MOLECULAR BASIS OF NON-NMDA GLUTAMATE RECEPTOR ASSEMBLEY

离子型谷氨酸受体介导快速兴奋性神经传递。谷氨酸受体亚基的异构化决定了关键的通道特性，而通道特性反过来又可以影响细胞的信号传递过程。然而，亚基齐聚只发生在谷氨酸受体亚家族中，这意味着决定亚基特异性组装的蛋白质结构域。这些实验的目的是确定决定谷氨酸受体亚单位齐聚的分子机制。该片段对AMPA的电流表达也有显性的负面影响，但对NMDA的表达没有影响。这些数据表明，AMPA受体的氨基末端参与了亚单位的结合，可能是组装的关键。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，应用了强大的酵母双杂交方法的结果。特定目的#1应用强大的酵母双杂交方法来定义和表征亚基联合的最小区域。特定目的#2回答了糖基化是否影响AMPA受体亚单位关联的问题。特定目标#3的分析性超速离心实验旨在确定氨基末端片段的分子量、化学计量比和结合性质，这些片段在共免疫沉淀实验中已被证明是自结合的。最后，特定的目的#4旨在确定AMPA受体的组装是发生在内质网还是发生在另一个细胞内隔室。结合以前的数据，这些实验将全面描述AMPA受体的生物发生。

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.