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Metalloproteinase- and γ-Secretase-mediated Cleavage of Protein-tyrosine Phosphatase Receptor Type Z

2008; Elsevier BV; Volume: 283; Issue: 45 Linguagem: Inglês

10.1074/jbc.m802976200

1083-351X

Jeremy P.H. Chow, Akihiro Fujikawa, Hidetada Shimizu, Ryoko Suzuki, Masaharu Noda,

Protease and Inhibitor Mechanisms

Protein-tyrosine phosphatase receptor type Z (Ptprz) is preferentially expressed in the brain as a major chondroitin sulfate proteoglycan. Three splicing variants, two receptor isoforms and one secretory isoform, are known. Here, we show that the extracellular region of the receptor isoforms of Ptprz are cleaved by metalloproteinases, and subsequently the membrane-tethered fragment is cleaved by presenilin/γ-secretase, releasing its intracellular region into the cytoplasm; of note, the intracellular fragment of Ptprz shows nuclear localization. Administration of GM6001, an inhibitor of metalloproteinases, to mice demonstrated the metalloproteinase-mediated cleavage of Ptprz under physiological conditions. Furthermore, we identified the cleavage sites in the extracellular juxtamembrane region of Ptprz by tumor necrosis factor-α converting enzyme and matrix metalloproteinase 9. This is the first evidence of the metalloproteinase-mediated processing of a receptor-like protein-tyrosine phosphatase in the central nervous system. Protein-tyrosine phosphatase receptor type Z (Ptprz) is preferentially expressed in the brain as a major chondroitin sulfate proteoglycan. Three splicing variants, two receptor isoforms and one secretory isoform, are known. Here, we show that the extracellular region of the receptor isoforms of Ptprz are cleaved by metalloproteinases, and subsequently the membrane-tethered fragment is cleaved by presenilin/γ-secretase, releasing its intracellular region into the cytoplasm; of note, the intracellular fragment of Ptprz shows nuclear localization. Administration of GM6001, an inhibitor of metalloproteinases, to mice demonstrated the metalloproteinase-mediated cleavage of Ptprz under physiological conditions. Furthermore, we identified the cleavage sites in the extracellular juxtamembrane region of Ptprz by tumor necrosis factor-α converting enzyme and matrix metalloproteinase 9. This is the first evidence of the metalloproteinase-mediated processing of a receptor-like protein-tyrosine phosphatase in the central nervous system. IntroductionReceptor-like protein-tyrosine phosphatases (RPTPs) 2The abbreviations used are: RPTP, receptor-like protein-tyrosine phosphatase (PTP); ADAM, a disintegrin and metalloproteinase; CHO, Chinese hamster ovary cells; HEK293 cells, human embryonic kidney cells; LTP, long term potentiation; MMP, matrix metalloproteinase; PMA, phorbol 12-myristate 13-acetate; PS, presenilin; PSD95, postsynaptic density-95; RIP, regulated intramembrane proteolysis; TACE, tumor necrosis factor-α (TNF-α) converting enzyme; WT, wild type; chABC, chondroitinase ABC; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase(s). are a structurally and functionally diverse family of enzymes comprised of eight subfamilies (1Tonks N.K. Nat. Rev. Mol. Cell Biol. 2006; 7: 833-846Crossref PubMed Scopus (1231) Google Scholar). Protein-tyrosine phosphatase receptor type Z (Ptprz, also called PTPζ or RPTPβ) is a RPTP classified in the R5 subfamily and expressed in neuronal and glial cells in the central nervous system (2Levy J.B. Canoll P.D. Silvennoinen O. Barnea G. Morse B. Honegger A.M. Huang J.T. Cannizzaro L.A. Park S.H. Druck T. Huebner K. Sap J. Ehrlich M. Musacchio J.M. Schlessinger J. J. Biol. Chem. 1993; 268: 10573-10581Abstract Full Text PDF PubMed Google Scholar, 3Nishiwaki T. Maeda N. Noda M. J. Biochem. (Tokyo). 1998; 123: 458-467Crossref PubMed Scopus (70) Google Scholar, 4Shintani T. Watanabe E. Maeda N. Noda M. Neurosci. Lett. 1998; 247: 135-138Crossref PubMed Scopus (74) Google Scholar). The physiological importance of this molecule has been demonstrated through studies of Ptprz-deficient mice (4Shintani T. Watanabe E. Maeda N. Noda M. Neurosci. Lett. 1998; 247: 135-138Crossref PubMed Scopus (74) Google Scholar, 5Fujikawa A. Shirasaka D. Yamamoto S. Ota H. Yahiro K. Fukada M. Shintani T. Wada A. Aoyama N. Hirayama T. Fukamachi H. Noda M. Nat. Genet. 2003; 33: 375-381Crossref PubMed Scopus (219) Google Scholar), which display impairments in hippocampal function in a maturation-dependent manner (6Niisato K. Fujikawa A. Komai S. Shintani T. Watanabe E. Sakaguchi G. Katsuura G. Manabe T. Noda M. J. Neurosci. 2005; 25: 1081-1088Crossref PubMed Scopus (58) Google Scholar, 7Tamura H. Fukada M. Fujikawa A. Noda M. Neurosci. Lett. 2006; 399: 33-38Crossref PubMed Scopus (76) Google Scholar). An independently generated knock-out mouse line suggests a fragility of myelin in the central nervous system (8Harroch S. Palmeri M. Rosenbluth J. Custer A. Okigaki M. Shrager P. Blum M. Buxbaum J.D. Schlessinger J. Mol. Cell. Biol. 2000; 20: 7706-7715Crossref PubMed Scopus (96) Google Scholar).It is known that three isoforms of Ptprz are generated by alternative splicing from a single Ptprz gene (on mouse chromosome 6; human chromosome 7), the two transmembrane isoforms Ptprz-A and Ptprz-B and the secretory isoform Ptprz-S (also known as 6B4 proteoglycan or phosphacan) (2Levy J.B. Canoll P.D. Silvennoinen O. Barnea G. Morse B. Honegger A.M. Huang J.T. Cannizzaro L.A. Park S.H. Druck T. Huebner K. Sap J. Ehrlich M. Musacchio J.M. Schlessinger J. J. Biol. Chem. 1993; 268: 10573-10581Abstract Full Text PDF PubMed Google Scholar, 9Krueger N.X. Saito H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7417-7421Crossref PubMed Scopus (201) Google Scholar, 10Maeda N. Hamanaka H. Shintani T. Nishiwaki T. Noda M. FEBS Lett. 1994; 354: 67-70Crossref PubMed Scopus (93) Google Scholar, 11Maurel P. Rauch U. Flad M. Margolis R.K. Margolis R.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2512-2516Crossref PubMed Scopus (258) Google Scholar, 12Sakurai T. Friedlander D.R. Grumet M. J. Neurosci. Res. 1996; 43: 694-706Crossref PubMed Scopus (78) Google Scholar), all of which are expressed as chondroitin sulfate proteoglycans in the brain (3Nishiwaki T. Maeda N. Noda M. J. Biochem. (Tokyo). 1998; 123: 458-467Crossref PubMed Scopus (70) Google Scholar). However, some inexplicable issues about the molecular profiles of Ptprz have remained in previous studies. For instance, although there exists substantial expression of the respective transcripts for all isoforms (11Maurel P. Rauch U. Flad M. Margolis R.K. Margolis R.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2512-2516Crossref PubMed Scopus (258) Google Scholar, 13Canoll P.D. Petanceska S. Schlessinger J. Musacchio J.M. J. Neurosci. Res. 1996; 44: 199-215Crossref PubMed Scopus (89) Google Scholar), full-length Ptprz-A has been scarcely observed at the protein level in the adult brain (3Nishiwaki T. Maeda N. Noda M. J. Biochem. (Tokyo). 1998; 123: 458-467Crossref PubMed Scopus (70) Google Scholar, 4Shintani T. Watanabe E. Maeda N. Noda M. Neurosci. Lett. 1998; 247: 135-138Crossref PubMed Scopus (74) Google Scholar). In addition, several lower molecular species have been detected with a specific antibody against Ptprz in wild-type mice (4Shintani T. Watanabe E. Maeda N. Noda M. Neurosci. Lett. 1998; 247: 135-138Crossref PubMed Scopus (74) Google Scholar). The technical difficulty in removal of the chondroitin sulfate chains to separate their core proteins by SDS-PAGE may induce variability in the signal patterns of this molecule in Western blotting among researchers.In this study we examined the molecular profile of Ptprz in the adult mouse brain at both protein and mRNA levels in detail and revealed that the proteolytic fragments are abundantly accumulated. The two receptor isoforms were found to undergo ectodomain cleavage by metalloproteinases, releasing their extracellular fragments. The membrane-tethered fragment of Ptprz was further cleaved by presenilin/γ-secretase to release the intracellular fragment, which was consequently detected in the cytoplasm and nucleus. These findings suggest a novel signaling mechanism of Ptprz by the regulated proteolytic processing in the central nervous system.EXPERIMENTAL PROCEDURESPharmacological Reagents—Phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. GM6001, compound E, lactacystin, and recombinant tumor necrosis factor-α (TNF-α)-converting enzyme (TACE) were from Calbiochem.Animal Experiments—Adult wild-type C57BL/6 mice and Ptprz-deficient mice (4Shintani T. Watanabe E. Maeda N. Noda M. Neurosci. Lett. 1998; 247: 135-138Crossref PubMed Scopus (74) Google Scholar) backcrossed with the inbred C57BL/6 strain for more than 10 generations were used. Mice were administered GM6001, suspended in saline containing 1.5% carboxyl methyl cellulose, intraperitoneally (100 mg per kg body weight). For intraventricular infusion of GM6001, anesthetized mice were placed in a stereotaxic apparatus, and brain infusion cannulas (brain infusion kit 3, Alza Corp.) were inserted in the cerebral ventricle. The cannula was secured to the skull with an anchoring screw and dental cement. The stereotaxic coordinates were 0.5 mm posterior and 1.0 mm lateral to the bregma and 2.4 mm below the skull surface. An osmotic mini-pump (model 1007D, flow rate = 0.5 μl/h, Alza Corp.) filled with 2.5 mm GM6001 in 50% DMSO was implanted between the scapulae and connected to the infusion cannulas. The brains were separated as described (14Glowinski J. Iversen L.L. J. Neurochem. 1966; 13: 655-669Crossref PubMed Scopus (5028) Google Scholar). All animal experiments were performed according to the guidelines of Animal Care with approval by the Committee for Animal Research, National Institutes of Natural Sciences.Expression Constructs for Ptprz Isoforms—Full-length rat Ptprz-A and Ptprz-S (10Maeda N. Hamanaka H. Shintani T. Nishiwaki T. Noda M. FEBS Lett. 1994; 354: 67-70Crossref PubMed Scopus (93) Google Scholar) were subcloned into the expression vector pZeoSV2 (Invitrogen) to yield pZeo-PTPζ-A and pZeo-PTPζ-S, respectively. The expression plasmid for rat Ptprz-B (pZeo-PTPζ) was described previously (15Kawachi H. Fujikawa A. Maeda N. Noda M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6593-6598Crossref PubMed Scopus (109) Google Scholar). This construct was used as a template to generate pZeo-PTPζ-G1631I (a mutant in which glycine at 1631 is substituted with isoleucine) by using a QuikChange multi-site-directed mutagenesis kit (Stratagene). The expression plasmid (pZeoSV-PtprzICR) for the entire intracellular region of rat Ptprz-A/-B (amino acid residues 1665–2316; GenBank™ accession number U09357) was prepared by PCR from pZeo-PTPζ with an initiative methionine encoding primer and cloning the fragment into the NotI site of pZeoSV2.Cell Culture and DNA Transfection—HEK293T cells (human embryonic kidney epithelial cells) were grown and maintained on dishes coated with rat tail collagen in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified incubator at 37 °C with 5% CO2. HEK293 cell lines stably expressing either human wild-type presenilin 1 (PS1 WT) or a dominant-negative PS1 variant (substitution of Asp at 385 for Ala, PS1 D385A) (16Kasuga K. Kaneko H. Nishizawa M. Onodera O. Ikeuchi T. Biochem. Biophys. Res. Commun. 2007; 360: 90-96Crossref PubMed Scopus (45) Google Scholar) were kindly provided by Takeshi Ikeuchi (Niigata University, Niigata, Japan). These cells were transfected with Ptprz expression plasmids by calcium-phosphate precipitation as described (17Fujikawa A. Chow J.P.H. Shimizu H. Fukada M. Suzuki R. Noda M. J. Biochem. (Tokyo). 2007; 142: 343-350Crossref PubMed Scopus (24) Google Scholar).CHO-M2 (TACE-deficient CHO cell line), CHO-(M2+TACE) (CHO-M2 cells rescued by expression of TACE), and parental CHO-WT cells (18Borroto A. Ruíz-Paz S. de la Torre T.V. Borrell-Pagès M. Merlos-Suárez A. Pandiella A. Blobel C.P. Baselga J. Arribas J. J. Biol. Chem. 2003; 278: 25933-25939Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) were kindly provided by Joaquín Arribas (University Hospital Vall d'Hebron, Barcelona, Spain). CHO-M2 and CHO-WT cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 500 μg/ml of G418. CHO-(M2+TACE) cells were maintained with G418 and hygromycin (500 μg/ml of each). These CHO cells were transfected by using Lipofectamine Plus reagent (Invitrogen; 1 μg of plasmid per 3.5-cm dish). Transfected cells were replated once on 3.5-cm dishes, cultured for 24 h, and then used for the experiments.Protein Extraction and Chondroitinase ABC (chABC) Treatment—Mouse tissues quickly separated on ice were homogenized with more than 10 volumes of a lysis buffer: 20 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 137 mm NaCl, 10 mm NaF, 1 mm sodium orthovanadate, and a EDTA-free protease inhibitor mixture (complete EDTA-free, Roche Applied Science). Supernatants were then collected by centrifugation at 15,000 × g for 15 min. Cultured cells were extracted with the same lysis buffer (300 μl per 3.5-cm dish) as above. The samples were stored at -85 °C before use.For chABC digestion, the protein concentration of each sample was adjusted with the lysis buffer (<4 mg/ml). Aliquots (10 μl) were then incubated with an equal volume of 0.2 m Tris-HCl, 4 mm sodium acetate, pH 7.5, with or without chABC (Seikagaku Co., Tokyo, Japan; the enzyme was added at 60 microunits/μg of protein) for 1 h at 37°C. Protein concentrations were determined with a Micro BCA protein assay kit (Pierce).Western Blot Analysis—Samples were mixed with an equal volume of 2× SDS-PAGE sample buffer (containing 4% mercaptoethanol), boiled for 5 min, and then separated on a 5–20% gradient polyacrylamide gel (E-R520L, Atto Corp., Tokyo, Japan). Proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp.) for 1 h using a conventional semidry electrotransfer (1.3 mA per cm2). The membrane was incubated for 1 h in a blocking solution (4% nonfat dry milk and 0.1% Triton X-100 in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl) and incubated overnight with anti-Ptprz-S rabbit serum (1:10,000) (3Nishiwaki T. Maeda N. Noda M. J. Biochem. (Tokyo). 1998; 123: 458-467Crossref PubMed Scopus (70) Google Scholar) in the blocking solution supplemented with 0.04% SDS to prevent nonspecific binding. Mouse monoclonal anti-RPTPβ (the epitope region is amino acid residues 2098–2307 of human Ptprz-A, 250 ng/ml, BD Biosciences) was incubated with the blots in the blocking solution. The binding of these antibodies was detected with an ECL Western blotting system (GE Healthcare).Subcellular Localization Analysis—Cells were washed 3 times with 10 mm phosphate buffer, pH 7.3, containing 150 mm NaCl, fixed with 10% neutral formalin, and then blocked with the blocking buffer as above and followed by overnight incubation with anti-RPTPβ (1 μg/ml) in the blocking buffer. Bound antibodies were visualized with Alexa488-conjugated anti-mouse antibody (Molecular Probes, Eugene, OR). For the nucleus labeling, the cells were incubated with TO-PRO-3 (Molecular Probes) and then analyzed with a Zeiss LSM-510 confocal scanning laser microscope (Carl Zeiss, Jena, Germany) using a Zeiss water-immersion objective (C-Apochromat 40×/1.20 W Korr).Northern Blot Analysis—Total RNA was isolated from mouse tissues using TRIzol (Invitrogen), and then poly(A)+ RNA was purified using the Dynabeads mRNA purification kit (Dynal) according to the manufacturer's instructions. Northern blotting was performed as described (19Suzuki R. Shintani T. Sakuta H. Kato A. Ohkawara T. Osumi N. Noda M. Mech. Dev. 2000; 98: 37-50Crossref PubMed Scopus (99) Google Scholar) with slight modifications in the electrophoresis. The poly(A)+ RNA was denatured in 1× MOPS buffer (20 mm MOPS, 2 mm sodium acetate, and 1 mm EDTA, pH 7.0) containing 6.8% formaldehyde, 50% formamide, and 50 μg/ml of ethidium bromide at 67 °C for 10 min, chilled on ice for 5 min, and then 2.5 μl of a loading buffer (50% glycerol, 0.25% bromphenol blue, and 0.25% xylene cyanol) was added. Electrophoresis was performed on a formaldehyde-denatured agarose gel (6.8% formaldehyde and 1% agarose in 1× MOPS buffer) at 5 V/cm for 4 h with the circulation of an electrophoresis buffer (6.8% formaldehyde in 1× MOPS buffer).Templates of complementary DNA probes were as follows: CAH-FNIII probe (nucleotide residues 93–1215 for rat Ptprz-A; GenBank™ accession number U09357), PTP-D1 probe (nucleotide residues 5047–6081 for rat Ptprz-A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (nucleotide residues 22–553 for mouse GAPDH; GenBank™ accession number BC096440). Signals of bands on the blot were detected using a BAS-MS 2025 imaging plate (Fuji Photo Film, Tokyo, Japan) and visualized using a Typhoon 9400 scanner (GE Healthcare).In Vitro Digestion Analyses—Peptides were synthesized on an Applied Biosystems ABI 433A peptide synthesizer by using the standard fluorenylmethoxycarbonyl protocol and purified by high pressure liquid chromatography on a C-18 reverse phase column. These peptides (2 pmol each) were incubated at 37 °C for 30 h in 25 mm Tris-HCl, pH 9.0, 2.5 μm ZnCl2, and 0.005% Brij-35 with or without recombinant TACE (250 ng) in a final volume of 10 μl. After purification using pipette tips packed with a C18 resin (ZipTip, Millipore), the samples were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (Reflex III, Bruker Daltonics) using α-cyano-4-hydroxycinnamic acid matrix (Sigma).RESULTSExpression Profile of Ptprz in the Adult Mouse Brain—It is well known that three splicing variants of Ptprz are expressed in the brain from a single gene (see Fig. 1A). All three isoforms expressed in the brain are highly glycosylated with chondroitin sulfate (3Nishiwaki T. Maeda N. Noda M. J. Biochem. (Tokyo). 1998; 123: 458-467Crossref PubMed Scopus (70) Google Scholar). Therefore, the removal of the chondroitin sulfate chains beforehand is necessary to resolve their core proteins by SDS-PAGE (20Maeda N. Hamanaka H. Oohira A. Noda M. Neuroscience. 1995; 67: 23-35Crossref PubMed Scopus (129) Google Scholar).When the chABC-treated extract of the wild-type mouse brain (+/+) was analyzed with anti-Ptprz-S, which recognizes the extracellular region of all three isoforms (see Fig. 1A), six bands (bands b–g) in the range from 300 to 70 kDa were clearly detected (Fig. 1B). Because these bands are not present in Ptprz-deficient mice (-/-), all these molecular species are considered to be derived from Ptprz gene products. Among them, the 300-kDa (band b) and 250-kDa (band c) species represent the core proteins of Ptprz-S and Ptprz-B, respectively (3Nishiwaki T. Maeda N. Noda M. J. Biochem. (Tokyo). 1998; 123: 458-467Crossref PubMed Scopus (70) Google Scholar); however, the other four species (bands d–g) have not been characterized; of note is that the band of Ptprz-A at 380 kDa is scarcely detected (the band a with an asterisk in Fig. 1E; see also Ref. 4Shintani T. Watanabe E. Maeda N. Noda M. Neurosci. Lett. 1998; 247: 135-138Crossref PubMed Scopus (74) Google Scholar). The signal intensity of the three lower molecular species (bands e–g) was not changed by chABC treatment (Fig. 1B), indicating that they are not modified with chondroitin sulfate. In contrast, the larger bands b–d were almost missing without chABC-treatment because they could not enter the gel.To identify the receptor isoforms and their derivatives, the same blot was reprobed with anti-RPTPβ, which recognizes the intracellular region (see Fig. 1A). As shown in Fig. 1C, the 250-kDa species (band c, Ptprz-B) was detected by anti-RPTPβ as expected along with additional bands at around 75 kDa. The enlarged view of the 75-kDa band (lower panel), demonstrated that the signal consists of two adjacent bands of 77 kDa (band h) and 73 kDa (band i). On the other hand, anti-RPTPβ did not recognize the other species (bands b, d, e–g) detected by anti-Ptprz-S.Although the uncharacterized species of Ptprz (bands d–i) appears to be processing products of the mature three isoforms of Ptprz, we addressed the possibility with the best of care that unknown novel Ptprz transcripts might be detected by Northern blotting. Probe 1 for the CAH-FNIII region, which should detect all Ptprz transcripts, demonstrated that the three transcripts of 8.5 kb (Ptprz-A), 7.5 kb (Ptprz-S), and 5.8 kb (Ptprz-B) are expressed only in the wild-type, not in the knock-out mice (Fig. 2). Probe 2 for the PTP-D1 region, which detects the transcripts for the receptor isoforms, showed the 8.5-kb (Ptprz-A) and 5.8-kb (Ptprz-B) transcripts as expected. Although full-length Ptprz-A protein (380 kDa) was hardly detected in the adult brain lysate, its mRNA was, thus, expressed in a significant amount. Importantly, other transcripts corresponding to the smaller Ptprz proteins such as the 180- or 75-kDa species were not detected.FIGURE 2Northern blot analyses of Ptprz transcripts in the adult mouse brain. Poly(A)+ RNA (2 μg) from the adult mouse brain was hybridized with a 32P-labeled cDNA probe for the CAH-FNIII region (probe 1). The same blot was stripped and then reprobed for the PTP-D1 region (probe 2). The amount of RNA loaded was confirmed with a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same blot. The figures are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ectodomain Shedding of Ptprz by Metalloproteinases—In our studies to exogenously express Ptprz-B in mammalian cells, we noticed that an immunoreactive species of 180 kDa is secreted into the culture medium. Because this size corresponded to that of the whole extracellular region of Ptprz-B (band d observed in the brain in Fig. 1B), we assumed that this is generated by the ectodomain shedding, a specialized type of limited proteolysis releasing the extracellular domain of a variety of cell surface receptors (21Arribas J. Borroto A. Chem. Rev. 2002; 102: 4627-4637Crossref PubMed Scopus (205) Google Scholar, 22Anders L. Mertins P. Lammich S. Murgia M. Hartmann D. Saftig P. Haass C. Ullrich A. Mol. Cell. Biol. 2006; 26: 3917-3934Crossref PubMed Scopus (87) Google Scholar); it occurs in the vicinity of the cell surface, generally dependent upon the actions of matrix metalloproteinases (MMPs) or adamalysins (ADAMs, a disintegrin and metalloproteinases).We tested this possibility by using a protein kinase C activator, PMA, which is known to trigger ectodomain shedding in various cells. The treatment of HEK293T cells expressing Ptprz-B with PMA resulted in an increase in the 180-kDa species in the conditioned medium, which was inversely correlated with the decrease in Ptprz-B in cell extracts (Fig. 3A). This event occurred dependent on the concentration of PMA (Fig. 3B), strongly suggesting that the band of 180 kDa represents the ectodomain (ZB-ECF) of Ptprz-B. Intriguingly, in the presence of a broad-spectrum metalloproteinase inhibitor, GM6001, the generation of the 180-kDa species was clearly inhibited under both unstimulated and stimulated conditions with PMA (Fig. 3A, right panel).FIGURE 3Metalloproteinase-mediated ectodomain cleavage of the receptor isoforms of Ptprz. A, HEK293T cells were transiently transfected with the expression construct of Ptprz-B or control vector (Moc). Twenty-four hours after transfection, cells were washed and incubated with or without PMA in fresh serum-free medium for 1 h. GM6001 was added 20 min before the stimulation by PMA or vehicle. The cell extracts (left panels) were analyzed by Western blotting (WB) using anti-Ptprz-S. The same membrane was then reprobed with anti-RPTPβ. Conditioned media (right panel) were analyzed with anti-Ptprz-S. B, cells were incubated with the indicated amount of PMA in fresh serum-free medium for 1 h and analyzed by Western blotting as above. C, HEK293T cells were transiently transfected with the Ptprz-A or Ptprz-S expression construct and treated as described in A. Before SDS-PAGE, the samples were treated with chABC. The figures are representative of three separate experiments, and the results of the densitometric analyses are shown in supplemental Fig. S1. The designations of the detected bands are shown in Fig. 1, A and E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Similar results were observed with the Ptprz-A isoform. Unlike Ptprz-B, when Ptprz-A was expressed in HEK293T cells, the mature receptor protein was highly modified with chondroitin sulfate (data not shown). Therefore, the samples were treated with chABC before SDS-PAGE. As with Ptprz-B, the release of the entire extracellular fragment of Ptprz-A, ZA-ECF (300 kDa), into the culture medium was clearly enhanced by PMA and suppressed in the presence of GM6001 (Fig. 3C). On the other hand, when the secretory isoform (Ptprz-S) was expressed, the full-length Ptprz-S was detected exclusively in the medium, and the amount was not affected by the treatment of cells either with PMA or with GM6001 (Fig. 3C). It is recognizable here that ZA-ECF is indistinguishable from Ptprz-S in size and antigenicity.TACE-mediated Shedding of Ptprz in Cultured Cells—Because GM6001 is a broad-spectrum metalloproteinase inhibitor, additional experiments were required to define the specific proteinase(s) involved in the ectodomain shedding of Ptprz-A/-B. TACE (also known as ADAM-17) is a GM6001-sensitive, membrane-anchored, zinc-dependent metalloproteinase. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins including TNF-α and Notch (23Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (909) Google Scholar). To determine whether TACE is involved in the ectodomain cleavage of Ptprz, we took advantage of CHO-M2 cells which are defective in TACE-mediated shedding (18Borroto A. Ruíz-Paz S. de la Torre T.V. Borrell-Pagès M. Merlos-Suárez A. Pandiella A. Blobel C.P. Baselga J. Arribas J. J. Biol. Chem. 2003; 278: 25933-25939Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Parental wild-type CHO cells (CHO-WT) and CHO-M2 cells which stably express a functional TACE, CHO-(M2+TACE), were also used for comparison.Transfection of the expression construct of Ptprz-B yielded similar expression levels of Ptprz-B in these cells (Fig. 4, left panels), and similar amounts of ZB-ECF were accumulated in the conditioned media during 1 h of incubation (Fig. 4, right panels); this basal level of accumulation of ZB-ECF was also observed in CHO-M2 and similarly suppressed by GM6001, indicating that the basal amount of ectodomain shedding of Ptprz-B is independent of TACE. PMA-stimulated ectodomain cleavage was reproduced in CHO-WT and CHO-(M2+TACE) cells and was inhibited by GM6001. However, the PMA-stimulated shedding was not observed in CHO-M2 cells. Similar results were observed with Ptprz-A (data not shown). TACE is, thus, highly responsible for the PMA-inducible cleavage of Ptprz for the generation of ZB-ECF and ZA-ECF but not for the constitutive cleavage in these cell lines.FIGURE 4Involvement of TACE in PMA-stimulated ectodomain cleavage of Ptprz. CHO-WT (wild-type), CHO-M2 (TACE defective), and CHO-(M2+TACE) (M2 cells expressing functional TACE) cells were transiently transfected with the Ptprz-B expression construct. Twenty-four hours after transfection cells were washed and incubated in fresh serum-free medium with or without PMA for 1 h. GM6001 was added 20 min before PMA or vehicle. The cell extracts (left panels) and conditioned media (right panels) were analyzed by Western blotting (WB) with anti-Ptprz-S. The arrows with asterisks indicate immature forms of Ptprz-B accumulated in cells. The designations are shown in Fig. 1, A and E. The figures are representative of three separate experiments, and the results of the densitometric analyses are shown in supplemental Fig. S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because the Ptprz-A and Ptprz-B isoforms have a common short sequence in the extracellular membrane-proximal region, the cleavage site was expected within this region. When a synthetic peptide corresponding to the juxtamembrane sequence was incubated with recombinant TACE in vitro, TACE indeed induced the cleavage of the substrate peptide into the two fragments. The molecular mass of them indicated that the enzymatic cleavage occurs between Gly at 1631 (P1 site) and Leu at 1632 (P1′ site) (see Fig. 5A). Previous studies with peptide substrates of TNF-α indicated that TACE has a strong preference for cleavage at Ala-Val sequences and cannot cleave a TNF-α-based peptide with the substitution of Ala with Ile at the P1 position (24Jin G. Huang X. Black R. Wolfson M. Rauch C. McGregor H. Ellestad G. Cowling R. Anal. Biochem. 2002; 302: 269-275Crossref PubMed Scopus (61) Google Scholar). Consistently, a mutant peptide, Zejm(G/I), in which Gly at P1 is replaced with Ile, was hardly cleaved by TACE (Fig. 5C). In contrast, the cleavage was highly enhanced by substitution with Ala at P1, Zejm (G/A), the same as TNF-α (Fig. 5D).FIGURE 5Identification of the TACE-cleavage site in Ptprz. A, amino acid sequence of the common extracellular membrane-proximal region in Ptprz-A and Ptprz-B. Amino acid numbers refer to the sequence of rat Ptprz-A. Three synthetic peptides are shown under the sequence. The arrowhead indicates the TACE cleavage site deduced by in vitro peptide digestion as below. TM, transmembrane segment. B–D, synthetic peptides only (left panels) or synthetic peptides with recombinant TACE (right panels) were incubated for 30 h at 37 °C, and the products were analyzed by mass spectrometry. T