Departments of Pharmacology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. .
J Biol Chem. 2012 Jul 20;287(30):25520-9. doi: 10.1074/jbc.M112.374397. Epub 2012 May 18.
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.
在过去的十年中，组织型纤溶酶原激活物（tPA）溶栓一直是治疗急性缺血性脑卒中患者的关键治疗方法。然而，最近的研究表明，这种溶栓蛋白酶在大脑生理和病理生理学谷氨酸依赖性过程中也发挥着各种作用，如突触可塑性和神经退行性变。此外，越来越多的证据表明 tPA 是 N-甲基-D-天冬氨酸（NMDA）受体的重要神经调节剂。在这里，我们证明重组人 tPA 可裂解 NMDA 受体的 NR2B 亚基。用 tPA 处理大鼠脑裂解物和皮质神经元后分析 NR2B 发现，NR2B 蛋白的浓度和时间依赖性降解。在 tPA 处理 NR2B 氨基末端结构域的重组融合蛋白获得的裂解产物上进行肽测序研究表明，tPA 介导的裂解发生在精氨酸 67（Arg（67））处。这种裂解是 tPA 特异性的，不依赖纤溶酶，并且从 NR2B 蛋白的氨基末端结构域中去除了一个预测的~4kDa 片段（Arg（27）-Arg（67））。将推定的切割位点 Arg（67）突变为 Ala（67）可阻止 tPA 介导的重组蛋白降解。该分析表明 NR2B 是 tPA 的一种新型底物，并表明可能形成含有 Arg（27）-Arg（67）截断的 NR2B 的 NMDA 受体。用 Gln（29）-Arg（67）缺失的 NR2B 进行异源表达是功能性的，但ifenprodil 抑制作用降低，甘氨酸 EC（50）增加，而谷氨酸 EC（50）不变。我们的结果证实了 NR2B 是 tPA 的一种新型蛋白水解底物，其中 tPA 可能与 NR2B 亚基直接相互作用，导致含有 NR2B 的 NMDA 受体的药理学特性发生变化。
