Molecular Anatomy of Mature and Immature Glutamate Receptors

成熟和未成熟谷氨酸受体的分子解剖学

• 批准号: 8627225
• 负责人: Terunaga Nakagawa
• 金额: $ 22.78万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-10 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8627225
• 关键词: Molecular; Anatomy; Mature; Immature; Glutamate

DESCRIPTION (provided by applicant): Subunit assembly and vesicle trafficking of AMPA receptors (AMPA-Rs) play central roles in synaptic plasticity. The molecular basis for AMPA-R function, trafficking, and biogenesis remains, however, poorly understood. Current data in the field suggest that AMPA-Rs have a dimer-of-dimers organization, asymmetric overall structure, and in the brain contain auxiliary stargazin/TARP subunits that modulate AMPA-R function. To obtain more insight into AMPA-R structure and assembly, which are critical for understanding synaptic plasticity, we propose to compare the 3D EM structure of the mature GluR2 tetramer with those of dimeric biosynthetic intermediates. We will DOX dependently express GluR2 subunits in stable HEK cell lines, purify the recombinant receptors and study their structures by single-particle electron microscopy. In Aim 1 we will test whether normal subunit assembly requires the subunits to adopt a specific conformation and whether point mutations interfering with subunit assembly change the subunit conformation. First, we will work towards obtaining an improved 3D structure of a fully assembled AMPA-R by imaging tetramers formed by recombinant GluR2 flip and locked into the non-desensitized state. We will also determine the structure of immature GluR2 dimers. By comparing the 3D structures of the mature and immature AMPA-Rs, we will unveil the inter-domain contacts that drive the assembly of dimers into tetramers. Finally, we will study the 3D structures of dimer intermediates of mutant GluR2 subunits and compare those with the structures of wild-type GluR2, providing insight into how mutations may interfere with subunit assembly. Because in the brain auxiliary stargazin/TARP subunits are associated with AMPA-Rs, in Aim 2 we will investigate when stargazin binds to GluR2 during maturation and how stargazin modulates trafficking and subunit assembly of GluR2. We have established stable HEK cell lines that constitutively express stargazin but GluR2 only in the presence of DOX. We will compare the time course of the DOX-induced expression of GluR2 in the presence and absence of stargazin to quantitatively test if stargazin is a molecular chaperone of GluR2. We will further use GFP-tagged GluR2 in neurons to examine the effect of stargazin on receptor dynamics using time-lapse fluorescent confocal mircroscopy. Results from these experiments will determine the contribution of stargazin to the trafficking and maturation of newly assembled AMPA-Rs. The NMDA receptor (NMDA-R) is another member of the glutamate receptor family that plays critical roles in synaptic plasticity. Evidence in the field suggests that the domain arrangement and mechanism of subunit assembly may differ between NMDA-Rs and AMPA-Rs. In Aim 3, we will therefore use the approaches we established for our studies on AMPA-Rs to NMDA-Rs. In particular, we will use EM to study the structure of heterotetrameric NMDA-Rs formed by NR1 and NR2B subunits. PUBLIC HEALTH RELEVANCE: Dysfunction of glutamate receptors causes a variety of neurological and psychiatric disorders and stroke. Mutations in glutamate receptor subunits have been found in patients with X-linked mental retardation, and thus altered glutamate receptor function and assembly is likely to be the direct cause of the disease. By revealing the detailed molecular basis for wild-type and mutant glutamate receptor function, trafficking and subunit assembly, this proposal intends to extend our understanding of neurological and mental disorders, which may especially lead to curing X-linked mental retardation.

描述(申请人提供)：AMPA受体的亚单位组装和囊泡运输在突触可塑性中发挥核心作用。然而，AMPA-R功能、运输和生物发生的分子基础仍然知之甚少。目前该领域的数据表明，AMPA-R具有二聚体结构，总体结构不对称，在大脑中含有辅助的Stargazin/TARP亚基，调节AMPA-R的功能。为了更深入地了解AMPA-R的结构和组装，这对理解突触的可塑性是至关重要的，我们建议比较成熟的GluR2四聚体和二聚体生物合成中间体的3D EM结构。我们将在稳定的HEK细胞系中DOX依赖性地表达GluR2亚基，纯化重组受体，并用单粒子电子显微镜研究其结构。在目标1中，我们将测试正常的亚基组装是否需要亚基采用特定的构象，以及干扰亚基组装的点突变是否会改变亚基的构象。首先，我们将致力于通过成像由重组GluR2翻转形成的并锁定到非脱敏状态的四聚体来获得完全组装的AMPA-R的改进的3D结构。我们还将确定未成熟的GluR2二聚体的结构。通过比较成熟和未成熟AMPA-R的3D结构，我们将揭示驱动二聚体组装成四聚体的域间接触。最后，我们将研究突变的GluR2亚基的二聚体中间产物的三维结构，并将其与野生型GluR2的结构进行比较，为突变如何干扰亚基组装提供洞察。由于在大脑中辅助的Stargazin/TARP亚基与AMPA-Rs相关，在目标2中，我们将研究Stargazin在成熟过程中何时与GluR2结合，以及Stargazin如何调控GluR2的运输和亚单位组装。我们已经建立了稳定的HEK细胞系，在DOX存在的情况下，该细胞系能够结构性地表达Stargazin，但GluR2。我们将比较DOX诱导的GluR2在有无Stargazin存在的情况下表达的时间进程，以定量检验Stargazin是否为GluR2的分子伴侣。我们将进一步使用GFP标记的GluR2在神经元中使用延时荧光共聚焦显微镜来检测Stargazin对受体动力学的影响。这些实验的结果将确定Stargazin对新组装的AMPA-R的运输和成熟的贡献。NMDA受体(NMDA-R)是谷氨酸受体家族中的另一个成员，在突触可塑性中起着关键作用。现场证据表明，NMDA-Rs和AMPA-Rs在结构域排列和亚基组装机制上可能有所不同。因此，在目标3中，我们将使用我们为研究AMPA-Rs到NMDA-Rs而建立的方法。特别是，我们将用EM研究由NR1和NR2B亚基形成的异四聚体NMDA-Rs的结构。 与公共卫生相关：谷氨酸受体功能障碍会导致多种神经和精神疾病以及中风。在X连锁智力低下患者中发现了谷氨酸受体亚单位的突变，因此谷氨酸受体功能和组装的改变可能是该病的直接原因。通过揭示野生型和突变型谷氨酸受体功能、转运和亚单位组装的详细分子基础，这一建议旨在扩大我们对神经和精神障碍的理解，这可能特别有助于治疗X-连锁智力低下。