Arginine 260 of the Amino-terminal Domain of NR1 Subunit Is Critical for Tissue-type Plasminogen Activator-mediated Enhancement of N-Methyl-D-aspartate Receptor Signaling
2004; Elsevier BV; Volume: 279; Issue: 49 Linguagem: Inglês
10.1074/jbc.m407069200
ISSN1083-351X
AutoresMónica Fernández‐Monreal, José P. López‐Atalaya, Karim Benchenane, Mathias Cacquevel, Fabienne Dulin, Jean‐Pierre Le Caër, Jean Rossier, Anne-Charlotte Jarrige, Eric T. MacKenzie, Nathalie Colloc'h, Carine Ali, Denis Vivien,
Tópico(s)Cell Adhesion Molecules Research
ResumoTissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-d-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59–64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function. Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-d-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59–64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function. Tissue-type plasminogen activator (tPA) 1The abbreviations used are: tPA, tissue-type plasminogen activator; LTP, long term potentiation; L-LTP, late phase LTP; NMDA, N-methyl-d-aspartate; ATD, amino-terminal domain; HEK, human embryonic kidney; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LIVBP, leucine-isoleucine-valine-binding protein; tPA-Stop®, 2,7-bis-(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride.1The abbreviations used are: tPA, tissue-type plasminogen activator; LTP, long term potentiation; L-LTP, late phase LTP; NMDA, N-methyl-d-aspartate; ATD, amino-terminal domain; HEK, human embryonic kidney; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LIVBP, leucine-isoleucine-valine-binding protein; tPA-Stop®, 2,7-bis-(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride. is one of the two mammalian serine proteases that activate the zymogen plasminogen into the broad spectrum serine protease plasmin. Apart from this primary function in the regulation of intravascular fibrinolysis, tPA has been implicated over the last decade in a variety of brain functions both during development and in adults. tPA is widely expressed in the developing brain, and it is prominently present in the hippocampus, cerebellum, and amygdala of the adult brain (2Sumi Y. Dent M.A. Owen D.E. Seeley P.J. Morris R.J. Development. 1992; 116: 625-637PubMed Google Scholar, 3Sappino A.P. Madani R. Huarte J. Belin D. Kiss J.Z. Wohlwend A. Vassalli J.D. J. Clin. Investig. 1993; 92: 679-685Crossref PubMed Scopus (303) Google Scholar, 4Carroll P.M. Tsirka S.E. Richards W.G. Frohman M.A. Strickland S. Development. 1994; 120: 3173-3183Crossref PubMed Google Scholar, 5Davies B.J. Pickard B.S. Steel M. Morris R.G. Lathe R. J. Biol. Chem. 1998; 273: 23004-23011Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) where both neurons and glial cells are sources of tPA (6Tsirka S.E. Rogove A.D. Bugge T.H. 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During onto-genesis, growing axons secrete tPA to modulate cell extracellular matrix interactions (2Sumi Y. Dent M.A. Owen D.E. Seeley P.J. Morris R.J. Development. 1992; 116: 625-637PubMed Google Scholar, 10Seeds N.W. Verrall S. Friedman G. Hayden S. Gadotti D. Haffke S. Christensen K. Gardner B. McGuire P. Krystosek A. Ann. N. Y. Acad. Sci. 1992; 667: 32-40Crossref PubMed Scopus (45) Google Scholar). In the adult brain, tPA has also been involved in processes such as synaptic plasticity and long term potentiation (LTP) (9Baranes D. Lederfein D. Huang Y.Y. Chen M. Bailey C.H. Kandel E.R. Neuron. 1998; 21: 813-825Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 11Qian Z. Gilbert M.E. Colicos M.A. Kandel E.R. Nature. 1993; 361: 453-457Crossref PubMed Scopus (639) Google Scholar). Indeed, mice deficient in tPA display a selective reduction in the late phase of LTP (L-LTP) (12Huang Y.Y. Bach M.E. Lipp H.P. Zhuo M. Wolfer D.P. Hawkins R.D. Schoonjans L. Kandel E.R. Godfraind J.M. Mulligan R. Collen D. Carmeliet P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8699-8704Crossref PubMed Scopus (277) Google Scholar, 13Frey U. Muller M. Kuhl D. J. Neurosci. 1996; 16: 2057-2063Crossref PubMed Google Scholar), whereas transgenic mice overexpressing tPA have increased and prolonged L-LTP (14Madani R. Hulo S. Toni N. Madani H. Steimer T. Muller D. Vassalli J.D. EMBO J. 1999; 18: 3007-3012Crossref PubMed Scopus (234) Google Scholar). Accordingly, a role for tPA has been established in learning and memory processes (12Huang Y.Y. Bach M.E. Lipp H.P. Zhuo M. Wolfer D.P. Hawkins R.D. Schoonjans L. Kandel E.R. Godfraind J.M. Mulligan R. Collen D. Carmeliet P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8699-8704Crossref PubMed Scopus (277) Google Scholar, 14Madani R. Hulo S. Toni N. Madani H. Steimer T. Muller D. Vassalli J.D. EMBO J. 1999; 18: 3007-3012Crossref PubMed Scopus (234) Google Scholar, 15Seeds N.W. Basham M.E. Ferguson J.E. J. Neurosci. 2003; 23: 7368-7375Crossref PubMed Google Scholar). Several mechanisms have been proposed to explain how tPA influences these processes. These include activation of extracellular proteolysis leading to a remodeling of the extracellular matrix and synaptic growth (9Baranes D. Lederfein D. Huang Y.Y. Chen M. Bailey C.H. Kandel E.R. Neuron. 1998; 21: 813-825Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 16Nakagami Y. Abe K. Nishiyama N. Matsuki N. J. Neurosci. 2000; 20: 2003-2010Crossref PubMed Google Scholar) or the cleavage of cell adhesion molecules (17Hoffman K.B. Martinez J. Lynch G. Brain Res. 1998; 811: 29-33Crossref PubMed Scopus (45) Google Scholar). Recently, it has been reported that the binding of tPA to the cell surface low density lipoprotein receptor-related protein could play a key role in L-LTP (18Zhuo M. Holtzman D.M. Li Y. Osaka H. DeMaro J. Jacquin M. Bu G. J. Neurosci. 2000; 20: 542-549Crossref PubMed Google Scholar). tPA has also been shown to play an important role in acute and chronic brain pathologies such as seizure (19Tsirka S.E. Gualandris A. Amaral D.G. Strickland S. Nature. 1995; 377: 340-344Crossref PubMed Scopus (587) Google Scholar, 20Wu Y.P. Siao C.J. Lu W. Sung T.C. Frohman M.A. Milev P. Bugge T.H. Degen J.L. Levine J.M. Margolis R.U. Tsirka S.E. J. Cell Biol. 2000; 148: 1295-1304Crossref PubMed Scopus (159) Google Scholar), ischemic brain injury (21Wang Y.F. Tsirka S.E. Strickland S. Stieg P.E. Soriano S.G. Lipton S.A. Nat. Med. 1998; 4: 228-231Crossref PubMed Scopus (553) Google Scholar, 22Benchenane K. Lopez-Atalaya J.P. Fernandez-Monreal M. Touzani O. Vivien D. Trends Neurosci. 2004; 27: 155-160Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and multiple sclerosis (23Gveric D. Hanemaaijer R. Newcombe J. van Lent N.A. Sier C.F. Cuzner M.L. Brain. 2001; 124: 1978-1988Crossref PubMed Scopus (108) Google Scholar, 24Lu W. Bhasin M. Tsirka S.E. J. Neurosci. 2002; 22: 10781-10789Crossref PubMed Google Scholar). The hippocampal neurons of tPA-deficient mice are resistant to in vivo excitotoxin-induced degeneration (19Tsirka S.E. Gualandris A. Amaral D.G. Strickland S. Nature. 1995; 377: 340-344Crossref PubMed Scopus (587) Google Scholar). Similarly, tPA potentiates the excitotoxic lesion induced following an intrastriatal injection of NMDA (1Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (608) Google Scholar, 25Liberatore G.T. Samson A. Bladin C. Schleuning W.D. Medcalf R.L. Stroke. 2003; 34: 537-543Crossref PubMed Scopus (152) Google Scholar). After cerebral ischemia, neuronal damage induced by middle cerebral artery occlusion is also reduced in tPA-deficient mice and exacerbated when exogenous tPA is administered (21Wang Y.F. Tsirka S.E. Strickland S. Stieg P.E. Soriano S.G. Lipton S.A. Nat. Med. 1998; 4: 228-231Crossref PubMed Scopus (553) Google Scholar). The idea that tPA contributes to the extent of the final damage induced by stroke has been later supported by other groups (26Nagai N. De Mol M. Lijnen H.R. Carmeliet P. Collen D. Circulation. 1999; 99: 2440-2444Crossref PubMed Scopus (212) Google Scholar, 27Zhang Z. Zhang L. Yepes M. Jiang Q. Li Q. Arniego P. Coleman T.A. Lawrence D.A. Chopp M. Circulation. 2002; 106: 740-745Crossref PubMed Scopus (114) Google Scholar). Different mechanisms have been proposed to explain the deleterious effect of tPA in these processes, including enhanced microglial activation (28Rogove A.D. Tsirka S.E. Curr. Biol. 1998; 8: 19-25Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), increased laminin degradation (29Chen Z.L. Strickland S. Cell. 1997; 91: 917-925Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar), and the potentiation of NMDA receptor-dependent signaling (1Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (608) Google Scholar). Here, we describe the molecular mechanism by which tPA modulates the NMDA receptor-evoked Ca2+ influx. We show that tPA cleaves the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor at arginine 260 and that this specific cleavage is a necessary event for tPA-induced potentiation of NMDA receptor signaling. This study constitutes an important step toward the understanding of how tPA influences several physiological and pathological processes involving the glutamatergic signaling.EXPERIMENTAL PROCEDURESMaterials—Horse serum and fetal bovine serum were purchased from Invitrogen. L(+)-amino-5-phosphonopentanoic acid was from Tocris (Bristol, United Kingdom). Human recombinant tPA was purchased from Boehringer Ingelheim (Paris, France). α-Casein was obtained from ICN Biomedicals (Aurore, OH), and human Lys-plasminogen was purchased from Calbiochem. Antibodies raised against the NR1 subunit (sc-9058) and His5 were purchased from Santa Cruz Biotechnology (Heidelberg, Germany) and Qiagen (Courtaboeuf, France), respectively. 2,7-Bis-(4-amidinobenzylidene)-cycloheptan-1-one dihydrochloride (tPA-Stop) and tPA substrate spectrozyme XF-444 were purchased from American Diagnostica (Greenwich, CT). Plasmin and all the other chemicals were obtained from Sigma.Cortical Cultures—Neuronal cortical cultures were prepared from fetal mice (embryonic day 15–16). Dissociated cortical cells were resuspended in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 5% horse serum, and 2 mm glutamine and plated in 24-well dishes previously coated with poly-d-lysine and laminin. After 3 days in vitro, the cells were exposed to 10 μm Ara-C to inhibit glial proliferation. Cultures were used after 14 days in vitro (30Buisson A. Nicole O. Docagne F. Sartelet H. MacKenzie E.T. Vivien D. FASEB J. 1998; 12: 1683-1691Crossref PubMed Scopus (104) Google Scholar).Immunoblotting—Protein samples were resolved on SDS-polyacryl-amide gel and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 5% dried milk in Tris-buffered saline containing 0.05% Tween 20 and incubated with primary antibodies. After incubation with the corresponding biotinylated secondary antibody and peroxidase-conjugated streptavidin reagent, proteins were visualized with an enhanced chemiluminescence ECL Plus immunoblotting detection system (PerkinElmer Life Sciences).Human Embryonic Kidney (HEK)-293 Cell Cultures and Transient Transfection—Human embryonic kidney 293 cells (ATCC 1573-CRL) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Low confluency cells were transfected by the calcium phosphate precipitation method (31Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4810) Google Scholar) with a mixture containing NR1–1a/b (or NR1–1aR260A), NR2A, and enhanced green fluorescent protein plasmids (1, 1, and 0.3 μg/coverslip, respectively). After transfection, NMDA antagonists (200 μm AP5, 2 mm MgCl2, 1 mm kynurenic acid) were added to the culture medium. Experiments were performed within 36–48 h after transfection.Calcium Videomicroscopy Analysis—Transfected HEK-293 cells were loaded in the presence of a HEPES-buffered saline solution containing 5 μm fura-2/AM plus 0.1% pluronic F-127 (Molecular Probes, Leiden, the Netherlands) (30 min, 37 °C) and incubated for an additional 30-min period in a HEPES-buffered saline solution. Experiments were performed at room temperature on the stage of a Nikon Eclipse inverted microscope equipped with a 75 W Xenon lamp and a Nikon 40×, 1.3 numerical aperture epifluorescence oil immersion objective. Fura-2 (excitation 340 and 380 nm, emission 510 nm) ratio images were acquired with a CCD camera (Princeton Instrument, Trenton, New Jersey) and digitized (256 × 512) using Metafluor 4.11 software (Universal Imaging Corporation, Cherter, Pennsylvania).Construction of His-tagged Recombinant ATD/Leucine-Isoleucine-Valine-binding Protein(LIVBP)-like Domain—The region of the NR1 subunit encoding amino acids 19–371 for NR1–1a or 19–389 for NR1–1b corresponding to the ATD was amplified from the full-length rat NR1–1a or NR1–1b cDNA, respectively, by using the upstream primer 5′-CGGGATCCCGCGCCGCCTGCGAC-3′ generating a BamHI restriction site and the downstream primer 5′-ATGGGTACCATTGTAGATGCCCAC-3′ containing an internal KpnI restriction site. PCR products were digested and inserted in pQE100-Double Tag vector (Qiagen), which encodes for His6 at the amino terminus of the insert. Recombinant proteins were purified from inclusion bodies of isopropyl 1-thio-β-d-galactopyranoside-induced bacterial cultures (Escherichia coli, M15 strain) on a nickel affinity matrix as described by the manufacturer (Qiagen).Site-directed Mutagenesis—Mutagenesis of either recombinant NR1-ATD (R260A and R217A) or full-length NR1–1a (R260A) was performed by using QuikChange® XL site-directed mutagenesis kit purchased from VWR International France (Fontenay-sous-Bois, France). All mutations were confirmed using an automated sequence analysis.Enzymatic Assay—Recombinant tPA (29 nm) was incubated in the presence of a tPA-specific fluorogenic substrate (5 μm) (Spectrozyme® XF444) and in the presence of either tPA-Stop® (10 nm) or recombinant ATD of NR1 (225 nm). The reaction was carried out at 25 °C in 100 mm Hepes (pH 8.0) containing 150 mm NaCl, and 0.01% Tween 80 in a total volume of 100 μl. The amidolytic activity of tPA was measured as the change in fluorescence emission at 440 nm (excitation at 360 nm). The apparent inhibition constant, Ki′, was determined as described by Petersen et al. (32Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Crossref PubMed Scopus (139) Google Scholar). The data sets on the inhibition of proteases were analyzed in terms of the equation Vi = V0/(1 + [I]0/Ki′), where Vi and V0 are the inhibited and uninhibited rates, respectively, and [I]0 is the total concentration of inhibitor. Ki values were obtained by correcting for the effect of substrate (S) with the equation Ki = Ki′ (1 + [S]/Km) (2Sumi Y. Dent M.A. Owen D.E. Seeley P.J. Morris R.J. Development. 1992; 116: 625-637PubMed Google Scholar)MALDI-TOF Analysis—Proteins in Coomassie-stained gels were subjected to acetonitrile washing and reductive alkylation by iodoacetamide in ammonium carbonate (0.1 m) (30 min in the dark). Then, in-gel trypsin digestion (Roche Applied Science, EC 3.421.4) was allowed as described by Shevchenko et al. (33Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7771) Google Scholar). Peptides were resuspended in 20 μl of formic acid 1%, desalted by using Zip Tip C-18 (Millipore), and eluted with 50 and 80% acetonitrile. The desalted peptide mixture was dried and dissolved in 3 μl of formic acid 1%. The matrix used was a saturated solution of 2,5-dihydroxybenzoic acid in trifluoroacetic acid 0.1%. The sample and the matrix (1:1, v/v) were loaded on the target using the dried droplet method. MALDI-TOF spectra of the peptides were obtained with a Voyager-DE STR Biospectrometry Work station mass spectrometer (PE Biosystems Inc.). Analyses were performed in positive ion reflector mode, with an accelerating voltage of 20,000 V, a delayed extraction of 200 ns, and ∼250 scans were averaged. For subsequent data processing, the Data Explorer software (PE Biosystems Inc.) was used. Spectra obtained for the whole protein were calibrated externally using the [M+H]+ ion from Des-Arg bradykinin peptide (904.4681 Da) and ACTH peptide (2465.1989 Da). The trypsin autoproteolysis products (fragment-(132–142), 1153.57 Da and fragment-(56–75), 2163.06 Da) were used as the external calibration standard. A mass deviation of 0.1 Da was allowed in the data base searches.Homology Modeling—The sequence of the ATD of NR1 has been aligned with an alignment deduced from the structural superposition of the extracellular domain of the metabotropic glutamate receptor 1 (1EWT, 1EWK) (34Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1096) Google Scholar) and four other proteins with a LIVBP fold (the atrial natriuretic peptide clearance receptor (1DP4), the amide receptor (1PEA), the ribose-binding protein (2DRI), and the leucine/isoleucine/valine-binding protein (2LIV)). The alignment and model have been built and optimized iteratively using Model Tool Server (35Douguet D. Labesse G. Bioinformatics. 2001; 17: 752-753Crossref PubMed Scopus (81) Google Scholar) and Modeler (36Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10390) Google Scholar).RESULTStPA Potentiates NMDA Receptor-mediated Ca2+Influx in HEK-293 Cells Expressing NMDA Receptors Containing Either NR1–1a or NR1–1b—We have previously shown that tPA enhances the NMDA-evoked Ca2+ influx in cultured cortical neurons (1Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (608) Google Scholar, 7Fernandez-Monreal M. Lopez-Atalaya J.P. Benchenane K. Leveille F. Cacquevel M. Plawinski L. MacKenzie E.T. Bu G. Buisson A. Vivien D. Mol. Cell Neurosci. 2004; 25: 594-601Crossref PubMed Scopus (57) Google Scholar). To investigate the molecular mechanism through which tPA is able to modulate the NMDA receptor signaling, we developed a heterologous expression system of functional NMDA receptors. We transiently transfected HEK-293 cells with the cDNA encoding for NR1–1a and NR2A subunits of the NMDA receptor. The expression of NMDA receptor subunits was confirmed by immunocytochemistry and immunoblotting analyses (data not shown). As shown in Fig. 1, in HEK-293 cells expressing NMDA receptors, tPA is able to potentiate NMDA receptor signaling, as it does in cortical neurons (1Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (608) Google Scholar). The mRNA encoding for the NR1 subunit exhibits an alternative splicing in its 5′-region, generating isoforms characterized by the absence (a forms) or the presence (b forms) of a 21-residue insert (N1 cassette) encoded by exon 5 (37Sugihara H. Moriyoshi K. Ishii T. Masu M. Nakanishi S. Biochem. Biophys. Res. Commun. 1992; 185: 826-832Crossref PubMed Scopus (479) Google Scholar). Because the exon 5 splicing is known to influence receptor properties (38Durand G.M. Bennett M.V. Zukin R.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6731-6735Crossref PubMed Scopus (305) Google Scholar, 39Hollmann M. Boulter J. Maron C. Beasley L. Sullivan J. Pecht G. Heinemann S. Neuron. 1993; 10: 943-954Abstract Full Text PDF PubMed Scopus (534) Google Scholar, 40Traynelis S.F. Hartley M. Heinemann S.F. Science. 1995; 268: 873-876Crossref PubMed Scopus (344) Google Scholar, 41Traynelis S.F. Burgess M.F. Zheng F. Lyuboslavsky P. Powers J.L. J. Neurosci. 1998; 18: 6163-6175Crossref PubMed Google Scholar), we have assessed its potential implication in tPA effect. We found that tPA increases NMDA-induced Ca2+ influx to the same extent in HEK-293 cells exhibiting NR1–1b/NR2A or NR1–1a/NR2A receptors (36.87 ± 10.9%, n = 32 and 31.37 ± 5.4%, n = 56, respectively, mean ± S.E.) (Fig. 1, A–C). Hence, the N1 cassette does not influence the tPA-induced potentiation of NMDA receptor signaling.tPA Induces a Single Cleavage of NR1 in Its ATD—To further investigate the mechanism of action of tPA on NMDA receptor signaling, we have examined whether tPA, per se, can cleave NR1. We have shown previously that the treatment of membrane preparations of cultured cortical neurons with tPA leads to the appearance of a cleaved form of NR1, with a molecular mass reduced to ∼25 kDa, recognized by an antibody raised against the carboxyl terminus of NR1 (1Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (608) Google Scholar). This suggests that the amino-terminal portion of the NR1 subunit is the region in which the cleavage occurs. Hence, to determine where tPA cleaves the amino terminus of NR1, we have generated a construct encoding for an amino terminus His-tagged corresponding to the first domain of NR1–1a (amino acid residues 19–371 of NR1, 39875 Da), termed ATD or LIVBP-like domain (Fig. 2A). Incubation of recombinant NR1-ATD with tPA shows that tPA is able to cleave NR1-ATD, in a dose-dependent manner, generating a cleaved fragment of 28 kDa detected by immunoblotting revealed with an antibody raised against the His-tag (Fig. 2B). In addition, tPA-Stop®, an inhibitor of tPA proteolytic activity, prevents the tPA-induced cleavage of both the full-length NR1 (of neuronal membrane preparations from cultured cortical neurons) and the recombinant NR1-ATD (Fig. 3A). Finally, as determined by the digestion pattern of the recombinant NR1-ATD generated by tPA or plasmin, we observed that although tPA induces a single cleavage of the ATD of NR1 subunit, plasmin leads to a complete degradation of the ATD (Fig. 3B).Fig. 2The proteolytic activity of tPA induces a single cleavage of the ATD of the NR1 subunit of NMDA receptor. A, map of the generated construct allowing the production of a recombinant form of the ATD of the NR1 subunit tagged with His6 at its amino terminus. B, immunoblotting was performed from the recombinant ATD-NR1 previously incubated with tPA (20 and 100 μg/ml) for 2 h at 37°C. Immunoblots were revealed with an antibody raised against His6 (WB 6xHIS).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Cleavage of the ATD of the NMDA receptor NR1 subunit is prevented by a synthetic tPA inhibitor, tPA-Stop®, and occurs independently of the plasminogen/plasmin system. A, immunoblots were performed from membrane preparations of mouse cultured cortical neurons and recombinant ATD-NR1 incubated in the presence of tPA alone or with the tPA inhibitor tPA-Stop®. Immunoblots were revealed with an antibody raised against either NR1 or His6, respectively. B, immunoblots were performed from the recombinant ATD-NR1 incubated with either tPA (20 μg/ml) or plasmin at increasing concentrations. Immunoblots were revealed with an antibody raised against either NR1 or His6, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ATD of NR1 Is a Substrate of tPA—The next question was to determine whether the ATD of NR1 could be a direct substrate for tPA. To address this question, we have used the recombinant ATD of NR1 to compete the ability of tPA to cleave a specific fluorogenic substrate (Spectrozyme®, XF444). Our data showed that the recombinant ATD of NR1 is able to compete with the tPA-specific substrate with a Ki of 0.234 ± 0.097 μm (Table I). Control experiments were performed in parallel in the presence of the tPA inhibitor tPA-Stop® with a Ki of 0.046 ± 0.015 μm (Table I).Table IKi values for the inhibition of tPA by the ATD of NR1 and tPA-Stop®[I][tPA]Substrate[S]KiμmμmATD-NR10.2250.029CH3SO2-d-Phe-Gly-Arg-AMC-AcOH50.239 ± 0.097tPA-Stop®0.0100.029CH3SO2-d-Phe-Gly-Arg-AMC-AcOH50.046 ± 0.015 Open table in a new tab tPA Cleaves the ATD of NR1 at the Arginine 260 —Next, to identify the exact location of the cleavage site, we analyzed both native (39 kDa) and tPA-cleaved (28 kDa) recombinant NR1-ATD using MALDI-TOF analysis. As summarized in Fig. 4, MALDI-TOF analysis allowed us to identify the putative cleavage site of the NR1–1a and NR1–1b subunits by tPA as the arginine in position 260 (Arg-260) and arginine 281 respectively (Fig. 4A). Sequence analyses revealed that the tPA-cleavage region "ISGNALRYAPDG" is highly conserved in NR1 subunits of NMDA receptors, whatever the species analyzed, and not found in other NMDA receptor subunits (data not shown). The analysis of the homology model of the ATD of the NR1 subunit (Fig. 4, B and C) shows that the Arg-260 is located close to the entry of a hydrophobic pocket for which no ligand has been described so far.Fig. 4Mass spectrometric analysis of both native and tPA-cleaved ATD of the NR1 subunit. A, recombinant ATD-NR1–1a and ATD-NR1–1b were incubated in the presence of tPA (20 μg/ml, 2 h, 37 °C) prior to SDS-PAGE and Coomassie staining. Mass spectrometry analyses of both full-length ATD-NR1–1a and ATD-NR1–1b and tPA-cleaved ATD-NR1–1a and ATD-NR1–1b were performed after in-gel digest procedure (tryptic digestion) as described under "Experimental Procedures." Peptides identified from corresponding full-length ATD-NR1 and tPA-cleaved ATD-NR1 are summarized in the Table I. The comparison of the peptidic profiles obtained between full-length ATD and tPA-cleaved ATD allows the identification of the tPA cleavage site. Peptide EISGNALR (bold characters) corresponds to the carboxyl-terminal peptide identified following cleavage induced by tPA. ISGNALRYAPDG was designed as the peptide containing the tPA cleavage site Arg-260 (recapitulative of three individual experiments for each isoform of NR1 subunit). B, ribbon-type representation of the calcium chain of the homology model of the ATD-NR1 constructed using the metabotropic glutamate receptor 1 structure showing the two lobes surrounding a hydrophobic pocket. The position of the arginine 260, in a loop overhanging the pocket domain is shown in ball-and-stick representation. C, alignment of ATD-NR1 and metabotropic glutamate receptor 1 sequences used to build the homology model, the resulting secondary structure of ATD-NR1 is shown above. Graphics were created using MOLSCRIPT (55Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and rendered by RASTER3D (56Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar), and alignment representations were made using ESPript software (57Gouet P. Courcelle E. Stuart D.I. Metoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2505) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mutation of the Arg-260 Prevents both tPA-induced Cleavage of NR1-ATD and Potentiation of NMDA-receptor Signaling—To validate mass spectrometry analyses, we performed a mutation of the Arg-260 (and Arg-217 as a negative control) of the NR1-ATD recombinant protein into alanine and tested the ability of tPA to cleave these proteins. Although tPA cleaved both wild-type and control R217A proteins, it failed to cleave the protein mutated
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