Cleavage of the NR2B subunit amino terminus of N-methyl-D-aspartate (NMDA) receptor by tissue plasminogen activator: identification of the cleavage site and characterization of ifenprodil and glycine affinities on truncated NMDA receptor

Abstract

Thrombolysis using tissue plasminogen activator (tPA) has been the key treatment for patients with acute ischemic stroke for the past decade. Recent studies, however, suggest that this clot-busting protease also plays various roles in brain physiological and pathophysiological glutamatergic-dependent processes, such as synaptic plasticity and neurodegeneration. In addition, increasing evidence implicates tPA as an important neuromodulator of the N-methyl-d-aspartate (NMDA) receptors. Here, we demonstrate that recombinant human tPA cleaves the NR2B subunit of NMDA receptor. Analysis of NR2B in rat brain lysates and cortical neurons treated with tPA revealed concentration- and time-dependent degradation of NR2B proteins. Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment on a recombinant fusion protein of the amino-terminal domain of NR2B revealed that tPA-mediated cleavage occurred at arginine 67 (Arg(67)). This cleavage is tPA-specific, plasmin-independent, and removes a predicted ~4-kDa fragment (Arg(27)-Arg(67)) from the amino-terminal domain of the NR2B protein. Site-directed mutagenesis of putative cleavage site Arg(67) to Ala(67) impeded tPA-mediated degradation of recombinant protein. This analysis revealed that NR2B is a novel substrate of tPA and suggested that an Arg(27)-Arg(67)-truncated NR2B-containing NMDA receptor could be formed. Heterologous expression of NR2B with Gln(29)-Arg(67) deleted is functional but exhibits reduced ifenprodil inhibition and increased glycine EC(50) with no change in glutamate EC(50). Our results confirmed NR2B as a novel proteolytic substrate of tPA, where tPA may directly interact with NR2B subunits leading to a change in pharmacological properties of NR2B-containing NMDA receptors.

FIGURE 1.

Representative immunoblots demonstrating the degradation of the NMDA receptor subunit NR2B upon recombinant tPA treatment. A, total rat brain lysate (RBL) (40 μg) was exposed to increasing concentrations of Calbiochem® tPA for 20 h at 37 °C. A Western blot was probed using anti-NR2B (Zymed Laboratories Inc.). No degradation of GAPDH was observed with increasing concentrations of tPA. B, shown is quantification of the blot seen in A. Data were normalized to control. n = 4; ** and *** denote p < 0.01 and p < 0.001, respectively (against control), one-way ANOVA with Tukey's post hoc test. The x axis was truncated to highlight the relative decrease in % NR2B immunoreactivity (≥1 μg/ml tPA). C, rat brain lysate (10 μg, ¼ of the amount used in A) was treated with 10 μg/ml Calbiochem® tPA for up to 3 h at 37 °C. A prominent decrease in NR2B immunoreactivity can be seen after tPA exposure for 30 min. Synthetic tPA inhibitor tPA-STOP^TM prevented degradation of NR2B proteins. D, % NR2B immunoreactivity of full-length NR2B was reduced gradually with time upon incubation with tPA (based on quantification of C). NR2B proteins rapidly degraded within 30 min and reduced to ∼8% after 3 h. Data were normalized to untreated control of the corresponding treatment time. n = 4; ** and *** denote p < 0.01 and p < 0.001, respectively (against control), and # and φ denote p < 0.001 and p < 0.01 (against tPA-only treatment), one-way ANOVA with Tukey's post hoc test. E, a 3-h Actilyse® treatment of day in vitro 10 cortical neurons led to a decrease in NR2B protein levels. F, shown is quantification of the blot seen in E. Data were normalized to control. n = 6; * denote p < 0.05 (against control), two-tailed paired t test. A single blot was probed for NR2B and GAPDH for A, C, and E.

FIGURE 2.

Plasmin and tPA degrade NR2B proteins (10 μg). A, rat brain lysate was treated with 0.01 unit of plasmin for 1 h at 37 °C and probed with anti-NR2B (Zymed Laboratories Inc.). A representative immunoblot shows that NR2B proteins were completely degraded by plasmin. The plasmin-induced degradation could be prevented in the presence of α₂-antiplasmin, a plasmin inhibitor (n = 3). B, rat brain lysate was treated with 10 μg/ml tPA in the presence of α₂-antiplasmin for 3 h at 37 °C and probed with anti-NR2B (Zymed Laboratories Inc.). C, shown is quantification of B. Data were normalized to untreated control. n = 3; n.s. and *** denote not significant and p < 0.001, respectively (against control unless otherwise labeled), one-way ANOVA with Tukey's post hoc test. Single blots were probed for NR2B and GAPDH for A and B.

FIGURE 3.

tPA cleaves the MBP-ATD2B fusion protein. A, a representative Coomassie Blue-stained SDS-PAGE gel shows that a 3-h tPA (8 μg/ml) treatment of MBP-ATD2B yielded cleaved fragments (F1–F4). † denotes a fragment sent for peptide identification by MS/MS peptide sequencing; †† denotes a fragment sent for amino-terminal peptide sequencing. The presence of the synthetic tPA inhibitor, tPA-STOP^TM (250 μm), prevented the tPA-induced degradation of MBP-ATD2B. B, shown is a representative Western blot analysis of tPA treatment of MBP-ATD2B (shown in A) using anti-MBP (left) and anti-NR2B (Santa Cruz Biotechnology, Inc) (right). Fragments F2 and F3 were revealed by anti-MBP and anti-NR2B, respectively. C, shown is a schematic representation (not drawn to scale) of tPA-induced-cleavage sites in MBP-ATD2B. MS/MS peptide mass spectroscopy and amino-terminal sequencing revealed two cleavage sites in the fusion protein. tPA cleaves the ATD of NR2B at arginine 67. Met represents the amino acid methionine. D, a representative Coomassie Blue-stained SDS-PAGE gel showed that mutant MBP-ATD2B (R67A) was not susceptible to tPA cleavage. Both wild-type (WT) and mutant recombinant MBP-ATD2B were subjected to tPA treatment for 1 h at 37 °C. The lack of cleavage fragments F1 and F4 after tPA treatment showed that MBP-ATD2B (R67A) was not susceptible to tPA cleavage.

FIGURE 4.

Electrophysiological characterization of heterologous NR1/NR2B-ΔATD-R67 receptors in Xenopus oocytes. A, (i) shown is a model of NR2B subunit consisting of ATD, agonist binding domain (S1S2), transmembrane domains, and the carboxyl terminus. Glutamate binds to the S1S2 domain, whereas critical amino acid residues affecting ifenprodil inhibition reside in the ATD. (ii) shown is a schematic representation of the full-length NR2B (NR2BWT) (upper panel) and truncated NR2B (deletion of Gln²⁹–Arg⁶⁷) (NR2B-ΔATD-R67) (lower panel) constructs. SP represents signal peptide, and M1, M3, and M4 represent transmembrane domain, whereas M2 represents the reentrant loop. B, the mean normalized concentration-response curve for glutamate in saturating concentrations of glycine (100 μm; pH 7.3) was obtained from oocytes expressing NR1/NR2BWT (open circles and solid line; n = 7) and NR1/NR2B-ΔATD-R67 (open triangle and dotted line; n = 6). Glutamate EC₅₀ for NR1/NR2BWT and NR1/NR2B-ΔATD-R67 are 0.85 ± 0.12 and 0.94 ± 0.16 μm, respectively. p > 0.05, two-tailed unpaired t test. C, shown is a mean normalized concentration-response curve for glycine in saturating concentrations of glutamate (100 μm; pH 7.3) obtained from oocytes expressing NR1/NR2BWT (open circles and solid line; n = 6) and NR1/NR2B-ΔATD-R67 (open triangle and dotted line; n = 7). Glycine EC₅₀ values for NR1/NR2BWT and NR1/NR2B-ΔATD-R67 are 0.28 ± 0.02 and 1.37 ± 0.19 μm, respectively, and are statistically significantly different. p < 0.001, two-tailed unpaired t test. D, shown is a mean normalized concentration-response curve for ifenprodil inhibition in saturating concentrations of glutamate and glycine (100 μm each; pH 7.3) obtained from oocytes expressing NR1/NR2BWT (open circles and solid line; n = 12) and NR1/NR2B-ΔATD-R67 (open triangle and dotted line; n = 4). Ifenprodil IC₅₀ values for NR1/NR2BWT and NR2B-ΔATD-R67 are 0.35 ± 0.10 and 24.09. ± 18.15 μm, respectively, and are statistically significantly different. p < 0.05, two-tailed unpaired t test.

FIGURE 5.

Crystal structure of apoNR2B_ATD. A, the strand representation of the crystal structure of apoNR2B_ATD (PDB code 3JPW) (47), which consists of two domains, R1 (light pink) and R2 (light purple) was created with Protein Workshop (60). Arg⁶⁷ (R67; residue highlighted in red with balls and sticks) is situated at the tip of R1 (light pink) and thus is proposed to be exposed to the surrounding aqueous milieu. Left, shown is the front view of the NR2B_ATD crystal structure. Right, shown is a view of the crystal structure when the protein molecule is rotated as denoted. B, several structures that may be critical for the tertiary structure of the ATD upon the removal of Gln²⁹–Arg⁶⁷ are highlighted. The tPA cleavage site Arg⁶⁷ (R67) is highlighted in red; Gly³⁶–Val⁴² fragment (β1 sheet), Val⁶⁸–Met⁷³ (β2 sheet), and Val⁹⁷–Asp¹⁰¹ (β3 sheet) are highlighted in dull yellow; Pro⁷⁸–Asp⁹¹ (α1 helix) and Leu²⁸⁹–His³¹¹ (α8 helix) are highlighted in light green. The hypervariable loop (HVL) is highlighted in dark purple, and the two cysteine residues, Cys⁸⁶ in α1 and Cys³²¹ in HVL (highlighted in orange with balls and sticks), form a disulfide bond that helps to stabilize the ATD structure. The peptide upstream of Val⁶⁸ can dissociate from the receptor after tPA treatment.