title: 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.
authors.affiliation: Departments of Pharmacology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. .
authors.forename: Kay-Siong
authors.initials: KS
authors.lastname: Ng
authors.forename: How-Wing
authors.initials: HW
authors.lastname: Leung
authors.forename: Peter T-H
authors.initials: PT
authors.lastname: Wong
authors.forename: Chian-Ming
authors.initials: CM
authors.lastname: Low
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005998:Glycine / Q000494:pharmacology*; D018684:Glycine Agents / Q000494:pharmacology*; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed; D010880:Piperidines / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D059748:Proteolysis / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D018691:Excitatory Amino Acid Antagonists; D018684:Glycine Agents; C121001:NR2B NMDA receptor; D010880:Piperidines; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator; C010739:ifenprodil; D005998:Glycine