Presynaptic Receptor Complexes

描述(由申请人提供)： 乳头状毒素是一种突触前神经毒素，能刺激大量突触小泡的胞吐。A-乳杆菌毒素通过与高亲和力的神经细胞表面受体结合来发挥作用，这些受体被认为代表突触前神经递质释放部位的特定标记。我们以前的研究发现了三种属于不同信号转导途径的α-拉曲霉毒素受体：环是G蛋白偶联受体，Neurexins是细胞黏附样蛋白，具有单一的跨膜区和短的细胞质尾巴，PTP6是一种受体样蛋白酪氨酸磷酸酶。为了了解α-乳杆菌毒素受体是如何将突触黏附与神经元胞吐作用结合起来的，我们建议鉴定受体相互作用的蛋白，并研究它们相互作用的机制。我们的实验方法是基于使用一种独特的工具--α-乳杆菌毒素，它以高亲和力结合受体，从而允许一步分离受体和相关蛋白质。我们提出了两组实验。第一个是鉴定亲和纯化的α-乳杆菌毒素受体制备中含量较低的蛋白质组分，并分析它们与受体相互作用的特异性。这将通过现代蛋白质组学技术实现，并辅之以受体特异性抗体和相关蛋白特异性抗体的免疫沉淀。作为关键的阴性对照，从缺乏α-乳杆菌毒素受体的基因敲除小鼠的大脑中获得的受体制剂将被使用。第二组实验是研究单个α-乳杆菌毒素受体与突触素和合成素的相互作用机制，这两种蛋白质参与了与α-乳毒素受体结合的胞吐作用。特别是，我们将确定这些蛋白质与哪些受体结合，并测试这种相互作用是否直接。我们还将确定受体及其相关蛋白的相互作用区域。获得更好的α-乳杆菌毒素受体复合体的生物化学定义是重要的，因为它有望提供对其功能所涉及的信号机制的洞察，并可能提供对突触前细胞黏附的洞察。这项研究将主要在俄罗斯Shemyakin-Ovchinnikov生物有机化学研究所与Alexander Petrenko合作进行，作为NIH赠款R37 MH5280的延伸。

DESCRIPTION (provided by applicant): a-Latrotoxin is a presynaptic neurotoxin that stimulates massive synaptic vesicle exocytosis. a-latrotoxin acts by binding to high-affinity neuronal cell-surface receptors that are thought to represent specific markers of presynaptic neurotransmitter release sites. Our previous studies identified three a- latrotoxin receptors that belong to different signaling pathways: The CIRLs are G protein-coupled receptors, neurexins are cell adhesion-like proteins with a single transmembrane domain and short cytoplasmic tail, and PTP6 is a receptor-like protein tyrosine phosphatase. To understand how a-latrotoxin receptors couple synaptic adhesion to neuronal exocytosis, we propose to identify receptor-interacting proteins and to study the mechanisms of their interactions. Our experimental approach is based on the use of a unique tool, a-latrotoxin, that binds the receptors with high affinity, thus allowing the one-stage isolation of the receptors together with associated proteins. We propose two sets of experiments. The first one is to identify less abundant protein components present in the preparations of affinity-purified a-latrotoxin receptors, and to analyze the specificity of their interaction with the receptors. This will be achieved by modern proteomics techniques complemented by immunoprecipitation with receptor-specific and associated protein-specific antibodies. As a key negative control, receptor preparations obtained from brains of knockout mice lacking a-latrotoxin receptors will be used. The second set of experiments is to study the mechanism of the interaction of individual a-latrotoxin receptors with synaptotagmin and syntaxin, proteins, involved in exocytosis that bind to a-latrotoxin receptors. In particular, we will identify to which receptors these proteins bind, and test if this interaction is direct. We will also determine the interacting domains of the receptors and their associated proteins. Obtaining a better biochemical definition of the a-latrotoxin receptor complexes is important because it promises to provide insight into the signaling mechanisms involved in their function, and may provide insight into presynaptic cell adhesion. This research will be performed primarily in Russia at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in collaboration with Alexander Petrenko as an extension of NIH grant R37 MH5280.