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Originally published In Press as doi:10.1074/jbc.M307974200 on November 24, 2003

J. Biol. Chem., Vol. 279, Issue 6, 4241-4249, February 6, 2004
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Evidence for a Critical Role of the Tumor Necrosis Factor {alpha} Convertase (TACE) in Ectodomain Shedding of the p75 Neurotrophin Receptor (p75NTR)*

Gisela Weskamp,a Johannes Schlöndorff,abc Lawrence Lum,abd J. David Becherer,e Tae-Wan Kim,f Paul Saftig,g Dieter Hartmann,h Gillian Murphy,i and Carl P. Blobelaj

From the aCell Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, the bTri-Institutional (Cornell University/Rockefeller University/Sloan-Kettering Cancer Center) MD-Ph.D. Training Program, New York, New York 10021, the eDepartment of Biochemical and Analytical Pharmacology, GlaxoSmithKline Inc., Research Triangle Park, North Carolina 27709, the fDepartment of Pathology, Center for Neurobiology and Behavior, Columbia University, New York, New York 10032, the gBiochemical Institute, Christian-Albrecht University, D-24098 Kiel, Germany, the hDepartment for Human Genetics, K.U. Leuven and Flanders Interuniversity Institute for Biotechnology (VIB-4), 3000 Leuven, Belgium, and the iCambridge Institute for Medical Research, Cambridge CB2 2XY, United Kingdom

Received for publication, July 22, 2003 , and in revised form, November 17, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-{alpha} converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75NTR shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein ectodomain shedding is emerging as an important post-translational mechanism for regulating the function of membrane-anchored proteins. Shedding involves the proteolytic processing of membrane-tethered proteins leading to the release of their extracellular- or ectodomain (reviewed in Refs. 1-4). About 2-4% of the proteins on the cell surface are released by metalloproteases in response to phorbol ester stimulation (5). These molecules comprise a variety of structurally and functionally distinct proteins, including epidermal growth factor receptor ligands such as TGF{alpha}1 and HB-EGF, TNF family members, and other cytokines, receptors such as the p55 and p75 TNF receptors and the interleukin-6 receptor, and several other proteins, including the amyloid precursor protein, Notch, and Delta (see Ref. 3 and references therein). Ectodomain shedding has been shown to be essential for proper signaling via epidermal growth factor receptor ligands (6-15), Notch-mediated lateral inhibition (16-23), limiting TNFR-mediated inflammatory reactions (24), and regulating axonal guidance (25-27). Even if the functional consequences of ectodomain release remain to be evaluated for most shed proteins, it seems likely that ectodomain shedding will affect the function of the majority of its substrate proteins.

Despite the growing number of proteins that are known to undergo ectodomain shedding, much remains to be learned about the mechanisms underlying this process and the responsible proteases. The TNF{alpha} convertase (TACE) is one of the first proteases shown to be involved in shedding. Its substrates include TNF{alpha} (28, 29), TGF{alpha} (6, 30, 31), HB-EGF (9, 31), L-selectin (6), MUC1 (32), interleukin-1R II (33), amyloid precursor protein (34), HER4 (35), Notch (23), neuregulin {alpha}2c (36), growth hormone receptor (37), and CD30 (38). In most of these instances, TACE is required for the increased shedding seen in response to stimulation by phorbol ester, although the TACE-mediated release of erbB-4 is also enhanced by pervanadate, a tyrosine phosphatase inhibitor (35). Additional proteases must be involved in stimulated shedding, as TACE is not responsible for the release of ACE (39), TRANCE (40), or syndecan-1 and -4 (41). Furthermore, for most ectodomain shedding events it remains an open question whether one or several proteases are involved.

Several intracellular signaling pathways have been implicated in mediating constitutive and stimulated shedding. Among these, the MEK/ERK MAP kinase pathway has been shown to mediate the receptor tyrosine kinase- and phorbol ester-stimulated shedding of the TACE substrates TGF{alpha} and TNF{alpha} (42), and of HB-EGF (43), syndecan-1 and -4 (41), and the L1 adhesion molecule (44). In contrast, the constitutive shedding of TGF{alpha} depends on the p38 MAP kinase pathway (42).

In this study, we evaluated the proteolytic and signaling activities involved in ectodomain shedding of the p75 neurotrophin receptor (p75NTR), which serves as a common receptor for all known neurotrophins. The p75NTR receptor has diverse functions, including the promotion of cell survival in some cell types, and triggering apoptosis in others (45-48). The p75NTR also refines ligand specificity of a second class of neurotrophin receptors, the TRK receptors (49). Furthermore, p75NTR reportedly regulates myelination by Schwann cells in the developing peripheral nervous system (50). Finally, recent work has demonstrated that p75NTR binds to the Nogo receptor, thereby inhibiting axon growth mediated by three myelin-associated proteins, MAG, OMgp, and Nogo-A (51, 52). One way to regulate p75NTR levels on the cell surface of Schwann cells was shown to occur via truncation by cellular metalloproteases (53). Whereas the relevance of p75NTR ectodomain shedding in vivo is unclear, it has been proposed that shedding regulates the amount of cell surface p75NTR and may therefore affect axonal outgrowth (54). Furthermore, recent studies demonstrate that p75NTR undergoes regulated intramembrane proteolysis (RIP), which may in turn affect the association with TRK receptors and regulate the function of p75NTR (55, 56). Like in other well characterized cases of RIP, such as in the cleavage of Notch, amyloid precursor protein, or sterol regulatory element-binding protein (57), a membrane proximal cleavage is considered a prerequisite for the subsequent intramembrane cleavage of p75NTR (55). These studies highlight the significance of identifying the enzyme responsible for membrane proximal cleavage and ectodomain shedding of p75NTR. Our results uncover two distinct activities involved in the release of p75NTR ectodomain, and define characteristic features of these two shedding activities. Furthermore, we evaluate the role of different candidate sheddases of the ADAM (a disintegrin and metalloprotease) family of metalloproteases in p75NTR ectodomain shedding.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Phorbol 12-myristate 13-acetate (PMA), hydrogen peroxide, and sodium vanadate were purchased from Sigma. Pervanadate was generated immediately prior to each experiment by mixing sodium vanadate and hydrogen peroxide to a final concentration of 100 mM each. Protein A- and Protein G-Sepharose were purchased from Pharmacia. Restriction enzymes, Hi-Fidelity Taq, and Klenow fragment were purchased from Roche Diagnostics. Protein kinase C inhibitor bisindolylmaleimide I, MEK inhibitor U0126, p38 MAP kinase inhibitor SB202190, compound E, lactacysteine, and EndoH and PNGaseF were purchased from Calbiochem. BB-94 metalloprotease inhibitor was kindly provided by Dr. J. D. Becherer (GlaxoSmithKline, Research Triangle Park, NC). Active recombinant human TIMP-1 and -2 were purified as described (58).

Constructs and Antibodies—The cDNA encoding for the rat p75NTR cDNA (59) was placed into pcDNA3 using EcoRI and XbaI restriction sites. The expression construct encoding full-length rat p75NTR with the NH2-terminal signal peptide followed by the Strep-tag II and hemagglutinin epitope tag (St75) was generated as previously described (56). Site-directed mutagenesis was performed with untagged wild-type p75NTR or St75 cDNA as a template and deletions were introduced with PCR and appropriate oligonucleotides using the QuikChange system (Stratagene). All of the resulting mutations were confirmed by cDNA sequencing. The anti-rat p75NTR polyclonal antibody (anti-REX) and monoclonal antibodies (mc192), and anti-TACE cytoplasmic domain polyclonal antibodies have been previously described (60-63). The p75NTR antibodies recognize the extracellular portion of the rat p75NTR receptor protein (60, 61). Affinity capture of secreted St75 ectodomain and Western blot analysis were performed as described (56).

Cell Culture, Transfection, and Metabolic Labeling—Mutant M2 CHO cells and control wild-type CHO cells (5) were grown in F-12 media supplemented with 5% fetal calf serum and 1% penicillin/streptomycin and glutamine. COS-7 cells were grown in Dulbecco's modified Eagle's medium with 5% fetal calf serum and 1% penicillin/streptomycin and glutamine. Rat pheochromocytoma cells (PC12 cells) and dorsal root ganglia from newborn mice (d1) were cultured as described (61). Primary mouse embryonic fibroblasts (wild-type, adam9/12/15-/-, adam17-/-) and immortalized adam10-/- mouse embryonic fibroblasts were isolated and cultured as previously described (40, 64, 65).

All cells were transfected in 6-well tissue culture plates with LipofectAMINE (Invitrogen) following the manufacturer's recommendations. Cells were allowed to recover for 6 to 18 h prior to metabolic labeling. For metabolic pulse labeling, cells were washed twice in Dulbecco's modified Eagle's medium lacking cysteine and methionine and then incubated in the same media supplemented with 10% dialyzed fetal calf serum for half an hour. 200 µCi/ml of [35S]Pro-mix (Amersham Biosciences) was then added for 30 min. For overnight labeling experiments, the 30-min starvation period was omitted and cells were labeled with 200 µCi/ml of [35S]Pro-mix for 10-14 h. In all cases, after labeling, the cells were washed twice with phosphate-buffered saline, pH 7.4, and immediately lysed in cell lysis buffer (TBS, pH 7.4, 1% (v/v) Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, and protease inhibitors 1 mM 1,10-phenanthroline, 2 µg/ml leupeptin, 0.4 mM benzamidine, 10 µg/ml soybean trypsin inhibitor, 0.5 mM iodoacetamide), or incubated in Opti-MEM (Invitrogen) for the indicated number of hours. Conditioned media was collected after 1 h and cell debris was removed by centrifugation at 13,000 x g for 20 min followed by a further 100,000 x g spin for 45 min. p75NTR protein was then immunoprecipitated from the media and lysates with anti-REX IgG. p75NTR protein from dorsal root ganglia cultures was immunoprecipitated with mc192 IgG. In all cases, immunoprecipitations were performed at 4 °C for at least 6 hours followed by washing, and samples were then analyzed by SDS-PAGE and autoradiography as described below.

In experiments in which PMA, pervanadate, or BB-94 were added, cells were first chased for 2 h followed by an additional chase of 15 min or 1 h in fresh Opti-MEM containing the indicated concentration of additive(s). In experiments examining the effect of bisindolylmaleimide I or of the MAP kinase inhibitors U0126 and SB202190, the indicated concentration of inhibitor was present both during the initial 2-h chase and during the following 1-h chase. Supernatants were then collected and cells lysed as before.

For immunoprecipitations, the appropriate antibody and Protein A-Sepharose were added to the lysate or supernatant and incubated at 4 °C from 2 h to overnight. The beads were then washed three times with lysis buffer (lysates) or PBS, pH 7.4, 0.05% (v/v) Nonidet P-40 (supernatant). 2x sample loading buffer supplemented with 10 mM dithiothreitol was added and the samples were incubated at 95 °C for 5 min prior to SDS-PAGE analysis. Gels were fixed in 50% (v/v) methanol, 10% (v/v) acetic acid for 15 min, rehydrated in water for 15 min, dried, and exposed to Kodak Bio-MAX MR film. Quantification was performed using a Fuji BAS2500 BioImaging analyzer.

Cell surface biotinylation of p75 was carried out as described (66, 67) and subsequent shedding was performed at 37 °C for 15 min. Deglycosylation experiments were carried out using endoglycosidase H (EndoH) and peptide N-glycosidase F as described (66, 67).

Determination of the Cleavage Site for TACE in Peptides Corresponding to the Juxtamembrane Domain of p75NTR—Two overlapping peptides corresponding to the juxtamembrane region of p75NTR were synthesized by Synpep (Dublin, CA). Both peptides contained a dinitrophenyl group at the their NH2 terminus to facilitate detection at 350 nM (peptide 1, dinitrophenyl-VTTVMGSSQPVVTRGT-CONH2; peptide 2, dinitrophenyl-VVTRGTTDNLIP-CONH2). Peptide cleavage assays were run as previously described (68). Briefly, peptides were incubated with the catalytically active purified recombinant extracellular domain of TACE (catalytic domain, disintegrin domain, and cysteine-rich region) and the cleavage products were analyzed by liquid chromatography matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Only peptide 1 was cleaved by TACE under these conditions in vitro, between residues QP and VV (data not shown).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the shedding of the p75 neurotrophin receptor (p75NTR), we assayed for the release of its 35S-labeled ectodomain into the tissue culture supernatant. Initially, we used wild-type CHO cells and the previously described M2 mutant CHO cells, which are defective in the metalloprotease-mediated shedding of several transmembrane proteins (5). The mutation in the M2 cells inactivates the TNF{alpha} convertase (TACE, ADAM17)2 and abolishes the stimulated shedding of all TACE substrates examined to date (69, 70). Cells transiently transfected with either a control vector or a plasmid encoding for rat p75NTR were pulse-labeled. Labeled proteins were recovered from the media and cell lysates immediately after the labeling period or after a 6-h chase (Fig. 1A). p75NTR is initially synthesized as a protein with a molecular mass of ~70 kDa (lanes 2 and 5) that is completely converted to the 75-kDa mature form after the 6-h chase period (lanes 3 and 6). This shift is likely because of differences in glycosylation (see below), and is not affected in the M2 mutant cells. After the 6-h chase period, a significant fraction of the labeled p75NTR was recovered as a 50-kDa ectodomain in the culture supernatant of wild-type CHO cells (lane 7), whereas no p75NTR ectodomain was detected in the media of the M2 mutant cells (lane 8).



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  		FIG. 1.
Constitutive release of p75NTR from wild-type and M2 mutant CHO cells. A, pulse-chase analysis of wild-type (wt, lanes 1-3 and 7) or M2 mutant (lanes 4-6 and 8) CHO cells transiently transfected with pcDNA3 (-, lanes 1 and 4)orp75NTR (+, lanes 2-3 and 5-8). Cells were chased for 6 h (lanes 3 and 6-8) or lysed immediately after metabolic labeling (0 h, lanes 1-2 and 4-5). Full-length p75NTR was immunoprecipitated from the lysate (lanes 1-6) and soluble p75NTR ectodomain was immunoprecipitated from culture supernatants (lanes 7 and 8). B, constitutive shedding of p75NTR is inhibited by BB-94 batimastat). Wild-type CHO cells transfected with the p75NTR expression vector were pulse-labeled and then chased for 6 h in the absence (lanes 1) or presence (lanes 2) of 2 µM BB-94. Soluble (upper panels) or full-length (lower panel) p75NTR was then immunoprecipitated from culture supernatants and lysates, respectively.

 
The shedding of numerous transmembrane proteins, including p75NTR (54, 55), is mediated by metalloproteases. To determine whether p75NTR shedding involves metalloproteases in CHO cells, constitutive shedding was examined in the presence or absence of the hydroxamate-based metalloprotease inhibitor, BB-94 (batimastat, Fig. 1B). The constitutive release of the p75NTR ectodomain was severely diminished in the presence of inhibitor (Fig. 1B, top panel), with a concomitant increase in the amount of labeled full-length protein recovered in the lysate (Fig. 1B, lower panel). Thus, similar to the behavior of p75NTR in Schwann cells (54, 55), the constitutive shedding of p75NTR is apparently dependent upon the action of one or more metalloproteases in CHO cells. Because BB-94 does not completely block p75NTR release (Fig. 2A), it is also possible that a small amount of shedding is because of activities that are not metalloprotease-dependent. Alternatively, it is also possible that the relevant metalloprotease activities cannot be completely inhibited by BB-94, for example, if some shedding originates from a location in the secretory pathway that is not completely accessible to exogenously added BB-94.



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  		FIG. 2.
Comparison of constitutive and PMA- and pervanadate-stimulated shedding of p75NTR in wild-type and M2 mutant CHO cells, and shedding of endogenous p75NTR from cultured primary mouse dorsal root ganglia cells and PC12 cells. A, loss of regulated shedding in M2 mutant CHO cells. Wild-type (wt; top panel) or M2 mutant (mut; lower panel) CHO cells were transfected with p75NTR expression vector. The cells were pulse-labeled and chased for 2 h. Supernatants were then replaced with fresh media with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5)2 µM BB-94 (batimastat) in addition to 25 ng/ml PMA (lanes 3 and 4), 100 µM pervanadate (lanes 5 and 6), or no further additives (lanes 1 and 2), and chased for 1 h. Soluble p75NTR was then immunoprecipitated from culture supernatants. B, specificity of pervanadate stimulation. Wild-type CHO cells transfected with the p75NTR expression vector were labeled and chased as in A, and then chased for an additional 1 h with fresh media alone (lane 1) or media containing 100 µM vanadate (lane 2), 100 µM hydrogen peroxide (lane 3), or 100 µM pervanadate (lane 4). Soluble p75NTR ectodomain was then immunoprecipitated from the culture supernatant. C, constitutive and stimulated shedding of endogenous p75NTR from cultured primary dissociated dorsal root ganglia cells (DRG, lanes 1 and 2) and a rat pheochromocytoma cell line (PC12, lanes 3 and 4). Both cell types were metabolically labeled and stimulated as described above. Endogenous p75NTR was immunoprecipitated from supernatants incubated for 1 h in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 25 ng/ml PMA.

 
Next, we examined the effect of either the phorbol ester PMA or pervanadate, a tyrosine phosphatase inhibitor, on the shedding of p75NTR (Fig. 2A). Both stimuli have been shown to up-regulate the metalloprotease-mediated shedding of several proteins (3, 40, 71). In wild-type CHO cells, both PMA and pervanadate are able to increase the release of the p75NTR ectodomain (Fig. 2A, top panel). Furthermore, the activated shedding events are also inhibited by BB-94. The effect of pervanadate on shedding is specific, as neither hydrogen peroxide nor vanadate enhances shedding (Fig. 2B). The stimulated shedding of numerous proteins, including several TACE substrates, in response to agents such as phorbol esters is absent in the M2 CHO cell line (5, 10, 70, 72). We therefore assessed the ability of this cell line to shed p75NTR when stimulated. M2 cells failed to release significant amounts of p75NTR ectodomain either constitutively or in response to either PMA or pervanadate (Fig. 2A, bottom panel). p75NTR is also shed in a constitutive and stimulated manner from several other cells, including transiently transfected COS-7 cells (see below), as well as two non-transfected cell types that endogenously express this receptor, cultured primary dissociated mouse dorsal root ganglia cells and the PC12 rat pheochromocytoma cell line (Fig. 2C).

Numerous signaling pathways have been implicated in metalloprotease-mediated ectodomain shedding. We used pharmacological inhibitors to assess the role of protein kinase C and the MEK/ERK and p38 MAP kinase pathways in the release of p75NTR ectodomains. The protein kinase C inhibitor bisindolylmaleimide I blocks the PMA-stimulated shedding of p75NTR, but does not affect either the constitutive or pervanadate-stimulated release of these proteins (Fig. 3A). The constitutive shedding of p75NTR is inhibited by SB202190, an inhibitor of the p38 MAP kinase pathway, but is not affected by inhibition of MEK 1 and 2 by U0126 (Fig. 3B). Both U0126 and SB202190 partially reduced the release of the p75NTR ectodomain in cells treated with PMA, the latter likely by inhibiting the underlying constitutive shedding pathway. However, even in the presence of both inhibitors, the shedding of the p75NTR ectodomain is still increased by PMA compared with unstimulated shedding levels, indicating that an additional shedding activity not dependent on the p38 or MEK/ERK MAP kinase pathways is activated. In contrast, the increase in p75NTR shedding seen in response to pervanadate treatment, compared with constitutive shedding, is completely abrogated by the MEK 1 and 2 inhibitor, U0126. Furthermore, pervanadate does not enhance the SB202190-sensitive shedding activity above what is seen in unstimulated cells.



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  		FIG. 3.
Effect of protein kinase C and MAP kinase inhibitors and of TIMP-1 and TIMP-2 on shedding of p75NTR. A, effect of the protein kinase C inhibitor bisindolylmaleimide I (bisind I) on constitutive, PMA-, and pervanadate-stimulated p75NTR ectodomain release. CHO cells transfected with p75NTR expression vectors were pulse-labeled and chased for 6 h in fresh media alone (lanes 1 and 2), with 25 ng/ml PMA (lanes 3 and 4), or with 100 µM pervanadate (lanes 5 and 6). For each condition, 5 µM bisindolylmaleimide I was either included (lanes 2, 4, and 6) or omitted (lanes 1, 3, and 5). B, effect of MAP kinase inhibitors on constitutive and stimulated shedding. CHO cells were transfected with p75NTR expression vector. Cells were metabolically labeled for 30 min and then chased for 2 h in media containing either carrier, 5 µM U0126 (a MEK 1 and 2 inhibitor), 10 µM SB202190 (a p38 MAP kinase inhibitor), or both inhibitors. Cells were then placed in fresh media containing the same inhibitors with no further additions (-), 25 ng/ml PMA (PMA), or 100 µM pervanadate (PV) and chased for an additional hour. Full-length and shed p75NTR were recovered from the lysate and conditioned supernatant, respectively, by immunoprecipitation and analyzed by SDS-PAGE. The amount of shed protein recovered from the media was quantified using a phosphorimager. Values were normalized to the amount observed in the untreated cells. Values represent the average of 3 to 5 experiments; error bars indicate one standard deviation. C, p75NTR expressing CHO cells were labeled overnight and chased for 1 h in Opti-MEM alone (lanes 1-3), with 25 ng/ml PMA (lanes 4-6), or with 100 µM pervanadate (lanes 7-9). TIMP-1 (15 nM) or TIMP-2 (18 nM) were present in the chase media as indicated. Soluble ectodomains were isolated from the media by immunoprecipitation and analyzed by SDS-PAGE followed by autoradiography.

 
To further characterize the proteolytic activities involved in p75NTR shedding, we assessed their sensitivity to tissue inhibitors of metalloproteases TIMP-1 and TIMP-2 (Fig. 3C). Neither inhibitor was able to significantly affect either the constitutive or stimulated shedding of p75NTR. In contrast, in parallel experiments, TIMP-2 was found to effectively inhibit pervanadate-stimulated TRANCE shedding (40). These results indicate that matrix metalloproteases are unlikely to be involved in this process, as most matrix metalloproteases are inhibited by one or both of these TIMPs. However, we cannot rule out that the sheddases involved are not accessible to the TIMPs.

To address the issue of where in the secretory pathway shedding of p75NTR occurs, we examined the sensitivity of membrane-anchored and soluble p75NTR to treatment with EndoH and PNGaseF. N-Linked carbohydrate residues acquire resistance to treatment with EndoH by conversion of high mannose glycans into complex carbohydrates during passage through the medial Golgi apparatus. When membrane-anchored p75NTR was immunoprecipitated from the cell lysate of transiently transfected COS-7 cells, we found that the 75-kDa form of p75NTR (marked by an asterisk in Fig. 4A, lanes 1 and 2) was mainly resistant to EndoH treatment, whereas the 70-kDa immature proform of the receptor was mostly or completely EndoH-sensitive (marked by an arrow in Fig. 4A, lanes 1 and 2). Soluble p75NTR immunoprecipitated from the supernatant was resistant to treatment with EndoH (Fig. 4A, lane 5), as would be expected following passage through the secretory apparatus and release from cells. Membrane anchored as well as soluble p75NTR could be deglycosylated with PNGaseF, which removes all N-linked carbohydrate residues (Fig. 4A, lanes 3 and 6). Identical results were obtained when cell-associated and shed p75NTR was immunoprecipitated from PC12 cells and subjected to treatment with EndoH and PNGaseF (data not shown). The EndoH resistance of most or all of the 75-kDa form of p75NTR suggests that shedding of p75NTR occurs after passage through the medial Golgi apparatus. Finally, biotinylated p75NTR can be immunoprecipitated from lysates of cell surface-biotinylated COS-7 cells, which confirms that p75NTR is present on the cell surface (Fig. 4B, lanes 1-3).



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  		FIG. 4.
Evaluation of the subcellular localization of p75NTR shedding. A, treatment of p75NTR with EndoH (lanes 2 and 4) or peptide N-glycosidase F (PNGaseF, lanes 3 and 5). p75NTR was immunoprecipitated from cell lysates of transiently transfected COS-7 cells (lanes 1-3), and soluble p75NTR was immunoprecipitated from the corresponding culture supernatant (lanes 4-6). Aliquots of the immunoprecipitated material were treated with EndoH (lanes 2 and 4) or PNGaseF (lanes 3 and 5). The migration of the 75-kDa form of p75NTR (lane 2, asterisk) is largely unaffected by EndoH treatment, whereas the immature 70-kDa form of the receptor (arrow in lane 1) is sensitive to deglycosylation by EndoH (compare lanes 1 and 2). Because passage through the medial Golgi compartment usually renders N-linked carbohydrate residues resistant to treatment with EndoH, this demonstrates that the 75-kDa form of p75NTR accumulates in an uncleaved form after passage through the medial Golgi apparatus (i.e. in the trans-Golgi network or on the cell surface). Treatment with PNGaseF removes all N-linked carbohydrate residues from p75NTR (lanes 3). Shed p75NTR in the supernatant is resistant to treatment with EndoH (lanes 4 and 5), but sensitive to treatment with PNGaseF (lane 6). Open arrowheads mark the position of untreated, fully glycosylated transmembrane or soluble p75NTR, and solid arrowheads mark the position of PNGaseF-treated forms of p75NTR. B, shedding of cell surface-biotinylated p75NTR. COS-7 cells expressing p75NTR were biotinylated with a non-membrane-permeable biotinylation reagent (see "Experimental Procedures" for details). After quenching the biotinylation reaction, the cells were incubated for 15 min in growth medium with no additions (lanes 1 and 4), with 25 ng/ml PMA (lanes 2 and 5), or with 100 µM pervanadate (lanes 3 and 6). Biotinylated p75NTR was immunoprecipitated from cell lysates (lanes 1-3), and shed p75NTR was immunoprecipitated from the corresponding supernatants (lanes 4-6). Arrowheads, membrane-bound p75NTR in lanes 1-3 and shed p75NTR ectodomain in lanes 4-6; asterisk, nonspecific background band in lanes 4-6.

 
When cell surface-biotinylated p75NTR expressing COS-7 cells were incubated in tissue culture medium for 15 min at 37 °C after cell surface labeling, soluble biotinylated p75NTR was released (Fig. 4B, lane 4, arrowhead marks position of soluble p75NTR). Shedding of cell surface-biotinylated p75NTR was slightly enhanced by treatment with PMA (Fig. 4B, lane 5), and strongly enhanced by treatment with PV (Fig. 4B, lane 6). The increased shedding of cell surface-labeled p75NTR following PV stimulation was also reflected in a concomitant decrease in cell-associated p75NTR in these cells (Fig. 4B, lane 3). Taken together, these results suggest that shedding of p75NTR occurs at or near the cell surface in transiently transfected COS-7 cells.

The stimulated shedding of several proteins is mediated by the TNF{alpha}-converting enzyme (TACE/ADAM17). To assess the role of ADAM17 as well as other candidate sheddases of the ADAM family in the shedding of p75NTR, we investigated release of the p75NTR ectodomain in murine fibroblasts isolated from adam9/12/15-/-, adam10-/-, or adam17-/- mice. When transfected into these cells, p75NTR is shed constitutively at low levels (Fig. 5). However, whereas stimulation with PMA strongly enhances p75NTR shedding in wild-type murine fibroblasts and in adam9/12/15-/- or adam10-/- cells, no PMA-dependent stimulation of p75NTR shedding was observed in adam17-/- cells. In separate experiments, we confirmed that pervanadate stimulates p75NTR shedding in mouse fibroblasts, and that this stimulation is also abolished in adam17-/- cells (data not shown). Thus, TACE is essential for both phorbol ester- and pervanadate-stimulated shedding of p75NTR in murine fibroblasts.



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  		FIG. 5.
Ectodomain shedding of p75NTR in wild-type or adam9/12/15-/-, adam17-/-, or adam10-/- mouse embryonic fibroblasts. Primary mouse embryonic fibroblasts from wild-type mice, adam9/12/15-/- triple knockout mice, adam17-/- mice, or immortalized adam10-/- embryonic cells (65) were transfected with p75NTR, metabolically labeled overnight and chased for 1 h in media with carrier (-), 25 ng/ml PMA, or 25 ng/ml PMA in the presence of 2 µM BB94 as indicated. Soluble p75NTR ectodomain was immunoprecipitated from the media and analyzed by SDS-PAGE. Full-length p75NTR was immunoprecipitated from the corresponding lysates to confirm that the expression levels were comparable in wild-type and adam-/- cells (data not shown).

 
What is the cleavage site that is processed to generate soluble p75NTR? This question was initially addressed by in vitro cleavage of peptides corresponding to the juxtamembrane domain of p75NTR by TACE, and then by assessing how mutagenesis of the membrane-proximal stalk domain of p75NTR affects its shedding from cells. When two peptides corresponding to partially overlapping fragments of the juxtamembrane domain of p75NTR were incubated with purified TACE in vitro, one of these peptides was cleaved between residues Pro234 and Val235 (data not shown, see "Experimental Procedures" for details). However, when two mutant forms of p75NTR carrying point mutations at this site (QPVV > QAAV, QPVV > QPAV) were expressed in COS-7 cells, no defect in constitutive and stimulated shedding compared with the wild-type receptor was observed (Fig. 6, lanes 19-22). Furthermore, deletion of 8 aa residues including the QPVV sequence ({Delta}8aa) also had little or no effect on constitutive or stimulated p75NTR shedding (Fig. 6B, lanes 9 and 10).



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  		FIG. 6.
Evaluation of the effect of mutations in the juxtamembrane region of p75NTR on its constitutive and stimulated shedding. A, schematic illustration of specific point mutations and deletions in the membrane-proximal region of p75NTR. The designation of each mutant is listed on the left and corresponds to the designations in panels B and C. Deleted aa residues are replaced by a hyphen, and the two point mutations are indicated by lowercase bold letters. Amino acid residues that are predicted to be part of the hydrophobic transmembrane domain of p75NTR are lowercase. The cytoplasmic domain is predicted to begin with the two charged aa residues Lys-Arg (KR). B, effect of mutations on constitutive and stimulated shedding of p75NTR. COS-7 cells expressing wild-type or mutant forms of p75NTR were labeled for 1 h, then chased for 1 h in fresh medium with carrier (-). This was followed by a 1-h chase in fresh medium containing 25 ng/ml PMA. p75NTR was immunoprecipitated from supernatants of unstimulated and PMA-stimulated cells as indicated, and separated on 10% SDS-polyacrylamide gels. The expression of wild-type and mutant forms of p75NTR was similar in all cases (data not shown). C, initial mapping of the position of the cleavage site of p75NTR. Western blot analysis of the culture supernatant (lanes 1-4) and cell lysate (lanes 5-8) of COS-7 cells expressing p75NTR. Samples were separated on a 4-20% SDS gel to visualize the effect of deleting 8 aa residues on soluble p75NTR and the corresponding COOH-terminal membrane stub. The presenilin inhibitor compound E (100 nM) and the proteasome inhibitor lactacysteine (1 µM) were included in the tissue culture medium to prevent degradation of the COOH-terminal stubs after shedding of the ectodomain. Shed wild-type p75NTR (lanes 1 and 3, open arrowhead) migrates slower than the shed {Delta}8aa mutant (lanes 2 and 4, solid arrowhead) in the presence of carrier (lanes 1 and 2) or 25 ng/ml PMA (lanes 3 and 4). The COOH-terminal stubs generated by ectodomain shedding of p75NTR in the presence of carrier (lanes 5 and 6) or 25 ng/ml PMA comigrate. This suggests that p75NTR is processed within the remaining membrane-proximal sequence of the {Delta}8aa mutant (TDNLIPV), even though mutations within this sequence do not abolish constitutive or stimulated p75NTR shedding.

 
One interpretation of these results is that the cleavage site for p75NTR peptides in vitro does not match the physiological cleavage site in cells. Alternatively, the cleavage site used to release p75NTR from cells may not have been present in the two peptide sequences, or may not be efficiently processed by TACE in vitro. To determine whether the cleavage site in cells is amino-terminal or carboxyl-terminal to the QPVV236 sequence, we compared the migration of the soluble forms of wild-type p75NTR and the {Delta}8aa mutant in culture supernatant, and of the remaining COOH-terminal membrane stubs in cell lysates of transiently transfected COS-7 cells. The samples were separated on 4-20% SDS-acrylamide gels to facilitate detection of a shift in migration caused by deletion of 8 aa residues. As shown in Fig. 6C (lanes 1-4), the shed form of {Delta}8aa p75NTR (solid arrowhead) migrates faster than the soluble wild-type receptor (open arrowhead), both with and without PMA stimulation. On the other hand, the membrane-anchored COOH-terminal stubs that remain after ectodomain release of wild-type and {Delta}8aa p75NTR co-migrate (Fig. 6C, lanes 5-8, position of COOH-terminal stubs marked by an arrowhead). At least three closely co-migrating COOH-terminal stubs are generated by shedding of wild-type and {Delta}8aa p75NTR, which indicates that more than one cleavage site may be used to release p75NTR. Taken together, these results suggest that the p75NTR cleavage site(s) in COS-7 cells are located between the deleted sequence in the {Delta}8aa mutant and the transmembrane domain (see Fig. 6A).

This possibility was addressed by generating additional mutant forms of p75NTR with various overlapping deletions in the very membrane-proximal region of p75NTR. Immunoprecipitations of p75NTR from cell lysates of transiently transfected COS-7 cells confirmed that all mutants were expressed at similar levels compared with wild-type p75NTR in the experiment presented in Fig. 6B (data not shown). When soluble mutant forms of p75NTR with short deletions (3-4 aa residues) or with longer deletions (up to 11 aa residues) were immunoprecipitated from the culture supernatant of these cells, no defect in constitutive or stimulated shedding was evident (Fig. 6B, lanes 1-4, 7, 8, and 13-16). Only a large deletion encompassing 15 aa residues of the juxtamembrane region of p75NTR substantially reduced both constitutive and stimulated shedding (Fig. 6B, lanes 5 and 6). Finally, removal of the cytoplasmic domain of p75NTR did not affect shedding, demonstrating that this domain is not required for regulated or constitutive release of p75NTR (Fig. 6, lanes 17 and 18).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Protein ectodomain shedding is a ubiquitous process that results in the release of a variety of functionally and structurally distinct proteins from cells. In this study, we evaluated the properties of the sheddases for the p75NTR, a neurotrophin receptor with diverse roles in the developing nervous system, ranging from axonal growth and retrograde transport to cell survival and apoptosis of different neuronal populations (45-48). We found that p75NTR is shed constitutively by a metalloprotease-dependent activity that requires an intact p38 MAP kinase pathway in CHO cells. Furthermore, metalloprotease-dependent p75NTR shedding can be stimulated by the phorbol ester PMA and the tyrosine phosphatase inhibitor pervanadate. In CHO cells, the phorbol ester-stimulated shedding of p75NTR requires protein kinase C, whereas the pervanadate-stimulated shedding does not. However, the pervanadate-stimulated shedding is blocked by MEK/ERK pathway inhibitors, which also partially reduce shedding in response to PMA.

The effect of MAP kinase pathway inhibitors on constitutive and activated shedding of p75NTR suggests that distinct signaling pathways affect these processes. The constitutive shedding of p75NTR is dependent on signaling via the p38 MAP kinase pathway in CHO cells. In contrast, stimulated shedding apparently acts independently of the p38 MAP kinase, as the absolute decrease in p75NTR release mediated by the p38 MAP kinase inhibitor is similar in the presence or absence of PMA or pervanadate. Conversely, most of the increase in p75NTR shedding seen in response to these stimuli can be blocked by inhibiting the MEK/ERK MAP kinase pathway.

These pharmacological inhibitors of ectodomain shedding raised the possibility that the constitutive and stimulated p75NTR sheddases may be distinct. We decided to further explore this possibility using cells that lack different candidate sheddases of the ADAM family of metalloproteases, and a mutant CHO cell line with a specific defect in the processing and activation of TACE (ADAM17) (70). Wild-type mouse fibroblasts released p75NTR constitutively, and this shedding could be strongly enhanced by stimulation with PMA and pervanadate. Mouse fibroblasts lacking ADAMs9, -12, and -15 (40) or ADAM10 (65) did not show any defect in constitutive or stimulated shedding, arguing against an essential role for these ADAMs in p75NTR shedding in these cells. However, in adam17-/- mouse embryonic fibroblasts (6), both PMA- and PV-stimulated p75NTR shedding was abrogated, whereas a small amount of BB94-insensitive constitutive shedding remained. Thus, whereas TACE is responsible for the PMA- and pervanadate-stimulated release of p75NTR in primary mouse embryonic fibroblasts and CHO cells, the role of TACE in constitutive shedding is unclear.

There are several plausible models to explain constitutive shedding of p75NTR in CHO cells. One model entails distinct sheddases responsible for constitutive and stimulated shedding. TACE would represent the MEK/ERK-dependent sheddase, whereas a second BB94-sensitive, TIMP-1- and TIMP-2-insensitive protease is responsible for constitutive shedding. The M2 mutant CHO cell line would then necessarily carry a defect that affects both of these sheddases. Alternatively, TACE could be responsible for constitutive and stimulated p75NTR shedding in CHO cells. This is plausible, because the basal release of two other TACE substrates, TGF{alpha} and TNF{alpha}, has been shown to depend on p38 MAP kinase activity in CHO cells (42).

To explore the cleavage site requirements for shedding of p75NTR, several point mutations and deletions were introduced into the juxtamembrane region of p75NTR, which is predicted to contain the cleavage site(s) for TACE and the constitutive sheddase. Stimulated shedding of p75NTR was not detectably affected by the point mutations or by overlapping deletions of up to 11 aa residues within the juxtamembrane domain. Only a large deletion of 15 aa residues led to a strong decrease in constitutive and PMA-dependent shedding of p75NTR. These results are reminiscent of studies of how mutations affect shedding of the TACE substrate TNF{alpha} (73), or of angiotensin-converting enzyme (74), which is not a TACE substrate (39). Shedding of TNF{alpha} and ACE was also only affected by relatively large deletions in the juxtamembrane domain, but not by shorter deletions. As has been suggested for TNF{alpha} and ACE, large deletions within the juxtamembrane domain of p75NTR may place its ectodomain so close to the plasma membrane that the cleavage site is no longer accessible. In this context it is noteworthy that mutagenesis of the juxtamembrane domains of other shed molecules, including L-selectin (75), p55 TNF receptor (76), and the amyloid precursor protein (77) has uncovered a relaxed sequence specificity of their respective sheddases (3). However, in these studies, certain mutations in or around the predicted cleavage site of each protein did abolish ectodomain shedding. This raises the question of why none of the shorter deletions in the juxtamembrane domain of p75NTR abolished its shedding? As shown in Fig. 6C, lanes 5-8, TACE and the constitutive sheddase(s) appear to cleave the receptor at several closely adjacent sites. Perhaps shorter deletions are not sufficient to abolish all of these potential cleavage sites. Another possibility is that TACE and the constitutive sheddase(s) have a highly relaxed sequence specificity toward p75NTR. Furthermore, the mutations generated in this study may have created new sites for TACE or other sheddases (an example of such a scenario is described in Ref. 78). Finally, a cleavage site may be COOH-terminal to the deleted regions, and thus within the predicted transmembrane domain of p75NTR. Further studies will be necessary to address these and other possibilities.

Ectodomain shedding is likely to occur in most or all cell types. In the case of the p75NTR, shedding may have several different functional consequences. It could conceivably lead to inactivation of the receptor and generation of soluble decoy receptors. This might serve to decrease the concentration of neurotrophins at the cell surface, or lower the concentration of soluble neurotrophins, or both. Furthermore, recent studies demonstrate that p75NTR undergoes RIP (55), which may in turn regulate the function of p75NTR (56). Like in other cases of RIP, luminal membrane-proximal cleavage may be a prerequisite for the intramembrane cleavage of p75NTR (55, 56). However, although p75NTR has been implicated in myelination, it was not possible to evaluate potential defects in this process in adam17-/- mice, because the perinatal lethality of these animals precludes proper analysis of myelination. Therefore future studies, including generation of a conditional TACE knockout mouse will be necessary to address the function of TACE-dependent p75NTR shedding in vivo.

In summary, we present the evaluation of the activities that are involved in the ectodomain shedding of p75NTR in different cell types, including CHO cells, COS-7 cells, and mouse embryonic fibroblasts. Pharmacological inhibitors were used to define different sheddases in these cells and a mutant CHO cell line. In addition, cells lacking ADAMs were used to identify TACE as the major stimulated p75NTR sheddase in these cells. Further studies will be necessary to determine the identity of the constitutive p75NTR sheddase, and to evaluate how shedding affects the function of p75NTR. In light of recent evidence suggesting that p75NTR is subjected to regulated intramembrane proteolysis, it will be interesting to learn whether this process is initiated and regulated by the membrane proximal cleavage by TACE or other metalloproteases in the same manner that RIP is controlled by membrane proximal proteolysis in other systems. The identification of TACE as a major p75NTR sheddase is thus likely to contribute to a better understanding of the regulation of this neurotrophin receptor.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant RO1 GM64750 (to C. P. B.), a grant from GlaxoSmithKline (to C. P. B.), Memorial Sloan-Kettering Cancer Center Support Grant NCI-P30-CA-08748, the Samuel and May Rudin Foundation, and the DeWitt Wallace Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

c Supported in part by a National Institutes of Health Medical Scientist Training Program Training Grant 5T32GM07739-17, the Louis and Rachel Rudin Family Foundation, and a Papanicolaou Medical Scientist Fellowship. Present address: Dept. of Medicine, Columbia Presbyterian Medical Center, 622 West 168th St., New York, NY 10032. Back

d Present address: Johns Hopkins University School of Medicine, 725 North Wolfe St., PCTB Rm. 714, Baltimore, MD 21205. Back

j To whom correspondence should be addressed: Cell Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Box 368, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2915; Fax: 212-717-3047; E-mail: c-blobel{at}ski.mskcc.org.

1 The abbreviations used are: TGF{alpha}, transforming growth factor {alpha}; HB-EGF, heparin-binding epidermal growth factor-like growth factor; ADAM, a disintegrin and metalloprotease; aa, amino acid(s); NTR, neurotrophin receptor; PMA, phorbol-12 myristate 13-acetate; PV, pervanadate; TACE, tumor necrosis factor {alpha} convertase; TIMP, tissue inhibitor of matrix metalloprotease; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRANCE, tumor necrosis factor-related activation-induced cytokine; CHO, Chinese hamster ovary; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; RIP, regulated intramembrane proteolysis; EndoH, endoglycosidase H. Back

2 J. Arribas, personal communication. Back


   ACKNOWLEDGMENTS
 
We are grateful to Drs. J. Peschon and R. Black for providing mice lacking functional TACE and Dr. A. Sehara-Fujisawa for providing mice lacking ADAM12. We thank B. Lee and T. Kacmarczyk for excellent technical assistance.



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