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• W2054978737 abstract "Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent γ-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx- and N-methyl-d-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor. Binding of EphB receptors to ephrinB ligands on the surface of adjacent cells initiates signaling cascades that regulate angiogenesis, axonal guidance, and neuronal plasticity. These functions require processing of EphB receptors and removal of EphB-ephrinB complexes from the cell surface, but the mechanisms involved are poorly understood. Here we show that the ectodomain of EphB2 receptor is released to extracellular space following cleavage after EphB2 residue 543. The remaining membrane-associated fragment is cleaved by the presenilin-dependent γ-secretase activity after EphB2 residue 569 releasing an intracellular peptide that contains the cytoplasmic domain of EphB2. This cleavage is inhibited by presenilin 1 familial Alzheimer disease mutations. Processing of EphB2 receptor depends on specific treatments: ephrinB ligand-induced processing requires endocytosis, and the ectodomain cleavage is sensitive to peptide inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal but insensitive to metalloproteinase inhibitor GM6001. The ligand-induced processing takes place in endosomes and involves the rapid degradation of the extracellular EphB2. EphrinB ligand stimulates ubiquitination of EphB2 receptor. Calcium influx- and N-methyl-d-aspartic acid-induced processing of EphB2 is inhibited by GM6001 and ADAM10 inhibitors but not by N-benzyloxycarbonyl-Val-Leu-leucinal. This processing requires no endocytosis and promotes rapid shedding of extracellular EphB2, indicating that it takes place at the plasma membrane. Our data identify novel cleavages and modifications of EphB2 receptor and indicate that specific conditions determine the proteolytic systems and subcellular sites involved in the processing of this receptor. The Ephrin (Eph) 2The abbreviations used are: ADAM, a disintegrin and metalloproteinase; BACE, β-secretase; CTF, C-terminal fragment; NTF, N-terminal fragment; Eph, ephrin receptor; FAD, familial Alzheimer disease; LC, lactacystin; MP, metalloproteinase; NMDA, N-methyl-d-aspartic acid; NMDAR, NMDA receptor; PS, presenilin; WB, Western blot; ZVLL, N-benzyloxycarbonyl-Val-Leu-leucinal; APP, amyloid precursor protein. receptors are the largest family of receptor tyrosine kinase proteins. They bind membrane ligand proteins, called ephrins, on adjacent cells forming multimeric clusters that bridge juxtaposed cells. These binding interactions trigger signaling cascades in both the receptor-expressing cells (forward signaling) and the ligand-expressing cells (reverse signaling) stimulating functions that modulate cell morphogenesis, tissue patterning, and angiogenesis (1.Adams R.H. Semin. Cell Dev. Biol. 2002; 13: 55-60Crossref PubMed Scopus (98) Google Scholar, 2.Kullander K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Crossref PubMed Scopus (954) Google Scholar, 3.McLaughlin T. Hindges R. O'Leary D.D. Curr. Opin. Neurobiol. 2003; 13: 57-69Crossref PubMed Scopus (188) Google Scholar, 4.Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (852) Google Scholar). In the developing central nervous system, binding of ephrin ligands to Eph receptors regulates axon guidance and synapse formation (5.Palmer A. Klein R. Genes Dev. 2003; 17: 1429-1450Crossref PubMed Scopus (228) Google Scholar, 6.Martinez A. Soriano E. Brain Res. Brain Res. Rev. 2005; 49: 211-226Crossref PubMed Scopus (92) Google Scholar). Paradoxically, depending on specific conditions such as the expression levels of Eph receptors and their ligands, signaling events initiated by the Eph-ephrin interactions can lead either to increased cell-cell adhesion or to repulsion and separation of the involved cells (4.Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (852) Google Scholar). In the adult brain, the Eph-ephrin systems regulate memory-related functions, including synaptic structure and long term potentiation (5.Palmer A. Klein R. Genes Dev. 2003; 17: 1429-1450Crossref PubMed Scopus (228) Google Scholar, 7.Ghosh A. Science. 2002; 295: 449-451Crossref PubMed Scopus (27) Google Scholar, 8.Goldshmit Y. McLenachan S. Turnley A. Brain Res. Brain Res. Rev. 2006; 52: 327-345Crossref PubMed Scopus (139) Google Scholar). There are two subclasses of Eph receptors, EphA and EphB, which are selectively activated by ephrinA and ephrinB ligands, respectively, although exceptions to this rule have been observed (4.Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (852) Google Scholar). The EphB-ephrinB system regulates the development of many tissues, including the vasculature and the central nervous system, where EphB-ephrinB interactions control axonal pathfinding and dendritic spine morphogenesis (reviewed in Refs. 5.Palmer A. Klein R. Genes Dev. 2003; 17: 1429-1450Crossref PubMed Scopus (228) Google Scholar and 6.Martinez A. Soriano E. Brain Res. Brain Res. Rev. 2005; 49: 211-226Crossref PubMed Scopus (92) Google Scholar). Furthermore, there is evidence that EphB-ephrinB binding regulates the function of excitatory synapses and synaptic plasticity by initiating forward signaling cascades that regulate phosphorylation of Src kinases and N-methyl-d-aspartic acid (NMDA) receptor (NMDAR) activity (5.Palmer A. Klein R. Genes Dev. 2003; 17: 1429-1450Crossref PubMed Scopus (228) Google Scholar, 7.Ghosh A. Science. 2002; 295: 449-451Crossref PubMed Scopus (27) Google Scholar, 9.Yamaguchi Y. Pasquale E.B. Curr. Opin. Neurobiol. 2004; 14: 288-296Crossref PubMed Scopus (130) Google Scholar). The cytosolic region of EphB receptors contains a tyrosine kinase domain (10.Ellis C. Kasmi F. Ganju P. Walls E. Panayotou G. Reith A.D. Oncogene. 1996; 12: 1727-1736PubMed Google Scholar) and a highly conserved sequence motif that is a major site for autophosphorylation by the EphB tyrosine kinase. This EphB motif interacts with Src-homology domain-containing factors, including Ras GTPase-activating proteins and Src kinases (4.Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (852) Google Scholar). Recent reports indicate that mechanisms by which adhesive and signaling interactions between EphB2 receptor and ephrinB2 ligands are terminated include endocytosis of the cell surface EphB-ephrinB complexes and cleavage of the ectodomain of ephrinB ligands (11.Zimmer M. Palmer A. Kohler J. Klein R. Nat. Cell Biol. 2003; 5: 869-878Crossref PubMed Scopus (292) Google Scholar, 12.Marston D.J. Dickinson S. Nobes C.D. Nat. Cell Biol. 2003; 5: 879-888Crossref PubMed Scopus (250) Google Scholar, 13.Hattori M. Osterfield M. Flanagan J.G. Science. 2000; 289: 1360-1365Crossref PubMed Scopus (464) Google Scholar). These processes reduce contact between the receptor and ligand-expressing cells and convert the initial cell-cell adhesion into a repulsion mediated by Rac signaling and actin polymerization. Ligand-induced endocytic vesicles internalized in the EphB-expressing cells contain EphB-ephrinB complexes and surrounding membranes deriving from both cells, but the fate of the internalized protein complex is not clear (11.Zimmer M. Palmer A. Kohler J. Klein R. Nat. Cell Biol. 2003; 5: 869-878Crossref PubMed Scopus (292) Google Scholar, 12.Marston D.J. Dickinson S. Nobes C.D. Nat. Cell Biol. 2003; 5: 879-888Crossref PubMed Scopus (250) Google Scholar). Like other substrates of the metalloproteinase (MP)/γ-secretase system (14.Marambaud P. Robakis N.K. Genes Brain Behav. 2005; 4: 134-146Crossref PubMed Scopus (61) Google Scholar), EphB receptors are cell surface proteins containing an extensive extracellular (ectodomain) region, a transmembrane sequence, and a cytoplasmic domain. We thus asked whether a MP/γ-secretase system participates in the processing of EphB2 receptor and its internalized complexes. Here we present evidence that two distinct pathways, one stimulated by calcium influx and the other by ephrinB2 ligands, regulate proteolytic processing of EphB2 receptor and its complexes. Both pathways involve a cleavage within the extracellular domain and processing of the remaining membrane bound fragment by the presenilin1 (PS1)-dependent γ-secretase system. Only ephrinB ligands, however, stimulate ubiquitination of EphB2 receptor. Materials and Antibodies−The γ-secretase inhibitor L-685,458, lactacystin (LC), bafilomycin, and epoxomicin were obtained from Calbiochem. MP inhibitor GM6001 was from Chemicon International and chloroquine from Sigma. Goat anti-human-Fc was obtained from Jackson ImmunoResearch Laboratories. Monoclonal antibody 33B10 against the C-terminal fragment of PS1 (PS1/CTF) and polyclonal antiserum 222 against the N-terminal fragment of PS1 (PS1/NTF) have been described (15.Georgakopoulos A. Marambaud P. Efthimiopoulos S. Shioi J. Cui W. Li H.C. Schutte M. Gordon R. Holstein G.R. Martinelli G. Mehta P. Friedrich Jr., V.L. Robakis N.K. Mol. Cell. 1999; 4: 893-902Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Polyclonal and monoclonal anti-EphB2 antibodies were purchased from Zymed Laboratories (San Francisco, CA). Anti-N-cadherin (C32) and anti-phospho-tyrosine (clone 4G10) antibodies were from BD Biosciences and Upstate Biotechnology (Charlottesville, VA), respectively. Anti-ubiquitin antibody P4D1 was from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated antibodies against extracellular EphB2 and recombinant mouse ephrin-B2/Fc Chimera were from R&D Systems (Minneapolis, MN). Streptavidin-Cy3 conjugate and anti-FLAG M2 affinity gels were obtained from Sigma. Recombinant Plasmids and Constructs−Murine EphB2 cDNA was kindly provided by Dr. G. Yancopoulos (Regeneron Pharmaceuticals). For retroviral gene expression, EphB2 cDNA was subcloned into the XhoI sites of pMX-IRES-GFP (provided by Dr. Kitamura, Tokyo, Japan). PMX/EphB2 construct carrying mutation K664M was generated by site-directed mutagenesis (sense primer, 5′-catcaagaccctcatgtccggatacacggag-3′; antisense primer, 5′-ctccgtgtatccggacatgagggtcttgatg-3′), and mutation was verified by sequencing. The sequence encoding a FLAG tag was inserted at the end of the EphB2 cDNA, and the resulting FLAG-EphB2 sequence was subcloned into the NotI sites of pQCXIP (Clontech). Mouse Embryo Preparation, Cell Cultures, Transfections, and Transduction−PS1+/+ and PS1–/– mouse embryos were collected at embryonic day 16 (E16), and brains were solubilized in radioimmune precipitation assay buffer as described (16.Baki L. Marambaud P. Efthimiopoulos S. Georgakopoulos A. Wen P. Cui W. Shioi J. Koo E. Ozawa M. Friedrich Jr., V.L. Robakis N.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2381-2386Crossref PubMed Scopus (158) Google Scholar). Eighty micrograms of extract was analyzed on Western blots (WBs). Primary neuronal cultures were prepared from E16 rat brains as described (17.Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Neurons were maintained 8 days in vitro in NeuroBasal medium (Invitrogen). Fibroblasts from PS1+/+ or PS1–/– mice were as described (16.Baki L. Marambaud P. Efthimiopoulos S. Georgakopoulos A. Wen P. Cui W. Shioi J. Koo E. Ozawa M. Friedrich Jr., V.L. Robakis N.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2381-2386Crossref PubMed Scopus (158) Google Scholar). Transient transfections were performed using FuGENE 6 transfection reagent (Roche Applied Science). Retroviral gene expression was performed as described (18.Georgakopoulos A. Litterst C. Ghersi E. Baki L. Xu C. Serban G. Robakis N.K. EMBO J. 2006; 25: 1242-1252Crossref PubMed Scopus (136) Google Scholar). Treatment with Inhibitors and Clustering of Ligand−Inhibitors were used at the following concentration in the indicated vehicle: L-685,458 (0.5 μm in Me2SO), ionomicin (2.5 μm in Me2SO), GM6001 (25 μm in Me2SO), ZVLL (30 μm in Me2SO), LC (10 μm in water), chloroquine (100 μm in water), epoxomycin (5 μm in Me2SO), bafilomycin (1.0 μm in Me2SO), monodansylcadaverine (125 μm in Me2SO and ethanol 1:1). Clustering of recombinant mouse ephrin-B2/Fc ligands and anti-Fc were performed as described and used at 1 μg/ml ephrin-B2/Fc for stimulation (19.Davis S. Gale N.W. Aldrich T.H. Maisonpierre P.C. Lhotak V. Pawson T. Goldfarb M. Yancopoulos G.D. Science. 1994; 266: 816-819Crossref PubMed Scopus (633) Google Scholar, 20.Wang H.U. Anderson D.J. Neuron. 1997; 18: 383-396Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Cell Lysates, Immunoprecipitation, SDS-PAGE, and Immunoblotting−Cell lysates for Western blotting (WB) were prepared in SDS lysis buffer (100 mm Tris/HCl, 20 mm NaCl, 10 mm EGTA, 10 mm EDTA, 1% SDS) containing complete protease inhibitor mixture (Roche Applied Science) and phosphatase inhibitor mixture I (Sigma). For immunoprecipitation, cells were lysed in immunoprecipitation buffer (20 mm Tris/HCl, pH 8, 150 mm NaCl, 1% Triton X-100 plus protease inhibitor, and phosphatase inhibitor mixtures). Lysates were processed for immunoprecipitation as described (15.Georgakopoulos A. Marambaud P. Efthimiopoulos S. Shioi J. Cui W. Li H.C. Schutte M. Gordon R. Holstein G.R. Martinelli G. Mehta P. Friedrich Jr., V.L. Robakis N.K. Mol. Cell. 1999; 4: 893-902Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). For separation of EphB2/CTF1 and EphB2/CTF2, 12% Protean II XL SDS-PAGE gels (Bio-Rad) were used. EphB2 fragments CTF1 and CTF2 were purified for sequencing from EphB2-transfected HEK293T cultures in the absence of ephrinB ligand. EphB2 Is Cleaved by PS1-dependent γ-Secretase−To investigate whether EphB2 receptor is processed by the γ-secretase system, we used WB to probe embryonic brain extract from PS1 null (PS1–/–) and WT (PS1+/+) mice. Antibodies against the cytoplasmic sequence of EphB2 receptor detected a peptide of an apparent molecular mass of ∼50 kDa (designated EphB2/CTF1) in the brains of PS1–/– but not in the brains of PS1+/+ mice (Fig. 1A). The immunoreactivity and apparent molecular mass of this peptide suggest that it is derived from EphB2 receptor and may contain all of its cytoplasmic and transmembrane sequences. Accumulation of EphB2/CTF1 in PS1 null mice suggests that this peptide is metabolized by the PS1-dependent γ-secretase system and may be produced following cleavage of the extracellular region of EphB receptor similar to a number of type I transmembrane proteins (14.Marambaud P. Robakis N.K. Genes Brain Behav. 2005; 4: 134-146Crossref PubMed Scopus (61) Google Scholar). A peptide similar to brain EphB2/CTF1 accumulates in EphB2 receptor-transfected PS1–/–, but not in PS1+/+, cells (Fig. 1B), further confirming the identity of EphB2/CTF1 and its degradation by the PS-γ-secretase system. To detect the fragment expected from the γ-secretase cleavage of EphB2/CTF1 (termed EphB2/CTF2), we employed the in vitro γ-secretase assay (17.Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 21.Pinnix I. Musunuru U. Tun H. Sridharan A. Golde T. Eckman C. Ziani-Cherif C. Onstead L. Sambamurti K. J. Biol. Chem. 2001; 276: 481-487Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) using membranes from EphB2-transfected PS1–/– and PS1+/+ fibroblasts. As expected, EphB2/CTF2 is produced in membranes from PS1+/+ but not from PS1–/– cells (Fig. 1C, lanes 2 and 4), and its production is sensitive to γ-secretase inhibitor L-685,458 (Fig. 1C, lane 5). Proteasomal inhibitor LC prevents degradation of γ-secretase-produced cytosolic peptides and is used for their in vivo detection (18.Georgakopoulos A. Litterst C. Ghersi E. Baki L. Xu C. Serban G. Robakis N.K. EMBO J. 2006; 25: 1242-1252Crossref PubMed Scopus (136) Google Scholar, 22.Kim 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). Because LC was toxic to our fibroblasts, we used HEK293T cells overexpressing EphB2 receptor to detect intracellular EphB2/CTF2 (Fig. 1D, lanes 1 and 2). Production of this peptide is inhibited by γ-secretase inhibitors with a concomitant accumulation of its precursor EphB2/CTF1 (Fig. 1D, lane 3). Conditioned media from overnight cultures contain a polypeptide of ∼90 kDa (termed EphB2/NTF) recognized by antibodies specific to extracellular EphB2 receptor (Fig. 1E). The apparent molecular mass of this peptide suggests it contains most of extracellular EphB2 receptor and may be the counterpart of EphB2/CTF1. Together, our data show that similar to other type I transmembrane receptors, EphB2 is cleaved within the ectodomain sequence to produce EphB2/NTF that is released to the medium and cellular EphB2/CTF1 that is further metabolized by the PS1-γ-secretase system to produce EphB2/CTF2 (Fig. 2A). Determination of the Processing Sites of EphB2 Receptor−EphB2/CTF1 and EphB2/CTF2 were affinity-purified from HEK293T cells overexpressing EphB2 receptor (Fig. 1D). Edman sequencing of EphB2/CTF1 through 10 cycles showed the following major sequence: SIKEKLPLIV. This is a unique sequence that corresponds to mouse EphB2 residues 544–553. These data show that the ectodomain cleavage site of EphB2 receptor is located seven amino acids upstream of its predicted transmembrane sequence (Fig. 2A). Thus, the 50-kDa EphB2/CTF1 is produced by a cleavage within the ectodomain region of EphB2 receptor after residue 543 (Fig. 2A, MP site) and contains the 451 C-terminal residues of EphB2 receptor, including all transmembrane and cytoplasmic sequence (Fig. 2A). Mass spectrometric analysis of lysine-C digests of EphB2/CTF2 (48.3 kDa) yielded peptide AIVCNRRGFERADSEYTDK (m/z 2287.00, +0.07). This is a unique sequence corresponding to mouse EphB2 receptor residues 570–588 located at the cytoplasmic juxtamembrane region of the receptor. The mass spectrometric analysis of EphB2/CTF2 yielded additional peptides all derived from the cytoplasmic sequence of EphB2 receptor (not shown). These results show that the γ-secretase-dependent cleavage of EphB2 receptor takes place between residues 569 and 570, three amino acids upstream of the C-terminal end of the predicted transmembrane sequence (Fig. 2A). This cleavage produces peptide EphB2/CTF2 containing the 425 C-terminal amino acids of EphB2 receptor (Fig. 2A). The close proximity of this cleavage site to the membrane/cytosol interface indicates it is analogous to the γ-secretase-catalyzed epsilon (ε)-cleavage sites observed in many PS-γ-secretase substrates (23.Robakis N.K. Marambaud P. Thiel G. Transcription Factors in the Nervous System. Wiley-VCH Verlag, Weinheim, Germany2006: 399-461Google Scholar). PS1 Familial Alzheimer Disease Mutants Inhibit the γ-Secretase Cleavage of EphB2/CTF1−Recent evidence shows that PS1 FAD mutations may inhibit the PS1-dependent ε-cleavage of many substrates suggesting that FAD mutations cause a loss of cleavage function at this site (17.Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 18.Georgakopoulos A. Litterst C. Ghersi E. Baki L. Xu C. Serban G. Robakis N.K. EMBO J. 2006; 25: 1242-1252Crossref PubMed Scopus (136) Google Scholar, 24.Chen F. Gu Y. Hasegawa H. Ruan X. Arawaka S. Fraser P. Westaway D. Mount H. St George-Hyslop P. J. Biol. Chem. 2002; 277: 36521-36526Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Fig. 2B shows that several PS1 FAD mutants, including P117L, ΔE9, A260V, P264L, and E280G as well as the γ-secretase dominant negative PS1 mutant D257A (25.Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar), are impaired in their ability to stimulate production of EphB2/CTF2 compared with WT PS1. PS1 FAD mutants A246E and M146L, however, showed no inhibition in their ability to increase EphB2/CTF2. These data show that certain PS1 FAD mutations interfere with the ability of PS1 to mediate cleavage of EphB2/CTF1 at the ε-site. Calcium Influx and Ligand Binding Stimulate Distinct Processing of EphB2 Receptor−Calcium influx stimulates a MP/γ-secretase processing of transmembrane proteins (26.Marambaud 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, 27.Maretzky T. Reiss K. Ludwig A. Buchholz J. Scholz F. Proksch E. de Strooper B. Hartmann D. Saftig P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9182-9187Crossref PubMed Scopus (540) Google Scholar, 28.Reiss K. Maretzky T. Ludwig A. Tousseyn T. de Strooper B. Hartmann D. Saftig P. EMBO J. 2005; 24: 742-752Crossref PubMed Scopus (399) Google Scholar). To investigate the effect of calcium influx on the processing of EphB2, cell cultures expressing this receptor were treated with the calcium ionophore ionomycin as described (26.Marambaud 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). This treatment resulted in the rapid decrease of full-length EphB2 and in a concomitant increase of both cellular EphB2/CTF1 and medium EphB2/NTF (Fig. 3, left panel), suggesting that extracellular EphB2 is rapidly released to the conditioned media following cleavage of cell surface receptor. The half-life of EphB2 in the presence of ionomycin was <10 min, and by 15 min most receptor was degraded (Fig. 3, lanes 1–5). To further explore the physiological significance of the EphB2 processing, we asked whether it is regulated by ligand-receptor interactions. To this end, we treated our cultures with a clustered ephrinB2-Fc construct containing the extracellular domain of ephrinB2 ligand fused to Fc portion of IgG. This construct binds to and activates the EphB2 receptor mimicking the ligand effects (19.Davis S. Gale N.W. Aldrich T.H. Maisonpierre P.C. Lhotak V. Pawson T. Goldfarb M. Yancopoulos G.D. Science. 1994; 266: 816-819Crossref PubMed Scopus (633) Google Scholar). Following 2 h of treatment, the levels of cellular full-length receptor decreased while EphB2/CTF1 increased suggesting that ephrinB ligand stimulates the extracellular cleavage of EphB2. By 4 h of ligand treatment, most EphB2 receptor had been degraded. Examination of the conditioned medium, however, failed to show any increase in EphB2/NTF, the extracellular counterpart of EphB2/CTF1 (Fig. 3, right panel, lanes 6–10). Furthermore, examination of cell extract from ligand-treated cultures failed to detect any cell-associated EphB2/NTF (data not shown) suggesting that EphB2/NTF produced in response to ligand binding is rapidly degraded. Together, these data show that ephrinB ligands as well as calcium influx induce processing of EphB2 receptor, but the kinetics of EphB2 decrease and the appearance of degradation products differ significantly between the two conditions suggesting that these two factors stimulate distinct processing pathways. Distinct Enzymes Cleave Extracellular EphB2 in Response to Calcium Influx or Ligand Treatment−Cleavage within the ectodomain sequence of a number of cell surface transmembrane proteins, including cadherins, APP, and Notch1, is often catalyzed by enzymes sensitive to the broad spectrum MP inhibitor GM6001. In addition, the ectodomain of APP is cleaved by β-secretase (BACE), an aspartyl protease sensitive to peptide inhibitor ZVLL (29.Abbenante G. Kovacs D.M. Leung D.L. Craik D.J. Tanzi R.E. Fairlie D.P. Biochem. Biophys. Res. Commun. 2000; 268: 133-135Crossref PubMed Scopus (39) Google Scholar). Fig. 4A (left panels) shows that GM6001 inhibits the ionomycin-stimulated metabolism of EphB2 and the production of both cell-associated EphB2/CTF1 and released EphB2/NTF suggesting that GM6001 blocks a MP cleavage of EphB2 ectodomain. GM6001, however, has no effect on the ligand-stimulated metabolism of EphB2 receptor (Fig. 4A, right panels). In contrast, peptide ZVLL inhibits the ligand-stimulated metabolism of EphB2 and the appearance of EphB2/CTF1 (Fig. 4B, right panels), but it has no effect on the ionomycin-stimulated metabolism of EphB2 and production of EphB2/CTF1 (Fig. 4B, left panels). Because ZVLL is not specific to BACE (29.Abbenante G. Kovacs D.M. Leung D.L. Craik D.J. Tanzi R.E. Fairlie D.P. Biochem. Biophys. Res. Commun. 2000; 268: 133-135Crossref PubMed Scopus (39) Google Scholar), we probed two additional BACE inhibitors, including Statin and the cell-permeable BACE inhibitor IV (30.Stachel S.J. Coburn C.A. Steele T.G. Jones K.G. Loutzenhiser E.F. Gregro A.R. Rajapakse H.A. Lai M.T. Crouthamel M.C. Xu M. Tugusheva K. Lineberger J.E. Pietrak B.L. Espeseth A.S. Shi X.P. Chen-Dodson E. Holloway M.K. Munshi S. Simon A.J. Kuo L. Vacca J.P. J. Med. Chem. 2004; 47: 6447-6450Crossref PubMed Scopus (278) Google Scholar). None of these was able to inhibit the ligand-induced processing of EphB2 (data not shown), suggesting that the activity responsible for the ligand-induced processing of EphB2 may differ from BACE. Together, these results show that, depending on the inducing agent, cleavage of the extracellular region of EphB2 involves two distinct activities, one sensitive to MP inhibitor GM 6001 and one sensitive to peptide inhibitor ZVLL. Because EphB2 interacts closely with NMDAR and regulates its activity (31.Takasu M.A. Dalva M.B. Zigmond R.E. Greenberg M.E. Science. 2002; 295: 491-495Crossref PubMed Scopus (429) Google Scholar), we asked whether calcium influx through NMDAR affects EphB2 processing. Fig. 4C (left panels) shows that treatment of rat embryo primary neuronal cultures (see “Experimental Procedures”) with NMDA, an agonist of NMDAR, increased production of EphB2/CTF1, and this increase was augmented by γ-secretase inhibitors (lanes 1–3). NMDA-induced production of EphB2/CTF1 was inhibited by the specific NMDAR antagonist d(–)-2-amino-5-phosphonovalerate (APV) and by GM6001 indicating that stimulation of NMDAR induces a MP-like cleavage of EphB2 receptor (Fig. 4C, left panels, lanes 4–7). Treatment of primary neurons with ephrinB ligand results in the degradation of EphB2 and in the accumulation of EphB2/CTF1 in the presence of γ-secretase inhibitor (Fig. 4C, right panels, lanes 1–3). As expected, this degradation was blocked by ZVLL (Fig. 4C, right panels, lane 4). Absence of EphB2/CTF1 accumulation upon ligand treatment may be due to rapid degradation of this peptide in primary neurons by γ-secretase. Other CTF1 fragments, including brain N-cadherin CTF1, also fail to accumulate due to γ-secretase activity (17.Marambaud P. Wen P.H. Dutt A. Shioi J. Takashima A. Siman R. Robakis N.K. Cell. 2003; 114: 635-645Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Together, our data show that stimulation of NMDAR induces a MP/γ-secretase processing of EphB2 receptor. In contrast, the ephrinB ligand-induced processing of this receptor is insensitive to MP inhibitors, but it is sensitive to ZVLL and to inhibitors of γ-secretase. Interestingly, the site of the ectodomain cleavage of EphB2 has the same relationship to the transmembrane region of EphB2 as the ectodomain cleavage site of E-cadherin to its transmembrane region. Both cleavage sites are located seven residues upstream of the putative outer face of the plasma membrane (Fig. 2A and Ref. 26.Marambaud 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). Because the cadherin cleavage is mediated by ADAM10 (a disintegrin and metalloproteinase 10) (27.Maretzky T. Reiss K. Ludwig A. Buchholz J. Scholz F. Proksch E. de Strooper B. Hartmann D. Saftig P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9182-9187Crossref PubMed Scopus (540) Google Scholar), we used the potent ADAM10 inhibitor GI254023X to examine whether this protease is involved in this cleavage. Fig. 4D shows that, at 50 nm, this inhibitor blocks the ionomycin-induced cleavage of EphB2 receptor" @default.
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• W2054978737 date "2007-06-01" @default.
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• W2054978737 title "Ligand Binding and Calcium Influx Induce Distinct Ectodomain/γ-Secretase-processing Pathways of EphB2 Receptor" @default.
• W2054978737 cites W1534001036 @default.
• W2054978737 cites W1553513611 @default.
• W2054978737 cites W1561206275 @default.
• W2054978737 cites W1572225000 @default.
• W2054978737 cites W1584018958 @default.
• W2054978737 cites W1606891122 @default.
• W2054978737 cites W1919241967 @default.
• W2054978737 cites W1972039621 @default.
• W2054978737 cites W1973629131 @default.
• W2054978737 cites W1986909516 @default.
• W2054978737 cites W1988567136 @default.
• W2054978737 cites W1989607398 @default.
• W2054978737 cites W1994979780 @default.
• W2054978737 cites W1995691453 @default.
• W2054978737 cites W1999910107 @default.
• W2054978737 cites W1999966549 @default.
• W2054978737 cites W2001363990 @default.
• W2054978737 cites W2003890494 @default.
• W2054978737 cites W2004464973 @default.
• W2054978737 cites W2005722151 @default.
• W2054978737 cites W2010710842 @default.
• W2054978737 cites W2017201573 @default.
• W2054978737 cites W2034064964 @default.
• W2054978737 cites W2039724703 @default.
• W2054978737 cites W2041108499 @default.
• W2054978737 cites W2056181922 @default.
• W2054978737 cites W2063733034 @default.
• W2054978737 cites W2072707337 @default.
• W2054978737 cites W2076444547 @default.
• W2054978737 cites W2081656219 @default.
• W2054978737 cites W2081733923 @default.
• W2054978737 cites W2085094002 @default.
• W2054978737 cites W2085532366 @default.
• W2054978737 cites W2090861667 @default.
• W2054978737 cites W2091169541 @default.
• W2054978737 cites W2093842873 @default.
• W2054978737 cites W2095020146 @default.
• W2054978737 cites W2106449226 @default.
• W2054978737 cites W2111314225 @default.
• W2054978737 cites W2114259322 @default.
• W2054978737 cites W2114737664 @default.
• W2054978737 cites W2127113850 @default.
• W2054978737 cites W2133019124 @default.
• W2054978737 cites W2138447190 @default.
• W2054978737 cites W2153471866 @default.
• W2054978737 cites W2157585888 @default.
• W2054978737 cites W2157638357 @default.
• W2054978737 cites W2167768159 @default.
• W2054978737 cites W4238999760 @default.
• W2054978737 cites W4377993708 @default.
• W2054978737 cites W2186135786 @default.
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