Lau Garrick C, Saha Shamol, Faris Ramona, Russek Shelley J
Laboratory of Molecular Neurobiology, Department of Pharmacology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Neurochem. 2004 Feb;88(3):564-75. doi: 10.1046/j.1471-4159.2003.02156.x.
Transcription mediated by protein kinase A and the cAMP response element binding protein (CREB) has been linked to the establishment of long-term memory and cell survival. However, all of the major targets for activated CREB have yet to be identified. Given the fact that CREB-mediated transcription is intimately involved in cellular processes of learning and memory and that CREB activity can be regulated by synaptic N-methyl-d-aspartate receptors (NMDARs) and metabotropic GABA receptors, we have studied the role of the cAMP-dependent signaling pathway in the regulation of the NMDA receptor subunit 1 (NMDAR1), a subunit required for functional receptor formation. We now report that levels of NMDAR1 subunit protein in primary neocortical cultures are increased 66% in response to forskolin, an activator of adenylyl cyclase. Up-regulation of NMDAR1 is paralleled by a twofold increase in mRNA levels and an 83% increase in NMDAR1 promoter/luciferase reporter activity that is dependent on protein kinase A. Three cAMP regulatory elements (CREs) in the rat NMDAR1 promoter (- 228, - 67, and - 39) bind CREB in vitro and forskolin increases binding to two of the sites (- 228 and - 67). Chromatin immunoprecipitation of neuronal rat genomic DNA reveals that CREB is bound in vivo to the endogenous NMDAR1 gene. Increased presence of the activated Ser133 phosphorylated form is dependent on the length of exposure to forskolin. Taken together with the results of mutational analysis, the findings strongly suggest that transcription of NMDAR1 is regulated by the c-AMP signaling pathway, most likely through the binding of CREB and its activation by signal-dependent phosphorylation.
由蛋白激酶A和环磷酸腺苷反应元件结合蛋白（CREB）介导的转录与长期记忆的建立及细胞存活有关。然而，激活的CREB的所有主要靶标尚未确定。鉴于CREB介导的转录密切参与学习和记忆的细胞过程，且CREB活性可由突触N-甲基-D-天冬氨酸受体（NMDARs）和代谢型GABA受体调节，我们研究了环磷酸腺苷依赖性信号通路在调节N-甲基-D-天冬氨酸受体亚基1（NMDAR1）中的作用，NMDAR1是功能性受体形成所需的一个亚基。我们现在报告，在原代新皮质培养物中，NMDAR1亚基蛋白水平因腺苷酸环化酶激活剂福斯高林而增加了66%。NMDAR1的上调与mRNA水平增加两倍以及NMDAR1启动子/荧光素酶报告基因活性增加83%相平行，该活性依赖于蛋白激酶A。大鼠NMDAR1启动子中的三个环磷酸腺苷调节元件（CREs）（-228、-67和-39）在体外与CREB结合，福斯高林增加了与其中两个位点（-228和-67）的结合。大鼠神经元基因组DNA的染色质免疫沉淀显示，CREB在体内与内源性NMDAR1基因结合。激活的Ser133磷酸化形式的增加存在取决于福斯高林暴露的时长。结合突变分析结果，这些发现强烈表明NMDAR1的转录受环磷酸腺苷信号通路调节，很可能是通过CREB的结合及其由信号依赖性磷酸化的激活。
