IMMUNOLOGICAL STUDIES OF MUSCARINIC RECEPTOR SUBTYPES

本申请寻求资金，以继续正在进行的生产项目 并鉴定了针对毒扁豆碱的定点抗肽抗体 受体(MR)亚型。该提案的目标是研究不同的 Mr亚型的结构、结合特性、功能和定位 哺乳动物的大脑。药理学基础上推测的MR亚型 结合数据已经被最近的分子生物学证据所证实 用于编码唯一初级序列的五个不同的MR基因。 然而，药理学、生物化学和生物化学之间的相关性 每种亚型的功能仍不清楚，主要是因为没有 区分这五种受体蛋白的可用方法。 目前可用的毒鼠碱配体仅区分三种MR亚型， 而核苷酸探针不识别基因产物或受体，而只能 DNA或RNA受体消息。因此，免疫学方法具有 优点：1)区分高度同源的受体 蛋白质和2)直接识别受体蛋白质。具体来说， 抗M1-M5 MR亚型独特序列的单抗和多克隆抗体 都会被制作出来。到目前为止，杂交瘤中有8种单抗 产生腹水的小鼠被培育成独特的多肽 对应于M1。也已经产生了多克隆抗体来 多肽M2和M3。免疫斑点、酶联免疫吸附试验、免疫印迹、 将使用免疫沉淀和免疫组织化学方法筛选和 根据免疫原、MR、 MR亚型和分子定位。磁共振的超微结构定位 啮齿类动物和人脑中的亚型将在光线和 电子显微镜水平。相关的G蛋白将被鉴定出来 可溶性MR-G蛋白的特异性免疫沉淀电泳法 复合体。MR亚型之间在一级结构上的区别将 经选择后用双向电泳法制作 免疫沉淀或免疫亲和层析。这些措施的结果 研究将提供对中枢性MR的更清晰的理解 记忆障碍治疗干预的重要靶点 与高龄和阿尔茨海默病有关。

This application seeks funds to continue the on-going project of producing and characterizing site-directed anti-peptide antibodies to muscarinic receptor (MR) subtypes. The goal of the proposal is to study the distinct structure, binding properties, function and location of MR subtypes in mammalian brain. MR subtypes postulated on the basis of pharmacological binding data have been verified by the recent molecular biological evidence for five distinct MR genes coding for unique primary sequences. Nevertheless, correlation between the pharmacology, biochemistry and function of each subtype remain unclear, primarily because there are no available methodologies which distinguish the five receptor proteins. Currently available muscarinic ligands distinguish only three MR subtypes, and nucleotide probes do not recognize gene product, or receptor, but only DNA or RNA receptor message. An immunological approach, therefore, has the advantage of 1) distinguishing between the highly homologous receptor proteins and 2) directly recognizing receptor proteins. Specifically, mono- and polyclonal antibodies to unique sequences of m1-m5 MR subtypes will be produced. Thus far, eight monoclonal antibodies from hybridomas and ascites-producing mice have been generated to peptide uniquely corresponding to m1. Polyclonal antibodies have also been generated to peptides m2 and m3. A combination of immunodot, ELISA, immunoblotting, immunoprecipitation and immunohistochemistry will be used to screen and characterize antibodies with respect to specificity for the immunogen, MR, MR subtype, and molecular location. Ultrastructural location of MR subtypes in the rodent and human brain will be assessed at the light and electron microscopic levels. Associated G proteins will be identified electrophoretically by specific immunoprecipitation of soluble MR-G protein complexes. Distinction in primary structure between the MR subtypes will be made using two-dimensional electrophoresis after selective immunoprecipitation or immunoaffinity chromatography. Results of these studies will provide a clearer understanding of central MR which remain important targets for therapeutic intervention in treating memory disorders associated with advanced age and Alzheimer's Disease.