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-RS）的亚基组装和囊泡运输在突触可塑性中起着核心作用。然而，AMPA-R功能，运输和生物发生的分子基础仍然很少了解。该领域的当前数据表明，AMPA-RS具有二聚体的组织，不对称的总体结构，并且大脑中包含调节AMPA-R功能的辅助Stargazin/TARP亚基。为了获得对了解突触可塑性至关重要的AMPA-R结构和组装的更多信息，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3D EM结构进行比较。我们将在稳定的HEK细胞系中依赖Dox表达Glur2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AIM 1中，我们将测试正常的亚基组装是否要求亚基采用特定构象，以及点突变是否干扰亚基组装改变了亚基构象。首先，我们将通过成像由重组GlUR2翻转并锁定到非敏化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-RS的3D结构，我们将揭露将二聚体组装到四聚体中的域间接触。最后，我们将研究突变Glur2亚基的二聚体中间体的3D结构，并将其与野生型Glur2结构进行比较，从而深入了解突变如何干扰亚基组装。因为在大脑辅助型星际加仑/气块亚基与AMPA-RS相关，因此在AIM 2中，我们将研究Stargazin在成熟过程中与Glur2结合时，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，它们组成型表达Stargazin，但仅在DOX存在下Glur2。我们将在存在和不存在Stargazin的情况下比较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。我们将在神经元中进一步使用GFP标记的GLUR2，以使用延时荧光共聚焦mircroscopicy检查Stargazin对受体动力学的影响。这些实验的结果将决定Stargazin对新组装AMPA-RS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，在突触可塑性中起关键作用。该领域的证据表明，NMDA-RS和AMPA-RS之间的域排列和亚基组装机理可能有所不同。因此，在AIM 3中，我们将使用我们为AMPA-RS进行NMDA-RS的研究所建立的方法。特别是，我们将使用EM研究由NR1和NR2B亚基形成的杂次NMDA-RS的结构。

项目成果

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

• DOI: 10.1126/science.aae0104
• 发表时间: 2016-07-15
• 期刊: Science (New York, N.Y.)
• 作者: Elegheert J;Kakegawa W;Clay JE;Shanks NF;Behiels E;Matsuda K;Kohda K;Miura E;Rossmann M;Mitakidis N;Motohashi J;Chang VT;Siebold C;Greger IH;Nakagawa T;Yuzaki M;Aricescu AR
• 通讯作者: Aricescu AR