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: 7991112
• 负责人: Terunaga Nakagawa
• 金额: $ 30.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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/7991112
• 关键词: AMPA Receptors; Adopted; Affect; Anabolism; Anatomy; Binding; Biochemical; Biochemistry; Biogenesis; Brain; Cell Line; Cells; Characteristics; Complex; Confocal Microscopy; Coupled; Cytoplasmic Tail; DNA Sequence Rearrangement; Data; Defect; Disease; Electron Microscopy; Family; Fluorescence; Functional disorder; Gene Expression; Glutamate Receptor; Human; Image; Individual; Ion Channel; Lead; Membrane; Membrane Proteins; Mental disorders; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; N-Methyl-D-Aspartate Receptors; NR1 gene; Neurologic; Neurons; Patients; Phenotype; Play; Point Mutation; Process; Recombinants; Resolution; Role; Stroke; Structure; Subcellular structure; Synaptic plasticity; System; Techniques; Testing; Time; Vesicle; Work; X-Linked Mental Retardation; base; dimer; improved; insight; interest; member; mutant; nervous system disorder; overexpression; particle; public health relevance; receptor; receptor function; research study; stargazin; three dimensional structure; time use; trafficking

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（由申请人提供）：AMPA受体（AMPA-R）的亚基组装和囊泡运输在突触可塑性中起核心作用。然而，AMPA-R功能、运输和生物发生的分子基础仍然知之甚少。该领域目前的数据表明，AMPA-R具有二聚体的二聚体组织，不对称的整体结构，并且在大脑中含有调节AMPA-R功能的辅助stargazin/TARP亚基。为了更深入地了解AMPA-R的结构和组装，这是理解突触可塑性的关键，我们建议比较成熟的GluR 2四聚体的三维EM结构与二聚体生物合成中间体。我们将在稳定的HEK细胞系中依赖DOX表达GluR 2亚基，纯化重组受体并通过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取特定的构象，以及干扰亚基组装的点突变是否会改变亚基构象。首先，我们将致力于通过成像重组GluR 2翻转形成的四聚体并锁定到非脱敏状态来获得完全组装的AMPA-R的改进的3D结构。我们还将确定未成熟的GluR 2二聚体的结构。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结构域间接触。最后，我们将研究突变型GluR 2亚基的二聚体中间体的三维结构，并与野生型GluR 2的结构进行比较，从而深入了解突变如何干扰亚基组装。因为在大脑中辅助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GluR 2结合，以及stargazin如何调节GluR 2的运输和亚基组装。我们已经建立了稳定的HEK细胞系，其仅在DOX存在下组成型表达stargazin但GluR 2。我们将比较在stargazin存在和不存在的情况下DOX诱导的GluR 2表达的时间过程，以定量测试stargazin是否是GluR 2的分子伴侣。我们将进一步使用GFP标记的GluR 2在神经元中使用延时荧光共聚焦显微镜检查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）是谷氨酸受体家族的另一个成员，在突触可塑性中发挥关键作用。该领域的证据表明，NMDA-R和AMPA-R之间的结构域排列和亚基组装机制可能不同。因此，在目标3中，我们将使用我们为AMPA-Rs到NMDA-Rs的研究建立的方法。特别地，我们将使用EM来研究由NR 1和NR 2B亚基形成的异源四聚体NMDA-R的结构。 公共卫生相关性：谷氨酸受体功能障碍可导致多种神经和精神疾病以及中风。谷氨酸受体亚单位的突变已在X连锁精神发育迟滞患者中发现，因此谷氨酸受体功能和组装的改变可能是该疾病的直接原因。通过揭示野生型和突变型谷氨酸受体功能，贩运和亚基组装的详细分子基础，该提案旨在扩展我们对神经和精神疾病的理解，特别是可能导致治愈X连锁精神发育迟滞。