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: 8733742
• 负责人: Terunaga Nakagawa
• 金额: $ 30.38万
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8733742
• 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; N-Methylaspartate; NR1 gene; 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; 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.

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 结构和组装（这对于理解突触可塑性至关重要），我们建议将成熟 GluR2 四聚体的 3D EM 结构与二聚体生物合成中间体的结构进行比较。我们将在稳定的HEK细胞系中DOX依赖性表达GluR2亚基，纯化重组受体并通过单颗粒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构象。首先，我们将通过对重组 GluR2 翻转形成的四聚体进行成像并锁定在非脱敏状态，努力获得完全组装的 AMPA-R 的改进 3D 结构。我们还将确定未成熟 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，我们将研究突变型 GluR2 亚基二聚体中间体的 3D 结构，并将其与野生型 GluR2 的结构进行比较，从而深入了解突变如何干扰亚基组装。由于在大脑中，辅助 stargazin/TARP 亚基与 AMPA-R 相关，因此在目标 2 中，我们将研究 stargazin 在成熟过程中何时与 GluR2 结合，以及 stargazin 如何调节 GluR2 的运输和亚基组装。我们已经建立了稳定的 HEK 细胞系，其组成型表达 stargazin，但仅在存在 DOX 的情况下才表达 GluR2。我们将比较在存在和不存在 stargazin 的情况下 DOX 诱导的 GluR2 表达的时间过程，以定量测试 stargazin 是否是 GluR2 的分子伴侣。我们将进一步在神经元中使用 GFP 标记的 GluR2，利用延时荧光共聚焦显微镜检查 stargazin 对受体动态的影响。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着关键作用。该领域的证据表明，NMDA-R 和 AMPA-R 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-R 到 NMDA-Rs 研究而建立的方法。特别是，我们将使用 EM 研究由 NR1 和 NR2B 亚基形成的异四聚体 NMDA-R 的结构。

项目成果

Engineering defined membrane-embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin.

工程定义的 AMPA 受体膜嵌入元件通过 cornichon 3 和 stargazin 诱导相反的门控调节。

• 发表时间: 2017
• 作者: Hawken, Natalie M;Zaika, Elena I;Nakagawa, Terunaga
• 通讯作者: Nakagawa, Terunaga

Structures of the AMPA receptor in complex with its auxiliary subunit cornichon.

AMPA 受体与其辅助亚基 cornichon 复合的结构。

• 发表时间: 2019
• 作者: Nakagawa; Terunaga
• 通讯作者: Terunaga

Structural basis for integration of GluD receptors within synaptic organizer complexes.

GluD 受体在突触组织复合体中整合的结构基础。

• 发表时间: 2016-07-15
• 作者: Elegheert, Jonathan;Kakegawa, Wataru;Clay, Jordan E;Shanks, Natalie F;Behiels, Ester;Matsuda, Keiko;Kohda, Kazuhisa;Miura, Eriko;Rossmann, Maxim;Mitakidis, Nikolaos;Motohashi, Junko;Chang, Veronica T;Siebold, Christian;Greger, Ingo H;Nakagaw
• 通讯作者: Nakagaw

The biochemistry, ultrastructure, and subunit assembly mechanism of AMPA receptors.

AMPA 受体的生物化学、超微结构和亚基组装机制。

• 发表时间: 2010-12
• 影响因子: 5.1
• 作者: Nakagawa, Terunaga