MOLECULAR MECHANISMS THAT REGULATE PROTEIN SYNTHESIS IN NEURONS.

调节神经元中蛋白质合成的分子机制。

• 批准号: 8081073
• 负责人: PAUL F WORLEY
• 金额: $ 46.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8081073
• 关键词: Agonist; Animal Model; Binding; Biological; Biological Assay; Bypass; Calmodulin; Cells; Cocaine; Cocaine Dependence; Complex; Confocal Microscopy; Corpus striatum structure; Coupled; Cyclic AMP-Dependent Protein Kinases; Dendrites; Dominant-Negative Mutation; Dopamine Receptor; Endosomes; Excitatory Synapse; Genetic Models; Glutamate Receptor; Grant; Hippocampus (Brain); Immediate-Early Genes; Knock-out; MAP Kinase Gene; Mediating; Messenger RNA; Methamphetamine; Methods; Molecular; Mus; N-Methylaspartate; Neurons; Oranges; Pathway interactions; Peptide Elongation Factor 2; Phase; Phenocopy; Phospho-Specific Antibodies; Phospholipase C; Phosphorylation; Phosphotransferases; Physiology; Play; Polyribosomes; Population; Process; Protein Biosynthesis; Proteins; Ras homolog enriched in brain; Receptor Activation; Regulation; Reporter; Reporting; Role; Signal Pathway; Signal Transduction; Sirolimus; Slice; Stimulus; Structure; Synapses; Synaptic plasticity; Time; Translations; Tuberous sclerosis protein complex; Up-Regulation; Vesicle; Viral; addiction; base; calmodulin-dependent protein kinase III; human FRAP1 protein; insight; interest; mutant; novel; prevent; receptor; response; synthetic protein; trafficking

The focus of this proposal is to examine molecular mechanisms that regulate protein synthesis in neurons in response to group 1 metabotropic receptor (mGluR) activation. mGluRI and 5 transduce excitatory synaptic stimuli to active phospholipase C and release Ca2+ from intracellular stores (1), and play an important role in regulating trafficking of AMPA and NMDA type ionotropic glutamate receptors (2, 3). mGluR 1 and 5 are also key regulators of protein synthesis at the excitatory synapse (4). Recently, it has been shown that mGluRS is essential for cocaine addiction (5) and other forms of protein synthesisdependent plasticity (6-10). Ongoing studies conducted during the previous grant period identified two distinct regulatory mechanisms that transduce mGluR activity to increase protein synthesis. The most rapid response is mediated by a molecular cascade that involves dynamic interactions between mGluR and eukaryotic elongation factor 2 kinase (eEF2K). Our interest in eEF2K began with the discovery that it binds both mGluR and Homer. Agonist stimulation of mGluR results in activation of eEF2K in a process that appears to involve calmodulin and requires intracellular stores of Ca2+. eEK2K phosphorylates elongation factor 2 (eEF2), which binds the polyribosome-mRNA complex and controls the elongation phase of protein translation (11). Phosphorylated eEF2 (p-eEF2) acts as a dominant negative and prevents translation of many mRNAs, although it is reported that p-eEF2 can selectively increase translation of certain mRNAs (12, 13). Time course studies indicate that activation of mGluR results in an immediate increase in p-eEF2 (should reduce elongation) that persists for ~5 min followed by a progressive decrease of p-eEF2 that persists >60 min. Our studies indicate that this pathway is essential for the rapid translation of the immediate early gene Arc. Neurons in which eEF2K is genetically deleted (ko) show an absence of the initial upregulation of Arc at 5 min after mGluR activation. Importantly, eEF2K ko mice phenocopy changes in plasticity seen in Arc ko mice. Arc is representative of a class of mRNAs (includes CamKII), that possess an internal ribosomal entry sequence (IRES) (14). Viral IRESs form a secondary structure that imitates the initiation complex required for ribosomal binding and therefore bypass regulation of initiation. We hypothesize that the IRES affords regulation by eEF2 in mGluR-dependent Arc translation, and that understanding of this process will reveal basic insights into synapse-specific protein synthesis. Arc is highly relevant for studies of addiction since it is strongly induced in accumbens/striatum by cocaine, methamphetamine, and dopamine receptor activation (15-20).

这项建议的重点是研究调节蛋白质合成的分子机制。 神经元对第1组代谢性受体(MGluR)激活的反应。MGluRI和5转导 兴奋性突触刺激激活磷脂酶C和细胞内钙释放(1)，并发挥作用 在调节AMPA和NMDA型离子型谷氨酸受体的运输中发挥重要作用(2，3)。 MGluR1和5也是兴奋性突触蛋白质合成的关键调节因子(4)。最近，它已经 已经证明mGluRs对于可卡因成瘾和其他形式的蛋白质合成依赖是必不可少的 可塑性(6-10)。在前一个赠款期间进行的持续研究确定了两个 不同的调节机制，转导mGluR活性以增加蛋白质合成。最快的 反应由分子级联介导，涉及mGluR和mGluR之间的动态相互作用 真核细胞延伸因子2激酶(EEF2K)。我们对eEF2K的兴趣始于它与 MGluR和荷马都是。激动剂刺激mGluR导致eEF2K激活，这一过程 似乎涉及钙调蛋白，需要细胞内储存钙离子。EEK2K使延伸磷酸化 因子2(EEF2)，它结合多聚核糖体-mRNA复合体，控制蛋白质的延伸期 译文(11)。磷酸化的eEF2(p-eEF2)起显性负性作用，阻止翻译 许多mRNAs，尽管据报道p-eEF2可以选择性地增加某些mRNAs的翻译(12， 13)。时程研究表明，激活mGluR会立即导致p-eEF2的增加 (应该减少伸长率)，持续约5分钟，然后p-eEF2逐渐减少， 持续&gt；60分钟。我们的研究表明，这条途径是快速翻译的关键 即刻早期基因Arc。EEF2K基因缺失(Ko)的神经元显示缺乏 在mGluR激活后5分钟，Arc开始上调。重要的是，eEF2K Ko小鼠的表型变化 在Arc Ko小鼠身上看到的可塑性。ARC是一类mRNAs(包括CaMKII)的代表，具有 一种内部核糖体进入序列(IRES)(14)。病毒IRESS形成一个二级结构，它模仿 核糖体结合所需的起始复合体，因此绕过起始调节。我们 假设IRES在mGluR依赖的Arc翻译中由eEF2提供调节，并且 对这一过程的理解将揭示对突触特异性蛋白质合成的基本见解。ARC是高度 与成瘾研究相关，因为可卡因在伏隔/纹状体强烈诱导成瘾， 甲基苯丙胺和多巴胺受体激活(15-20)。