro and is inhibited by DAPT. 293 cells were transfected with 5 μg of pcDNA3.1-LRP (expression plasmid for LRP) in 100-mm dishes. 48 h later the cells were lysed, and membranes were isolated as described under “Experimental Procedures.” Resuspended membranes were incubated at 0 °C or at 37 °C for 1 h in the absence or presence of 500 nmDAPT. Pellet (P100) and supernatant (S100) fractions were prepared by centrifugation at 100,000 × g for 1 h and analyzed by immunoblotting with an antibody directed against the carboxyl terminus of LRP. C, treatment of 293 cells with DAPT leads to accumulation of a membrane-bound 25-kDa fragment of LRP. 293 cells were transfected with 5 μg of pcDNA3.1-LRP in 100-mm dishes. 12 h after transfection the cells were treated with the indicated concentrations of DAPT for 24 h. Membranes were isolated and analyzed by immunoblotting with an antibody directed against the carboxyl terminus of LRP. D, a 25-kDa LRP carboxyl-terminal fragment accumulates in N2a cells stably transfected with a dominant negative PS1-mutant cDNA. Membranes were prepared from N2a cells that expressed (PS1-D385A) or did not express (PS1-wt) a dominant negative acting PS1 cDNA (31Kim S.H. Leem J.Y. Lah J.J. Slunt H.H. Levey A.I. Thinakaran G. Sisodia S.S. J. Biol. Chem. 2001; 276: 43343-43350Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and analyzed as described in C. E, PS1-wt- and PS1-D385A-expressing N2a cells were transfected with 1.5 μg of pG5E1B-Luc, 0.05 μg of pCMV-β-Gal, and 1 μg of pcDNA3.1-LRP-Gal4/VP16 (LRP-Gal4/VP16) using the MBS kit (Stratagene, La Jolla, CA). After 2 days cell lysates were assayed for reporter gene activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Activation of reporter gene activity is an indirect measure of LRP tail release from cellular membranes. To directly demonstrate this release of the cytoplasmic domain of LRP, we transfected 293 cells with an LRP expression plasmid and incubated isolated membranes for 1 h in vitro in the presence or absence of DAPT at 0 °C or at 37 °C in the absence of cytoplasm (Fig. 5 B). Incubation at 37 °C, but not at 0 °C, caused the release of an ∼12-kDa fragment that could be detected with an antibody directed against the extreme carboxyl terminus of the LRP tail from the membranes (indicated by the arrow). The appearance of this fragment was completely blocked by DAPT. Furthermore, inhibition of γ-secretase activity by culturing 293 cells in the presence of increasing concentrations of DAPT was accompanied by the accumulation of the carboxyl-terminal 25-kDa fragment in the membrane fraction (Fig. 5 C). Increased levels of this 25-kDa fragment were also observed in membrane preparations of N2a cells that stably express a dominant negative presenilin-1 mutant (PS1-D385A) (31Kim S.H. Leem J.Y. Lah J.J. Slunt H.H. Levey A.I. Thinakaran G. Sisodia S.S. J. Biol. Chem. 2001; 276: 43343-43350Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), compared with control cells (Fig. 5 D). LRP tail cleavage-dependent reporter gene activity was reduced by ∼75% in PS1-D385A-expressing N2a cells compared with PS1-wt cells (Fig. 5 E). Taken together, these results suggest that γ-secretase is involved in the processing of LRP but that one or more additional γ-secretase-insensitive, and presumably cytoplasmic, proteases can also participate.Modulation of LRP Cytoplasmic Domain-dependent Reporter Activity by Tail Binding ProteinsThe cytoplasmic tails of LRP and APP contain binding sites for a variety of adaptor and scaffold proteins, including FE65, a scaffolding protein that stimulates the nuclear import of the APP cytoplasmic domain and is likely also involved in the transcription of APP target genes (26Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1045) Google Scholar), and Dab1, an adaptor protein that mediates signaling through the VLDL receptor and the apo-ER2 (11Trommsdorff M. Gotthardt M. Hiesberger T. Shelton J. Stockinger W. Nimpf J. Hammer R.E. Richardson J.A. Herz J. Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopu" @default.
• W2074701223 created "2016-06-24" @default.
• W2074701223 creator A5037865809 @default.
• W2074701223 creator A5042496262 @default.
• W2074701223 creator A5072196146 @default.
• W2074701223 date "2002-05-01" @default.
• W2074701223 modified "2023-10-09" @default.
• W2074701223 title "Proteolytic Processing of Low Density Lipoprotein Receptor-related Protein Mediates Regulated Release of Its Intracellular Domain" @default.
• W2074701223 cites W1502785574 @default.
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