CHEMOKINE RECEPTOR EXPRESSION IN THE CNS

这是第二次重新申请，以调查的类型和功能， 大鼠体内的趋化素受体已经被一种 集中注意力，提高成功率。 它有三 来自先前提案的具体目标，但规模较小 范围内。 第一个具体目标是确定 配体结合趋化因子受体CKR 2、CKR 5和RBS 11。 这一目标有两个部分。 在第一部分中，他将横切HEK 293 细胞使用pcDNA 3载体。 他将评估 通过北方印迹分析mRNA进行转染。 他将 通过暴露转染的细胞或其 放射性标记的MCP-1。 他将决定是否 这种结合的特异性可以与多种其它CC竞争， 胆碱;将确定解离常数。 他也会做 MIP-1a的CKR 5也是如此。 在这个目标的第二部分，他 将尝试定义跨膜信号传导的选择性， 各种细胞因子通过这些特定的受体。 COS、HEK 293或 K562与他将要研究的大鼠趋化因子受体。 的 信号的有效性将通过确定 花生四烯酸生成，细胞内钙增加，肌醇 磷酸盐产生和cAMP积累[初步数据 这表明调查员有能力这样做]。 如果他没发现 他将使用与人ga 14共转染的细胞， 在这些细胞中已经定义了有效信号传导的Ga 15 系统. 特异性受体的剂量依赖性反应 将为许多趋化因子配体建立配体。 在第二个目标中，他将测试大鼠小胶质细胞 在体外表达有效的chomokine受体。 第一部分 他将检测小胶质细胞对趋化因子的反应。 控制和 细胞因子（INFg或LPS）刺激的小胶质细胞将暴露于4 CC趋化因子和信号转导的参数确定 上面提到。 细胞内信号传导增加的机制 钙将被研究。 信号传递的机制增加了 将研究细胞内钙。 小胶质细胞预处理 百日咳毒素（通过前ADP-gibosylating G- 蛋白质的α亚基）将被研究，以确定 细胞内钙积累。 如果在Ca耗尽的情况下信号传导失败， 介质，源必须是外部的。 这将允许调查人员 为了定义来自多种药物同时结合的协同效应， chemolines受体。 在第二部分的目标中， 趋化因子受体在培养的小胶质细胞中的表达 对多种细胞因子的影响。 chomokine 将比较对造血细胞最有效的浓度 影响小胶质细胞的剂量和时间。 水平的变化 通过全细胞的北方印迹来确定表达的量 mRNA和/或通过RNA保护测定。 在最终的目标中，他将使用体内系统。 这将考验 小胶质细胞表达功能性趋化因子受体的假说。 这将通过在特定CNS组织上进行原位杂交来完成 在已知激活小胶质细胞的特定操作之后。 要研究的系统是面神经挤压，或EAE。 细胞 调节趋化因子受体将通过原位 杂交，而特异性细胞分型将通过 免疫细胞化学染色。 在原地证明意志 针对特定的趋化因子受体mRNA， Ga 15分子的mRNA。 这些在同一个表达 将定义操作后的细胞类型。

This is a second reapplication to investigate the type and function of chemoline receptors in the rat has been greatly improved by a narrowed focus and improved probability of success. It has three specific aims which come from prior proposals, but which are smaller in scope. The first specific aims are to define the specificity of the ligand binding to the chemokine receptors CKR2, CKR5 and RBS11. This aim has two parts. In the first part he will transect into HEK293 cells using the pcDNA3 vector. He will assess the effectiveness of the transfection by assaying for mRNA by Northern blotting. He will determine ligand binding by exposing transfected cells or their membranes to radiolabeled MCP-1. He will determine if the specificity of this binding can be competed with a variety of other CC chomolines; dissociation constants will be determined. He will also do the same for the CKR5 for MIP-1a. In the second part of this aim, he will attempt to define the selectivity for transmembrane signaling of various cytokines through these specific receptors. COS, HEK293, or K562 with the rat chemokine receptors he will study. The effectiveness of signaling will be assessed by determining the arachidonic acid production, intracellular calcium increased, inositol phosphate production and cAMP accumulation [preliminary data shows the ability of the investigator to do this]. If he fails to detect signaling, he will use these cells cotransfected with human ga14 or Ga15 for which effective signaling has been defined in these cell systems. The dose dependent response of the receptors for specific ligands will be established for a number of the chemokine ligands. In the second aim he will test the hypothesis that rat microglial cells express effective chomokine receptors in vitro. In the first part of this aim he will examine microglial responses to chemokines. Control and cytokine (INFg or LPS) stimulated microglial cells will be exposed to 4 CC chemokines and signal transduction determined by the parameters noted above. The mechanism of signaling increased intracellular calcium will be studies. The mechanism of signaling increases intracellular calcium will be studies. Microglia pre-treated with pertussis toxin (to blunt response by pre-ADP-gibosylating the G- protein's alpha subunit) will be studied to determine the source of intracellular calcium accumulating. If signaling fails in a Ca depleted medium, the source must be external. This will permit the investigator to define synergistic effects from the simultaneous binding of multiple chemolines to receptors. In the second part of the aims, the level of expression of chemokine receptors in cultured microglial cells exposed to a variety of cytokines will be studied. The chomokine concentrations most effective on hematopoetic cells will be compared to the dose and time of effect on microglial cells. The change in levels of expression will be determined by northern blotting of whole cell mRNA and/or by RNA protection assay. In the final aim, he will use an in vivo systems. This will test the hypothesis that microglial cells express functional chomokine receptors. This will be done by in situ hybridization on specific CNS tissues following defined manipulations known to activate microglial cells. The systems to be studies are facial nerve crush, or EAE. The cells modulating chemokine receptors will be defined by in situ hybridization while specific cells typed will be delineated by immunocytochemistry on adjacent croystat sections. In situ proves will be directed against specific chemokine receptor mRNAs and the mRNA for the Ga15 molecule. The expression of these in the same cell type following manipulation will be defined.