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Originally published In Press as doi:10.1074/jbc.M407069200 on September 23, 2004

J. Biol. Chem., Vol. 279, Issue 49, 50850-50856, December 3, 2004
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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*

Mónica Fernández-Monreal{ddagger}§, José P. López-Atalaya{ddagger}§||, Karim Benchenane{ddagger}**, Mathias Cacquevel{ddagger}||, Fabienne Dulin{ddagger}, Jean-Pierre Le Caer{ddagger}{ddagger}, Jean Rossier§§, Anne-Charlotte Jarrige{ddagger}, Eric T. MacKenzie{ddagger}, Nathalie Colloc'h{ddagger}, Carine Ali{ddagger}, and Denis Vivien{ddagger}¶¶

From the {ddagger}CNRS UMR 6185, University of Caen, Centre Cyceron, Bd. Henri Becquerel, BP 5229, 14074, Caen cedex, France, {ddagger}{ddagger}Ecole Polytechnique CNRS UMR 7651, 91128 Palaiseau Cedex, France, and §§ESPCI, CNRS UMR 7637, 75231 Paris cedex 5, France

Received for publication, June 24, 2004 , and in revised form, September 20, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue-type plasminogen activator (tPA)1 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 (2–5) where both neurons and glial cells are sources of tPA (6, 7). Furthermore, it is now well known that membrane depolarization induces a calcium-dependent secretion of tPA by neuronal cells (7–9). During onto-genesis, growing axons secrete tPA to modulate cell extracellular matrix interactions (2, 10). In the adult brain, tPA has also been involved in processes such as synaptic plasticity and long term potentiation (LTP) (9, 11). Indeed, mice deficient in tPA display a selective reduction in the late phase of LTP (L-LTP) (12, 13), whereas transgenic mice overexpressing tPA have increased and prolonged L-LTP (14). Accordingly, a role for tPA has been established in learning and memory processes (12, 14, 15). 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 (9, 16) or the cleavage of cell adhesion molecules (17). 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 (18). tPA has also been shown to play an important role in acute and chronic brain pathologies such as seizure (19, 20), ischemic brain injury (21, 22), and multiple sclerosis (23, 24). The hippocampal neurons of tPA-deficient mice are resistant to in vivo excitotoxin-induced degeneration (19). Similarly, tPA potentiates the excitotoxic lesion induced following an intrastriatal injection of NMDA (1, 25). 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 (21). The idea that tPA contributes to the extent of the final damage induced by stroke has been later supported by other groups (26, 27). Different mechanisms have been proposed to explain the deleterious effect of tPA in these processes, including enhanced microglial activation (28), increased laminin degradation (29), and the potentiation of NMDA receptor-dependent signaling (1). 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 PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—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). {alpha}-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 (30).

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 (31) 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 40x, 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 x 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-{beta}-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. (32). 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) (2)

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. (33). 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) (34) and four other proteins with a LIVBP fold (the atrial natriuretic peptide clearance receptor (1DP4 [PDB] ), the amide receptor (1PEA), the ribose-binding protein (2DRI [PDB] ), and the leucine/isoleucine/valine-binding protein (2LIV [PDB] )). The alignment and model have been built and optimized iteratively using Model Tool Server (35) and Modeler (36).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
tPA 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 (1, 7). 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 (1). 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 (37). Because the exon 5 splicing is known to influence receptor properties (38–41), 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.



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  		FIG. 1.
tPA potentiates NMDA receptor signaling in HEK-293 cells exhibiting either NR1–1a or NR1–1b and NR2A subunits. A, representative records of NMDA-evoked Ca2+ influx prior to and after the addition of tPA (20 µg/ml) for 10 min in HEK-293 cells transiently transfected with NR1–1a/NR2A. Each value represents the mean ± S.E. of 15–20 cells from a representative experiment of three independent experiments. B, similar experiments were performed in HEK-293 cells transiently transfected with NR1–1b/NR2A. Each value represents the mean ± S.E. from 15–20 cells from an experiment representative of three independent experiments. C, data are shown as the percentage of tPA effect on NMDA-induced calcium influx. No significant difference in tPA-mediated potentiation was observed between receptors exhibiting NR1-a and NR1-b isoforms. The histogram represents values of three independent experiments pooled.

 
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 (1). 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).



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  		FIG. 2.
The 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).

 



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  		FIG. 3.
Cleavage 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.

 
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).


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  		TABLE I
Ki values for the inhibition of tPA by the ATD of NR1 and tPA-Stop®

 
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.



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  		FIG. 4.
Mass 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 (55) and rendered by RASTER3D (56), and alignment representations were made using ESPript software (57).

 
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 at the arginine in position 260 (Fig. 5A). Moreover, wild-type NR1-ATD inhibited the ability of tPA to cleave its specific fluorogenic substrate (40%, p < 0.01). In contrast NR1R260A-ATD had no effect on tPA activity (Fig. 5B). Next, to determine whether a point mutation of this arginine at the position 260 could prevent the tPA-induced potentiation of NMDA receptor-mediated signaling, we transiently co-transfected HEK-293 cells with NR2A in the presence of either wild-type or R260A mutated NR1–1a, and performed calcium videomicroscopy experiments. When co-transfected with NR2A, both wild-type and R260A mutated NR1–1a subunits displayed the same responses to increasing concentrations of NMDA (Fig. 6, A and B). In contrast, although the addition of exogenous tPA potentiated NMDA-induced Ca2+ influx in HEK-293 cells co-transfected with wild-type NR1–1a and NR2A, tPA failed to potentiate NMDA receptor signaling in transfected cells containing the mutated NR1–1a R260A subunit and NR2A (27.57 ± 3.7%, n = 102 and 2.49 ± 1.7%, n = 110, respectively) (Fig. 7, A and B).



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  		FIG. 5.
Mutation of the Arg-260 into alanine prevents tPA-induced cleavage of the ATD of NR1 subunit. A, immunoblots (WB) were performed from both wild-type and mutated recombinant ATD-NR1 (R260A and R217A) incubated in the presence of tPA (20 µg/ml) and revealed with an antibody raised against His6. B, histogram showing the ability of recombinant ATD-NR1 or recombinant ATD-NR1R260A to inhibit tPA amidolytic activity, assessed by the cleavage of Spectrozyme® XF444 (tPA = 29 nM, ATD-NR1 = ATD-NR1R260A = 225 nM).

 



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  		FIG. 6.
Mutation of the Arg-260 into alanine does not influence basal NMDA receptor signaling. A, representative NMDA dose-dependent responses assessed by digital quantitation of [Ca2+]i with fura-2 performed in HEK-293 cells transiently transfected with NR1–1a/NR2A (left panel) or NR1–1aR260A/NR2A receptors (right panel). B, data are the mean (± S.E.) of NMDA responses in HEK-293 cells transiently transfected with NR1–1a/NR2A (n = 2, n (cell number) = 56) or NR1–1aR260A/NR2A receptors (n = 2, n (cell number) = 47). Both heterologously expressed receptors exhibit a dose-dependent NMDA-evoked calcium influx (p < 0.0001), and no difference was observed between NR1–1a/NR2A and NR1–1aR260A/NR2A transfected cells (p > 0.56 by two-way analysis of variance followed by Bonferroni correction).

 



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  		FIG. 7.
Mutation of the Arg-260 into alanine prevents tPA-induced potentiation of NMDA receptor-evoked calcium influx. A, representative records of NMDA-evoked [Ca2+]i show an increase prior to and after addition of tPA (20 µg/ml) to the perfusion solution for 10 min in HEK-293 cells transiently transfected with NR1–1a/NR2A (left panel) or NR1–1aR260A/NR2A receptors (right panel). B, tPA exposure enhances NMDA responses in NR1–1a/NR2A-transfected cells of 27.6 ± 3.66% (n = 5, n (cell number) = 102), whereas no potentiation is observed in cells expressing receptors containing R260A mutated NR1–1a subunit (2.5 ± 1.7%, n = 5, n (cell number) = 110). Data presented are the percentage of increase of NMDA-induced calcium influx (mean ± S.E.; *, indicates significantly different from stimulations before tPA application; #, indicates difference between NR1 and NR1R260A by one-way analysis of variance followed by a Bonferroni-Dunn's test for multiple comparison (p < 0.0001)).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Fast excitatory neurotransmission in the mammalian central nervous system is mediated by ionotropic glutamate-gated receptors. When overstimulated, ionotropic glutamate-gated receptors, particularly the NMDA subtype, cause excitotoxicity. We have shown previously that the serine protease tPA potentiates NMDA receptor signaling, which might be of particular relevance in several physiological and pathological brain conditions (1). However, whether this effect is the direct consequence of the ability of tPA to bind to and then cleave the NMDA receptor NR1 subunit remained to be demonstrated. Here, we demonstrated that tPA cleaves NR1 within a particular extracellular region, the ATD, in which we identified the arginine in position 260 as a critical residue for the tPA-induced potentiation of NMDA signaling.

First, by using transfected HEK cells, we show that tPA potentiates NMDA signaling indifferently in receptors containing either NR1a or NR1b splice variants. Both isoforms differ by the absence or presence of a amino-terminal N1 cassette, known to modulate some properties of the NMDA receptor (38–41). Our results thus demonstrated that the N1 cassette is not involved in the potentiating activity of tPA on NMDA receptor signaling. We also demonstrated that tPA can interact with and cleave the ATD of both NR1a and b subunits. The ATD of NR1 also LIVBP-like domain is a conserved domain found in all metabotropic and ionotropic glutamate receptor subunits described so far. Mutagenesis analyses within the ATD of NMDA receptor subunits have suggested that this domain might regulate the functional features of this receptor, by controlling subunit-subunit interactions (42) and/or altering allosteric modulations (43–45). Our present data provide a novel function for the ATD of NR1. Indeed, we showed that this ATD of NR1 is a binding site for tPA and that the cleavage of its arginine 260 in NR1-a (arginine 281 for NR1-b) is a necessary event for tPA-induced potentiation of NMDA signaling.

As previously suggested (46, 47) whether tPA could form a direct complex with NR1 or through the participation of its classical substrate plasminogen remained to be determined. Although the presence of locally synthesized tPA in the central nervous system has been well characterized (3, 5) that of plasminogen is still a matter of debate (3, 5, 6, 48, 49). Hence, we can not exclude the idea that tPA could cleave the NMDA receptor via the activation of plasminogen into plasmin. Indeed, activated plasminogen has been shown to lead to a complete degradation of the NR1 subunit of NMDA isolated from brain lysates (47). Accordingly, we showed that plasmin leads to a complete degradation of the ATD of NR1. However, this proteolytic pattern differs from that of tPA, which induces a single cleavage at the Arg-260. Thus, these data demonstrated that tPA-induced cleavage of NR1 is not mediated by plasmin, which is in agreement with our previous observations that in contrast to tPA, plasmin does not influence NMDA receptor-mediated neuronal death (1).

Another important issue is to know whether the endogenous concentrations of tPA are high enough to lead to the cleavage of the NR1 subunit of the NMDA receptor and subsequent potentiation of NMDA-mediated signaling in in vivo situations. In a previous study, we have shown that in cultured cortical neurons, NMDA receptor activation is sufficient to promote both tPA release and subsequent cleavage of the NR1 subunit of NMDA receptor. In addition, this NMDA-dependent cleavage of NR1 is inhibited by exogenous plasminogen activator inhibitor-1 and does not occur in tPA-deficient cortical neurons (1). Additionally, we have demonstrated that the blockade of the proteolytic activity of endogenous tPA reduces NMDA-induced excitotoxic death (1, 30). Nevertheless, the concentration of tPA not only in the brain parenchyma but also at the synaptic cleft remain actually undetermined.

In physiological conditions, an impairment or increase of L-LTP has been observed in tPA-deficient or tPA-overexpressing mice, respectively (12–14). In addition to hippocampal functions, tPA has also been involved in phenomena such as cerebellar motor learning (15, 50), visual cortex plasticity (51), and fear conditioning (52). During the last several years, several reports have also demonstrated a connection between tPA and brain disorders. tPA has been involved in many pathological processes including stroke (21, 25), seizure (19, 20, 53), and multiple sclerosis (23, 24). For instance, tPA-deficient mice display a high resistance to neuronal damages induced through either excitotoxic paradigms (19, 24) or experimental ischemia (21, 25). Conversely, plasminogen activator inhibitor-1-deficient mice show an increased volume of infarct following cerebral ischemia (25). In addition, mice overexpressing neuroserpin, another tPA inhibitor, have a reduced volume of lesion following ischemic brain injury (54). Here, we described a molecular mechanism through which tPA can influence the glutamatergic signaling. Although this mechanism might not explain all of the functions of tPA in the brain, it highlights the importance of this protease in physiological and pathological situations. Hence, these data should represent the basis of the development of new therapeutic strategies targeting the interaction between tPA and NMDA receptors.


   FOOTNOTES
 
* This work was supported by grants from the CNRS, University of Caen and European Council (FEDER). 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

§ Both authors contributed equally to this work. Back

Supported by the Fondation pour la Recherche Medicale. Back

|| Supported by the Regional Council of Lower Normandy. Back

** Supported by the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche. Back

¶¶ To whom correspondence should be addressed. Tel.: 33-231-56-6039; Fax: 33-231-56-61-99; E-mail: d.vivien{at}neuro.unicaen.fr.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank the CRIHAN, the Centre Européen de Bioprospectives, and the FEDER for the use of the visualization software Insight II (Accelrys, San Diego, CA).



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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