GABAA RECEPTOR - INTERACTING PROTEINS

GABAA 受体 - 相互作用的蛋白质

• 批准号: 6540104
• 负责人: ANGEL L DE BLAS
• 金额: $ 28.6万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6540104
• 关键词: GABA receptor; confocal scanning microscopy; guanine nucleotide exchange factors; protein localization; protein protein interaction; protein sequence; protein structure function; receptor binding; synapses; yeast two hybrid system

DESCRIPTION: (Applicant's Abstract) It has been estimated that 40 percent of the brain synapses are GABAergic. However, little is known about the mechanisms by which GABAA receptors (GABAAR) are concentrated at inhibitory GABAergic Gray's type 2 synapses. The long-term objective of this proposal is to reveal the molecular components of the GABAergic Gray's type 2 postsynaptic density and to understand the molecular mechanisms involved in clustering and anchoring GABAAR at the postsynaptic membrane. The specific aims of this proposal are directed towards revealing the cytoplasmic proteins that directly bind to GABAAR and to test whether these proteins are responsible for the clustering and/or localization of the GABAAR at synapses and/or other membrane domains. The aims will test the hypotheses that 1) a novel GABAAR protein is involved in GABAAR clustering and/or anchoring at GABAergic synapses; 2) in neurons, the interaction of the beta3 GABAAR subunit with a guanine nucleotide exchange protein is necessary for GABAAR transport to the cell surface; and 3) the novel proteins that interact with various GABAAR subunits in the yeast two-hybrid screens also interact with the GABAAR in neurons and are involved in receptor clustering or anchoring at synapses. Yeast two-hybrid assays are being used in our laboratory to identify GABAAR binding proteins. The functionality of the interaction between GABAAR and binding proteins will be tested by various in vitro biochemical assays as well as by in vivo (in culture) assays including primary hippocampal neuronal cultures and cotransfected host cells. The synaptic colocalization of the GABAA receptors and interacting proteins will be studied by both light microscopic immunofluorescence and electron microscopic immunogold immunocytochemistry using specific antibodies to GABAAR subunits and interacting protein. The GABAAR and interacting protein amino acid sequences involved in protein-protein interactions will be identified. These studies will help to better understand the molecular components and interactions involved in the structure, function and modulation of the inhibitory GABAergic synaptic transmission as well as the mechanisms involved in neuronal connectivity and synaptic plasticity. It is predicted that the inappropriate synaptic and extrasynaptic localization of the GABAAR, affects GABAergic synaptic function and brain development, leading to neurological and mental disorders.

描述：（申请人的摘要） 据估计，40％的大脑突触是GABA能。 但是，对于GABAA受体（Gabaar）的机制知之甚少 集中在抑制性GABA能灰色的2型突触上。长期 该提议的目的是揭示分子成分 GABA能灰色的2型突触后密度，并了解分子 涉及聚类和锚定Gabaar的机制在突触后 膜。该提案的具体目的是针对揭示 直接与Gabaar结合并测试这些是否直接结合的细胞质蛋白 蛋白质负责GABAAR的聚类和/或定位 在突触和/或其他膜域。目的将检验假设 1）一种新型的Gabaar蛋白参与Gabaar聚类和/或 锚定在GABA能突触； 2）在神经元中，beta3的相互作用 具有鸟嘌呤核苷酸交换蛋白的GABAAR亚基是必要的 Gabaar转运到细胞表面； 3）相互作用的新型蛋白质 酵母双杂交屏幕中的各种Gabaar亚基也与 神经元中的Gabaar，并参与受体聚类或锚定 突触。我们的实验室正在使用酵母两杂交测定 GABAAR结合蛋白。 Gabaar之间相互作用的功能 结合蛋白将通过各种体外生化测定法测试 以及通过体内（培养）测定法，包括原发性海马神经元 培养物和共转染的宿主细胞。 GABAA的突触共定位 两种光显微镜将研究受体和相互作用的蛋白质 免疫荧光和电子显微镜免疫细胞化学化学 使用针对Gabaar亚基和相互作用蛋白的特定抗体。这 GABAAR和相互作用的蛋白质氨基酸序列参与蛋白质蛋白 将确定相互作用。这些研究将有助于更好地理解 在结构，功能中涉及的分子成分和相互作用 并调节抑制性GABA能突触传播以及 涉及神经元连通性和突触可塑性的机制。这是 预测，不适当的突触和突触外定位 Gabaar，影响GABA能突触功能和大脑发育，导致 神经和精神障碍。