MOLECULAR BASIS OF NON-NMDA GLUTAMATE RECEPTOR ASSEMBLEY

非 NMDA 谷氨酸受体组装体的分子基础

• 批准号: 6140039
• 负责人: CHRISTINE T SCHULTEIS
• 金额: $ 3.75万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-10-03 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6140039
• 关键词: AMPA receptors; electrophysiology; gene deletion mutation; glycosylation; immunoprecipitation; molecular assembly /self assembly; protein structure function; receptor binding; receptor expression; tissue /cell culture; yeast two hybrid system

Ionotropic glutamate receptors mediate fast excitatory neurotransmission. Heteromultimerization of glutamate receptor subunits determines critical channel properties which in turn can influences cell signaling processes. Subunit oligomerization, however, occurs only within glutamate receptor subfamilies, suggesting a protein domain that determines subunit-specific assembly . The objective of these experiments is to define molecular mechanisms that determine oligomerization of glutamate receptor subunits. This fragment also exerts a dominant negative effect on AMPA, but not NMDA, current expression. These data, among others, demonstrate that the amino terminus of AMPA receptors is involved in subunit association and may be critical for assembly. Experiments proposed herein are designed to complement these results by answering several specific questions. Specific Aim #1 applies the powerful yeast- two-hybrid approach results by answering several specific questions. Specific Aim #1 applies the powerful yeast-two-hybrid approach to define and characterize minimal regions of subunit association. Specific Aim #2 answers the question of whether glycosylation influences subunit association in AMPA receptors. Analytical ultracentrifugation experiments in Specific Aim #3 are designed to determine the molecular weight, stoichiometry, and association properties of amino-terminal fragments, which have been shown to self-associated in co- inmmunoprecipitation experiments. Finally, Specific Aim #4 is designed to determine whether AMPA receptor assembly occurs in the endoplasmic reticulum or in another intracellular compartment. In combination with previous data, these experiments will provide a comprehensive description of AMPA receptor biogenesis.

嗜离子性谷氨酸受体介导快速兴奋性神经传递。谷氨酸受体亚单位的异多聚化决定了关键的通道特性，进而可以影响细胞信号传导过程。然而，亚基寡聚化仅发生在谷氨酸受体亚家族中，表明一个蛋白质结构域决定亚基特异性组装。这些实验的目的是确定谷氨酸受体亚基寡聚化的分子机制。该片段对AMPA的当前表达也有主要的负作用，而对NMDA的当前表达没有影响。这些数据，除其他外，表明AMPA受体的氨基端参与亚基结合，可能对组装至关重要。本文提出的实验旨在通过回答几个具体问题来补充这些结果。具体目标#1通过回答几个具体问题来应用强大的酵母-双杂交方法结果。特异性目标#1应用强大的酵母-双杂交方法来定义和表征亚基关联的最小区域。特异性目标#2回答了糖基化是否影响AMPA受体亚基结合的问题。特异Aim #3中的分析性超离心实验旨在确定氨基末端片段的分子量，化学计量学和关联特性，这些片段已在共免疫沉淀实验中显示出自关联。最后，Specific Aim #4旨在确定AMPA受体组装是否发生在内质网或另一个细胞内腔室。结合以往的数据，这些实验将提供一个全面的描述AMPA受体的生物发生。